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Alterations in C3 Activation and Binding
Caused by Phosphorylation by a Casein
Kinase Released from Activated Human
Platelets
Kristina Nilsson Ekdahl and Bo Nilsson
J Immunol 1999; 162:7426-7433; ;
http://www.jimmunol.org/content/162/12/7426
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Copyright © 1999 by The American Association of
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References
Alterations in C3 Activation and Binding Caused by
Phosphorylation by a Casein Kinase Released from Activated
Human Platelets1
Kristina Nilsson Ekdahl2†* and Bo Nilsson*
A
number of plasma proteins have been shown to contain
covalently bound phosphate in vivo. These proteins include members of both the coagulation and the complement cascades, fibrinogen (1), coagulation factor V (2), vitronectin
(3), and complement component C3 (4, 5) among others. Phosphorylation in vitro with various protein kinases has been demonstrated to alter the functional properties of these proteins; for example, human fibrinogen has been shown to act as substrate for
protein kinase A (PKA),3 protein kinase C (PKC), casein kinase 1
(CK1), and CK2 (6). Phosphorylation of fibrinogen in vitro leads
to changes in fibrin bundle thickness after treatment of phosphofibrinogen with thrombin (7, 8). Coagulation factor Va becomes
more easily cleaved by activated protein C after phosphorylation
by a CK that is released from activated human platelets (9), and
phosphorylation of vitronectin with PKC attenuates its cleavage by
plasmin (10).
*Department of Clinical Immunology and Transfusion Medicine, University Hospital,
Uppsala, Sweden; and †Department of Natural Sciences, University of Kalmar, Kalmar, Sweden
Received for publication January 11, 1999. Accepted for publication March 31, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Göran Gustafsson Research Foundation, King Gustaf V’s Research Foundation, the Swedish Rheumatism Association,
Prof. Nanna Svartz Research Foundations, and Grants 5647 and 11578 from the
Swedish Medical Research Council.
2
Address correspondence and reprint requests to Dr. Kristina Nilsson Ekdahl, Department of Clinical Immunology, University Hospital, S-751 85 Uppsala, Sweden.
E-mail address: [email protected]
3
Abbreviations used in this paper: PKA, cAMP-dependent protein kinase; PKC, calcium- and phospholipid-dependent protein kinase; CK, casein kinase; C3a, C3b, C3c,
C3d, and iC3b, proteolytic fragments of C3; HAGG, heat-aggregated g-globulin;
ATS, activated thiol Sepharose; CKI-7, N-(2-aminoethyl)-5-chloro-isoquinoline-8sulfonamide; A3, N-(2-aminoethyl)-5-chloronaphtalene-1-sulfonamide HCl; PRP,
platelet-rich plasma; VBS, veronal-buffered saline.
Copyright © 1999 by The American Association of Immunologists
Complement component C3 has been shown to be phosphorylated in vitro by at least five different protein kinases, in different
domains of the molecule and with different biologic effects. The
C3a moiety of C3 is known to be phosphorylated by PKA and
PKC (11) as well as by an ecto-protein kinase from the human
parasite Leishmania major (12). The effect of this phosphorylation
is to protect the molecule against cleavage at the Arg77-Ser78 bond
either by trypsin (all three protein kinases) or by both the classical
and the alternative pathway convertases (PKA and PKC). In contrast, CK2 phosphorylates both polypeptide chains of C3 and increases its susceptibility to cleavage by elastase (5).
We have recently demonstrated that C3 is phosphorylated in
vitro by a CK1 that is released from activated human platelets (13).
This CK phosphorylates a Thr residue in the C3d moiety, which
increases the resistance of C3b to cleavage by factor I, acting in
concert with factor H (13). In addition to C3, the platelet CK also
phosphorylated fibrinogen, vitronectin, and serum albumin (14).
We have also shown that plasma proteins in healthy blood donors contain covalently bound phosphate at a level of approximately 0.09 mol phosphate/mol protein; among these phosphorylated proteins is C3 (14). Activation of isolated platelets by heataggregated g-globulin (HAGG) under the conditions used to
release platelet CK was sufficient to support the phosphorylation of
exogenously added proteins (e.g., C3) without further addition of
ATP or divalent cations (14). In the same study we also demonstrated that the phosphate content of plasma proteins as a whole
and of C3 and fibrinogen in particular are substantially increased in
systemic lupus erythematosus patients during exacerbation, as a
result of platelet activation. Taken together, these observations
strongly suggest that platelet CK-mediated phosphorylation of
plasma proteins, including C3, is likely to occur in the blood as a
result of platelet activation. However, direct evidence linking
platelet activation in vitro to extracellular phosphorylation of proteins in whole blood has not yet been established.
0022-1767/99/$02.00
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A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that
activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins,
including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s)
on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15
to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was
distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and
immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage
by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect
the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in
purified systems and in serum was increased by 1.6 – 4.5 times over that of unphosphorylated C3. A covariation was seen between
the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0
for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3
by platelet casein kinase is to enhance the opsonization of immune complexes. The Journal of Immunology, 1999, 162: 7426 –7433.
The Journal of Immunology
7427
The present study was undertaken to characterize this platelet
protein kinase and to further elucidate the effect(s) of phosphorylation on C3 functions.
ciable dephosphorylation of C3 was found when samples were stored under
these conditions.
Materials and Methods
Platelet CK was analyzed by the dot-blot technique using Abs against CK1
and CK2. Recombinant rat CK1 served as a positive control when platelet
CK was detected by biotinylated polyclonal chicken Abs against CK1,
followed by HRP-conjugated streptavidin. Human CK2 on U937-derived
microparticles served as a positive control when platelet CK was analyzed
using mAb anti-CK2, followed by HRP-conjugated anti-mouse Igs.
In separate experiments, ATS-C3b was phosphorylated with platelet CK
in the presence of 10 mM Mn21 and 0.1 mM ATP (Sigma) and with three
different protein kinase inhibitors, present in concentrations of up to 1 mM.
The compounds tested were N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7; Seikagaku, Tokyo, Japan), which is specific for CK1
(24); 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (Calbiochem, La
Jolla, CA), which is specific for CK1 (25); and N-(2-aminoethyl)-5-chloronaphtalene-1-sulfonamide HCl (A3; Calbiochem), which is reported to
inhibit CK1 and CK2; protein kinases A, C, and G; and myosin light chain
kinase (26). After phosphorylation, Sepharose was washed three times with
PBS containing 0.1% Tween-20, and the amount of protein-bound radioactivity was measured in a beta counter.
The ion dependence of platelet CK was compared with that of rat recombinant CK1 (Calbiochem) using casein (3 mg; Sigma) and C3 (20 mg)
as substrates in the presence of 0.1 mM [g-32P]ATP and 5 mM of Mn21,
Mg21, or Ca21. To facilitate comparison of the two enzymes, the recombinant CK1 was diluted in albumin-free Tyrode’s medium to yield a sp. act.
similar to that of the platelet CK preparation, giving final concentrations of
0.25 mM Mg21 and 0.5 mM Ca21, respectively. After phosphorylation at
37°C for 30 min, the samples were subjected to 10% SDS-PAGE under
reducing conditions (27). Exposure of the dried gels and scanning were
performed in a PhosphorImager using ImageQuant software (Molecular
Dynamics, Sunnyvale, CA) for calculations.
Antibodies
Polyclonal rabbit Abs against human C3a and C3c and HRP-conjugated
polyclonal Abs against human C3c and mouse Igs were purchased from
Dako (Glostrup, Denmark). Chicken Abs against rat recombinant CK1
were purchased from Immunsystem (Uppsala, Sweden), a mouse monoclonal IgG1 Ab (mAb) clone 1AD9 against the a subunit of human protein
kinase CK2 was obtained from Boehringer Mannheim (Mannheim Germany). Polyclonal rabbit Abs against phosphoserine and phosphothreonine
were bought from Zymed (San Francisco, CA). HRP-conjugated streptavidin was purchased from Amersham (Slough, U.K.),
Falcon tubes from (Becton Dickinson, Meylan, France) were furnished
with Corline heparin surface (Corline, Uppsala, Sweden) according to the
manufacturer’s recommendation (15). The surface concentration of heparin
was 0.5 mg/cm2, corresponding to approximately 0.1 IU/cm2, with an antithrombin III binding capacity of 2– 4 pmol/cm2.
C3 and factor B were purified from human plasma according to the procedures described by Hammer et al. (16), and Lambris and Müller-Eberhard (17), respectively. Factor D was purified from peritoneal fluid from
patients with renal failure, as described by Catana and Schifferli (18). C3
was digested with trypsin (Sigma, St. Louis, MO) to C3a and C3b (1%,
w/w, for 5 min at room temperature), and the fragments were separated on
Sephadex G-100 (Pharmacia Upjohn, Stockholm, Sweden) equilibrated in
PBS. Nascent C3b, which exposes a free sulfhydryl group, was covalently
bound to activated thiol Sepharose (ATS; Pharmacia Upjohn) (19): 80 mg
trypsin was added to 10 mg of C3 and 2 ml of ATS and incubated at 37°C
for 15 min. The digestion was interrupted by incubation with 0.8 mg of
soybean trypsin inhibitor (Sigma) for 15 min, followed by extensive washing of the Sepharose.
U937-derived microparticles were prepared according to the procedure
of Paas and Fishelson (20). We have previously demonstrated the presence
of CK2 on these membrane vesicles (5).
Human g-globulin (Pharmacia Upjohn; 1 mg/ml in PBS) was heat aggregated to HAGG by incubation for 30 min at 63°C. Chicken polyclonal
Abs against rat recombinant CK1, rabbit Abs against human C3a, and
phosphoserine and phosphothreonine were biotinylated using biotin-amidocaproate N-hydroxysuccimide ester (Sigma) as previously described
(21). Polyclonal rabbit Abs to human C3c were coupled to cyanogen bromide-activated Sepharose (Pharmacia Upjohn) according to the manufacturer’s instructions.
For substitution experiments, serum from a patient deficient in C3 was
used. The patient’s parents were cousins and were both of Turkish origin.
High voltage electrophoretic analysis indicated that both parents had C3S
only, and no C3 could be detected in samples from the patient. Serum from
healthy blood donors was used as a control. Serum samples were collected
within 1 h after blood was drawn and were stored at 270°C until used for
analysis.
Phosphorylation of C3 and C3b
Platelets were precipitated from citrated blood and washed as described by
Mustard et al. (22), with the modifications described previously (13, 23).
Platelet protein kinase was prepared by resuspending the final platelet pellet to one-third of is original plasma volume in albumin-free Tyrode’s
medium (pH 7.45) containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2,
0.36 mM NaH2PO4, 12 mM NaHCO3, 2 mM CaCl2, and 5.5 mM glucose.
The platelets were activated with 10 mg/ml of HAGG for 15 min at room
temperature and were centrifuged at 1100 3 g for 10 min. After centrifugation, the supernatant was frozen in aliquots at 270°C. The major protein
kinase activity released from platelets under these conditions is CK (13).
C3 and C3b were phosphorylated with the protein kinase preparation in
the presence of 10 mM Mn21 or Ca21 and 0.1 mM ATP (final concentrations) at 37°C for 30 min. The reaction was terminated by passing the
samples through PD10 columns (Pharmacia Upjohn). Unphosphorylated
controls were treated identically, except for the addition of ATP. The degree of phosphorylation was estimated from parallel samples with added
[g-32P]ATP of known specific activity (Amersham). Levels of 0.7– 0.9 mol
of phosphate/mol protein were measured in the preparations of C3 and C3b
used in this study. The phosphorylated and unphosphorylated preparations
of C3 and C3b were divided into aliquots and stored at 270°C. No appre-
Activation of platelets in whole blood or in platelet-rich plasma
(PRP)
Twenty milliliters of blood was collected in a 50-ml Falcon tube (Becton
Dickinson), which had been furnished with a Corline heparin surface and
contained 20 IU of soluble heparin (Bio Iberica, Barcelona, Spain). PRP
was produced from blood by centrifugation for 15 min at 150 3 g and was
used immediately. Platelets in PRP or whole blood were stimulated with
100 mM ADP (Sigma) or HAGG (up to 100 mg/ml) for up to 15 min
without agitation at room temperature. After incubation, 10 mM EDTA
(final concentration) was added, and the cells were removed by centrifugation at 1100 3 g for 10 min. Blood collected in Vacutainer tubes containing EDTA (Becton Dickinson, Rutherford, NJ) was used as a control.
C3 was precipitated removed from these samples by incubation with
Sepharose-coupled anti-human C3c (13). The samples were then subjected
to SDS-PAGE followed by Western blot analysis. A standard curve for the
quantification of phosphate content in isolated proteins, ranging from 0.1–
150 pmol of phosphate, was constructed with mixed histone type II-AS
(Sigma). By using a colorimetric technique (28), this preparation of histone
was shown to contain 0.2 mol of phosphate/mol protein. Nitrocellulose
membranes with precipitated proteins and histone were incubated in parallel with biotinylated rabbit Abs against phosphoserine and phosphothreonine, followed by HRP-conjugated streptavidin as described previously
(14). The membranes were scanned using Studio Scan II (Agfa-Gaevert,
Antwerp, Belgium), the intensity of the bands was quantified using NIH
Image 1.54 for Macintosh, and the phosphate content of C3 from the samples was evaluated from the standard curve.
Activation of C3 in serum on an IgG-coated surface
Wells of microtiter plates were coated with 10 mg of human IgG/ml. Normal human serum or C3-deficient serum was diluted 3-fold (final concentration) in veronal-buffered saline (VBS; 1.8 mM sodium barbiturate, 3.1
mM barbituric acid, and 0.15 M NaCl, pH 7.4) containing 0.75 mM Ca21
and 2.5 mM Mg21. Before dilution, the C3-deficient serum was supplemented with 1 mg/ml of either phosphorylated or unphosphorylated C3.
After incubation at 37°C for 20 min the serum was transferred to test tubes
containing EDTA (10 mM, final concentration) and stored at 270°C until
used for the detection of generated C3a.
C3 binding to IgG. After removal of the serum, the amount of C3 bound
to the IgG-coated surface was detected by HRP-conjugated anti C3c. A
sandwich ELISA (29) was used to assess the relative amount of C3/C3
fragments present on the IgG surface. This ELISA, which used a polyclonal anti-C3c Ab for capture and a HRP-conjugated polyclonal anti-C3c
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Preparation of reagents
Partial characterization of platelet CK
7428
FUNCTIONAL PROPERTIES OF PHOSPHORYLATED C3
Ab for detection, was performed in parallel, with identical Ab dilutions and
staining times. This technique had a lower detection limit of 0.5 ng C3/C3
fragments/ml, which corresponds to 0.05 ng of C3/C3 fragments/well.
ELISA for detection of C3a. Frozen serum samples were diluted 1/2000
(final dilution) and analyzed as described previously (29). mAb 4SD17.3
was used for capture, and bound C3a was detected with biotinylated antiC3a followed by HRP-conjugated streptavidin. Zymosan-activated serum,
calibrated against a solution of purified C3a, served as the standard.
Cleavage of C3 by trypsin
Phosphorylated or unphosphorylated C3 (10 mg) in PBS was incubated
with trypsin in serial dilutions up to 0.5 mg for 30 min at 37°C. The
reaction was then stopped by boiling the samples in electrophoresis sample
buffer containing 15% 2-ME, and the samples were analyzed by SDSPAGE, followed by densitometric scanning of the gels.
Activation of C3 by the alternative pathway convertase
Activation of C3 by the classical pathway convertase
Activation by the classical pathway was measured by incubating phosphorylated or unphosphorylated C3 diluted in VBS containing 0.75 mM Ca21
and 2.5 mM Mg21 with EAC14oxy2 cells bound to microtiter plates as
described previously (30); microtiter plates were coated with EAC14oxy2
or EA (control) cells. Phosphorylated or unphosphorylated C3 in 3-fold
serial dilutions from 0.04 –30 mg/ml was incubated at 37°C for 60 min, and
the bound C3 fragments were visualized by incubation with HRP-conjugated Abs to human C3c followed by staining. EDTA-serum served as a
positive control. The deposition of C3/C3 fragments was estimated using
the sandwich ELISA described above, and nonspecific binding, i.e., binding to plates with control cells, was subtracted from that to EAC14oxy2
cells.
Binding of trypsin-generated C3 fragments to IgG
Wells of microtiter plates were coated with 10 mg of human IgG/ml. Purified phosphorylated or unphosphorylated C3 (0.5 mg/ml) in PBS was
Binding of factor B-generated C3 fragments to IgG
Purified phosphorylated or unphosphorylated C3 (0.5 mg/ml) in VBS containing 2.5 mM Mg21 was activated in IgG-coated microtiter plates for 30
min at 37°C by incubation with factors B and D in serial dilution from 50
and 0.4 mg/ml, respectively. Detection was performed as described above,
and quantitation of the deposited C3 fragments was performed.
Covalent binding of C3 to glycerol and glycine
The binding of glycerol and glycine to C3 was measured by a modification
of the technique described by Dodds and Law (31). Phosphorylated or
unphosphorylated C3 (10 mg) in PBS containing 1 mM EDTA was activated with trypsin (0.03 mg) in the presence of either 2.5 mM [2-3H]glycine
(41.1 Ci/mmol) or 10 mM [2-3H]glycerol (200 mCi/mmol; both from New
England Nuclear, Boston, MA) for 60 min at 37°C. Control samples of
phosphorylated and unphosphorylated C3 were incubated in parallel, but
without the addition of trypsin. After incubation, the reaction was terminated by boiling the samples in electrophoresis sample buffer, followed by
separation by SDS-PAGE. Under these conditions, C3 was quantitatively
activated, while no fragments beyond C3b were visible. Thereafter, the gels
were cut, and the band in each lane corresponding to the a9-chain of C3b
was incubated with Biolute-S tissue solubilizer (Zissner Analytic, Frankfurt, Germany), and the radioactivity in the samples was determined after
the addition of Aquasafe 300 Plus scintillation mixture (Zissner Analytic).
The sp. act. of the added glycine and glycerol was determined, and the
degree of binding to the C3 samples, expressed as moles per mole of
protein, was calculated.
Statistical analyses
The results are expressed as the mean 6 SEM. Statistical significance was
calculated with Student’s t test for unpaired samples, using StatView 4.01
(Abacus Concepts, Berkeley, CA) for Macintosh.
Results
Partial characterization of platelet CK
We first compared platelet CK to recombinant rat CK1 with regard
to its immunological reactivity, dependence on divalent cations,
and inhibition by protein kinase inhibitors (Table I). The immu-
Table I. Comparison of platelet CK and recombinant rat CK1
Platelet CK
Rat CK1
a
Immunological reactivity
Antibody
Anti-CK1
Anti-CK2
(1)
2
111
2
Effect of protein kinase inhibitorsb
Inhibitor (specificity)
CKI-7 (CK1)
DRB (CK2)
A3 (CK1, CK2, MLCK, PKA, PKC, PKG)
110c
No inhibition
800c
10d
Not reported
80d
Dependence on divalent cationse
Substrate
Casein
C3
Mn21$Ca21.Mg21.Bufff
(2.7) (2.6) (1.9) (1.0)
Mn21.Ca21.Mg21.Bufff
(3.2) (2.7) (2.1) (1.0)
Mg21.Ca21.Bufff..Mn21
(3.2) (1.9) (1.0) (0.8)
Mg21.Ca21.Bufff..Mn21
(1.5) (1.2) (1.0) (0.7)
a
Tested by dot blot using biotinylated polyclonal chicken Abs against rat CK1, HRP-conjugated streptavidin or a mouse mAb against human CK2, and HRP-conjugated
anti-mouse Ig.
b
Inhibition of platelet CK activity, using ATS-bound C3b as substrate, as described in Materials and Methods.
c
Concentration required to achieve 50% inhibition (mM).
d
Ki (mM) as reported by the manufacturers.
e
Protein kinases were diluted in Tyrode’s buffer to give final concentrations of 0.25 mM Mg21 and 0.5 mM Ca21, respectively. Thereafter 5 mM of either Mg21, Mn21,
or Ca21 was added, together with 0.1 mM [g-32P]ATP. After phosphorylation, the samples were subjected to SDS-PAGE under reducing conditions, and the dried gels were
exposed as described in Materials and Methods.
f
Tyrode’s buffer diluted 1/4; 0.25 mM of Mg21 and 0.5 mM of Ca21 (final concentrations).
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Activation by the alternative pathway was assessed by incubating phosphorylated or unphosphorylated C3 (16 mg) with factor B (2.1 mg) and
factor D (10 ng) diluted in VBS containing 2.5 mM Mg21 for up to 5 min
at 37°C. In an alternative procedure, phosphorylated or unphosphorylated
C3 was added to preformed alternative pathway convertase complex molecules obtained by incubating 2 mg of unphosphorylated C3b in VBS containing 2.5 mM Mg21 with factors B and D. The resulting cleavage was
analyzed by SDS-PAGE, as described above.
incubated with trypsin in serial dilutions from 3.1 mg/ml for 30 min at
37°C. The deposited C3 was detected with HRP-conjugated anti-C3c.
Quantitation of the relative amount of bound C3 fragments was made using
the sandwich ELISA as described above.
The Journal of Immunology
nological identity of platelet CK was checked by means of dot
blots using Abs against CK1 and CK2 (Table I). Chicken Abs
against CK1 reacted weakly with platelet CK and strongly with rat
CK1. In contrast, a rat anti-CK2 mAb reacted with CK2 on U937derived microparticles (not shown), but failed to react with both
platelet CK and CK1.
The activity of platelet CK was then measured in the presence of
three protein kinase inhibitors in concentrations up to 1 mM, using
ATS-bound C3b as a substrate (Table I). Platelet CK was inhibited
by CKI-7 (specific for CK1) and A3 (which inhibits multiple protein kinases, including CK1 and CK2), but not by 5,6-dichloro-1b-D-ribofuranosylbenzimidazole (which is specific for CK2). The
Ki50 values obtained were 110 mM for CKI-7 and 800 mM for A3,
respectively. These values are substantially higher than those cited
by the manufacturers for these inhibitors against CK1.
Mn21 and Ca21 were more potent stimulators of platelet CK
activity than Mg21 when equimolar amounts of casein and C3
were used as substrates (Table I). In contrast, activation of CK1
was most pronounced in the presence of Mg21, whereas inhibition
was seen in the presence of Mn21.
Phosphorylation of C3 after activation of platelets in whole
blood or in PRP
Western blot analysis (using a mixture of Abs against phosphoserine and phosphothreonine) of C3 that had been isolated from
FIGURE 2. Activation of C3 in serum by interaction with an IgGcoated surface. C3-deficient serum, which was supplemented with 1 mg/ml
of phosphorylated (E) or unphosphorylated (M) C3 and diluted from 1/3
was incubated in an IgG-coated ELISA plate C3 at 37°C for 20 min. The
relative amount of bound C3/C3 fragments was assessed by a sandwich
ELISA that was performed in parallel as described in Materials and Methods. A shows binding of C3 fragments to the IgG-coated plate. The serum
was analyzed for C3a (B). The presented data represent the mean 6 SEM
of three experiments.
EDTA plasma had a phosphate content of 0.14 6 0.02 mol/mol
protein. The level was increased in C3 that had been isolated from
activated whole blood or PRP. In whole blood the phosphate content in C3 increased after incubation with HAGG (from 0.14 6
0.02 to 0.52 6 0.08 mol/mol; n 5 7) or incubation with ADP (to
0.60 6 0.14 mol/mol; n 5 7). A similar increase was seen in PRP
from 0.15 6 0.02 to 0.56 6 0.09 mol/mol (n 5 7) after incubation
with HAGG or to 0.65 6 0.14 (n 5 7) after incubation with ADP
(Fig. 1A).
The phosphate content of C3 in whole blood that had been incubated with 100 mg of HAGG/ml increased continuously during
the 15-min incubation, reaching a level more than 4 times the
starting level (Fig. 1B).
Activation of C3 in serum on an IgG-coated surface
When the activation of C3 and binding of C3 fragments to an
IgG-coated surface were assessed by ELISA, significantly higher
amounts of split products from phosphorylated than from unphosphorylated C3 were detected in all serum concentrations tested
(Fig. 2A). At the highest serum concentration tested (1/3), substitution with unphosphorylated C3 resulted in deposition of approximately 10 ng of C3 fragments/well as assessed by sandwich
ELISA, whereas substitution with phosphorylated C3 resulted in
deposition of 4 times higher amounts of C3 fragments/well (n 5 3;
p , 0.0001).
This higher level of binding of fragments from phosphorylated
C3 was accompanied by higher complement activation, as assessed
by the generation of C3a (Fig. 2B). At a serum dilution of 1/3 the
levels of C3a reached 3000 ng/ml (for phosphorylated C3) and
2200 ng/ml (for unphosphorylated), respectively (n 5 3; p 5
0.0286).
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FIGURE 1. Phosphorylation of C3 in whole blood and PRP. A, Increased phosphate content after incubation of whole blood or PRP containing 1 IU of heparin/ml with 100 mg of HAGG/ml (hatched bars), 100
mM ADP (open bars), or without additions (filled bars) for 15 min at room
temperature. B, Continuous increase in the phosphate content of C3 after
incubation of whole blood with 100 mg of HAGG/ml at room temperature.
C3 was precipitated with Sepharose-bound Abs and subjected to Western
blot analysis as described in Materials and Methods. The data represent the
mean 6 SEM of seven (A) or two (B) separate experiments.
7429
7430
FUNCTIONAL PROPERTIES OF PHOSPHORYLATED C3
FIGURE 3. Trypsin cleavage of phosphorylated
and unphosphorylated C3. Phosphorylated or unphosphorylated C3 (10 mg) was cleaved by trypsin at the
concentrations indicated for 30 min at 37°C. Digestion was followed by SDS-PAGE and densitometric
scanning of the gels. A, A Coomassie-stained gel containing unphosphorylated (lanes 1 and 3) or phosphorylated (lanes 2 and 4) C3 incubated without trypsin (lanes 1 and 2) or in the presence of 0.5 mg of
trypsin. B, The remaining a- and a9-chain of unphosphorylated (M) or phosphorylated (E) C3. The data
represent the mean 6 SEM of three experiments.
Cleavage of C3 by trypsin
There was no significant difference in the rate of trypsin cleavage
after phosphorylation of C3 (Fig. 3B). In addition, cleavage generated very similar or identical polypeptide fragments from phosphorylated and unphosphorylated C3 (Fig. 3A).
Alternative pathway activation of phosphorylated and unphosphorylated C3 was studied by incubation of mixtures of purified factor
B, factor D, and C3. The cleavage of phosphorylated C3 occurred
at a slightly (but not significantly) lower rate that of unphosphorylated C3 (Fig. 4, A and B). After 3 min of incubation, 60% of the
unphosphorylated C3 remained intact compared with 75% of the
phosphorylated C3.
In experiments designed to expose phosphorylated and unphosphorylated C3 to identical amounts of preformed convertase, factor
B and factor D were incubated with (unphosphorylated) C3b before the addition of C3. Phosphorylated C3 was marginally more
resistant to cleavage than was unphosphorylated C3, with approximately 40% cleavage as compared with 50% for unphosphorylated C3 after 3 min of incubation (Fig. 4, C and D).
Activation of C3 by the classical pathway convertase
Physiological activation of C3 by the classical pathway convertase
was measured after incubation of phosphorylated or unphosphor-
Binding of trypsin-generated C3 fragments to IgG
The capacity of C3 to bind to IgG was assessed by incubating C3
together with trypsin in wells of microtiter plates coated with IgG.
The level of binding of phosphorylated C3 was significantly higher
at all concentrations of trypsin tested (Fig. 6A). When trypsin concentrations of 1.5 mg/ml or higher were used, the binding of C3
fragments reached a plateau at approximately 10 ng of C3 fragments/well for unphosphorylated C3 (as estimated by sandwich
ELISA) and 1.6 times higher for phosphorylated C3, respectively
(n 5 4; p , 0.0001).
Binding of factor B-generated C3 fragments to IgG
The capacity of C3 to bind to IgG was further assessed by incubating C3 together with factor B and factor D in wells of microtiter
plates coated with IgG. The level of binding of phosphorylated C3
FIGURE 4. Alternative pathway activation of C3. A and B, C3 (16 mg), factor B (2.1
mg), and factor D (10 ng) were incubated
together at 37°C for up to 5 min and then
analyzed as described in Fig. 3. C and D,
Generation of alternative pathway convertase complexes by preincubation of factor B
and factor D with 2 mg of unphosphorylated
C3b before the addition of C3. A and C, Coomassie-stained gels with unphosphorylated
(lanes 1 and 2) or phosphorylated (lanes 3
and 4) C3, incubated in the absence (lanes 1
and 3) or presence (lanes 2 and 4) of factor B
and factor D. B and D, The remaining
a-chain of unphosphorylated (M) or phosphorylated (E) C3. Data represent the
mean 6 SEM of three experiments.
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Activation of C3 by the alternative pathway convertase
ylated C3 with EAC14oxy2 cells fixed to microtiter plates. Activation was monitored by measuring the binding of C3 fragments
on the EAC14oxy2 cell surface. Approximately 2-fold higher binding occurred when fragments were generated from phosphorylated
C3 than from unphosphorylated C3 after addition to the cells at
concentrations .3 mg/ml (Fig. 5; n 5 4; p 5 0.0006 for 30 mg/ml).
Lower concentrations of added C3 resulted in the same degree of
binding.
The Journal of Immunology
7431
was significantly higher at all tested concentrations of factor B
(Fig. 6B). When factor B at concentrations of 6 mg/ml or higher
were used, the binding of C3 fragments reached a plateau at an
approximately 1.6 times higher level for phosphorylated C3 compared with unphosphorylated C3 (n 5 4; p 5 0.0001).
Covalent binding of C3 to glycerol and glycine
The capacity of C3 to form ester and amide bonds was assessed by
examining the binding of radiolabeled glycerol and glycine to unphosphorylated and phosphorylated C3 when trypsin was used to
activate the C3. The ratio of trypsin to C3 (indicated by an arrow
in Fig. 6A) was the lowest amount that gave maximal binding of
C3 fragments to the IgG surface. Under these conditions unphosphorylated C3 was found to bind 0.454 6 0.047 mol of glycerol/
mol of protein, whereas phosphorylated C3 bound 0.726 6 0.065
mol of glycerol/mol of protein (n 5 10; p 5 0.0148).
The binding of glycine decreased after phosphorylation, from
0.224 6 0.025 to 0.149 6 0.011 mol of glycine/mol of protein,
respectively (n 5 10; p 5 0.0036). Consequently, the glycerol/
glycine ratio increased from 2.0 for unphosphorylated C3 to 4.9 for
phosphorylated C3 (Table II). The background levels of glycine
binding was 0.010 6 0.002 mol/mol for unphosphorylated C3 and
0.011 6 0.002 mol/mol for phosphorylated C3 (n 5 4). The corresponding values for glycerol binding to control and phosphorylated C3 were 0.020 6 0.002 and 0.021 6 0.002 mol/mol (n 5 4),
respectively.
Discussion
We have previously shown that activation of washed platelets
leads to the release of a CK that is able to phosphorylate a number
of plasma proteins in vitro, including C3, after the addition of ATP
and ions. The endogenous supplies of ATP and ions in the platelets
are sufficient to support phosphorylation of exogenously added
substrates, suggesting that activation of platelets in whole blood
might lead to extracellular phosphorylation. Indeed, the phosphate
content of plasma proteins increased in systemic lupus erythematosus patients in parallel to platelet activation in vivo, but the direct
line of evidence linking platelet activation in vitro to extracellular
phosphorylation of proteins in whole blood has been missing until
now. In the present study we have demonstrated that the phosphate
content of plasma C3 increases by .4-fold after platelet activation,
thus confirming that platelet activation in PRP or intact blood indeed leads to extracellular phosphorylation of plasma proteins,
without the addition of exogenous ATP or cations. The initial
value of 0.14 mol of phosphate/mol of C3 reported here is in good
agreement with earlier reports of 0.15 and 0.30 mol of phosphate/
mol of C3 detected using other techniques (4, 13).
The protein kinase that is released from platelets and phosphorylates human C3 is a Ser/Thr protein kinase that has CK1-like
properties, in that it phosphorylates casein and is not inhibited by
heparin; however, in contrast to CK1, its activity is dependent on
Mn21 and Ca21 (13, 23). Phosphorylation by platelet kinase made
Table II. Covalent binding of glycerol and glycine to C3ba
C3b (activated)
C3 (nonactivated)
C3-Pb (activated)
C3-P (nonactivated)
p value
Glycerol Binding
(mol/mol)
Glycine Binding
(mol/mol)
Glycerol/Glycine
Ratio
0.454 6 0.047
0.020 6 0.002
0.726 6 0.065
0.021 6 0.002
0.224 6 0.025
0.010 6 0.002
0.149 6 0.011
0.011 6 0.002
2.0
2
4.9
2
0.0148
0.0036
Phosphorylated or unphosphorylated C3 (10 mg) was activated by 0.03 mg of trypsin in the presence of 10 mM [2-3H]glycerol or 2.5 mM [2-3H]glycine. Quantitation of the bound molecules was carried out as described in Materials and Methods.
The data represent the means 6 SEM of 10 experiments and 4 controls.
b
C3-P, phosphorylated C3.
a
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FIGURE 5. Classical pathway activation of C3. Analysis of bound C3
fragments on EAC14oxy2 cells treated with phosphorylated or unphosphorylated C3. Physiological activation of phosphorylated (E) and unphosphorylated (M) C3 by the classical pathway convertase, as monitored after
incubation with EAC14oxy2 cells fixed to microtiter plates as described in
Materials and Methods. The relative binding of C3/C3 fragments was estimated as described in Fig. 2. Data represent the mean 6 SEM of four
experiments.
FIGURE 6. Binding of C3 fragments to IgG. A, Binding of trypsingenerated C3 fragments to IgG. Purified phosphorylated (E) or unphosphorylated (M) C3 was incubated for 30 min at 37°C with equal volumes
of trypsin at the concentrations given. Data represent the mean 6 SEM of
four experiments. The arrow indicates the ratio of trypsin to C3 that was
subsequently used in the experiments presented in Table II. B, Alternative
pathway activation of C3. Binding to IgG of C3 fragments generated by the
alternative pathway convertase. Purified phosphorylated (E) or unphosphorylated (M) C3 was incubated with equal volumes of factor B at the
concentrations given together with factor D for 30 min at 37°C. Data represent the mean 6 SEM of four experiments. The relative binding of C3/C3
fragments in A and B was estimated as described in Fig. 2.
7432
significantly (;1.6 times) higher than that for fragments from unphosphorylated C3.
The putative phosphorylation site for platelet CK in C3 is located within a tryptic fragment, between Lys979 and Lys1014,
which also comprises the thiol ester as we have reported previously (13). After extensive trypsin digestion of phosphorylated
ATS-bound C3b, this fragment containing 90% of the total radioactivity still bound to the ATS. After hydrolysis of the ATS-bound
C3 fragment, only Thr-P was detected. Based on these results we
concluded that the most likely phosphorylation site for platelet CK
is Thr1009, 19 aa residues toward the C terminus of the C3 a-chain
from the thiol ester. The sequence DETEQWE surrounding
Thr1009 (34) contains several acid amino acid residues, which
makes it a potential phosphorylation site for CKs (35). One possible explanation for the difference in binding efficiency reported
in the present study is that phosphorylation of C3 in some way
alters the binding properties of the thiol ester. To test whether this
was the case, we used trypsin to cleave phosphorylated or unphosphorylated C3 in the presence of either [2-3H]glycerol or [2-3H]glycine. Phosphorylation of C3 increased the glycerol binding capacity (by ;1.6-fold), while it decreased glycine binding, resulting
in an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. These changes
are sufficient to account for the increased binding of C3 fragments
to IgG, since it has been demonstrated that the binding of C3b to
IgG mainly occurs via ester bonds that are formed with hydroxyl
group-containing residues in the heavy chain of IgG (36 –38). It
should also be noted that in the present study phosphorylation of
C3 brought about an analogous increase in glycerol binding and in
binding of generated C3 fragments to IgG after activation by trypsin (Fig. 6), emphasizing that the binding to IgG is indeed ester
linked. A recently published crystal structure of human C3d (39)
confirms that Thr38 of C3d (which corresponds to Thr1009 of intact
C3) is exposed on the exterior of the molecule, in close proximity
to a cluster of acidic amino acid residues. In C3d, Thr38 and the
thiol ester are located at opposite ends of the a1 helix, with Thr38
at the C terminus. For the formation of ester bonds the thiol ester
of C3 requires the sequence Cys1010, Gln1013, and His1126 (40). In
C3d, these amino acid residues correspond to Cys17, Gln20, and
His133, which are located at opposite ends of the T3 segment. The
mechanism by which phosphorylation of Thr1009 alters the binding
properties of C3 remains to be established; one possibility is that
the negatively charged phosphate group alters the distance between the thiol ester and His1126, thereby contributing to the formation of ester bonds instead of amides.
The overall effect of the phosphorylation of C3 by platelet CK
should be to enhance the opsonization of immune complexes in
whole blood. Handling of immune complexes involves a sequence
of events that includes complement activation and covalent binding of C3b to the immune complex (41). One can propose a pathway in vivo by which platelets that become activated by immune
complexes (triggered by Fc receptors, C1q, and/or C5b-9 complexes) mediate the phosphorylation of fluid phase C3 and immune
complex-bound C3b. The consequence is an amplification effect
by which immune complexes indirectly potentiate their own
opsonization.
Acknowledgments
We thank Foozieh Ghazi for her excellent technical assistance
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bound C3b less susceptible to cleavage by factor I, with factor H
as a cofactor (13). To characterize the platelet Ser/Thr protein kinase, we have now compared this protein kinase to commercially
available rat recombinant CK1 with regard to its response to specific protein kinase inhibitors, immunological reactivity, and dependence on various divalent cations. Platelet CK was partially
inhibited by compounds reported to be specific for CK1, but with
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Phosphorylation of C3 by PKA and PKC has previously been
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trypsin (11). We have now conducted similar experiments with
purified C3 phosphorylated by platelet CK; exposure of phosphorylated C3 to the classical pathway convertase on EAC14oxy2 cells
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C3. This hypothesis was confirmed by our observation of increased
binding to surface-bound IgG of fragments generated from phosphorylated C3 by either C3bBb convertase- or trypsin-mediated
cleavage. In these experimental systems, as in EAC14oxy2 cells,
the binding of fragments generated from phosphorylated C3 was
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