Download When 1 plus 1 equals 3 in VWD

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The studies by Graham et al in this issue of
Blood reveal the importance of dense granule
secretion for in vivo platelet accumulation
following laser injury of cremaster arterioles.8
The most striking observation is that dense
granule deficient mice (ruby-eye mice) show
almost no platelet accumulation at injury sites,
indicating that molecules released by dense
granules are necessary for efficient thrombus
formation. In VAMP-8⫺/⫺ mice, thrombus
formation is delayed and decreased (but not
absent) following laser injury, suggesting that
the v-SNARE Endobrevin/VAMP-8 is an
important component of in vivo platelet granule secretion. The incomplete inhibition of
platelet accumulation at sites of injury in
VAMP-8⫺/⫺ mice was somewhat surprising
because VAMP-8 mediates the release of all
platelet granules, hence an additive effect
might have been expected. The results can be
explained by functional redundancy of
SNAREs, whereby VAMP-2 or -3 can mediate granule release in the absence of VAMP-8.7
The authors observe that VAMP-2 and
VAMP-8 levels are comparable in mouse
platelets, whereas VAMP-3 and VAMP-8
levels are comparable in human platelets. As
VAMP-2 levels are greatly diminished in human platelets, this suggests that the major alternative granule v-SNARE in humans
is VAMP-3.
The dense granule constituents responsible for mediating platelet accumulation in
vivo were examined in vitro via aggregation
studies of plasma-free washed platelets. Compared with wild-type, platelets from VAMP8⫺/⫺ and ruby-eye mice required 1.5- to
10-fold higher concentrations of thrombin for
complete aggregation, while addition of exogenous ADP produced normal aggregation responses at low thrombin concentrations. The
function of PKC␣⫺/⫺ mouse platelets (which
have impaired dense granule biogenesis and
defective secretion of both dense and
␣-granules) can also be restored by ADP in
vitro.9 Thus, ADP is a key dense granule component, and its release during activation is
likely required for efficient platelet accumulation at vascular injury sites. The thrombus
formation defects observed in ruby-eye mice
indicate that in vivo thrombin generation following laser injury cannot compensate for the
absence of dense granule secretion.
Although these studies provide important
new insights into the role of platelet granule
secretion in vivo, some questions do remain.
blood 3 0 J U L Y 2 0 0 9 I V O L U M E 1 1 4 , N U M B E R 5
For example, which SNAREs and regulatory
proteins control selective secretion from granules, including the recently discovered distinct
populations of ␣-granules?10 What contribution does ␣-granule secretion make to thrombus formation in vivo? The availability of
megakaryocyte-restricted gene knockout mice
will undoubtedly shed light on some of these
questions. Importantly, the current studies
suggest that antiplatelet therapies targeting
granule secretion could attenuate acute thrombus formation and also reduce the development of chronic atherosclerotic lesions by inhibiting the release of inflammatory and
immune-modulating factors from platelets.
Conflict-of-interest disclosure: The author
declares no competing financial interests. ■
REFERENCES
1. Lo B, Li L, Gissen P, et al. Requirement of VPS33B, a
member of the Sec1/Munc18 protein family, in megakaryocyte and platelet ␣-granule biogenesis. Blood. 2005;106:
4159-4166.
2. Nurden P, Nurden AT. Congenital disorders associated
with platelet dysfunctions. Thromb Haemost. 2008;99:
253-263.
3. Flaumenhaft R. Molecular basis of platelet granule secretion. Arterioscler Thromb Vasc Biol. 2003;23:1152-1160.
4. Ren Q, Ye S, Whiteheart SW. The platelet release reaction: just when you thought platelet secretion was simple.
Curr Opin Hematol. 2008;15:537-541.
5. Reed GL. Platelet secretory mechanisms. Semin Thromb
Hemost. 2004;30:441-450.
6. Südhof TC, Rothman JE. Membrane fusion: grappling
with SNARE and SM proteins. Science. 2009;323:474-477.
7. Ren Q, Barber HK, Crawford GL, et al. Endobrevin/
VAMP-8 is the primary v-SNARE for the platelet release
reaction. Mol Biol Cell. 2007;18:24-33.
8. Graham GJ, Ren Q, Dilks JR, Blair P, Whiteheart SW,
Flaumenhaft R. Endobrevin/VAMP-8 – dependent dense
granule release mediates thrombus formation in vivo. Blood.
2009;114:1083-1090.
9. Konopatskaya O, Gilio K, Harper MT, et al. PKC␣
regulates platelet granule secretion and thrombus formation
in mice. J Clin Invest. 2009;119:399-407.
10. Italiano JE Jr, Richardson JL, Patel-Hett S, et al. Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood. 2008;
111:1227-1233.
● ● ● THROMBOSIS & HEMOSTASIS
Comment on Sutherland et al, page 1091
When
1 plus 1 equals 3 in VWD
---------------------------------------------------------------------------------------------------------------Anne C. Goodeve
UNIVERSITY OF SHEFFIELD
In this issue of Blood, Sutherland and colleagues describe an unusual in-frame deletion of exons 4-5 of the VWF gene associated with both dominantly inherited
type 1 and with type 3 VWD.1
he mutation was initially identified in homozygous type 3 von Willebrand disease
(VWD) patients, subsequently found in further compound heterozygous patients with
type 3 VWD, and then sought in a previously
studied cohort of type 1 VWD cases.2 It has
thus been identified in 8 of 24 (33%) alleles of
the type 3 VWD cohort of 12 apparently unrelated index cases (2 homozygotes and 4 compound heterozygotes) and additionally identified in 2 of 34 (6%) type 1 VWD index cases.
Most mutations identified in type 3 VWD
are “null” mutations, resulting in a lack of expression due to non-sense, frameshift, splice
site, and large deletion mutations. Large deletions typically result in a lack of von Willebrand factor (VWF) expression, and penetrance of bleeding symptoms in simple
heterozygous individuals with such null mutations is very low. The deletion currently described is unusual in that a truncated VWF
T
protein is produced, albeit in very low quantity, that has a dominant negative effect on
expression of the second allele. The small
group of heterozygous type 1 VWD patients
with the mutation have mean VWF levels of
around 25 IU/dL, and the mutation appears
to result in bleeding in the majority of individuals investigated.
All but one family where the mutation was
identified shared a common haplotype associated with the deletion comprising 25 SNP
throughout VWF. A second related haplotype
found in heterozygous form in only one type 3
family appeared to have arisen from a subsequent recombination 3⬘ to the deletion. The
distribution of the deletion is not yet known;
however, its presence in a number of presumed independent type 3 and type 1 VWD
families suggests that it may be widespread in
families of British origin, possibly including
emigrant families. Analysis of the other 2 large
933
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
plicated in the recombination event could also
currently likely to be
missed, where a hetbe investigated.
erozygous deletion is
Conflict-of-interest disclosure: A.C.G. remasked by the presence
ceived sponsorship from CSL-Behring for mainof an intact second altenance of ISTH-SSC on VWF website. ■
lele. Initial analysis of
the partial cloned seREFERENCES
quence of VWF indi1. Sutherland MS, Cumming AM, Bowman M, et al. A
cated that there were
novel deletion mutation is recurrent in von Willebrand dis14 Alu sequences idenease types 1 and 3. Blood. 2009;114:1091-1098.
tified,7 but there are
2. Cumming A, Grundy P, Keeney S, et al. An investigation of the von Willebrand factor genotype in UK patients
likely to be many more
Alu-mediated VWF deletion followed by recombination in one family. The top
diagnosed to have type 1 von Willebrand disease. Thromb
than
this
in
the
entire
portion of the figure shows the VWF gene intron-exon structure. Unequal homoloHaemost. 2006;96:630-641.
gous recombination between Alu Y repeats in introns 3 and 5 removes 8631bp and
gene. Close sequence
3. Goodeve A, Eikenboom J, Castaman G, et al. Phenotype
results in an in-frame deletion of exons 4-5. The deletion is located on an ancestral
similarity between reand genotype of a cohort of families historically diagnosed
haplotype (Hap 1), present in all but one of the alleles investigated by Sutherland et
with type 1 von Willebrand disease in the European study,
al.1 A subsequent recombination event (panels 2 and 3) results in generation of
peat sequences can
Molecular and Clinical Markers for the Diagnosis and Manhaplotype 2 (Hap 2). The recombination location is likely to lie between intron 5 and
help
promote
unequal
agement of Type 1 von Willebrand Disease (MCMDMexon 8. Professional illustration by Debra T. Dartez.
1VWD). Blood. 2007;109:112-121.
homologous recombi4. James PD, Notley C, Hegadorn C, et al. The mutational
nation, resulting in deletion of the interventype 1 VWD cohorts collected in Europe and
spectrum of type 1 von Willebrand disease: results from a
ing sequence. In the current example, Alu Y
Canada3,4 will help provide insight into the
Canadian cohort study. Blood. 2007;109:145-154.
sequences surrounding the breakpoint had
mutation’s distribution. If it is sufficiently
5. Rudiger NS, Gregersen N, Kielland-Brandt MC. One
over 80% sequence similarity.
short well conserved region of Alu-sequences is involved in
widespread, then the mutation should be
human gene rearrangements and has homology with proThe
subsequent
recombination
event
idensought first in any mutation screening strategy
karyotic chi. Nucleic Acids Res. 1995;23:256-260.
tified
in
one
family
by
Sutherland
et
al
is
also
for types 1 and 3 VWD, prior to VWF DNA
6. Bernardi F, Patracchini P, Gemmati D, et al. In-frame
of interest. SNP analysis suggests that the resequence analysis. A specific long polymerase
deletion of von Willebrand factor A domains in a dominant
type of von Willebrand disease. Hum Mol Genet. 1993;
chain reaction (PCR) described by the authors, combination site lies between intron 5 and
2:545-548.
exon
8.
There
may
now
be
sufficient
density
of
which produces a deletion-specific 1084 bp
7. Mancuso DJ, Tuley EA, Westfield LA, et al. Structure
VWF SNP reported that the region involved
amplicon along with a wild-type product of
of the gene for human von Willebrand factor. J Biol Chem.
1989;264:19514-19527.
could be identified and sequence features im1694 bp, will facilitate this analysis.
Examination of the sequence surrounding
the deletion breakpoint implicated unequal
● ● ● VASCULAR BIOLOGY
homologous recombination between 2 Alu Y
Comment on Wei et al, page 1123
repeat elements in introns 3 and 5 as the mutation mechanism. There are approximately
100 000 Alu repeats in the human genome,
and these have been repeatedly involved in the
---------------------------------------------------------------------------------------------------------------generation of deletion mutations. As the auPeter Oettgen BETH ISRAEL DEACONESS MEDICAL CENTER
thors highlight, this is at least the third example of an Alu-mediated deletion in type 3
In this issue of Blood, Wei and colleagues investigate the overlapping roles of Ets1
VWD and in early reports of VWF deletions,
and Ets2 in the regulation of endothelial cell function and survival during embrymutation mechanism was not investigated so
onic angiogenesis.1
the mechanism is likely underrecognized. Alu
ts1 and Ets2 are known to regulate
morphic mutant of Ets2, in which the conrepeats may be involved in both homologous
endothelial-restricted
gene
expression
served threonine 72 phosphorylation site is
and non-homologous recombination, as a core
and endothelial function. For example, Ets1
mutated, Ets2T72A/T72A mice, are viable and
26 bp repeat sequence is reported to be recomhas been shown to regulate the expression of
appear normal.5 In an elegant series of experibogenic.5 A previously reported type 2 VWD
several genes that regulate endothelial funcments, Wei et al uncover the overlapping roles
patient (disease subtype not specified) had a
tion
and
angiogenesis
including
VEGF,
of Ets1 and Ets2 during vascular development.
large in-frame deletion of 31 kb (exons 26-34)
2 However, homozygous
VEGF-R2,
and
Tie2.
First, they cross Ets1⫺/⫺ mice with Ets2T72A/T72A
associated with recombination between an Alu
inactivation of Ets1 is associated with defects
to derive double mutant Ets1⫺/⫺; Ets2T72A/T72A
repeat in intron 25 and a short stretch of ho6
in
T-cell
function,
but
no
defects
in
vascular
mice. The double mutants are embryonal lemologous sequence in intron 34. These ex3 Similarly,
development
or
angiogenesis.
thal and begin to develop vascular abnormaliamples suggest that many more large delewhile Ets2 is a critical regulator of trophoblast
ties at day E10.5, including a marked reductions, both in-frame and resulting in null
alleles, will be identified as techniques for gene function during extraembryonic development, tion in vascular complexity, defective
it is dispensable for development of the embranching, with the remaining vessels being
dosage (large deletion or duplication) analysis
dilated. To further validate that the phenotype
bryo proper.4 Mice with a homozygous hypoare developed. Many of these mutations are
Functional redundancy of Ets1 and Ets2
E
934
30 JULY 2009 I VOLUME 114, NUMBER 5
blood
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2009 114: 933-934
doi:10.1182/blood-2009-04-218099
When 1 plus 1 equals 3 in VWD
Anne C. Goodeve
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