Download factor xiii: a coagulation factor with multiple

Physiol Rev 91: 931–972, 2011
doi:10.1152/physrev.00016.2010
FACTOR XIII: A COAGULATION FACTOR WITH
MULTIPLE PLASMATIC AND CELLULAR
FUNCTIONS
László Muszbek, Zsuzsanna Bereczky, Zsuzsa Bagoly, István Komáromi, and Éva Katona
Clinical Research Center and Thrombosis, Haemostasis and Vascular Biology Research Group of the Hungarian
Academy of Sciences, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
INTRODUCTION
GENERAL OVERVIEW ON STRUCTURE,...
ACTIVATION AND REGULATION OF...
THE ACTION OF ACTIVATED FACTOR XIII...
FACTOR XIII IN WOUND HEALING AND...
OTHER EFFECTS OF FACTOR XIII
FACTOR XIII AS AN INTRACELLULAR...
SUMMARY, CONCLUSIONS, AND...
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became apparent only after Duckert et al. (94) demonstrated that the severe bleeding diathesis of a patient was
due to the deficiency of fibrin stabilizing factor. In 1963,
soon after this clinical finding, the International Committee
on Blood Clotting Factors acknowledged this protein as a
clotting factor and termed it factor XIII. In 2007 the International Society of Thrombosis and Haemostasis, Scientific
and Standardization Committee published a recommendation on the terms and abbreviations concerning FXIII (258),
which will be followed throughout this review.
I. INTRODUCTION
The history of blood coagulation factor XIII (FXIII) started
more than 70 years ago, when Barkan and Gaspar (33)
observed that fibrin clots formed in the presence of Ca2⫹
become insoluble in weak bases. Some 20 years later, Robbins (314) performed the experiment with purified fibrinogen and concluded that in addition to Ca2⫹, a “serum factor” is also needed to render the clot insoluble in weak acids
and bases. Laki and Lóránd (196) and Lóránd (221, 222)
were the first who realized that the serum factor, which
made the fibrin clot insoluble in concentrated urea solution,
was thermo-labile and nondialyzable, and they called this
protein fibrin stabilizing factor. Fibrin stabilizing factor was
then purified from plasma, and the enzymatic nature of it
was first revealed and characterized by Loewy and co-workers (215–219). The clinical significance of these findings
For a relatively long time, FXIII did not raise much interest
among scientists, and research on this field was practiced
only by a few devotees, although their contributions were
essential for later developments. It was revealed that
FXIII is a zymogen, and in contrast to all other zymogen
clotting factors, its active form (FXIIIa) is a transglutaminase (TG; protein-glutamine: amine ␥-glutamyltransferase, EC 2.3.2.13) that forms ⑀(␥-glutamyl)lysyl crosslinks between two polypeptide chains. The mechanism of
the activation of zymogen FXIII and the basic enzymology
of FXIIIa were clarified. FXIII was also found in platelets,
and structural differences between plasma FXIII (pFXIII)
and cellular factor (cFXIII) were revealed. Clinical data on
the consequences of inherited FXIII deficiency provided important pieces of information on features of the disease and
on the physiological role of FXIII in hemostasis. Neverthe-
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Muszbek L, Bereczky Z, Bagoly Z, Komáromi I, Katona E. Factor XIII: A Coagulation
Factor With Multiple Plasmatic and Cellular Functions. Physiol Rev 91: 931–972,
2011; doi:10.1152/physrev.00016.2010.—Factor XIII (FXIII) is unique among clotting factors for a number of reasons: 1) it is a protransglutaminase, which becomes
activated in the last stage of coagulation; 2) it works on an insoluble substrate; 3) its
potentially active subunit is also present in the cytoplasm of platelets, monocytes, monocyte-derived
macrophages, dendritic cells, chondrocytes, osteoblasts, and osteocytes; and 4) in addition to its
contribution to hemostasis, it has multiple extra- and intracellular functions. This review gives a
general overview on the structure and activation of FXIII as well as on the biochemical function and
downregulation of activated FXIII with emphasis on new developments in the last decade. New
aspects of the traditional functions of FXIII, stabilization of fibrin clot, and protection of fibrin against
fibrinolysis are summarized. The role of FXIII in maintaining pregnancy, its contribution to the wound
healing process, and its proangiogenic function are reviewed in details. Special attention is given to
new, less explored, but promising fields of FXIII research that include inhibition of vascular permeability, cardioprotection, and its role in cartilage and bone development. FXIII is also considered as
an intracellular enzyme; a separate section is devoted to its intracellular activation, intracellular
action, and involvement in platelet, monocyte/macrophage, and dendritic cell functions.
MUSZBEK ET AL.
less, FXIII used to be considered a stepchild among clotting
factors for a number of reasons: 1) it falls outside the mainstream of the clotting cascade, and it only starts working
after the visible end result, fibrin formation, had been completed; 2) as opposed to other clotting enzymes, it is making
rather than breaking peptide bonds; and 3) it is working on
an insoluble substrate matrix that is rather unattractive for
enzymologists.
This review, besides giving a short overview on earlier discoveries concerning structure and biochemical functions of
FXIII, mainly deals with recent developments on the regulation of FXIII, with its role in fibrinolysis, and with its
functions which partially or completely lie outside hemostatic mechanisms. The latter topic includes the cellular effects of FXIII and its involvement in angiogenesis and
wound repair as well as in maintaining pregnancy. The
intracellular activation and function of FXIII will also be
discussed.
II. GENERAL OVERVIEW ON STRUCTURE,
GENE, POLYMORPHISMS, AND
EXPRESSION OF FACTOR XIII
The structure and synthesis of FXIII have been reviewed
earlier in detail (124, 263); here only a general overview
with emphasis on new discoveries will be provided. FXIII is
a pro-TG that circulates in plasma in tetrameric form
(FXIII-A2B2) (124, 259, 263). It consists of two potentially
active, catalytic A subunits (FXIII-A) and two carrier/inhibitory B subunits (FXIII-B). FXIII-B is in excess; in the
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A. Factor XIII A Subunit and Cellular Factor
XIII
FXIII-A, a protransglutaminase, belongs to the family of
TGs. Eight members of the family (FXIII-A and TG1-TG7)
possess potential enzymatic activity; while an additional
protein, erythrocyte band 4.2, has a similar domain structure but, due to the replacement of active site amino acid,
lacks enzymatic activity. (A more detailed review on TGs is
provided in Reference 95.) All TGs are monomers; only
FXIII-A forms a dimeric structure. The three-dimensional
structure of three human TGs, FXIII-A2, TG-2, and TG-3,
has been resolved (14, 213, 377, 383). It is interesting that
despite the modest sequential agreement, their secondary
structure shows striking similarity. A detailed comparison
of the structure of the three TGs is given in Reference 187.
FXIII-A consists of 732 amino acids including an initiator
methionine (http://www.uniprot.org/uniprot/P00488); its
molecular mass is 83 kDa. Serine is in the penultimate position, which favors the removal of initiator methionine and
then N-acetylation. The NH2-terminal amino acid in the
mature molecule is, indeed, an acetylated serine residue. In
the following, amino acid numbering starting with the Ser
residue will be used. No hydrophobic leader sequence could
be identified in the primary structure of FXIII-A (144).
There are nine cysteine residues in the protein, including the
active site cysteine (Cys314), none of which forms disulfide
bonds. Although the molecule has potential sites for N-glycosylation, no carbohydrate residue is detected. FXIII-A is
expressed in all vertebrates investigated so far. A phylogenetic tree and sequence identity in different species are
shown on FIGURE 1. Recombinant FXIII-A2 (rFXIII) has
been expressed in Escherichia coli (45), in Saccharomyces
cerevisiae (42, 157), in Schizosacharomyces pombe (52),
and even in tobacco plant cells and whole tobacco plants
(108). Features of rFXIII are identical to those of cFXIII.
The first atomic resolution structure of FXIII-A2 was published (383) one and a half decades ago. Two crystal forms,
orthorhombic (383) and monoclinic (377), were obtained
for FXIII-A2, and X-ray crystallography of both crystal
forms resulted in essentially the same structure. Studies on
the X-ray structure of rFXIII-A2 (102, 377, 382, 383) revealed numerous structural details of FXIII-A2 and provided invaluable information on the structure-function relationships. The X-ray structure was also a great help in
understanding the structural consequences of mutations
causing FXIII deficiency (173).
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Discoveries starting from the 1980s changed this trend, and
since then FXIII has become an increasingly attractive field
of research. The milestones of FXIII research were the following: 1) the realization that FXIII plays a highly important role in the regulation of fibrinolysis. 2) The discovery of
cFXIII in monocytes and different types of macrophages,
including tissue macrophages, revealed that this proenzyme
is present in most organs and tissues of the body. 3) The
primary protein structure and the genomic structure of
FXIII subunits were published in the second half of the
1980s followed by the exact identification of molecular genetic defects in an increasing number of FXIII deficient patients. In 1994 the three-dimensional structure of cellular
FXIII has also been published. 4) The availability of recombinant cFXIII (rFXIII) significantly promoted basic biochemical studies. 5) New, relatively simple reliable methods
for the determination of FXIII activity and concentration in
the plasma became available and made large-scale clinical
studies possible. 6) The discovery of FXIII polymorphisms
induced biochemical and clinical studies to explore their
consequences. 7) It was realized that FXIII is a multifunctional protein that, beside hemostasis, plays an important
role in a wide variety of physiological and pathological
processes.
plasma, ⬃50% of it exists in free noncomplexed form.
FXIII-A dimer (FXIII-A2), but not FXIII-B, is also present in
the cytoplasm of certain cells, particularly of platelets and
monocytes/macrophages. The primary structure of both
subunits has been determined by cDNA cloning and amino
acid sequence analysis (127, 145, 146, 359).
MULTIPLE FUNCTIONS OF FACTOR XIII
FXIII-A consists of four main structural domains, ␤-sandwich (amino acid 38 –184), catalytic core (185–515), ␤-barrel 1 (516 – 628), and ␤-barrel 2 (629 –731) domains, plus
an NH2-terminal activation peptide (AP-FXIII) (FIGURE 2).
The latter is cut off by thrombin during activation (see sect.
IIIA for details). The sandwich and barrel domains almost
exclusively consist of ␤-sheets with only a few small helical
structural elements in the sandwich and ␤-barrel 1 domains.
The core domain contains both ␤-sheets and helices. On the
basis of thermal denaturation studies (92, 193), the core
domain can be further divided into NH2- (189 –332) and
COOH-terminal (333–515) subdomains. Two nonproline
cis peptide bonds were observed in the core domain (377)
that also exists in human TG-2 (213) and TG-3 (14). The
FXIII-A subunit monomers are arranged in a dimeric structure resembling a hexagon with C2 symmetry, in which the
two central core domains are surrounded by the six ␤-sheet
domains (FIGURE 2).
The X-ray studies also revealed that the arrangement of the
amino acid residues playing a key role in the catalytic process (Cys314, His373, Asp396) resembles the catalytic triad
in cysteine proteases (FIGURE 3A). Therefore, it was assumed that the catalytic reaction corresponds to a reverse
proteolysis (294). However, due to the active site specificity
of the enzyme, it catalyzes interchain peptide bond formation between the glutamine and lysine side chains instead of
peptide chain elongation. In the reaction catalyzed by cys-
teine proteases, the stabilization of the intermediates by the
so-called oxyanion hole is of crucial importance. Based on
the structural analogy with papain, it has been assumed that
the NH group of the indole side chain of Trp279 and the
NH group of Cys314 backbone form the oxyanion hole
(294) (FIGURE 3A). Trp279 is highly conserved in vertebrate
TGs, and its direct involvement in the TG reaction has been
shown by kinetic study with TG-2 (147).
Although the geometrical arrangement of the catalytic triad
and the oxyanion hole in the catalytic domain corresponds
to the arrangements in cysteine proteases (288), X-ray
structure also revealed that the catalytic cysteine residue is
completely buried by AP-FXIII (FIGURE 3A). A strong saltbridge between the side chain of Arg11= in AP-FXIII extending over from the opposite subunit and the side chain of
Asp343 of the core domain enforces AP-FXIII into a position
that makes Cys314 inaccessible for the substrate and even for
the solvent molecules. Therefore, the removal or disposition of
AP-FXIII is a prerequisite of activation. However, this is not
sufficient since the accessibility of the active site to the substrate is also prevented by the strong hydrogen bond that exists
between the O␩ atom of Tyr560 and S␥ atom of Cys314
residues (FIGURE 3A). Tyr560 is located on a loop of the ␤-barrel 1 domain, which means that the transposition of this loop
from the immediate vicinity of the active site is also required
for the interaction with the substrate. Considering the extreme
stability of barrel domains (193) and the size of the natural
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FIGURE 1 Phylogenetic tree and sequence
identity of A subunit of factor XIII (FXIII-A) in
different species. A: phylogenetic tree of
FXIII-A was constructed by the BIONJ version
(109) of neighbor-joining algorithm using the
SplitsTree4 software (141) downloaded from
the www.splitstree.org website. FXIII-A protein sequences were aligned by ClustalX software (198). Distance scale represents the
number of differences between sequences
in species since their divergence; 0.1 means
10% difference between two sequences. B:
identity percent represents the degree of homology in different organisms compared with
human FXIII-A protein and coding DNA. Data
were obtained from the HomoloGene database (www.ncbi.nlm.nih.gov/sites/homologene). The amino acid sequence of salmon
FXIII-A was downloaded from the Universal
Protein Resource (UniProt) web server
(http://www.uniprot.org), and its identity
percent value was calculated by BLAST software (www.blast.ncbi.nlm.nih.gov) using
the alignment obtained by ClustalX software
(198).
MUSZBEK ET AL.
FIGURE 2 Ribbon and tube representation of the zymogen
rFXIII-A2 homodimer based on its X-ray structure (377) and visualized by the Chimera software (299). The relative positions of the
constituting domains are marked for only one of its monomeric
FXIII-A subunit. Helical and ␤-sheet structural elements are colored
orange and purple, respectively. The disordered (random coil like)
structural elements and loops are shown in cyan, and the activation
peptides are depicted in bright green.
substrates, the removal of the whole ␤-barrel 1 from its original position is an even more likely option.
Ca2⫹ binding sites on the surface of FXIII-A2 are essential for
the activation of FXIII. FXIII-A, but not FXIII-B, tightly binds
The gene coding for human FXIII-A (F13A1) spans over 160
kb and has been localized to chromosome 6p24 –25 (46). It is
transcribed into a 3.9-kb mRNA, with an 84-bp 5=-untranslated region, a 2.2-kb open reading frame, and a 1.6-kb 3=untranslated region. F13A1 contains 15 exons and 14 introns
(144). Exon I consists of the 5= noncoding region, and exon II
encodes AP-FXIII (FIGURE 4). The ␤-sandwich domain, the
catalytic core domain, and the two ␤-barrel domains are encoded by exons II-IV, exons IV-XII, exons XII-XIII, and exons
XIII-XV, respectively.
shows the polymorphisms and their location in
the coding region of FXIII-A gene. Data provided by the
International HapMap project (www.hapmap.org) on the
frequency of the polymorphisms in the Caucasian, African,
FIGURE 4
FIGURE 3 Structures around the catalytic triad (A) and the main Ca2⫹ binding site (B) of FXIII-A. Structural
elements are shown by “ribbon and tube” representation; helical and ␤-sheet structural elements are colored
orange and purple, while disordered (random coil like) structural elements and loops are shown in light green.
Side chains of selected amino acid residues are represented by balls and sticks. The N, C, O, and S atoms are
colored blue, gray, red, and yellow, respectively. A: the amino acids of the catalytic triade (Cys314-His373Asp396) and Trp279 participating in the formation of oxyanion hole are shown together with interacting amino
acid residues that fix the structure in inactive conformation and bury the active site cysteine. The X-ray
structure published by Weiss et al. (377) was used for the preparation of the picture (377). Arg11= represents
the Arg residue in AP-FXIII of the opposite subunit. B: amino acid residues implicated in the binding of Ca2⫹
(green ball) at the main Ca2⫹ binding site. The structure based on the X-ray coordinates published by Fox et al.
(102) was visualized by the Chimera software (299).
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one Ca atom with a Kd of 0.1 mM as determined by equilibrium dialysis and fluorescence studies (209), while a Kd of 0.51
mM was determined by 43Ca NMR experiments (19). These
reports also suggest the existence of additional low-affinity
Ca2⫹ binding sites. From X-ray structural studies of FXIII-A2
cocrystallized with Ca2⫹, Str2⫹, and Yb3⫹, a single main cation binding site per subunit was identified (102). The carboxylate group of Asp438, Glu485, and Glu490 side chains, the
carbonyl O atom of Asn436, as well as the backbone carbonyl
O atom of the Ala457 residue participate in cation binding
(either directly or through a water bridge) to a different extent
depending on the nature of the cation (FIGURE 3B). The backbone carbonyl O atom of Ala457 and the carboxylate groups
of Glu485 and Glu490 side chains have been reported to be
directly involved in Ca2⫹ binding (102, 195).
MULTIPLE FUNCTIONS OF FACTOR XIII
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FIGURE 4 Distribution of SNPs published so far within the coding region of F13A1 gene. The “A” of the
translation initiation codon “ATG” is considered as nucleotide one, and nucleotide positions within cDNA are
given accordingly. The amino acid numbering is given according to Ichinose et al. (144). Exons encoding a
particular domain are color-coded. The mutations resulting in amino acid replacements are depicted in purple,
while silent mutations are shown in black.
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MUSZBEK ET AL.
tive effect against venous thromboembolism (378) and
myocardial infarction (370). The complex interrelationship of FXIII polymorphisms with the risk of thrombotic
disease is beyond the scope of this review; interested
readers should consult a most recent review (259). The
biochemistry of other common FXIII-A polymorphisms
has not been investigated in such details as that of
Val34Leu polymorphism. The Phe204 allele of FXIII-A
Tyr204Phe polymorphism was reported to be associated
with decreased pFXIII level and activity, whereas the
Leu564 allele of the Pro564Leu variant resulted in lower
FXIII plasma level with increased FXIII activity (22,
107). However, these results still need to be confirmed.
FXIII-A is expressed primarily in cells of bone marrow origin. It is present in platelets in huge quantity (55, 228). The
average FXIII-A content of a single platelet was estimated to
be 60 ⫾ 10 fg, which corresponds to 3% of total platelet
proteins (176). The concentration of FXIII-A is ⬃100- to
150-fold higher in platelet cytoplasm than in plasma.
FXIII-A is also present in megakaryocytes (10, 179, 231,
256) and in their precursor cells (10). Megakaryocytes synthesize FXIII-A and package it, together with its encoding
Table 1 Racial variations of the polymorphisms in factor XIII-A and factor XIII-B subunit genes
Nucleotide Variation
F13A1
c.103G⬎T
c.614A⬎T
c.996A⬎C
c.1652C⬎T
c.1694C⬎T
c.1704A⬎G
c.1696T⬎A
c.1951G⬎A
c.1954G⬎C
F13B
c.344G⬎A
c.456G⬎A
c.765C⬎T
c.1049A⬎G
c.1707T⬎G
c.1806T⬎C
c.1952 ⫹ 144 C⬎G
Allele Frequency
Amino Acid
Variation
CEU
YRI
JPT
HCB
p.V34L
p.Y204F
p.P331P
p.T550I
p.P564L
p.E567E
p.L588Q
p.V650I
p.E651Q
0.767/0.233
0.983/0.017
0.853/0.147
1
0.758/0.242
0.890/0.110*
0.975/0.025
0.950/0.050
0.775/0.225
0.883/0.117
1
0.933/0.067
0.992/0.008
0.864/0.136
0.991/0.009
1
0.942/0.058
0.742/0.258
1
1
1
1
0.705/0.295
1
1
0.909/0.091
0.909/0.091
1
1
0.989/0.011
1
0.733/0.267
1
1
0.911/0.089
0.911/0.089
p.R95H
p.T132T
p.C235C
p.H330R
p.D549E
p.N582N
-
0.075/0.925
0.925/0.075
1
1
1
0.492/0.508
0.85/0.142
0.725/0.275
0.307/0.693
0.983/0.017
0.933/0.067
0.908/0.092
0.842/0.158
0.98/0.017
0.034/0.966
1
1
0.989/0.011
0.966/0.034
0.244/0.756
0.42/0.578
0.044/0.956
1
1
0.989/0.011
0.956/0.044
0.189/0.811
0.35/0.644
In the case of F13A1 and F13B polymorphisms, amino acid positions are numbered according to Ichinose et
al. (144) and according to Bottenus et al. (50), respectively. Allele frequency data of the International HapMap
project (www.hapmap.org) were used; the first numbers represent the frequency of ancestral allele. CEU, Utah
residents with ancestry from Northern and Western Europe; YRI, African population in Yoruba in Ibadan,
Nigera; JPT, Japanese in Tokyo, Japan; HCB, Han Chinese in Beijing, China. *As in the case of c.1704A⬎G
mutation, no HapMap data for CEU were available; data were taken from the Centre d’Etude du Polymorphisme
Human (CEPH) project (www.cephb.fr).
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and Asian populations demonstrate considerable racial
variation (TABLE 1). Detailed studies on the biochemical
and clinical consequences of the polymorphisms were carried out only in the case of Val34Leu polymorphism, a
primarily Caucasian mutation first described by Mikkola et
al. (244). This mutation increases the rate of FXIII activation (23, 31, 374) (see sect. IIIA for details). The structure of
fibrin clots is also influenced by FXIII-A Val34Leu polymorphism (23), and this effect is modulated by fibrinogen
concentration (211). At high fibrinogen levels, plasma samples from homozygotes for the Leu34 allele form clots having looser structure, thicker fibers, and increased permeability, while at low fibrinogen concentrations fibrin
meshwork had thinner, more tightly packed fibers, and
lower permeability. Practically no fibrinogen concentration-dependent changes were observed in the plasma
samples of wild-type individuals. The effect of the
Val34Leu polymorphism on the specific activity of
FXIIIa was controversial for a while; it is now clear that
the specific activity of fully activated pFXIII (23, 31),
cFXIII (374), and rFXIII-A2 (201) of different FXIII-A
Val34Leu genotypes are identical. Although there are
contradictory results on the clinical implications of this
polymorphism, meta-analyses demonstrated its protec-
MULTIPLE FUNCTIONS OF FACTOR XIII
mRNA, into newly formed platelets (5, 6, 231). It is now
clear that FXIII-A in platelets is essentially of cytoplasmic
localization (51, 220, 278, 346). Platelets could take up a
tiny amount of FXIII-A2B2 from the plasma which appears
in ␣-granules (233); this amount is negligible compared
with the amount of FXIII-A in the cytoplasm.
The presence of FXIII-A mRNA and the synthesis of
FXIII-A have also been verified in the above cell types (4, 5,
190, 271). The transcriptional regulation of cell type-specific expression of FXIII-A was investigated in monocytoid
(U937) and megakaryocytoid (MEG-01) cell lines containing FXIII-A mRNA (178). Reporter gene assays revealed that
a 5=-fragment was sufficient to support the basal expression in
the above cells, but not in other cell types. Promoter elements
for a myeloid-enriched transcription factor (MZF-1) and for
the ubiquitous transcription factors, SP-1 and NF-1, seem to
be important for the basal FXIII-A expression. Another region
had enhancer activity in MEG-01 cells but silencer activity in
B. Factor XIII B Subunit
FXIII-B is a glycoprotein consisting of 641 amino acids
and containing 8.5% carbohydrate; its molecular mass is
⬃80 kDa. It is a typical mosaic protein consisting of 10
short tandem repeats, called sushi domains or GP-I structures, each containing ⬃60 amino acids and held together by a pair of internal disulfide bonds (FIGURE 5).
The same structures have also been found in more than
20 other proteins, including ␤2-glycoprotein I, many of
which are encoded by genes clustered in chromosome 1
band q32 (143). By electron microscopy, the B subunit
appears as a thin, flexible, and kinked strand (58).
FXIII-B has not been crystallized, and no reliable information on its three-dimensional structure is available.
While FXIII-A in the absence of FXIII-B forms homodimer, contradictory publications concerning the
state of noncomplexed FXIII-B were obtained. Gel filtration of purified FXIII-B suggested the presence of FXIII-B
dimers (335), while sedimentation analysis revealed monomeric form (58). In a most recent report, gel filtration
of recombinant FXIII-B supported the former finding
(350). There is no good explanation for the discrepancy,
and in plasma conditions, the state of free FXIII-B remains to be explored.
The gene of FXIII-B subunit (F13B) is located at position
1q31–32.1; it is ⬃28 kb in length and composed of 12
exons producing a 2.2 kb mRNA (146). Exons are interrupted by 11 introns. Exon I encodes a 20-amino acid
leader sequence characteristic for proteins that are secreted
via the classical secretory pathway (50, 169, 375). Exon
II-XI code for the sushi domains, each of which is encoded
by a single exon. The sushi domains show a high degree of
homology, suggesting gene duplication and exon shuffling
during evolution (292, 293). The last exon codes for a
COOH-terminal region of FXIII-B, for the 3=-untranslated
region, and for the polyA tail. F13B is directly regulated by
transcription factor HNF1␣ and HNF4␣ (143, 214). Accordingly, FXIII-B is expressed in the liver and secreted by
hepatocytes (146, 149, 266).
The polymorphic nature of FXIII-B was revealed a long
time ago (43, 81, 207). On the basis of isoelectric focusing experiments, three major population-associated phenotypes were described: FXIII-B*1, FXIII-B*2, and
FXIII-B*3, characteristic of European, African, and
Asian populations, respectively. Since the first report of a
F13B gene polymorphism by Board (43), several new
polymorphisms have been described. FXIII-B polymorphisms and their frequency in different races are summarized on FIGURE 6 and in TABLE 1. By screening all the
exons of F13B, Komanasin et al. (186) found three poly-
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FXIII-A was also detected in monocytes (257) and in their
bone marrow precursor cells (10). Like platelets, monocytes
also lack FXIII-B. A most recent study revealed that FXIII-A
may be differently expressed in monocyte subsets; higher
FXIII-A mRNA expression was demonstrated in Ly-6Chigh
monocytes than in their Ly-6Clow counterparts (358).
FXIII-A in monocytes is also of cytoplasmic localization,
although during differentiation into macrophages it also
appears in the nucleus (7). The results of the first discovery
were soon confirmed, and FXIII-A was also detected in a
number of monocyte-derived macrophages including macrophages of different serous cavities, alveolar macrophages,
tumor-associated macrophages, histiocytic and dendritic
reticulum cells of lymph nodes, connective tissue histiocytes, perivascular dendritic macrophages, dermal dendrocytes etc. (reviewed in Refs. 6, 263). FXIII-A has also been
demonstrated in chondrocytes, osteoblasts, and osteocytes
(279) (see sect. VID). In early studies, FXIII-A was also
identified in placenta and in uterus (48, 61). With the use of
monocyte and macrophage differentiation markers, it
has been clearly demonstrated that FXIII-A is confined to
monocyte-derived tissue macrophages in the placenta (9,
171) and in the uterus (11) as well. The expression of
FXIII-A in monocytes is retained after malignant transformation; in fact, its expression in the respective cells is
upregulated in acute myelomonocytic, monocytic leukemias and in chronic myelomonocytic leukemias (172). It
turned out that the detection of FXIII-A by flow cytometry in leukemic cells is a useful intracellular marker in
the classification of acute myeloid leukemias. It is interesting that FXIII-A becomes expressed in part of the lymphoblasts in acute B cell lymphoid leukemia, while it is
absent in lymphocytes, normal lymphoid precursors, and
mature lymphocytes from B cell chronic lymphoid leukemia (183).
U937 cells, and the GATA-1 element was found to be responsible for the enhancer activity.
MUSZBEK ET AL.
morphisms in healthy Caucasian subjects. An A to G
transversion within exon III (rs6003) leads to His to Arg
amino acid exchange at codon 95 in the second sushi
domain of the mature protein. The frequency of FXIII-B
Arg95 carriers among healthy Caucasians is ⬃15%, it is
more frequent among Africans (also named as FXIII-B*2
at the phenotypic level), and it is missing from the Asian
population (155, 186). The polymorphism did not influence
FXIII-A, FXIII-B, or pFXIII antigen levels. Increased subunit
dissociation was found in plasma from subjects possessing the
Arg allele. However, when the variants were purified to homogeneity and binding was analyzed by steady-state kinetics,
no difference was observed (186).
Most recently, a C-to-G change at position 29756 in
intron K (IVS11⫹144) leading to a novel splice acceptor
site was described (155, 321). This polymorphism results
in allele-specific splicing products, and a protein 15
amino acids longer at the COOH terminus than its wildtype counterpart is synthesized. The variant sequence
includes two additional lysine and one glutamic acid res-
938
idues. These charged amino acids change the pI of the
protein. The polymorphism characteristically occurs in
Asians; it corresponds to FXIII-B*3 at the phenotype
level. Its frequency is less in Caucasian populations, and
it does not seem to be present among Africans. Although
such a profound structural change would be expected to
alter some of the biochemical features of the molecule,
this possibility has not been explored, yet.
C. Factor XIII Complex in Plasma
The origin of FXIII-A in pFXIII complex has been a debate
for a long period of time. Studies characterizing phenotypes
of plasma FXIII subunit A after bone marrow and liver
transplantation clearly indicated that the major source of
plasma FXIII-A is a cell population or cell populations of
bone marrow origin (303, 379). In one of the studies, a
minor source of recipient FXIII-A, independent of donor
hematopoesis, was also detected (303); in theory, such
source(s) could be long-persisting tissue macrophages or
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FIGURE 5 Schematic structure of FXIII-B consisting of 10 sushi domains each held together by two pairs of
disulfide bonds. Amino acids in the leader sequence are depicted in purple, and those in the mature protein are
shown in cyan. The solid stars point to the site of N-glycosylation, and the empty star points to a potential
glycosylation site to which no carbohydrate is attached.
MULTIPLE FUNCTIONS OF FACTOR XIII
liver cells. However, FXIII-A and its encoding mRNA could
be detected in hepatocytes only in trace amounts using
highly sensitive techniques (5, 6), and there is no evidence
on the contribution of tissue macrophages to FXIII-A in the
plasma. Bone marrow ablation in patients undergoing autologous peripheral blood stem cell transplantation decreased platelet count to a very low level (⬎90% decrease),
while the reduction of plasma FXIII level was only ⬃25%
(152). It has also been shown that in long-standing severe
thrombocytopenia, the reduction of plasma FXIII was less
than expected (179). These findings suggest that in the case
of highly impaired production of FXIII-A by bone marrow
cells, synthesis of FXIII-A could be taken over by other cell
types. It is to be noted that in early embryonic life, well
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FIGURE 6 Distribution of SNPs published
so far within the coding region of F13B
gene. The “A” of the translation initiation
codon “ATG” is considered as nucleotide
one, and nucleotide positions within cDNA
are given accordingly. The amino acid numbering is given according to Bottenus et al.
(50). The mutations resulting in amino acid
replacements are depicted in purple, and
silent mutations are in black. The intronic
mutation resulting in an alternative splice
site is shown in green.
MUSZBEK ET AL.
Only the liver has been demonstrated as the source of
FXIII-B in plasma (see above). This conclusion was also
supported by phenotyping plasma FXIII-B following liver
transplantation. In this case, the phenotype of recipient’s
FXIII-B changed to the donor’s phenotype, while the phenotype of FXIII-A remained unchanged (379).
FXIII subunits derived from separate cellular sources form
tetrameric complex in the circulating blood. A reference
interval of 14 –28 mg/l has been established for FXIII-A2B2
in human plasma (175). It is surprising that there is still
uncertainty concerning the affinity of the two types of FXIII
subunits. Apparent binding constants of 4 ⫻ 10⫺7 M (305)
and 8.06 ⫻ 10⫺8 M (186) were reported. However, when
calculating with these binding constants, a significant part
of FXIII-A should be in free form, which is evidently not the
case. In our laboratory one magnitude lower binding constant (2.73 ⫻ 10⫺9 M) was measured; this value fits better
with the fact that FXIII-A is present in the plasma in practically fully complexed form. In normal conditions, FXIII-B
is in excess in the plasma, and ⬃50% of it circulates in free,
noncomplexed form (384). In patients with FXIII-A deficiency, the total amount of FXIII-B in the plasma is reduced
while the concentration of free B subunits remains constant.
The FXIII-A domain(s) that are responsible for the association with FXIII-B have not been identified. Most recently,
binding analysis with various truncated forms of rFXIII-B
suggested that the first sushi domain was responsible for the
binding of FXIII-B to FXIII-A (350).
In the plasma, practically all FXIII molecules are bound to
fibrinogen (Kd ⬃10⫺8 M) and the association is independent of the presence of calcium ions (123, 125, 215). The
␥=-chain in ␥A␥= fibrinogen (fibrinogen 2) provides the major binding site for FXIII (91, 250). The ␥=-chain is present
in ⬃15% of plasma fibrinogen. Compared with the ␥A
chain, the variant chain contains a 20-amino acid exten-
940
sion, which is the binding site for FXIII through its B subunit and also for thrombin exosite 2.
III. ACTIVATION AND REGULATION OF
FACTOR XIII: THE BIOCHEMICAL
ACTION OF ITS ACTIVE FORM
A. Activation of Factor XIII in the Plasma
and Within Cells
The activation of pFXIII occurs in the final phase of the
clotting cascade by the concerted action of thrombin and
Ca2⫹ (FIGURE 7). In the initial step of the reaction, thrombin
cleaves off AP-FXIII from the NH2 terminus of FXIII-A by
hydrolyzing the Arg37-Gly38 peptide bond. Then, in the
presence of Ca2⫹, the inhibitory B subunits dissociate,
which is a prerequisite for the truncated FXIII-A dimer
(FXIII-A2=) to assume an enzymatically active conformation (FXIII-A2*). The conformational change of FXIII-A2=
resulting in an active TG also requires Ca2⫹. The proteolytic cleavage of FXIII-A by thrombin considerably weakens the interaction between A and B subunits (224, 305).
The binding of Ca ions to the high-affinity Ca2⫹ binding
sites on the A subunits is sufficient to dissociate the subunits
and to activate the released A= dimer (138, 210). Interestingly, in the presence of Ca2⫹, complete activation of the
FXIII-A dimer occurs if only one AP-FXIII is released from
one of the two A subunits: in other words, the dimer of a
cleaved and an uncleaved A subunit (FXIII-A*A°) possesses
full enzymatic activity (138). It is to be noted that Siebenlist
et al. (342) reported a low innate activity of the zymogen
FXIII-A2B2 that can slowly cross-link fibrin(ogen) in the presence of Ca2⫹, but it is ineffective on low-molecular-weight
substrates (342). It is not clear if such an activity is due to the
presence of a small fraction of free FXIII-A2 that remains uncomplexed or dissociates from the complex and undergoes
nonproteolytic activation (see below) or it is the intrinsic property of the fibrin(ogen)-bound tetrameric zymogen.
Thrombin is not the only serine protease that could cleave
and activate FXIII in the presence of Ca2⫹. Several other
proteases, including batroxobin marajoensis (371), thrombocytin (273), trypsin (188, 334), and activated factor X
(FXa) (236) have been reported to be able to activate FXIII.
Although the cleavage site of these enzymes on FXIII-A has
not been identified, based on their substrate specificity and
on the Mr of the truncated FXIII-A, it is assumed that they
cleave the same Arg37-Gly38 peptide bond as thrombin,
and the active form of FXIII-A produced by these proteases,
like the thrombin activated form, also has an NH2-terminal
glycine. In contrast to thrombin, the proteolytic cleavage of
FXIII-A by FXa requires Ca2⫹. No evidence has been provided on the physiological implication of FXa-induced
FXIII activation. The mannan-binding lectin associated serine protease 1 (MASP1) that is involved in the complement
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before the development of bone marrow, FXIII-A is present
in mesenchymal histiocytes, and by week 20, FXIII-A also
appears in liver cells (170). In summary, in normal conditions, FXIII-A present in pFXIII is predominantly synthesized by cells of bone marrow origin; however, in the case of
impaired bone marrow function, other not yet identified cell
types might take over the production of FXIII-A. Another
unresolved question is how FXIII-A is released from cells
that synthesize it. In the absence of leader sequence, the
classical secretory pathway does not operate. In a most recent paper, cFXIII in macrophages was found in association
with podosomes and other structures adjacent to the plasma
membrane, and FXIII-A was present in intracellular vesicles
positive for Golgi matrix protein 130, which has been implicated in the delivery of nonclassically secreted proteins to the
plasma membrane (75). Although this finding raises the possibility of a nonclassical secretory pathway, clear-cut evidence
supporting this hypothesis is still to be provided.
MULTIPLE FUNCTIONS OF FACTOR XIII
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FIGURE 7 Different mechanisms of FXIII activation. FXIII-A=, A subunit of FXIII from which the activation
peptide was cleaved off; FXIII-A*, FXIII-A= transformed into an active transglutaminase in the presence of Ca2⫹;
FXIII-A°, noncleaved FXIII-A transformed into an active transglutaminase by the nonproteolytic mechanism in the
presence of Ca2⫹. Green and orange cylinders represent ␤-barrel and ␤-sandwich domains of FXIII-A, respectively. The central core domains in FXIII-A are depicted as horseshoes in magenta. The activation peptides are
shown as red loops. The elongated bended structure consisting of 10 pearls surrounding FXIII-A2 corresponds
to FXIII-B.
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MUSZBEK ET AL.
system and possesses thrombin-like activity also cleaves
FXIII-A at Arg37-Gly38, although at a much slower rate
than thrombin (191). It is not likely that FXIII activation by
MASP1 has any physiopathological importance. Recently,
it has been shown that limited cleavage of FXIII by human
neutrophil elastase resulted in the activation of both pFXIII
and cFXIII with TG activities of ⬃50% of thrombin-activated FXIII (28). MALDI-TOF analysis of the cleaved fragments and NH2-terminal Edman degradation of the truncated protein identified Val39-Asn40 as the primary cleavage site, and the NH2-terminal amino acid of this novel
active form of FXIII is Asn.
The presence of polymerizing fibrin enhances the activation
of pFXIII by ⬃100-fold (122, 123, 159, 210, 272). The
binding to fibrin makes the orientation of both pFXIII and
thrombin favorable for the proteolysis of FXIII-A at Arg37Gly38. Both fibrin I, which is devoid of fibrinopeptide A,
and fibrin II, which is devoid of fibrinopeptides A and B, are
effective accelerators. This effect is partly attributed to the
FXIII-B-driven optimal orientation of the tetramer molecule. It facilitates the interaction with thrombin and accelerates the thrombin-induced cleavage of FXIII-A in the
presence of fibrin (122, 137). In the absence of FXIII-B, i.e.,
in the case of cFXIII, no fibrin-induced promotion of the
removal of AP-FXIII could be observed (121, 137). Experiments with thrombin mutants demonstrated that His66,
Tyr71, and Asn74, surface residues of thrombin, are involved in high-specificity fibrin-enhanced factor XIII activation. These residues, representing a distinct interaction
site on thrombin (within exosite 1), are also employed by
thrombomodulin in the cofactor-enhanced activation of
protein C (300). Enhancement of factor XIII activation by
fibrin was competitively inhibited by thrombomodulin.
When the degree of fibrin ␥-chain cross-linking exceeds
942
Results concerning the effect of FXIII binding to fibrinogen ␥=-chain on FXIII activation are contradictory.
Moaddel et al. (247) reported that ␥A␥A fibrin accelerated the activation of pFXIII less efficiently than ␥A␥=
fibrin. In contrast, Siebenlist et al. (342) demonstrated
more rapid FXIII activation in the presence of ␥A␥A
fibrin(ogen) than with ␥A␥= fibrin(ogen). Atroxin, a
thrombin-like snake venom enzyme that does not bind to
the ␥= sequence, activated FXIII in the presence of ␥A␥A
and ␥A␥= fibrin(ogen) to the same extent. This finding
indicates that rate differences observed with thrombin
are related to the binding of thrombin rather than to the
binding of FXIII to the ␥= sequence.
In the plasma FXIII becomes effectively activated only on
the surface of newly formed fibrin, and the truncated active
dimer remains associated with its substrate; no thrombincleaved FXIII could be detected in the serum (341), while
following dissociation, FXIII-B becomes detached from fibrin. Fluorescence studies showed that cFXIII also binds to
fibrin, but with less affinity than pFXIII. The affinity, however, is increased upon cFXIII activation (137). Fibrin(ogen) also regulates the Ca2⫹ requirement for the activation
of truncated FXIII-A2= (78, 79). It brings down the optimal
Ca2⫹ concentration from ⬎10 mM to the physiological
interval present in the plasma.
Considering that the location of FXIII-A Val34Leu polymorphism is just 3 amino acids upstream from the thrombin
cleavage site, one would expect its influence on thrombininduced FXIII activation. Indeed, it was demonstrated with
both cFXIII (374) and pFXIII (23, 31) that the thrombininduced release of AP-FXIII from the Leu34 FXIII-A variant, as well as the consequent activation of FXIII, proceed
at a 2.5-fold higher rate than in the case of the Val34 variant. Faster activation of FXIII results in accelerated fibrin
cross-linking and in a higher rate of ␣2-PI incorporation
into fibrin (23, 31, 330, 374). The introduction of
Val34Leu and Val35Thr double mutations into FXIII-A
increased its rate of activation even further (by 7.6-fold)
(20). Investigation of the effect of FXIII-A Val34Leu genotype on thrombin-induced FXIII activation in the more
complex environment of human plasma led to a similar
conclusion (341). In plasma, the onset of FXIII activation
was initiated by fibrin polymerization independently of the
polymorphism; however, after initiation, the release of
Leu34 AP-FXIII occurred significantly faster than that of
Val34 AP-FXIII.
A few pieces of biochemical evidence suggest major conformational changes of FXIII during the process of activation;
however, the active form of FXIII has not been crystallized
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The question whether AP-FXIII is released into plasma
after thrombin cleavage or it remains noncovalently associated with the truncated FXIII molecule has been addressed by X-ray crystallography and by biochemical
techniques. In the crystal prepared from FXIII-A2 treated
with thrombin and Ca2⫹, the AP-FXIII appeared in the
same position as it was in the zymogen (382). It is also
known that in certain conditions the proteolytic removal
of AP-FXIII is not a prerequisite of the active configuration (79, 223, 302). However, as discussed earlier, the
data obtained by X-ray crystallography do not fit well
with the biochemical data obtained with FXIIIa in solution. Recent findings obtained with antibodies specific
for free AP-FXIII indicate that in plasma AP-FXIII dissociates from the truncated parent molecule and appears in
the serum (287, 332). While noncleaved AP-FXIII is
strongly hydrogen bonded to the adjacent ␤-sandwich
and to the ␤-barrel 1 domain of opposite FXIII-A monomer, according to NMR and circular dichroism studies
its released form seems unstructured (287).
⬃40%, the promoter effect of fibrin is lost (210), i.e., the
formation of cross-linked product downregulates the activation of cross-linking enzyme.
MULTIPLE FUNCTIONS OF FACTOR XIII
While X-ray crystallographic study could not reveal significant difference in the structure of nonactivated and thrombin and Ca2⫹-activated FXIII-A2 (382), the above findings
suggest a considerable conformational change during the
transformation of FXIII-A2 into FXIII-A2*. It is interesting
that Pinkas et al. (301) trapped TG-2 in complex with an
inhibitor peptide, Ac-P(DON)LPF, that mimics a natural
Gln substrate and solved the X-ray crystal structure (301).
They demonstrated that the substrate analog stabilizes active TG-2 in an extended conformation, dramatically different from earlier TG structures. No such crystal structure
of FXIIIa has been produced. Based on the homology with
TG-2, a molecular model of FXIII-A2* with extended conformation has been developed, and this model showed good
correlation with the biochemical data (187).
Although in physiological conditions pFXIII is generally
activated by the concerted action of a protease (thrombin)
and Ca2⫹, proteolytic cleavage is not an absolute requirement for the activation of the tetramer molecule. In nonphysiological conditions, i.e., at extremely high Ca2⫹ concentrations (ⱖ100 mM), the A2B2 complex dissociates and
the nontruncated FXIII-A2 becomes transformed into an
active TG (FXIII-A2°) (79, 223). The occupancy of lowaffinity Ca2⫹ binding sites might play a role in this process.
In sharp contrast to the nonproteolytic activation of pFXIII,
low Ca2⫹ concentration is sufficient to induce a slow progressive activation of cFXIII at physiological ionic strength
(302). The rate of this nonproteolytic activation of cFXIII is
greatly enhanced by increasing the ionic strength, and complex formation with FXIII-B fully abrogates the activation
process (302). This finding suggests that in the case of
pFXIII, high Ca2⫹ concentration is only required for the
dissociation of FXIII-B from FXIII-A2.
In extracellular conditions, cFXIII could be activated by
thrombin and Ca2⫹ the same way as pFXIII, excluding the
dissociation of FXIII-B. It was suggested that cFXIII on
platelets is activated by the cysteine protease calpain (21).
However, in the intracellular environment, cFXIII does not
need proteolytic cleavage for activation, and during platelet
activation, no proteolytic truncation of FXIII-A occurs
(261, 262). The elevation of intracellular Ca2⫹ concentration, as a consequence of cell activation, seems sufficient to
bring about the enzymatically active configuration (FXIIIA2°). The activation of cFXIII by the nonproteolytic mechanism (i.e., by the elevation of intracellular Ca2⫹ concentration) represents the physiological way of cFXIII activation in platelets (261, 262) and most likely in monocytes (1)
as well. In platelets, elevation of intracellular Ca2⫹ concentration, as a result of activation induced by thrombin and Ca2⫹
ionophore, initiated the intracellular activation of cFXIII without the involvement of proteolysis (261, 262). Similarly, elevation of intracellular Ca2⫹ and stimulation of monocytes by
angiotensin II resulted in nonproteolytic activation of FXIII,
which catalyzed the dimerization of angiotensin receptor 1
(AT1) (1) (see more details in sect. VIIB).
B. Enzymatic Reaction Catalyzed by
Activated Factor XIII: Substrate
Specificity
TGs, including FXIIIa, catalyze an acyl transfer reaction
that consists of two major steps (124, 148, 263). First, the
peptide-bound glutamine substrate forms a binary complex
with the enzyme (thioester linkage between the carboxamide group of the glutamine and the active site cysteine). The
reaction proceeds through an oxyanion intermediate, and
ammonia is released as the acyl-enzyme intermediate is
formed. In the second step, if a substrate primary amine
group is present, the acyl group is transferred to the acyl
acceptor amine through a second oxyanion intermediate.
The amine becomes attached to the ␥-glutamyl residue via
peptide (“isopeptide”) bond, and the active-site cysteine becomes deacylated. In the absence of a substrate amine, the end
result of the reaction is the deamidation of the substrate glutamine residue. If an ⑀-amino group of a peptide-bound lysine
residue is the acyl acceptor primary amine for the reaction,
⑀(␥-glutamyl)lysyl is formed and the two peptide bonds become cross-linked. A more detailed description of the reactions catalyzed by TG is given in References 147 and 148.
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and X-ray structural studies could not be carried out. The
active-site cysteine, originally buried within the catalytic
core domain, becomes unmasked during activation and
available for reaction with its substrates and also with alkylating agents (301). Hydrogen-deuterium exchange and
chemical modification of amino acid side-chains (cysteine
alkylation and lysine acetylation) provided further insight
into structural changes occurring during the activation process. Hydrogen-deuterium exchange evaluated by MALDITOF mass spectrometry showed that FXIII activation exposes the potential substrate recognition region in the catalytic core domain (residues 220 –230), while decreased
deuteration occurred in regions 98 –104 of the ␤-sandwich
and 526 –546 of ␤-barrel 1 (364). Results from chemical
modifications demonstrated that Lys156 and Lys221, the
active sites Cys314 and Cys409 (both cysteines in the catalytic core domain), as well as Cys695 in the ␤-barrel 2
become exposed upon activation by thrombin and Ca2⫹
(365). In a further study, the exposure of Cys238, Cys327,
and Lys68 was also revealed (322). Experiments using an
inhibitory analog of FXIIIa substrate K9 DON (a ␤-caseinderived nonapeptide in which the substrate Gln residue was
replaced by the Gln isostere, 6-diazo-5-oxo-norleucine;
DON) provided further insight into the changes of FXIII-A2
structure upon activation and upon the binding of the substrate analog. K9 DON inhibited FXIIIa and provided even
greater protection of the ␤-sandwich and the ␤-barrel 1
from deuteration. At the same time, the K9 DON was able
to block the alkylation of Cys409 in the catalytic core near
the dimer interface (322).
MUSZBEK ET AL.
The substrate specificity of FXIIIa is more restricted than
that of other TGs. The primary physiological substrates of
FXIIIa are fibrin and ␣2-PI (225, 324). Twenty-three additional FXIIIa substrate proteins are enumerated in the
TRANSDAB database (http://genomics.dote.hu/wiki/).
The substrate proteins can be divided into the following
categories: coagulation factors, components of the fibrinolytic system, adhesive and extracellular matrix proteins, intracellular cytoskeletal proteins, and others (TABLE 2). The interaction of FXIIIa only with part of these
proteins has physiopathological implications; these interactions will be discussed in the respective sections of this
review.
Most recently, an interesting study demonstrated that, in
addition to the TG activity of FXIIIa, FXIII, just like TG-2,
also possesses protein disulfide isomerase (PDI) activity
944
(194). Although both activities are located on FXIII-A, they
are distinct. FXIII without being transformed into an active
TG as well as active-site alkylated FXIIIa exert PDI activity.
The physiological importance of PDI activity of FXIII still
needs to be explored.
C. Inactivation of Activated Factor XIII
Within the Clot
Extensive cross-linking of fibrin increases the resistance of
fibrin clot to fibrinolysis (103, 104). This finding suggests
that noncontrolled cross-linking by FXIIIa results in overcross-linked fibrin, and extensive cross-linking of other
plasma proteins to fibrin, that could lead to undesired prolonged persistence of thrombi. A gradual decrease of FXIIIa
activity, which has been demonstrated within the fibrin clot
and in an experimental pulmonary emboli model (catalytic
half-life: ⬃20 min) (315), suggests the existence of inactivation mechanism(s) that operate in whole plasma clot and
in thrombi. The inactivation of FXIIIa is much less investigated than its activation. Activated clotting factors are inactivated by two mechanisms (70). Proteolytically active
factors are inhibited by specific serine protease inhibitors,
serpins (like antithrombin III or tissue factor pathway inhibitor), or by less specific protease inhibitors, like ␣2-macroglobulin. The other way of inactivation of active factors is
performed by proteolytic enzymes. The latter can be carried
out by highly specific proteases, like the cleavage of activated factor V (FVa) and factor VIII (FVIIIa), by activated
protein C, or by a proteases with much broader substrate
specificity, like plasmin. Plasmin degrades fibrin, fibrinogen, FVa, and FVIIIa. As no plasma protein inhibitor of
FXIIIa has been discovered, one has to consider the proteolytic inactivation mechanism. The fibrinolytic enzyme plasmin could be a possible candidate for such a role. However,
both plasma and cellular FXIII and their activated forms are
highly resistant to plasmin (310). In addition, the powerful
inhibitory effect of fibrin-linked ␣2-PI also makes it unlikely
that plasmin could effectively be involved in the inactivation of FXIIIa in the fibrin clot. In contrast, polymorphonuclear granulocytes (PMNs) became activated and released proteases [for instance, elastase, cathepsin G, and
matrix metalloproteinase (MMP-9)] in the fibrin clot. The
released PMN proteases proteolytically degraded both
FXIII subunits and inactivated FXIIIa within the fibrin clot
(29). Such a mechanism also operated in clots made from
whole plasma, and the main physiological inhibitor of
PMN proteases, ␣1-antitrypsin, provided only limited protection. The time course of FXIIIa degradation by PMNs
suggests that the proteolytic degradation of FXIIIa by the
released proteases does not interfere with the initial crosslinking events; however, it could prevent the formation of
over-cross-linked plasma-clot and facilitate the elimination
of fibrin when it is no longer needed. It would be interesting
to investigate if this mechanism also plays a role in other
processes, in which FXIII is implicated and PMN leukocytes
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As glutamine-containing peptides with different primary
structure around the reactive Gln residue could serve as acyl
donor substrate for FXIIIa-catalyzed TG reaction (80, 296,
356) (http://genomics.dote.hu/wiki/), the substrate-enzyme
interaction could only be described for individual peptides.
Oligopeptides corresponding to the NH2-terminal amino
acid sequence of Asn1-␣2-plasmin inhibitor (Asn1-␣2-PI),
an isoform of ␣2-PI, the major plasmin inhibitor and an
excellent glutamine substrate of FXIIIa (see sect. IV for
details), are the most well-studied acyl donor peptide substrates in this respect. Enzyme kinetic and NMR experiments with such 15-mer and 12-mer oligopeptides and with
their chemical mutants shed some light on the role of amino
acid side chains around the Gln2 substrate site in the substrate-enzyme interaction (63, 64, 232, 296). Kinetic experiments with peptides, in which Asn1 residue was either
truncated or replaced by Ala, and proton NMR analysis of
FXIII-A2*-N1-␣2-PI(1–12) complex demonstrated that
Asn1 residue is essential for effective enzyme-substrate interaction (296). Further studies demonstrated the supportive role of Glu3, Gln4, and Lys12 side chains in the reaction
(64, 296). It was shown by solution NMR methods that the
reactive peptide glutamines are encountering a distinctive
environment within the FXIIIa active site and the glutamines and residues located COOH-terminally come in
direct contact with the enzyme and adopt an extended conformation (232, 296). It is interesting that the replacement
of Gln4 by Ser or Leu doubles the catalytic efficiency of the
reaction, indicating that the FXIIIa subsite for this residue
must be relatively broad (63). Experiments with COOHterminally truncated peptides proved that amino acid residues 7–12 are essential for the interaction of N1-␣2-PI(1–
12) with the enzyme and suggested the existence of a secondary binding site on FXIII-A2*. Hydrophobic residues,
particularly Leu10, and the COOH-terminal Lys12 seemed
especially important in this respect, and direct interaction
between hydrophobic COOH-terminal residues and FXIIIA2* has been demonstrated by STD NMR (296).
MULTIPLE FUNCTIONS OF FACTOR XIII
Table 2
Protein
Coagulation factors
Substrate proteins of activated factor XIII
Substrate
Sequence Around Reactive
Gln
Fibrin(ogen) ␣ chain
77, 234, 312, 348
2, 60
105, 140, 372
181, 204
49, 212
37, 38
367
229
328, 347
99, 238
168, 349
27
65
69
1
298
39
249
ND, not determined.
accumulated. For instance, wound healing could be a target
of such an investigation.
IV. THE ACTION OF ACTIVATED FACTOR
XIII ON FIBRIN: ITS ROLE IN THE
REGULATION OF FIBRINOLYTIC
PROCESS
FXIIIa cross-links fibrin ␥- and ␣-chains into ␥-chain dimers
and ␣-chain polymers, respectively. (For detailed information on fibrinogen and fibrin clot structure, see References
93, 250, and 376.) The extremely rapid ␥-dimer formation
is the result of reciprocal intermolecular bond formation
between ␥406 lysine of one ␥-chain and ␥398/399 glutamine residue of another aligning ␥-chain (60). Cross-linking of ␣-chains, a much slower process, occurs among multiple glutamine and lysine residues, resulting in ␣-oligomers
and high Mr ␣-polymers. A small amount of ␣-␥-chain heterodimers and ␥-chain trimers or tetramers have also been
identified in fibrin (251, 343), and their presence was related to increased resistance to fibrinolysis (344). Although
␣-chain cross-linking confers the final stability to the fibrin
clot allowing strength, rigidity, and resistance to fibrinolysis
(103, 106), ␥-chain dimerization also contributes to clot
stiffness (117, 353). It has been reported that ␥A␥= fibrin
becomes more extensively cross-linked by activated FXIII
than fibrin containing only ␥A homodimers (97, 247, 248).
In sharp contrast to these findings, Siebenlist et al. (345)
found that the overall rate of ␣-polymer formation was
slower for ␥A␥= fibrin than for ␥A␥A fibrin and suggested
that the presence of ␥=-chain suppresses FXIII-induced fibrin cross-linking.
An interesting question is whether cFXIII, present in platelets incorporated into thrombi, contributes to the crosslinking of the fibrin clot. Elevated platelet count and pFXIII
concentration both increase the formation of highly crosslinked ␣-polymers (104). In the presence of normal platelets, the fibrin clot formed from FXIII-free fibrinogen becomes cross-linked (308); however, the contribution of
platelet FXIII, compared with the contribution of plasma
FXIII, was only negligible (132). Platelet FXIII is neither
secreted nor exposed to the platelet surface during platelet
activation (167, 265). The tiny amount of pFXIII, taken up
by platelets and packed into the ␣-granules, could be released during clot formation (233), but its involvement in
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-S-Q221-L-Q-K-V-P-P-Q237-M-R-M-E-L-N-Q328-N-P-G-S-P-G-Q366-W-H-S-E-S-G-Q398-Q399-H-H-L-G-GFibrin(ogen) ␥ chain
Factor V
N.D.
␣
-Plasmin
inhibitor
(
␣
-antiplasmin)
-N-Q
-E-Q-V-S-PFibrinolytic proteins
2
2
2
Lipoprotein A
ND
Plasminogen
-S-Q322-V-R-W-E-Y
Procarboxypeptidase B/U
-F-Q2-S-G-Q5-V-L-A-A-L-S-Q292-H-I-V-F-P(thrombin-activable fibrinolysis
inhibitor; TAFI)
Adhesive/matrix proteins Thrombospondin
ND
Vitronectin
-E-Q93-T-P-V-L-KFibronectin
-A-Q3-Q-I-V-Q-POsteopontin
-S-Q34-K-Q36-N-L-L-A-PCytoskeletal proteins
Vinculin
ND
Actin
-H-Q41-G-V-M-V-GMyosin
ND
-L-Q315-L-L-K-Y-IOthers
AT1 receptor
Semenogelins I and II
ND
Protein synthesis initiation factor
ND
5A
␣2-Macroglobulin
-Q-Q670-Y-E-M-H-G-
Reference Nos.
MUSZBEK ET AL.
the cross-linking reaction is insignificant. Platelets, however, have another effect on the cross-linking reaction that is
independent of their FXIII content. When fibrin cross-linking was induced by highly purified plasma FXIII, the formation of ␣-chain polymers and the incorporation of ␣2-PIfibrin ␣-chain heterodimer into these polymers was significantly accelerated by intact FXIII-free platelets obtained
from patients with severe FXIII-A deficiency (132). The results indicate that, in physiological conditions, platelets promote the cross-linking reaction by providing a catalytic surface, on which the cross-linking of fibrin polymers is enhanced. In addition, activated platelets bind pFXIII through
fibrinogen (265) and could deliver it to the thrombus.
Cross-linking of fibrin chains by FXIIIa, in the absence of
␣2-PI, decreased the rate of lysis induced by exogenous plasmin (103). FXIII, in excess to its normal plasma concentration, increased the extent of fibrin cross-linking and inhibited plasmin-induced degradation of fibrin (103, 104).
However, the lysis rate for cross-linked fibrin prepared
from plasma was much slower than the lysis rate of crosslinked fibrin prepared from purified fibrinogen, and 10-fold
higher plasmin concentration was needed to bring the lysis
rate of plasma-derived fibrin to that of fibrin formed from
purified fibrinogen. This finding emphasizes that plasma
component(s) incorporated into the clot are required for the
effective protection of fibrin from fibrinolysis. The impaired
binding of plasminogen to cross-linked fibrin (237, 326) might
also play a role in the FXIII-induced resistance to fibrinolysis.
␣2-PI, the main physiological inhibitor of plasmin, is an
excellent substrate of FXIIIa, and it becomes cross-linked to
fibrin ␣-chains during the initial phase of cross-linking reaction (324). It is synthesized in the liver and becomes secreted as a protein of 491 amino acids starting with a methionine (Met1-␣2-PI). In the plasma, a protease, antiplasmin cleaving enzyme (APCE), cleaves off the NH2-terminal
propeptide of 12 amino acid residues (202) and transforms
Met1-␣2-PI into Asn1-␣2-PI (32, 357). The transformation
is only partial; in plasma, the ratio of Met1-␣2-PI to Asn1␣2-PI is 3:7. Only the Asn1-␣2-PI isoform is a good substrate for FXIIIa (357). It provides a Gln residue penultimate to Asn1 (Gln2; Gln14 in Met1-␣2-PI) to the crosslinking reaction. The same Gln residue is sheltered by the
NH2-terminal 12-residue peptide of Met1-␣2-PI and makes
it inaccessible for FXIIIa (203). In fact, the addition of only
three COOH-terminal amino acids of the propeptide makes
Asn-␣2-PI a poor substrate for the TG reaction (136). With
the use of recombinant Gln2Ala ␣2-PI mutant, three further
946
During the formation of the fibrin clot, the Gln2 site becomes cross-linked to Lys303 residue of ␣-chain of fibrin
(181). It is interesting that this Lys residue is not involved in
the formation of ␣-chain polymers. The formation of ␣2-PIfibrin ␣-chain heterodimers is a rapid reaction; it only
slightly lags behind fibrin ␥-chain dimerization and precedes fibrin ␣-chain cross-linking. It can also be cross-linked
to fibrinogen, although at a significantly slower rate. The
existence of ␣2-PI cross-linked to plasma fibrinogen has also
been reported (252). There is no acyl acceptor lysyl residue
in the ␣2-PI molecule; therefore, no ␣2-PI dimer is formed
(360). Only Asn1-␣2-PI is cross-linked to fibrin, which explains that part of ␣2-PI remains in the serum. Cross-linked
␣2-PI retains its full inhibitory activity and makes fibrin
strikingly resistant to digestion by plasmin (202, 325). The
concerted action of FXIII and ␣2-PI plays a key role in the
protection of fibrin clots against prompt elimination by fibrinolysis. The dodecapeptide with the NH2-terminal sequence of Asn1-␣2-PI was shown to inhibit the cross-linking of ␣2-PI to fibrin, resulting in accelerated fibrinolysis
(182). ␣2-PI deficiency is a very rare, but severe bleeding
diathesis with symptoms similar to those of FXIII-A deficiency (56). Phenylglyoxal inactivated form of ␣2-PI (205)
or the active site Arg364Ala mutant (206) enhanced urokinase-induced plasma and whole blood clot lysis significantly by competing with the native ␣2-PI for FXIIIa catalyzed incorporation into fibrin. Jansen et al. (158) reported
that the tissue plasminogen activator (tPA)-induced lysis of
whole blood clot from a patient with ␣2-PI deficiency was
not or only slightly increased by an antibody that inhibited
FXIIIa. This finding suggests a primary role of ␣2-PI to
fibrin cross-linking in the reduction of clot lysis by FXIII.
However, Sakata et al. (326) demonstrated in ␣2-PI depleted plasma, and in purified system without ␣2-PI, that
fibrin cross-linking itself and the consequent decrease in the
binding of plasminogen to fibrin also contribute to the reduction of fibrinolysis by FXIII.
The above findings clearly prove that FXIII has a major role
in protecting fibrin from degradation by the fibrinolytic
system. Most recently, the protective effect of FXIII against
fibrinolysis was also demonstrated in clots formed under
flow (264). Fibrin cross-linking and the binding of ␣2-PI to
fibrin by FXIIIa might work together or consecutively. Using a unique antibody, which inhibited FXIIIa-fibrin and
FXIIIa-fibronectin interaction but did not influence the
cross-linking of ␣2-PI to fibrin, McDonagh and Fukue (235)
demonstrated that both ␣2-PI-fibrin cross-linking and
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FXIII may influence fibrinolysis by two mechanisms: 1) the
cross-linking of fibrin ␣-chains into high-Mr ␣-polymers
may render the clot more resistant to fibrinolysis, and 2) the
binding of ␣2-PI, and perhaps also other plasma components, to fibrin may protect the fibrin clot and prevent its
prompt elimination by the powerful fibrinolytic system.
glutamine donor sites were found in the molecule at positions 21, 419, and 447. However, even in the mutant protein,
their kinetic efficiency was much lower than that of the Gln2
residue (204). It has been shown that the Arg6Trp polymorphism in Met1-␣2-PI affects its cleavage rate by APCE in favor
of the Arg6 variant, but this polymorphism does not affect the
poor cross-linking of Met1-␣2-PI to fibrin (203).
MULTIPLE FUNCTIONS OF FACTOR XIII
␣-chain polymerization were necessary to regulate the rate
of fibrinolysis. In experimental pulmonary embolism, both
FXIIIa-mediated fibrin cross-linking and ␣2-PI-fibrin crosslinking increased the resistance to endogenous and tPAinduced fibrinolysis (307). In summary, the above studies
indicate that the cross-linking of ␣2-PI to fibrin occurs at an
early stage of thrombus formation and provides protection
of the newly formed fibrin clot against the prompt elimination
by the fibrinolytic system, while the increased resistance to
thrombolysis of matured thrombi could be the consequence of
the much slower, extensive cross-linking of ␣-chains, including ␣-chains to which ␣2-PI had been attached.
Thrombin activatable fibrinolysis inhibitor (TAFI; also
known as procarboxypeptidase U, procarboxypeptidase B,
or procarboxypeptidase R) plays an important role in the
regulation of fibrinolysis (71, 253, 311). TAFI is activated
by thrombin, a process that is greatly enhanced in the presence of thrombomodulin. Activated TAFI inhibits fibrinolysis by abrogating the following plasmin-mediated positive-feedback mechanisms of fibrinolysis. Once formed,
plasmin starts to digest the clot by catalyzing cleavages after
selected arginine and lysine residues. These cleavages provide extra binding sites to Glu plasminogen and tPA by
exposing COOH-terminal lysine residues. This way the cofactor activity of fibrin in Glu plasminogen activation increases. In addition, the partially cleaved fibrin serves as a
cofactor for the conversion of Glu plasminogen into Lys
plasminogen by plasmin. The activation of Lys plasminogen by tPA is ⬃20-fold more efficient than the activation of
Glu plasminogen. TAFI interferes with these positivefeedback mechanisms by removing the newly exposed
COOH-terminal lysine residues of degraded fibrin, and
thereby diminishing the catalytic efficiency of plasmin
formation. TAFI has been shown to be a substrate for
FXIIIa (367), which catalyzed the polymerization of
TAFI and its cross-linking to fibrin. TAFI contains both
acyl donor Gln and acyl acceptor Lys residues; Gln2,
Gln5, and Gln294 are the preferred acyl donor sites. It
has been suggested that the cross-linking may facilitate
the activation of TAFI, stabilize the enzymatic activity,
FXIIIa catalyzed the incorporation of labeled amines and a
Gln-containing substrate peptide into plasminogen, demonstrating that it contains both glutamine and lysine donor
sites (37). FXIIIa can polymerize plasminogen into high Mr
multimers and form fibronectin-plasminogen heteropolymers. The physiological significance of these findings, if
any, remains to be elucidated. The protein part of lipoprotein (a) [Lp(a)], an atherogenic risk factor, strikingly similar
to plasminogen, also contains acyl donor glutamine residues, for the FXIIIa catalyzed TG reaction (49). In native
Lp(a), part of the substrate glutamyl sites are buried and
inaccessible for FXIIIa. Lp(a) competes with plasminogen
and tPA for binding sites on fibrin(ogen) and inhibits plasmin generation (227). FXIIIa cross-links Lp(a) to fibrinogen
(317), but the concentration of FXIII in this experiment was
well above normal plasma concentration, and the incubation times were too long to support the physiological relevance of this interaction.
In summary, FXIII is an essential component of the clotting
system; its deficiency causes severe bleeding diathesis. The
main function of FXIII in hemostasis is the stabilization of
newly formed fibrin. By cross-linking fibrin ␥-chains into
dimers and fibrin ␣-chains into high-molecular-weight
polymers, FXIII increases the rigidity and strength of the
fibrin clot and protects it against shear stress in the circulation. FXIII also protects fibrin from the prompt elimination
by the fibrinolytic system. This mechanism involves the covalent cross-linking of ␣2-PI, the major plasmin inhibitor,
to fibrin strands, the diminished binding of plasminogen to
cross-linked fibrin, and the reduced lysis of cross-linked
fibrin by plasmin.
V. FACTOR XIII IN WOUND HEALING AND
ANGIOGENESIS
A. Clinical Data and Animal Experiments
Supporting the Involvement of Factor XIII
in Wound Healing
The first FXIII-deficient patient, recognized by Duckert et
al. in 1960, demonstrated impaired wound healing and abnormal scar formation, in addition to the severe bleeding
complications (94, 331). The frequency of poor wound
healing in severe FXIII deficiency was reported between
14 –36% in various reviews (22, 44, 154, 336). The essential role of FXIII in the wound healing process was clearly
proven in transgenic mice, in which the targeted deletion of
FXIII-A gene caused complete FXIII-A deficiency (199). In
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There are also other interactions between FXIII and components of the fibrinolytic system. It has been shown that
plasminogen activator inhibitor 2 (PAI-2), an inhibitor of
urokinase-type plasminogen activator (uPA), is cross-linked
to fibrinogen or fibrin by FXIIIa (163, 313). Cross-linked
PAI-2 retained its activity and effectively inhibited uPAmediated fibrin clot lysis. PAI-2 is detectable in the plasma
only during pregnancy; although it is mainly of cellular
localization, peripheral blood monocytes incorporated into
the fibrin clot are capable of secreting it. The cross-linking
of PAI-2 to fibrin may serve compartmentalization of the
inhibitor. Monocytes incorporated into thrombus could
therefore play a role in the downregulation of plasminogen
activation (313).
and protect the active enzyme from further degradation.
However, no experimental support has been provided to
this hypothesis, and the long incubation time required for
the cross-linking questions the physiological implications
of this mechanism.
MUSZBEK ET AL.
the FXIII-A-deficient mice, the healing of excisional wound
was considerably delayed (FIGURE 8); incomplete reepithelization, persisting necrotized fissure, and abnormal scar
formation were observed at day 11 after excision, when
wild-type mice demonstrated complete closure of the
wound (150). Substitution with human pFXIII restored the
normal healing process (FIGURE 8).
The influence of FXIII polymorphisms on the prevalence
and presentation of CVLU has been intensively studied by
Gemmati and co-workers (111-113, 361) FXIII-A Val34Leu,
Tyr204Phe, Pro564Leu, and FXIII-B Arg95His did not influence the prevalence of CVLU (361). However, the presence of Leu34 and Leu564 alleles was associated with
smaller ulcer surface, and the size of ulcer inversely related to the number of Leu34 alleles (111, 113, 361).
Healing time was increased in patients with the Val34Val
genotype compared with Leu34 carriers (111). Furthermore, FXIII-A Leu34 beneficially influenced the healing
time after superficial venous surgery (112). The above
results suggest that FXIII is involved in the pathomechanism of the healing process of venous leg ulcer, its topical
application is beneficial, and the accelerated rate of FXIII
activation and altered fibrin structure in FXIII-A
Val34Leu polymorphism decrease the severity of the disease. Further details on this topic can be found in an
excellent recent review (386).
Early sporadic reports demonstrated the beneficial effect
of FXIII supplementation on the healing of surgical
wounds in postoperative situations (114, 115, 246).
In inflammatory bowel disease, ulcerative colitis, and
Crohn’s disease, the compensation of consumption and
loss of FXIII into inflamed tissue by substitution seems to
improve the healing tendency (133, 226, 337). Treatment
948
When acquired FXIII deficiency was induced in rats by CCl4
treatment, the healing of microvascular anastomosis and
burn injury were impaired, but the healing process could be
normalized by the administration of FXIII (153, 284). The
healing of osteotomies in sheep could also be stimulated by
FXIII, and the bone formed in this condition had higher
tensile strength (62).
B. Effect of Factor XIII on Fibroblasts,
Macrophages, and Extracellular Matrix
Fibrin gel formation, macrophage invasion, fibroblast migration/proliferation, production of extracellular matrix,
and angiogenesis are the key events in wound healing. Earlier and recent studies enlightened certain details of the
involvement of FXIII in these mechanisms. FXIII exerts its
effect by cross-linking extracellular matrix proteins, by affecting fibroblasts and macrophages, and by promoting the
complex mechanism of angiogenesis.
The first publication in this field came directly after discovering the first FXIII-deficient patient. Beck et al. (36) demonstrated that growth of fibroblasts in the plasma of this
patient was impaired, the contact between cells was lost,
their normal elongated cell shape became irregular, and the
cells failed to produce collagen fibers. Supplementation
with normal plasma or with partially purified FXIII corrected the defects; however, a higher amount of FXIII was
required for the correction of growth impairment than for
restoring normal fibrin cross-linking. The enhancement of
fibroblast proliferation by cross-linking fibrin matrix (366)
and by direct proliferation-inducing effect of FXIII (54)
were both reported in early preliminary studies. Another
study indicated that FXIII binds to the extracellular matrix
and matrix assembly sites, where it remains active (34). Its
binding to a putative cell surface receptor, which mediates
internalization and degradation, was also suggested. The
migration of fibroblasts into fibrin gel was greatly enhanced
by extensive cross-linking of fibrin ␣-chains, while the
dimerization of ␥-chains was not sufficient to support fibroblast migration (53). Fibronectin, an important component
of extracellular matrix, is cross-linked by FXIIIa at cellular
matrix assembly sites (35), which complements disulfidebonded multimer formation in the stabilization of assembling fibronectin molecules. Fibronectin covalently incorporated into the fibrin clot by FXIIIa supports fibroblast
adherence and promotes the migration of cells into the gel
(126). Cell attachment and spreading on composite fibrin/fibronectin clot significantly decreased when recombinant fibronectin with mutations in the FXIIIa substrate
glutamine sites was used, i.e., fibronectin must be crosslinked to fibrin by FXIIIa to ensure maximal cell attachment (74). In addition to fibrin and fibronectin, several
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Chronic venous leg ulcer (CVLU) represents a peculiar form
of impaired wound healing with multicausative background. The possible involvement of FXIII in the pathomechanism of this disease is of particular interest. An earlier
study demonstrated decreased FXIII level in a small group
of patients with CVLU (369), while in a more recent report
FXIII-A antigen level did not differ significantly between
CVLU patients and matched controls (n ⫽ 91 in both
groups) (113). However, in the latter study, 11% of the
cases had moderate FXIII deficiency (FXIII-A antigen
ⱕ0.65 U/mL). Although only a relatively small number of
patients were recruited for the four studies evaluating the
effect of topical treatment of CVLU with FXIII concentrate,
the studies unanimously reported a beneficial effect (131,
134, 297, 380). Accelerated wound surface reduction,
shorter healing time, improved availability of granulation
tissue, and decreased secretion and bleeding tendency were
observed following daily local administration of the concentrate in CVLU, but not in arterial-venous mixed disease
(380).
with recombinant FXIII also improved established experimental colitis in rats (83).
MULTIPLE FUNCTIONS OF FACTOR XIII
other adhesive proteins, vitronectin, osteopontin, thrombospondin, and von Willebrand factor, are substrates of
FXIIIa (TABLE 2). Their cross-linking to other connective
tissue matrix components might have further impact on
the attachment of cells to the matrix and on the healing of
injuries.
Macrophages have a well-known important role in the
wound repair process (3, 118, 119). FXIII of tissue macrophages might play a role in maintaining and modulating the
connective tissue matrix in normal conditions. This hypothesis, however, still lacks experimental support. In theory,
FXIII of the extracellular compartment might act on monocytes/macrophages by influencing their activation, differentiation, and migration, while cFXIII present in these cells
might play a role in the mechanism of macrophage migration and phagocytosis. Like in the case of fibroblasts, FXIIIa
enhanced the proliferation of peripheral blood monocytes,
accelerated their migration through the filter of a Boyden
chamber by twofold, and significantly inhibited monocyte
apoptosis (85). These changes were related to the downregulation of thrombospondin-1 and to the upregulation of
c-Jun and Egr-1 in monocytes. Active-site blocked FXIIIa
was ineffective, suggesting that the TG activity of FXIIIa is
essential for its effects on monocytes (85). The above results
seem to be in contradiction to the finding that anti-FXIII-A
antibodies did not influence the binding of THP-1 cells (a
human monocytic leukemia cell line) to fibronectin and
their migration on fibronectin surface (16). However, the
leukemic cells might behave differently from normal monocytes, and the inhibitory effect of the antibody was not
proven in this publication. It was also shown that in fibrin
gel containing cross-linked fibronectin the migration of
monocytes slowed down (197). Although the relation of
migration in fibrin gel to the migration required for wound
healing is questionable, the adhesion to cross-linked fibronectin might represent an anchoring mechanism that
keeps macrophages where they are needed for the wound
healing process.
The involvement of monocyte/macrophage cFXIII in the
wound healing process has not been explored to a full
extent. FXIII-A2 cannot be secreted by the classical pathway, and there is no proof for its secretion by other
secretory mechanisms. Nevertheless, leakage of cFXIII from
damaged cells might contribute to the local cross-linking of
proteins. The activation of monocytes/macrophages by the
alternative pathway, for instance, by interleukin-4, highly increased FXIII-A expression (362). Alternatively activated or
M2 macrophages express scavenger receptors, bind apoptotic
cells, and are important in mediating wound healing following
tissue damage (96). cFXIII is involved in receptor-mediated
phagocytosis by monocytes (6, 329). Removal of cell debris
and apoptotic cells is an important step in the wound healing process, and the decreased phagocytosis of monocytes/
macrophages in FXIII-A deficient patients might contribute
to the impaired tissue repair. Another intracellular mechanism involving cFXIII in wound healing could operate
through the angiotensin II-induced release of monocytes
from their splenic reservoir and migration to the site of
injury (358). Binding of angiotensin II to its receptor (AT1)
could lead to cFXIII activation and consequently to the
covalent dimerization of AT1 by FXIIIa, which induces the
activation and mobilization of monocytes (1). (The expression and activation of cFXIII in activated monocytes and
the involvement of cFXIII in the function of these cells are
detailed in section VIIB.)
C. Proangiogenic Effect of Activated Factor
XIII
Angiogenesis is a complex process that includes remodeling and sprouting of new capillaries from existing
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A recent study confirmed and extended sporadic preliminary results on the direct effect of FXIII on fibroblasts using
more elaborate techniques; this study also shed light on the
mechanism by which FXIII exerts its effect (85). FXIIIa, but
not nonactivated FXIII or inactivated FXIIIa, bound to skin
fibroblast, enhanced their migration in a wound-migration
assay, increased the incorporation of [3H]thymidine into
the cells, and decreased fibroblast apoptosis. The finding
that an antibody against ␣v␤3-integrin, the vitronectin receptor, blocked the binding of FXIIIa to fibroblasts implicated this receptor in the effect exerted by FXIIIa (85). In
addition, FXIII counteracted the toxic effect of metalloproteinases on dermal fibroblasts, although it is not clear from
this study if FXIII added to the cell culture became activated
or not (385).
Another interesting aspect of the relationship between
macrophages and FXIII is the FXIII-dependent generation of a complement C5-derived monocyte chemotactic
factor during clotting (286). The major monocyte chemotactic factor identified in the serum had a Mr (75 kDa)
by gel filtration that is much higher than that of the active
complement fragment C5a or C5a des-Arg (8 kDa). This
finding suggests that C5a was cross-linked to another
plasma protein, although no direct evidence has been
provided. Most recently, a plasma protein with the features of ribosomal protein S19 (RP S19) was identified,
which was dimerized by FXIIIa on the surface of activated platelets during coagulation and converted into a
monocyte-selective chemoattractive factor (339). Interestingly, RP S19 showed immunological cross-reactivity
with C5a. However, the Mr of the active dimeric form
was only 32 kDa. It remains to be seen what is the exact
relationship between the cross-linked C5a fragment and
the active ribosomal protein S19-like factor. Such monocyte specific chemotactic factor(s) formed during clotting
could be involved in recruiting monocytes/inflammatory
macrophages to the site of injury.
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MULTIPLE FUNCTIONS OF FACTOR XIII
blood vessels. It is involved in a wide range of physiopathological processes from wound healing to tumor
growth. The results published in the last decade strongly
suggest that the role of FXIII in tissue repair and wound
healing is at least in part mediated by its proangiogenic
effect. The interaction of FXIII with immortalized human
endothelial cells was first reported a decade ago by Dallabrida et al. (84) and by Dardik et al. (89). The binding
itself did not require TG activity, nonactivated FXIII,
FXIIIa, and inactivated FXIIIa all bound to the cells via
␣v␤3 integrin (vitronectin receptor) and activation of the
receptor by MnCl2 highly increased the binding (84, 89).
In addition, FXIII seems to be capable of mediating endothelial cell-platelet interaction (89).
Dardik et al. (90) also demonstrated in the Matrigel tube
formation model that FXIIIa caused a dose-dependent enhancement array formation of HUVECs. No effect was observed on the addition of FXIII or iodoacemide inhibited
FXIIIa, or when the TG activity of FXIIIa was blocked by a
polyclonal antibody (90). Interestingly, an opposite effect
was observed by Dallabrida et al. (84) using a simian virus
40 (SV40) transformed human dermal microvascular endothelial cell line (HMEC-1) and fibrin gel. Differences in the
features of the two cell types, e.g., in their TSP-1 content
The role of FXIII in angiogenesis has also been verified in
several in vivo animal models. Injection of FXIIIa into the
cornea subepithelially induced the formation of a rich network of capillaries within 36 h. At the same time, the positive staining for TSP-1 observed in the fibers of the stroma
and in keratocytes of nontreated cornea disappeared (90).
Topical application of FXIIIa produced a significantly
higher number of new vessels in neonatal heterotopic
mouse heart allograft model and increased the contractile
performance of the allograft compared with untreated normal mice (86). Here again, FXIIIa decreased TSP-1 mRNA
and protein expression. These findings also demonstrate
that FXIIIa exerts its proangiogenic effect, at least in part,
through the downregulation of TSP-1. In FXIII-A knockout
mice, the formation of new vessels in subcutaneously injected
Matrigel plug dramatically decreased compared with normal
mice (86). If FXIIIa was added to the Matrigel, the number of
vessels almost reached the normal level. When a critical-sized
bone defect in the tibia of rat was filled with hydroxyapatite,
the addition of FXIII stimulated the in-growth of microvessels
into the implant (180). Although FXIII added to hydroxyapatite was not preactivated, it was transformed somehow into
its active form locally as demonstrated by staining with an
antibody specific for the active form.
The mechanism of the proangiogenic effect of FXIIIa was
partly enlightened in Inbal’s laboratory. FXIIIa bound to
␣v␤3 receptor enhances its noncovalent interaction with
VEGFR-2, and even resulted in partial cross-linking between the ␤3 subunit of vitronectin receptor and
VEGFR-2 (88). FXIIIa then induced tyrosine phosphorylation and activation of VEGFR-2 that led to the phosphorylation and activation of Akt and the extracellular
signal-regulated protein kinases (ERKs) and to the upregulation of the transcription factors c-Jun and Egr-1.
Akt promotes growth factor-mediated cell survival and
has been implicated in angiogenesis. The activation of
ERKs together with the zinc-finger transcription factor,
Egr-1-mediated pathway promotes cell proliferation. As
mentioned above, the downregulation of TSP-1 is a key
event in the proangiogenic effect of FXIIIa. Overexpres-
FIGURE 8 Impaired wound healing in FXIII-A knock out mice. A: representative views of the gross appearance of excisional wounds of control,
FXIII-A-deficient, and FXIII-A-deficient/pFXIII-treated mice on days 0, 8, and 11. B: follow up of wound scores at different time points of control
(squares), FXIII-A-deficient mice (open triangles), and FXIII-A-deficient/pFXIII-treated mice (black triangles). Each group included 10 animals. Data
are presented as means ⫾ SD. Mean ⫾ SD area under the curve (AUC) was calculated from the wound scores. AUC was significantly higher
in either the control or FXIII-treated groups compared with the FXIII-A-deficient animals (P ⬍ 0.001 in both cases). [From Inbal et al. (150), with
permission.]
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Although the binding of the nonactivated form might be
important for targeting FXIII to endothelial cells, its active form is required to exert proangiogenetic effects.
FXIIIa, but not the zymogen FXIII or active-site blocked
FXIIIa, enhanced human umbilical endothelial cell
(HUVEC) migration in two different migration assays
(90). FXIIIa also increased [3H]thymidine incorporation
into HUVECs and significantly suppressed the apoptosis
of these cells as demonstrated in TUNEL assay (90).
FXIIIa had no effect on the secretion of vascular endothelial growth factor (VEGF) into the medium, and neither VEGF protein level nor steady-state VEGF receptor-2 (VEGFR2) mRNA levels were affected by FXIIIa. In
contrast, FXIIIa induced an almost complete disappearance of thrombospondin-1 (TSP-1) mRNA from the cells
and markedly reduced the amount of TSP-1 in the conditioned medium. TSP-1, a homotrimeric multifunctional glycoprotein produced and secreted by many cells,
is involved in the inhibition of angiogenesis by inhibiting
endothelial cell migration and proliferation and by inducing apoptosis (200, 245, 276). The interference of
TSP-1 with wound healing-associated angiogenesis was
also demonstrated in transgenic mice with targeted overexpression of TSP-1 in the skin (355).
(90), opposing response in VEGFR-2 and VEGF expression
to ␣v integrin suppression (87), might be partly responsible
for the discrepancy. Another significant difference was the
composition of gel matrix; Matrigel, a gelatinous protein
mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse
sarcoma cells, resembles the complex extracellular environment found in many tissues as opposed to fibrin gel consisting of a single component.
MUSZBEK ET AL.
sion of c-Jun in HUVECs and in cardiac allografts has
been linked to the downregulation of TSP-1 (86, 88), and
this effect is mediated by Wilm’s tumor-1 (WT-1), another transcription factor (88). As the level of WT-1 is
not influenced by FXIIIa, its effect seems to be exerted
through the modulation of WT-1 to TSP-1 association.
VI. OTHER EFFECTS OF FACTOR XIII
A. Role of Factor XIII in Maintaining
Pregnancy
The levels of fibrinogen, factors VII, VIII, IX, and X progressively increase during pregnancy (354). A similar pattern is followed by FXIII-B but not FXIII-A. FXIII-A concentration and consequently also FXIII-A2B2 concentration
and FXIII activity start to decrease between the fifth to
eighth gestational week (wG) and following a gradual reduction reaches ⬃50% of normal average (41, 73, 129,
277, 368). FXIII then rises with the onset of labor and falls
FIGURE 9 A hypothetic scheme for the involvement of FXIII in wound healing. pFXIII activated in the coagulation pathway plays a pleiotropic role that involves antifibrinolytic effect, modulation of extracellular matrix,
promotion of monocyte/fibroblast migration, proliferation, survival, and a complex effect on endothelial cells
leading to angiogenesis. cFXIII in monocytes/macrophages recruited to the site of injury becomes upregulated
and activated. Activated cFXIII of monocytes/macrophages promotes phagocytosis. This way it is involved in
the removal of cell debris and apoptotic cells. The evidence for individual pathways shown is discussed and
evaluated in the text.
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The above findings leave no doubt that FXIIIa is a proangiogenic factor, and this function of FXIIIa is important for
the promotion of wound healing. The biochemical mechanism of FXIIIa-induced angiogenesis and its participation in
wound healing have been partly clarified (FIGURE 9), but
there are still a number of missing links that need to be
explored. Further basic and clinical research in this area
may lead to the exploitation of this proangiogenic effect in
clinical practice. Topical application of FXIII concentrate
might be used to promote the angiogenesis in poorly healing
wounds, and the inhibition of FXIIIa-induced angiogenesis
might have a potential in antitumor therapy.
MULTIPLE FUNCTIONS OF FACTOR XIII
again postpartum. Whether the reduction of plasma FXIII
during pregnancy represents decreased synthesis of
FXIII-A, increased utilization/consumption, or simple dilution by expanded plasma volume is not clear.
The concentration of pFXIII required for maintaining pregnancy has not been established. The observation that FXIIIB-deficient women, who have FXIII level in the range of
5–10%, do not experience miscarriage (116, 323) suggests
that pFXIII concentration significantly lower than normal is
sufficient to maintain pregnancy without substitution therapy. A patient with pFXIII activity, as low as 0.3%, was
able to carry two pregnancies until wG 31 and 37, and only
then did ablation of placenta with bleeding complications
occur that needed replacement therapy and caesarian sections (243). Present recommendations for FXIII substitution of FXIII-deficient patients suggest that 10% presubstitution FXIII level is ideal to prevent miscarriages (26, 139,
173). Ogasawara et al. (283) investigated FXIII level in 424
patients with a history of first trimester miscarriages. No
difference in the subsequent miscarriage rates was found
between patients with normal and moderately decreased
level of FXIII. The above findings clearly demonstrate that
pFXIII is essential to carry out normal pregnancy, but the
level required to prevent miscarriages is significantly below
the reference interval of pFXIII.
FIGURE 10 Distribution of FXIII-A in the definitive placenta. The major components, related to the role of
definitive placenta, are indicated. FXIII-A-containing cellular and structural elements are depicted in maroon.
These include macrophages in the placental villi (Hofbauer cells) and in the decidua and the Nitabuch’s fibrinoid
layer. This layer is located between the zona spongiosa and zona compacta where the release of placenta will
take place. The fibrinoid deposits contain fibrin and fibronectin cross-linked by FXIIIa.
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FXIII-A is not required for conception, but it is essential for
maintaining pregnancy; patients with severe FXIII-A deficiency and without replacement therapy experience recurrent pregnancy losses in the first trimester (see reviews in
Refs. 26, 142, 151, 173). Homozygous female FXIII-A
knock out mice were also capable of becoming pregnant;
however, most of them died of excessive vaginal bleeding
(189). Massive placental hemorrhage with subsequent necrosis developed in the uteri by day 10. As embryonic development of the FXIII-A-deficient fetus is normal, the defect should be at the maternal side. FXIII-A has been identified in histiocytes of the uterus (11), in macrophages of the
implantation tissue (25), and in the placenta (9). In the
placenta small mononuclear round-shaped FXIII-A-containing cells appear at the fifth wG and then they rapidly
proliferate and differentiate into large stellate cells reaching
⬃30% of total cell number (171). From the eighth wG they
tend to accumulate in the peripheral part of chorionic villi.
The location of FXIII-A-containing cells in the definitive
placenta is demonstrated in FIGURE 10. Although the presence of FXIII-A-containing cells both in the placenta and
the implantation tissue suggests the involvement of these
FXIII-A-containing macrophages in the development of
normal pregnancy, the simple fact that replacement by
pFXIII concentrate is sufficient to carry FXIII-A-deficient
patients successfully through pregnancy (47, 100, 185,
316) proves the primary importance of pFXIII. The function of cFXIII present in uterine and placental macrophages remains unclear; it might be involved in the crosslinking of extracellular matrix proteins in these tissues.
MUSZBEK ET AL.
It is very likely that activated FXIII cross-links and stabilizes fibrin (and fibronectin) of the Nitabuch’s layer,
and protects it from fibrinolysis. The urokinase-type
plasminogen activator (uPA) is abundant in placental
cells (98, 101) and, together with its cellular receptor and
inhibitors (PAI-1 and PAI-2), FXIII may act as a regulator of uPA-induced fibrinolysis. The fibrinoid of the Nitabuch’s layer seems to play a complex role. In addition to
its sealing effects, it also acts as a protective immunologic
“barrier” between feto-maternal tissues and participates
in anchoring the placenta. The absence of its cross-linking by FXIIIa could compromise all these functions. The
angiogenic effect of FXIIIa may contribute to the angiogenesis during placentation. Insufficient formation of cytotrophoblastic shell was also reported in woman with
FXIII deficiency (24). Although the essential role of
pFXIII in maintaining pregnancy has been firmly established, we are only beginning to understand its precise
role and the mechanisms through which it ensures normal placental development as well as prevents peri-placental bleeding, detachment of the placenta from the
uterus, and subsequent spontaneous fetal loss.
B. Effect of Factor XIII on Vascular
Permeability
The first report concerning the influence of FXIII on vascular permeability came from Hirihara et al. (135). In their
experiments the vascular permeability enhancement induced by intradermal injection of anti-endothelial cell antiserum was demonstrated by the leakage of Evans blue from
vascular compartment after 15 min and by measuring the
swelling after 1, 2, or 3 days, respectively. Local administration of pFXIII suppressed both the acute and the subacute increase of permeability significantly. It was not explored if FXIII became activated following administration
or the inactive form was sufficient to exert the inhibitory
effect. A more detailed in vitro study used porcine endothelial cell culture and ischemic-reperfused rat heart in a Lan-
954
gendorf perfusion system to address the effect of FXIII on
endothelial barrier function (275). Physiological concentration of activated pFXIII or cFXIII decreased the permeability of endothelial cell monolayer for Trypan blue-labeled
albumin in a two-compartment system separated by a porous filter. In addition, they were able to prevent the rapid
rise of macromolecule permeability that was induced by an
inhibition of endothelial mitochondrial and glycolytic energy production. Likewise, FXIIIa prevented the increase of
myocardial water content induced by low-flow ischemia
and subsequent reperfusion on rat heart (275). Nonactivated pFXIII and cFXIII, inactivated FXIIIa and FXIII-B
failed to exert any effect in these experiments, suggesting
that the effect of FXIIIa is related to its TG activity. Immunohistological investigations demonstrated the deposition
of FXIIIa at interfaces of adjacent endothelial cells and between the cells and the filter support. The mechanism of the
effect of FXIIIa on endothelial cell permeability has not
been revealed. A variety of FXIIIa substrate adhesive proteins, like fibronectin and vitronectin, reside in the intracellular clefts and subcellular matrix; their cross-linking might
influence the paracellular pathway for passage of macromolecules through the monolayer.
A few clinical and experimental animal studies also support
the effect of FXIII on vascular permeability. In a small prospective investigation on newborns and young children
with congenital heart disease undergoing open-heart surgery, peri-operative cardiac edema formation was related to
the decrease of pFXIII concentration (381). Preoperative
treatment with FXIII concentrate reduced the extent of
myocardial swelling and the extension of generalized
edema. In another small study on children with open-heart
surgery, a single intravenous dose of pFXIII reduced severe
pleural effusion (333). Czabanka et al. (82) demonstrated
that the administration of pFXIII together with endotoxin
reduced the increase of vascular permeability in the rat, and
leukocytes were not needed for this effect.
The findings published, so far, clearly indicate that FXIIIa
reduces the permeability of endothelial cell monolayers to
macromolecules. Revealing the mechanism by which FXIIIa
exerts its effect seems to be a promising and interesting field
of future investigations. A few data also suggest that FXIII
decreases the enhanced vascular permeability induced by
various stimuli in experimental and clinical setting. However, the results of these sporadic observations and their
possible clinical impact need to be confirmed by wellplanned and focused clinical and animal studies.
C. Cardiac Effect of Factor XIII
In 2006 a surprisingly novel aspect of FXIII-A deficiency
was reported by Nahrendorf et al. (268). They compared
the survival of homozygous and heterozygous FXIII-A
knock out mice (FXIII-A⫺/⫺ and FXIII-A⫹/⫺, respectively),
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The exact role of FXIII in maintaining pregnancy and the
cause of pregnancy losses in women with FXIII deficiency is
unknown. Two substrates of FXIIIa, fibrin(ogen) and fibronectin, are important components of the Nitabuch’s fibrinoid layer, the layer in the decidua basalis between the
zona compacta and zona spongiosa, where the placenta
detaches itself from the uterus at birth (FIGURE 10). Kobayashi et al. (184) reported that FXIII-A was present in the
extracellular space of the extravillous cytotrophoblast
forming the cytotrophoblastic shell adjacent to Nitabuch’s
layer. FXIII-A colocalized with fibrinogen and fibronectin
at the Nitabuch’s layer next to the cytotrophoblastic shell
(24). The localization of FXIII-A supports its plasmatic origin, while the absence of FXIII-B at these locations suggests
that pFXIII became activated and FXIII-B dissociated from
FXIII-A.
MULTIPLE FUNCTIONS OF FACTOR XIII
were detected in the myocardium of patients with infarct
rupture (267), and moderately decreased pFXIII levels
(57 ⫾ 8%) were reported in three patients presenting with
acute myocardial rupture following myocardial infarction
(270). These interesting findings could provide a basis for
controlled clinical studies on the risk of cardiac rupture
following myocardial infarction in individuals with moderately decreased FXIII, including heterozygous FXIII-A-deficient patients.
An imbalance in extracellular matrix turnover was also observed. The expression of mRNA for collagen was lower in
the infarct area of FXIII-A⫺/⫺ mice, while the level of
MMP-9 in the myocardial tissue of FXIII-deficient mice was
sevenfold higher than in wild-type mice (268). Substitution
with human pFXIII preparation reversed most, but not all,
changes observed in FXIII-A⫺/⫺ mice. Postmyocardial infarction remodeling was enhanced and left ventricule enddiastolic volume was increased in spite of replenishment
with pFXIII (268). The collagen synthesis remained impaired, and the scar was significantly thinner in these animals. These findings suggest that, although the lack of
pFXIII plays the dominant role in the development of cardiac rupture following ligation-induced myocardial infarction in FXIII-A knockout animals, cFXIII present in the cells
of cardiac tissue also contributes to the pathomechanism.
FXIII-A mRNA and protein have been detected in murine
cardiac tissue, and FXIII-A antigen was localized to spindleand round-shaped cells positive for monocyte/macrophage
marker by immunohistochemical methods (351, 352).
FXIII activity becomes gradually elevated in the myocardial
infarct of wild-type mice with a maximum on day 3 (267,
268). Treatment of these animals by extra pFXIII resulted in
a series of changes. It attenuated left ventricular dilation,
enhanced the recruitment of macrophages in the myocardium, upregulated VEGF, and increased the formation of
new microvessels and the synthesis of collagen (267).
The same survival rate of homozygous and heterozygous
FXIII-A knockout animals after coronary artery ligation is
rather unexpected. However, FXIII has been shown to be
used up during the acute phase of myocardial infarction
(18, 110), and further decrease of the already-low FXIII
level in FXIII-A⫹/⫺ mice could result in a situation similar to
FXIII-A⫺/⫺ animals. Furthermore, diminished FXIII levels
Spontaneous pathological alterations in the cardiac tissue
were also observed in FXIII-A⫺/⫺ mice (352). Interestingly,
these changes seemed to be gender specific. Only ⬃50% of
male FXIII-A⫺/⫺ mice survived 10 mo, while 90% of female
mice of the same genotype remained alive. A quarter of
deceased mice died of severe intra-thoracic hemorrhage,
and large hematomas were found in their hearts. Hemorrhage was also found in the heart of other deceased mice,
and all FXIII-A⫺/⫺ males showed widespread hemosiderin
deposition and fibrosis in the cardiac tissue. Heart structure
and function of FXIII-A knockout mice did not differ significantly from their wild-type counterparts. Fibrosis and
hemosiderin deposition were also present, although to a
much lower extent, in FXIII-B⫺/⫺ males, but not in females.
There is no clear explanation for the gender difference; it
was supposed that the more aggressive fighting behavior of
male animals could be one of the contributing factors.
The above findings suggest that FXIII exerts its cardiac
effect by multiple mechanisms. It upregulates the synthesis
of extracellular matrix components, and in the case of ischemic tissue damage, it enhances microvessel angiogenesis
and modulates the influx of inflammatory cells. Additional
mechanisms might also be revealed in the near future. It also
remains to be seen to what extent the mechanisms operating
in the coronary artery ligation-induced murine infarction
model are valid for human myocardial ischemic damage
caused by atherothombotic events. Acute myocardial infarction developed on the basis of atherosclerosis is a more
complex process in which ischemic cell death is only the
end-stage of a series of events. Clinical findings suggest that
FXIII might contribute to the events leading to coronary
occlusion. Gene expression profiling revealed increased expression of FXIII-A in peripheral blood cells in patients with
coronary artery disease (230), and an elevated FXIII level
was found to be a risk factor of myocardial infarction in
women (40). Detailed discussion of the interrelationship
between FXIII and myocardial infarction is beyond the
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FXIII-A⫺/⫺ mice supplemented with human pFXIII and
wild-type mice following myocardial infarction caused by
left coronary artery ligation. FXIII-A⫺/⫺ and FXIII-A⫹/⫺
mice subjected to coronary ligation died within 5 days due
to the rupture of the left ventricle wall. No death occurred
in sham-operated mice, and none of the wild-type coronary
ligated mice died within 5 days (only 40% of them died
within 40 days). Daily intravenous replenishment of FXIIIA⫺/⫺ mice with human pFXIII restored survival to the level
seen in wild-type controls. Immunohistochemistry demonstrated the presence of FXIII-A in the healing infarct of
wild-type and pFXIII supplemented FXIII-A⫺/⫺ mice.
Within the infarct of these animals, considerable FXIIIa
activity was detected by molecular imaging using 111Inlabeled NH2-terminal ␣2-PI dodecapeptide as FXIIIa substrate (156, 268). In FXIII-A⫺/⫺ mice, a reduced inflammatory response was observed at the site of infarct; on day 2
following ligation, the recruitment of neutrophil granulocytes and macrophages to the infarct decreased by 56 and
54%, respectively (268, 269). The diminished inflammatory response is related to the impaired migration of monocytes from their splenic reservoir to the site of the injury
(358). Normally the recruitment of monocytes to the site of
myocardial injury operates through angiotensin II-AT1 receptor signaling (358) that very likely involves the intracellular activation of FXIII (1) (see also sects. VB and VIIB).
The lack of FXIII could abrogate such a mechanism.
MUSZBEK ET AL.
scope of this review; for a most recent overview on this
topic, see Reference 260.
D. Factor XIII in Cartilage and Bone
The function of chondrocyte FXIII seems to change according to the location of the cells and the stage of bone development. In the embryonic avian and mouse growth plate of
long bones, it is not present in cells of proliferative zone and
appears first within the cells of the zone of maturation. Its
expression progressively increases until the prehypertrophic
zone, and in hypertrophic cells, the expression remains at a
high level (280 –282). In articular cartilage, FXIII-A-containing chondrocytes are predominantly localized in the
condylar region of tarsus, suggesting that FXIII is also
needed in regions that will be exposed to mechanical stress
and abrasive forces. On the basis of experiments using cultured hypertrophic chondrocytes transfected with FXIII-Agreen fluorescence protein construct, it was surmised that
cell death and lysis are responsible for the externalization of
FXIII (280). However, this theory could not be verified by
experiments on human embryonic growth plate and articular chondrocytes (165). The incorporation of a lysine-containing rhodamine-labeled tetrapeptide, an amine substrate
of FXIIIa, into intracellular proteins and into extracellular
matrix components indicated that, at least part of chondrocyte FXIII became activated both in the intracellular and
extracellular environment (281, 282). No clear evidence
was provided for its intracellular proteolytic activation in
chondrocytes. However, just like in platelets, nonproteolytic activation of the zymogen, induced by increased Ca2⫹
concentration, might have been sufficient to bring about TG
activity. On the basis of SDS-PAGE analysis, FXIII-A of
avian chondrocyte was mistakenly considered as a monomer (282); it is very likely a homodimer, like cFXIII of other
cell origin.
956
In normal human knee articular cartilage, some expression
of TG-2 and cFXIII was detected in the superficial zone and
in the central (chondrocytic) zone of medial menisci (165).
Markedly upregulated expression of both TGs by enlarged
chondrocytes was observed in the superficial and deep
zones of articular cartilage obtained from patients with severe osteoarthritis. A similar finding was obtained with medial meniscal cartilage. Antibody against FXIII-A had a
greater neutralizing effect on the TG activity of meniscal cell
lysate than anti-TG-2 antibody, which suggests the predominance of cFXIII over TG-2. Because the cell lysates
were not treated with thrombin, the actual cFXIII-to-TG-2
ratio could have been significantly higher than that measured by the authors.
Interleukin-1␤ (IL-1␤) and transforming growth factor-␤
(TGF-␤), whose activities are upregulated in osteoarthritis,
induced increased TG-2 and FXIII-A immunostaining in
knee articular and meniscal cartilage slices carried in organ
culture (165). In cartilage with severe osteoarthritis, IL-1␤
was more effective than TGF-␤. The mechanism of IL-1␤induced TG expression is not clear. IL-1␤ induces nitric
oxide (NO) production, and NO donors also increase TG
expression, although to a lesser extent than IL-1␤. The results suggest that the effect of IL-1␤, at least in part, is
exerted via increased NO generation.
When TC28 cells, an immortalized cell line of human juvenile costal chondrocytes, or cultured meniscal chondrocytes
were transfected with FXIII-A or TG-2, the markedly increased TG activity was accompanied by marked increase
of calcium precipitates in the matrix of cultured cells (165).
This effect of elevated cFXIII and TG-2 did not seem to be
attributable to changes in PPi metabolism or cell apoptosis.
Increased TG activity was not associated with apoptosis of
the TC28 cells or meniscal chondrocytes and failed to in-
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FXIII-A has been detected by immunohistochemical and
Western blotting methods in hypertrophic chondrocytes of
chicken and mouse embryonic long bone’s growth plate
(281, 282), as well as in avian, porcine, and human articular
chondrocytes (165, 282, 318). Chondrocytes express TG
activity, which is due to TG-2 and cFXIII that coexist in
these cells (165, 280 –282). The highly increased TG activities following treatment with thrombin also prove the presence of functional cFXIII zymogen in these cells (280, 319).
The detection of FXIII-A mRNA indicates that chondrocytes actively synthesize this protein (280, 282, 318).
FXIII-A was detected in the cytosol and membrane fractions of cultured porcine chondrocytes, more in those cells
obtained from old than from young animals (318). FXIII-A
also appeared in the cell culture media and in articular
cartilage vesicles that participate in calcium pyrophosphate
crystal formation. The mechanism of its externalization is
unclear.
More recent experiments reported by Johnson et al. (166)
shed some more light on the mechanism by which FXIII is
involved in the hypertrophic differentiation of chondrocytes. There seems to be an intriguing interplay between
cFXIII and coexisting TG-2. Treatment of human articular
chondrocytes with cFXIII or with TG-2 increased the expression of VEGF and MMP-13 genes, typically expressed
by hypertrophic chondrocytes, and type X collagen synthesis was also enhanced. cFXIII was shown to be a ligand of
␣1-integrin, and extracellularly administered cFXIII induced ␣1␤1-integrin-dependent signaling leading to drastic
translocation of TG-2 from the cytosol to the cell surface
and to consequent p38 MAP kinase and FAK phosphorylation. The latter events have a central role in transducing
maturation to hypertrophy in cultured chondrocytes (241,
373, 387). As it was shown on chondrocytes from FXIII-A
and TG-2 knockout mice, exogenous cFXIII required both
endogenous cFXIII and TG-2 for its effect on chondrocyte
differentiation (166).
MULTIPLE FUNCTIONS OF FACTOR XIII
duce significant changes in the extracellular PPi or nucleoside triphosphate pyrophosphohydrolase or alkaline phosphatase activity. The cross-linking and stabilization of pericellular calcium binding proteins by TGs (12, 13) might
contribute to this process.
The data obtained on MC3T3-E1 cells were confirmed and
extended on mouse bone by the same group (271). The
expression of FXIII-A protein in osteoblasts and osteocytes
of long bones formed by endochondrial ossification and flat
bones formed primarily by intramembraneous ossification
was demonstrated by immunohistochemistry and in situ
hybridization. FXIII-A mRNA was also detected in the osteoblasts of human tibial subchondral bone plates (327).
Interestingly, its expression became significantly upregulated in sclerotic osteoblasts compared with nonsclerotic
cells.
It is rather surprising that FXIII-A in the bone extract and in
MC3T3-E1 cells was found to be a 37 kDa protein (271), as
opposed to the 80 kDa FXIII-A in platelets and monocytes.
The expression of full-length FXIII-A mRNA in MC3T3-E1
cells and in mouse primary osteoblasts, moreover, the presence of 80 kDa protein in forskolin-treated MC3T3-E1
cells, suggest that the 37 kDa band on the Western blot
represents a product of posttranslational proteolytic fragmentation (271). It is a question if the 37 kDa product is the
result of a controlled, limited intracellular proteolysis or it is
produced during the extraction procedure by proteolysis in
the cell lysate. There are several disturbing findings concerning the former possibility. The authors also found, almost exclusively, 37 kDa FXIII-A in mouse hypertrophic
chondrocytes. Similarly, a 37 kDa band together with even
In conclusion, the studies of the last 8 years, described
above, extended FXIII research to a new field, cartilage and
bone physiopathology. Now, it is clear that chondrocytes
and osteoblasts express FXIII-A, and the expression is influenced by their stage of differentiation, location, and agonists of different cellular functions. In these cells, FXIII-A
coexists with another TG, TG-2, and there is an interesting
functional interplay between the two enzymes. cFXIII of
chondrocytes and osteoblasts becomes externalized and activated, although the biochemistry of neither of these events
has been clarified. Experiments with cultured cells suggest
that externalized FXIIIa (and TG-2) is involved in extracellular matrix formation, assembly, and stabilization, which
seem to be important prerequisites for the process of mineralization. Investigations on FXIII-deficient patients and
on FXIII-A knockout mice concerning the structure/development of bone and cartilage has not been carried out in
this respect. The few available clinical data support a pathophysiological role of FXIII related to hypertrophic differentiation of chondrocytes and osteoarthritis. In the coming
years, more extensive research is expected on this area that
might even lay down the scientific foundation of topical
recombinant FXIII therapy in certain pathological conditions or in traumatic bone injuries.
VII. FACTOR XIII AS AN INTRACELLULAR
ENZYME
A. Involvement of Factor XIII in Platelet
Function
Although the presence of FXIII-A in platelets has been
known for half a century, only sporadic pieces of information became available on the intracellular function of cFXIII
in these cells. Platelet FXIII-A used to be considered only as
a source of FXIII-A in pFXIII. However, in the light of
slowly accumulating data on the intracellular activation of
cFXIII and on the cross-linking of proteins within platelets,
this former view could hardly be maintained any more.
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In addition to chondrocytes, cFXIII and TG-2 were also
detected in cultured MC3T3-E1 (pre)osteoblasts both at
mRNA and protein levels (17). Abundant TG activity was
observed in the lysate of cells undergoing differentiation,
which was highly increased after thrombin treatment, suggesting that cFXIII was the dominant TG in these cells.
Treatment of the cells with ascorbic acid, which stimulated
collagen synthesis and assembly, greatly enhanced the externalization of cFXIII, although the mechanism of externalization has not been explored. The covalent incorporation of monodansyl cadaverine (MDC), a TG amine substrate, into the extracellular matrix proved that at least part
of externalized FXIII existed in active form. Fibronectin
was identified as the major TG substrate matrix component. Abrogation of TG activity by cystamine decreased
matrix accumulation and mineralization induced by the addition of ascorbic acid plus ␤-glycerophosphate (17). Cystamine also blocked cell differentiation program as indicated by the low level of alkaline phosphatase. It is very
likely that the lack of sufficient matrix assembly and alkaline phosphatase level both contributed to the blockade of
mineralization.
lower molecular weight breakdown products are shown on
the Western blot of mouse macrophage lysate. In sharp
contrast to these findings, full-length FXIII-A has been demonstrated in chondrocytes (282) and in mouse macrophages
(320) without significant proteolytic fragmentation. Furthermore, it was also claimed that the 37 kDa FXIII-A fragment is active without proteolysis by thrombin (271), while
in a previous report from the same group thrombin highly
increased the TG activity in MC3T3-E1 cell extracts (17).
Unless a 37 kDa FXIIIa is isolated from these cells and
becomes biochemically well characterized (which sequence
of FXIII-A is represented, what is its activity compared with
the full-length enzyme, etc.), its intracellular existence and
function remain questionable.
MUSZBEK ET AL.
Agonist-induced platelet aggregation in platelet-rich plasma
from patients with FXIII deficiency and from FXIII-A knockout mice was normal (174, 199, 289), and washed FXIIIA-deficient human or mouse platelets showed normal aggregation response to thrombin (199, 262). Although fibrinogen binding to thrombin receptor agonist peptide
(TRAP) activated FXIII-A-deficient platelets was somewhat
decreased (160), this decrease does not seem to be sufficient
to influence the aggregation process. Platelet aggregation is
a relatively fast process; it is over in 5 min, while the crosslinking of platelet proteins is much slower (262). This suggests that cross-linked polymers are required only for the
later phases of platelet activation, e.g., for spreading of
platelets following adhesion or for clot retraction. The
cross-linking of contractile proteins, like actin, myosin, vinculin, and filamin (27, 67, 68, 340), supports such a hypothesis. The role of platelet FXIII in clot retraction is not clear.
Platelet clot retraction of FXIII-deficient patients was found
to be normal (162, 306) or even enhanced (274). In contrast, reduced contractile force was measured in plateletrich plasma clot from a FXIII-deficient patient (57), and the
combination of FXIII-A-deficient plasma with normal
platelets was sufficient to impair isometric contraction of
platelet-rich plasma clot (66). Impaired retraction of clot
formed in platelet-rich plasma of FXIII-A knockout mice
was observed by Kasahara et al. (174). In the latter experiments, the addition of human pFXIII or rFXIII-A2 to the
platelet-rich plasma of FXIII-A knockout mice only partially restored clot retraction, suggesting that cFXIII also
contributes to the process (174). However, in an earlier
report, FXIII-deficient platelets induced normal clot retraction (306). The controversies concerning the involvement of
pFXIII and/or cFXIII in clot retraction might be due to
958
species difference and to the considerable difference between contractile force measurement and clot retraction in
a test tube.
Most recently, an interesting observation was reported concerning the spreading of platelets on fibrinogen-coated surface (160). FXIII-A-deficient platelets demonstrated an altered spreading phenotype compared with normal platelets.
The altered phenotype of FXIII-A-deficient platelets is characterized by higher percentage of platelets emitting filopodial versus lamellipodial extension and by a delay in the
spreading process. The involvement of cFXIII in the spreading process is further supported by experiments in which
MDC induced a dose-dependent increase in the proportion
of control platelets showing filopodial processes (160).
MDC might also influence platelet function by means other
than the inhibition of protein cross-linking by FXIIIa; it can
inhibit calmodulin-dependent mechanisms (76) and modify
proteins at their glutamine residues. However, its ineffectiveness on the spreading of FXIII-A-deficient platelets indicates that the alteration of platelet spreading by this compound was due to the inhibition of FXIIIa-mediated protein
cross-linking.
Kulkarni and Jackson (192) studied the involvement of
FXIII in the late stage of platelet spreading on collagen I
coated surface. Fully spread platelets undergo a specific sequence of morphological changes, which the authors term
sustained calcium-induced platelet (SCIP) morphology. It
started with the marked contraction of lamellipodial membranes leading to microvesiculation and eventual fragmentation of the cells into a larger central membrane structure
surrounded by smaller membrane bodies. SCIPs bind annexin V, indicating the translocation of phosphatidylserine,
the procoagulant phospholipid, to the outer membrane surface. SCIP formation of platelets from a FXIII-A-deficient
patient was decreased, and MDC also attenuated this process. It was claimed that a membrane-impermeable FXIIIa
inhibitor also exerted an inhibitory effect; however, no evidence was given for the extracellular presence of FXIIIa. It
seems that the activation of FXIII and calpain act synergistically in this process.
The above findings indicate that the interaction of FXIII
with cytoskeletal elements plays a role in its intracellular
function. Results published on this area have not been assembled into a comprehensive coherent view; however, important pieces of the puzzle have been revealed. In resting
platelets, FXIII-A is uniformly distributed in the platelet
cytosol (340, 346, 389); however, during activation or
spreading on an adhesive surface, at least part of it becomes
translocated to the cytoskeletal fraction (340, 389). In
platelets activated by thrombin or Ca2⫹ ionophore, cFXIII
translocated to the periphery of the cells within 1 min and
became associated with thin ruffle like extensions and/or
pseudopods, then gradually diffused back into the central
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Experiments with FXIII-A-deficient platelets provided important data on the role of cFXIII in platelet function. As
discussed in section IIIA, FXIII becomes activated by the
nonproteolytic pathway in human platelets during activation induced by thrombin or Ca2⫹ ionophore (261, 262)
and cross-links platelet proteins into high Mr polymers (67,
68, 128, 262). Although only a smaller portion of platelet
FXIII becomes activated, due to the very high concentration
of cFXIII in these cells, this active fraction is quite sufficient
to bring about the cross-linking reaction (261, 340). It is to
be emphasized that in human platelets FXIII is the only TG.
TG-2 protein and TG-2 mRNA could not be detected in
FXIII-A-deficient or normal human platelets, TG activity is
negligible in FXIII-A-deficient platelets (160, 243, 255),
and no highly cross-linked protein polymers are formed in
these cells (262). There might be a species difference in this
respect; TG activity in the lysate of platelets from FXIIIA⫺/⫺ mice was higher than in platelet lysate from normal
mice, suggesting a compensatory upregulation of another
TG (164). It is clear that no such compensatory mechanism
exists in humans, and protein cross-linking observed in activated platelet is entirely due to the TG activity of FXIIIa.
MULTIPLE FUNCTIONS OF FACTOR XIII
located to the cytoskeleton formed during platelet activation. 4) Intracellular FXIIIa is very likely involved in certain
phases of the platelet spreading process and might be involved in clot retraction, at least in mice.
cFXIII may interact with platelet proteins in two different
ways: 1) its inactive or active form may associate with other
platelet proteins without using them as substrate and move
together during translocation, and 2) FXIIIa can utilize
some proteins as glutamine and/or lysine donor substrates.
Zhu et al. (389) demonstrated the association of FXIII with
the low Mr heat shock protein HSP27 in platelets. In resting
platelets, HSP27 was also found in the cytosol, but upon
activation, it becomes phosphorylated and associated with
the activation-dependent cytoskeleton (240). As opposed to
resting platelets, in adhered platelets HSP27, in colocalization with cFXIII, was preferentially confined to the circumferential region consistent with the outermost extensions of
spread platelets and to the central region corresponding to
condensed granules and contractile elements (389). It is
possible that HSP27 escorts FXIII to specific sites within the
cells, for instance, to the cytoskeleton assembled de novo
during the activation of platelets. Four other proteins, focal
adhesion kinase (FAK), gelsolin, myosin IIA, and thrombospondin I, coimmunoprecipitated with cFXIII from the
platelet lysate and found in colocalization with FXIII-A
both in resting and activated platelets (309). Gelsolin and
myosin IIA are cytoskeletal proteins, while the tyrosine kinase FAK translocates to the cytoskeleton in the second
wave of cytoskeletal rearrangement (285). Direct association between cFXIII and these proteins should be confirmed
in binding experiments with purified proteins.
Most recently, the involvement of cFXIII in certain functions of megakaryocytes has also been reported (231). It
was shown that megakaryocyte adhesion to type I collagen
promoted the spreading of these cells and inhibited proplatelet formation through the release and relocation to the
plasma membrane of cellular fibronectin. cFXIII was also
shown to be translocated to the outer surface of plasma
membrane and cross-linked fibronectin, although the mechanism of its translocation was not revealed. The inhibition
of FXIIIa significantly decreased megakaryocyte spreading
on type I collagen and reduced actin stress fiber formation
and the assembly of fibronectin. These findings suggest that
FXIII participates in the mechanism by which type I collagen, in the osteoblastic niche, suppresses megakaryocyte
maturation and prevents premature platelet release.
The cross-linking of two cytoskeletal elements, filamin and
vinculin, during platelet activation has been analyzed in
detail (340). A high portion of filamin was cross-linked to at
least four different nonidentified platelet proteins in thrombin-activated platelets within 10 min. Blocking TG activity
by the -SH reagent iodoacetamide completely abolished filamin cross-linking. Inhibition of fibrinogen binding to
platelets and, consequently, platelet aggregation by the peptide Gly-Pro-Arg-Pro decreased the cross-linking of filamin,
suggesting that it is an aggregation-induced process. The
cross-linking of vinculin was less effective, and it was a
platelet aggregation-dependent slower process.
In summary, although the exact biochemical mechanism by
which FXIII participates in platelet function still needs clarification, the available data allow the following conclusions: 1) a part of platelet FXIII becomes transformed into
an active TG during platelet activation by the nonproteolytic activation mechanism. 2) Activated FXIII, the only TG
in human platelets, cross-links platelet proteins in the later
phases of platelet activation. 3) A few cytoskeletal proteins
have been identified as substrates for FXIIIa in the crosslinking reaction, and at least part of FXIII becomes trans-
B. Involvement of Factor XIII in Monocyte/
Macrophage Function
Since the discovery of FXIII-A in monocytes and macrophages in 1985 (8, 130, 257), its expression in various
monocyte-derived mobile and fixed macrophages has been
intensively studied and turned out to be a marker reaction
for various subsets of these cells in physiological and pathological settings (6, 304). The possible coexistence of cFXIII
with TG-2 in monocytes and macrophages is a somewhat
debated question. We could not measure any detectable TG
activity in monocytes from four patients with severe
FXIII-A deficiency (255), and only negligible TG activity
was measured in such monocytes by the highly sensitive
[3H]putrescine incorporation assay (72). No TG-2 was
found in normal peripheral blood monocytes by Seiving et
al. (338), and only minute TG activity was measured in
nonactivated human monocyte cell lysate without thrombin activation (239, 242, 254). In the latter studies, Western
blotting also detected a small amount of TG-2. The amount
of TG-2 was much less than that of FXIII-A in the antigenspecific immunoprecipitates from nonactivated monocytes
(16). Most recently, similar FXIII-A and TG-2 mRNA levels
were measured in human monocytes; however, in this
study, no attempt was made to measure the translated protein levels (161). In conclusion, overwhelming pieces of evidence suggest that in resting nonactivated monocytes,
cFXIII is the major TG, and the amount of TG-2, if any, is
not significant. However, during differentiation into macrophages and following activation by various agents, major
changes occur in the intracellular level of both TGs.
In two early studies, opposing results were obtained on the
changes of cFXIII in monocyte culture. A three- to fourfold
increased FXIII-A activity and antigen levels were measured
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area (340). Cytochalasin D that inhibits the polymerization
of actin in intact cells abolished the translocation of cFXIII
to the platelet periphery. Part of cytoskeleton-associated
cFXIII existed in active form.
MUSZBEK ET AL.
on day 8 (72), while decreasing cFXIII with increasing TG-2
was reported in long-term culture of monocytes (338).
More recently, a significant elevation of both FXIII-A
mRNA and protein level was observed during the course of
monocyte/macrophage differentiation in cell culture that
peaked on day 3 (329). It is to be noted that the expression
of TG-2 also becomes upregulated during culturing of
monocytes (239, 242, 254). It is interesting that in monocytic leukemia cells, cFXIII level was also increased compared with normal peripheral blood monocytes (172).
IL-4 is also involved in the in vitro differentiation of monocytes into antigen-presenting dendritic cells. Induction of
such differentiation by granulocyte/macrophage-colony
stimulating factor (GM-CSF) plus IL-4 resulted in very high
intracellular levels of FXIII-A mRNA and protein (15, 161,
362). If dendritic cells were treated by BCG, stimulating the
classical pathway, FXIII-A expression returned to the basal
level within 2 days (362). Subsets of dendritic cells present
in tissues, like subsets of dermal dendrocytes, also express
FXIII-A. The characterization of these subsets is reviewed in
Reference 304.
cFXIII of perivascular macrophages has been implicated in
the flow-dependent remodeling of small arteries in mice (30,
388). In this process, the perivascular accumulation of
FXIII-A-positive macrophages was dependent on CXCchemokine receptor 3 (CXCR3) signaling by the CXCR3
960
Considering the high number of reports on the expression
of FXIII-A in monocytes, macrophages, histiocytes, and
dendritic cells, it is surprising that only a few studies have
concerned with the intracellular function of cFXIII in these
cells. Monocytes from FXIII-deficient patients, which are
exempt of cFXIII, showed an impaired capacity of Fc␥,
complement, and lectin-like receptor-mediated phagocytosis (329). In cell culture, the expression of FXIII-A and the
phagocytosing capacity of monocytes/macrophages increased in parallel, and the competitive FXIIIa substrate,
MDC, significantly inhibited receptor-mediated phagocytosis by these cells. DD cells (a human myelomonocytic cell
line) were incapable of phagocytosis and did not express
FXIII-A antigen. Partial differentiation of the cells, achieved
by phorbol ester treatment, resulted in increased phagocytosis in parallel with the expression of FXIII-A (177). Jayo
et al. (161) investigated the role of cFXIII in the locomotion
of monocyte-derived dendritic cells. MDC inhibited both
basal and chemokine (C-C motif) ligand 19 (CCL19)-induced migration of mature dendritic cells, and FXIII-Adeficient dendritic cells showed a reduced chemotactic response to CCL19. CHO cells overexpressing FXIII-A
showed enhanced mobility both in trans-well and scratch
wound migration assays and displayed membrane blebs
and dynamic cell protrusions involved in cell movement. As
both phagocytosis and cell locomotion heavily involve intracellular contractile elements, the interaction of cFXIII or
its activated form with cytoskeletal components in monocytes/macrophages seems rather feasible. However, this theory still lacks experimental support.
An interesting aspect of the intracellular function of cFXIII
in monocytes was reported by AbdAlla et al. (1). In monocytes from hypertensive patients, covalently cross-linked
angiotensin receptor 1 (AT1) dimers were detected; this
dimerization was carried out by nonproteolytically activated cFXIII. The covalent cross-linking of AT1 could be
reproduced by the combined addition of angiotensin II and
ionomycin, which activated AT1 and, by increasing the intracellular Ca2⫹ concentration, cFXIII. No cross-linking of
AT1 was observed in monocytes from FXIII-A-deficient patients, and it was also abolished by angiotensin convertase
inhibitor. Cross-linked AT1 dimers displayed enhanced
G␣q/11-stimulated signaling, increased internalization, and
desensitization. Gln315 in the cytoplasmic side of AT1 provided the Gln substrate for the FXIIIa-catalyzed cross-linking. The appearance of cross-linked AT1 dimers correlated
with the enhanced angiotensin II-dependent adhesion of
monocytes to endothelial cells and endothelial dysfunction.
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Torocsik et al. (362) investigated the changes of FXIII-A
expression after activation of monocytes through the classical or alternative pathway. Induction of the classical activation pathway by interferon (IFN)-␥ or Mycobacterium
bovis BCG downregulated FXIII-A mRNA and protein expression in cultured macrophages, while activation by IL-4
through the alternative pathway exerted the opposite effect.
In the latter case, on days 4 and 5, the increase of FXIII-A
mRNA level was more than 40-fold, and FXIII-A protein
level became elevated 11-fold on day 5. The expression of
TG-2 did not change in these conditions. Similar upregulation of FXIII-A mRNA by IL-4, and to a lesser extent by
IL-13, was observed by another group, as well (59), while
priming monocytes by lipopolysaccharide decreased
FXIII-A expression (290). In another study, the effect of
IL-4 on the expression of FXIII-A at the mRNA and protein
level was highly increased by the addition of dexamethasone (120). Results of in vitro investigations were also supported by the detection of FXIII-A in tumor-associated
macrophages that are considered as alternatively activated,
and its absence in macrophages of tuberculotic granuloma
that go through the classical activation pathway (362). The
expression of FXIIIa substrate fibronectin is also augmented by IL-4/IL-13. The parallel upregulation of cFXIII
expression and tissue inhibitor of metalloproteinase 3
(TIMP3) could protect the deposited extracellular matrix
protein from degradation (59).
ligand, interferon inducible protein-10 (IP-10). This mechanism also contributed to the highly increased production
of FXIII-A in CRCR3-positive macrophages (388). However, it is not clear how elevated cFXIII in perivascular
macrophages is involved in vascular remodeling and if such
a mechanism also operates in humans.
MULTIPLE FUNCTIONS OF FACTOR XIII
The inhibition of angiotensin II generation or intracellular
FXIII activity suppressed the appearance of cross-linked
AT1 receptors and symptoms of atherosclerosis in apoEdeficient mice. Follow-up studies confirming these exciting
findings are awaited.
There is not much information on the release or surface
expression of monocyte/macrophage cFXIII. Although
there is a general agreement on the cytoplasmic localization
of FXIII-A in these cells, a few studies indicated that macrophages in cell culture translocate it to the surface (16, 72,
190). Increased surface staining of U937 cells (a human
promonocytic tumor line) for FXIII-A was observed after
incubation with phorbol myristate acetate, lipopolysaccharide, and IFN-␥ (190). In a single study, FXIII-A was detected in the culture medium of dendritic cells differentiated
from peripheral blood monocytes (15). However, in these
studies, dying or dead cells were not excluded as the source
of cFXIII appearing on the cell surface or in the medium.
Although the surface expression or release of cFXIII from
macrophages, especially from resident macrophages and its
involvement in organization and remodeling of extracellular matrix by the cross-linking matrix proteins is quite plausible, this hypothesis has not been supported by experimental proof. As mentioned in section IIC, there is evidence for
cFXIII entering the alternative secretory pathway in human
macrophages (75), but its actual secretion has not been
proven.
In addition to the possible translocation of cFXIII from the
cytoplasm to the cell surface, another type of cFXIII translocation has also been revealed in macrophages. The transient appearance of cFXIII in the nuclei was demonstrated
by confocal laser scanning microscopy, Western blotting,
and immunoelectron microscopy in the early phase of
monocyte/macrophage differentiation (7). On day 2 of culturing, 90% of the cells expressed FXIII-A in the nucleus in
association with electrodense areas, but this phenomenon
was not observed on any other day. cFXIII in the nucleus
somehow became activated, and its TG activity was demonstrated by the incorporation of MDC into nuclear proteins. Nuclear localization of TG-2 has also been observed
(208, 291, 295), but the function of neither TG has been
revealed in the nuclear environment. Follow-up studies on
VIII. SUMMARY, CONCLUSIONS, AND
PERSPECTIVES
pFXIII is a tetrameric zymogen protransglutaminase, which
consists of two potentially active A and two inhibitory/
carrier B subunits (FXIII-A2B2). In the plasma it is bound to
the ␥=-chain of fibrinogen through FXIII-B subunits. The
synthesis of the two subunits occurs in different cells, and
they form complex in the plasma. There is little doubt that
the major part of FXIII-A is synthesized by cells of bone
marrow origin, while FXIII-B is produced and secreted by
hepatocytes. However, it is still unclear what is the contribution of megakaryocytes and monocytes/macrophages to
FXIII-A present in pFXIII. The secretory pathway by which
FXIII-A is released from the cells also remains to be elucidated, although most recently evidence has been provided
on its entering into the nonclassical secretory pathway in
macrophages. FXIII-B is in excess in the plasma; ⬃50% of
it circulates in free, uncomplexed form. The dimer of
FXIII-A, but not FXIII-B, is also present in cells (platelets,
monocytes, macrophages, chondrocytes, osteoblasts, osteocytes). The three-dimensional structure of FXIII-A2 has
been revealed by X-ray crystallography, and four major
structural domains (␤-sandwich domain, central core domain, and two ␤-barrel domains) and AP-FXIII were identified. The three-dimensional structure of FXIII-B is not
known; it contains 10 sushi domains each held together by
two pairs of disulfide bonds.
pFXIII becomes activated in the final phase of coagulation
cascade by thrombin and Ca2⫹. Thrombin cleaves off APFXIII from FXIII-A, which, as demonstrated most recently,
dissociates from the truncated molecule (FXIII-A=). The
newly formed fibrin ⬃100-fold accelerates this process. In
the presence of Ca2⫹, FXIII-B leaves FXIII-A=2, and the
latter assumes an enzymatically active configuration (FXIIIA*2). In plasma, the activation of FXIII occurs on the surface of fibrin, and FXIIIa remains associated with it, while
FXIII-B is released into the serum. The release of AP-FXIII
is not a prerequisite of FXIII activation. At very high unphysiological Ca2⫹ concentration (ⱖ100 mM), FXIII-B dissociates from the nontruncated FXIII-A2, which then becomes enzymatically active (FXIII-A°2). The main role of
the truncation of FXIII-A is to bring down the Ca2⫹ requirement for the dissociation step into physiological range. In
the absence of FXIII-B, cFXIII can be transformed into
FXIII-A°2 at low Ca2⫹ concentration. The main role of
FXIII-B seems to prevent FXIII-A2 from going through a
slow progressive activation in plasmatic environment. Several pieces of evidence suggest that the nonproteolytic pathway is the main form of cFXIII activation in platelets and in
monocytes/macrophages. No major conformational change
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In a most recent paper it was demonstrated that cFXIII is
involved in the regulation of gene expression in alternatively activated human macrophages (363). In microarray
experiments, a significant difference was demonstrated in
the IL-4-sensitive gene expression profile of cultured macrophages derived from FXIII-deficient and nondeficient individuals. The most prominent differences were related to
immune functions and wound response, suggesting that the
functional impairment of macrophages at the level of gene
expression regulation plays a role in the wound healing
defect of FXIII-deficient patients.
the involvement of cFXIII in nuclear processes are required
to extend and clarify the significance of this intriguing finding.
MUSZBEK ET AL.
was detected in the structure of FXIII-A2 during transformation into a TG by X-ray crystallography; however, biochemical evidence and the analogy with the activation of
TG-2 indicate that in solution the activation process induces considerable structural changes.
The downregulation of FXIIIa has been explored only most
recently. Polymorphonuclear granulocytes incorporated in
the clot become activated and release several proteolytic
enzymes into their surroundings. These proteases, primarily
elastase, break down FXIIIa within the plasma clot, even in
the presence of serine protease plasma inhibitors. The kinetics of FXIIIa inactivation and fibrinolytic effect of granulocyte proteases ensure the formation of stable fibrin clot,
but prevent the production of over-cross-linked plasma
clot, which would be difficult to eliminate, when it is not
needed any more. Further research on this important field
would broaden our knowledge on what is happening with
FXIIIa in the plasma clot and in the thrombus.
The main function of FXIIIa in the plasma is to cross-link
fibrin chains and ␣2-PI to fibrin. Fibrin ␥-chains are crosslinked into dimers at a very early stage of clotting, while
fibrin ␣-chains form high-molecular-weight polymers at a
much slower rate. The cross-linking of ␣2-PI to fibrin
␣-chains is also rather fast, and the heterodimer becomes
progressively incorporated into the highly cross-linked
␣-chain polymers. Fibrin cross-linking stabilizes fibrin and
makes it more resistant against shear stress. Both fibrin
cross-linking and the covalent incorporation of ␣2-PI into
962
The involvement of FXIII in wound healing has been supported by sporadic clinical data from studies with FXIII-Adeficient patients. Its role in wound healing is now well
proven by the impaired wound healing of FXIII-A knockout
mice. The promotion of wound healing by FXIII is a complex process; it involves its effects on fibroblasts, macrophages, extracellular matrix, and angiogenesis. FXIIIa enhanced the migration of fibroblasts and macrophages, increased the rate of fibroblasts proliferation, and decreased
their apoptosis. Cross-linking of extracellular matrix proteins might modulate these direct effects. The FXIIIa-induced biochemical mechanisms in these events have not
been enlightened, yet. The downregulation of TSP-1 has
been shown to contribute to the effect of FXIIIa on monocytes.
In the last couple of years, the proangiogenic effect of FXIII
has been explored to a significant extent. Although nonactivated FXIII can bind to endothelial cells, the proangiogenic
effect is exerted only by the active form. FXIIIa enhances endothelial cell migration and proliferation and suppresses their
apoptosis. The role of FXIII in angiogenesis has also been
verified in several animal models. The effect of FXIIIa on endothelial cells involves binding to ␣v␤3 receptor, the interaction of this receptor with VEGFR-2, VEGFR-2 phosphorylation, phosphorylation and activation of Akt and ERKs, the
upregulation of the transcription factors c-Jun and Egr-1, and
the downregulation of TSP-1. Further exploration of this
mechanism is expected, and the proangiogenic effect of FXIIIa
or its inhibition could have a prospect of clinical utilization.
Clinical findings and experiments with FXIII-A knockout
mice prove, without any doubt, that pFXIII is essential for
maintaining pregnancy. It is surprising that, in spite of the
importance of these observations, hardly any studies explored the mechanism by which pFXIII prevents spontaneous abortions. As fibrinogen deficiency also leads to early
pregnancy loss, the cooperation of the two clotting factors
seems essential. The cross-linking of fibrinoid components
in the placenta (fibrin, fibronectin) is very likely important
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FXIIIa, like other TGs, catalyzes a double displacement acyl
transfer reaction, in which the acyl donor peptide-bound
glutamine residue forms thioester with the active site
Cys314 of the core domain, and ammonia is released. In the
second step, an acyl acceptor primary amine is cross-linked
to the glutamine residue through isopeptide bond, and the
active site cysteine becomes deacylated. If the acyl acceptor
amine is provided by a peptide-bound lysine residue, two
peptide chains become covalently cross-linked. The glutamine substrate specificity of FXIIIa is rather restrictive,
only less than two dozen proteins have been identified as
FXIIIa substrates. However, the amino acid sequences
around the substrate glutamine site show considerable variation, and no consensus sequence could be derived from the
known primary structures. The structural requirement of
the interaction with FXIIIa was studied only in the case of
two peptide substrates. One can conclude from the NMR
studies and kinetic experiments with chemical mutant peptides that, in addition to the active site, binding of the substrate peptide to a second interacting site, involving hydrophobic interaction, on the surface of FXIII-A*2 is also a
prerequisite of the enzymatic action. Experiments with a
series of individual glutamine donor peptide substrate are to
be carried out to clarify the structural elements required for
effective enzyme-substrate interaction.
fibrin protect fibrin against fibrinolysis. The cross-linking of
␣2-PI to the fibrin network seems to play the dominant role
in preventing newly formed fibrin from the prompt elimination by plasmin, while fibrin ␣-chain cross-linking could
contribute to the increased resistance of mature thrombi to
fibrinolysis. pFXIII is essential for maintaining hemostasis;
its deficiency results in very severe bleeding diathesis.
pFXIII is now considered as a key player in the process of
fibrinolysis, which exerts its effect through the two mechanisms discussed above. In addition to ␣2-PI, FXIIIa also
cross-links other components of fibrinolysis, like PAI-2,
TAFI, and plasminogen, to fibrin or fibronectin; however,
the significance of these cross-linking reactions remains to
be explored. Further intensive research is expected on this
field in the near future.
MULTIPLE FUNCTIONS OF FACTOR XIII
for the sealing/anchoring effect and barrier function of the
Nitabuch’s layer. The proangiogenic effect of FXIIIa might
contribute to normal placenta development. Elaboration of
these issues could lead to highly important pieces of information and to better understanding of the mechanisms required for maintaining pregnancy.
The active form of FXIII-A2 is also an intracellular enzyme.
Although its presence in platelets is known for more than a
half century and their existence in monocytes/macrophages
for a quarter of century, its intracellular function started to
be addressed only most recently. During platelet activation
part of its cFXIII becomes activated by the nonproteolytic
mechanism in the later phase of the activation process. During this process, part of FXIII becomes translocated to the
cytoskeleton and cross-links platelet proteins, particularly
cytoskeletal proteins. Activated FXIII is involved in certain
phases of platelet spreading. Information on the involvement of cFXIII in clot retraction is controversial. It is not
clear if partial contribution of cFXIII to the clot retraction
in mice could be extrapolated to humans.
Intriguing novel findings were reported on monocyte/macrophage cFXIII in the last couple of years. The expression of
cFXIII in monocytes activated by the alternative pathway
was highly upregulated, while activation through the classical pathway resulted in the opposite effect. The results
obtained with cultured monocytes were also confirmed
with in situ activated tissue macrophages. The transient
appearance of cFXIII in the nucleus on day 2 of monocyte
culturing is an intriguing finding that warrants further investigation. Differentiation of monocytes into dendritic
cells resulted in very high intracellular level of cFXIII. Impaired receptor-mediated phagocytosis of monocytes from
FXIII-deficient patients suggests the involvement of cFXIII
in the biochemical mechanism of this process. cFXIII has
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: L.
Muszbek, Clinical Research Center, Univ. of Debrecen,
Medical and Health Science Center, 98 Nagyerdei krt., Debrecen 4032, Hungary (e-mail: [email protected]).
GRANTS
This review was made possible through research funding
from the Hungarian National Research Fund (OTKANKTH CNK 80776), the Hungarian Academy of Sciences
(TKI 227), and the Hungarian Ministry of Health (ETT).
The work was also supported by the TÁMOP 4.2.1./B-09/
1/KONV-2010-0007 project. The project is implemented
through the New Hungary Development Plan, cofinanced
by the European Social Fund.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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In addition to its role in hemostasis, wound healing/angiogenesis, and maintaining pregnancy, studies in the last decade drew attention to other less evident, less expected, and
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