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Intracellular events in platelet activation induced by antiphospholipid antibodies in the presence of low doses of thrombin.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 9, September 2004, pp 2911–2919
DOI 10.1002/art.20434
© 2004, American College of Rheumatology
Intracellular Events in Platelet Activation Induced by
Antiphospholipid Antibodies in the Presence of
Low Doses of Thrombin
Mariano Vega-Ostertag, E. Nigel Harris, and Silvia S. Pierangeli
Objective. Thrombosis and thrombocytopenia are
features of the antiphospholipid syndrome (APS), suggesting that antiphospholipid antibodies (aPL) may
bind platelets, causing activation and aggregation of
platelets and thrombosis. The intracellular events involved in aPL-mediated platelet activation are not fully
understood and are therefore the subject of this study.
Methods. IgG fractions and their F(abⴕ)2 fragments were purified from the sera of 7 patients with APS
and from the pooled sera of 10 healthy subjects (as
controls). Phosphorylation of p38 MAPK, ERK-1/2, and
[Ca2ⴙ]-dependent cytosolic phospholipase A2 (cPLA2)
was determined in lysates of washed platelets pretreated
with low doses of thrombin and aPL or control IgG or
their F(abⴕ)2 fragments, by immunoblot. The effects of
aPL on platelet aggregation in the presence or absence
of a p38 MAPK inhibitor, SB203580, were examined.
Thromboxane B2 (TXB2) production was detected by
enzyme-linked immunosorbent assay on gel-filtered
platelets treated with aPL and thrombin, with or without SB203580. Calcium mobilization studies were done
utilizing a fluorometric assay.
Results. Treatment of platelets with IgG aPL, or
their F(abⴕ)2 fragments, in conjunction with subactivating doses of thrombin resulted in a significant increase
in phosphorylation of p38 MAPK. Neither the IgG aPL
nor their F(abⴕ)2 fragments increased significantly the
phosphorylation of ERK-1/2 MAPKs. Furthermore, pre-
treatment of platelets with SB203580 completely abrogated the aPL-mediated enhanced aggregation of the
platelets. Platelets treated with F(abⴕ)2 aPL and thrombin produced significantly larger amounts of TXB2 when
compared with controls, and this effect was completely
abrogated by treatment with SB203580. In addition,
cPLA2 was also significantly phosphorylated in platelets
treated with thrombin and F(abⴕ)2 aPL. There were no
significant changes in intracellular [Ca2ⴙ] when platelets were treated with aPL and low doses of thrombin.
Conclusion. The data strongly indicate that aPL
in the presence of subactivating doses of thrombin
induce the production of TXB2 mainly through the
activation of p38 MAPK and subsequent phosphorylation of cPLA2. The ERK-1/2 pathway does not seem to be
involved in this process, at least in the early stages of
aPL-mediated platelet activation.
Antiphospholipid antibodies (aPL) have been
associated with recurrent thrombosis (arterial and/or
venous) and recurrent pregnancy losses in patients with
systemic lupus erythematosus and in those with the
antiphospholipid syndrome (APS) (1). Thrombocytopenia is a frequent feature of APS, giving rise to the
speculation that aPL may play a pathogenic role in
thrombosis by binding platelets and causing platelet
activation and aggregation (2).
Studies have demonstrated binding of affinitypurified aPL to platelets (2–5). Lellouche and colleagues
(6) reported that urinary secretion of the major thromboxane metabolite, 11-dehydrothromboxane B2 (TXB2),
was significantly increased in patients with lupus anticoagulant (LAC) as compared with normal controls. Studies from our group have also shown that affinity-purified
anticardiolipin antibodies (aCL) from patients with
APS, but not from patients with syphilis, enhanced
activation of platelets treated with suboptimal doses of
ADP, thrombin, or collagen (7). In a recent study by our
Supported in part by a Research Center in Minority Institutions grant from the NIH (G12-RR-03034) and a Minority Biomedical
Research support grant from the NIH (S02-GM-08248).
Mariano Vega-Ostertag, BS, E. Nigel Harris, MD, Silvia S.
Pierangeli, PhD: Morehouse School of Medicine, Atlanta, Georgia.
Address correspondence and reprint requests to Silvia S.
Pierangeli, PhD, Antiphospholipid Standardization Laboratory, Department of Microbiology, Biochemistry and Immunology, Morehouse
School of Medicine, Gloster Building 332, 720 Westview Drive SW,
Atlanta, GA 30310-1495.
Submitted for publication February 24, 2004; accepted in
revised form April 13, 2004.
2911
2912
VEGA-OSTERTAG ET AL
Figure 1. Possible intracellular signaling platelet pathways activated by antiphospholipid antibodies (aPL).
TRAP ⫽ thrombin receptor agonist peptide; PAR ⫽ protein-activated receptor; gp ⫽ glycoprotein; Rc ⫽
receptor; PLC␤ ⫽ phospholipase C␤; PLC␥ ⫽ phospholipase C␥; DG ⫽ diacylglycerol; IP3 ⫽ inositol triphosphate;
PKC ⫽ protein kinase C; cPLA2 ⫽ [Ca2⫹]-dependent cytosolic phospholipase A2; AA ⫽ acetylsalicylic acid; TXB2 ⫽
thromboxane B2; TXRc ⫽ thromboxane receptor.
group, platelets pretreated with suboptimal doses of
thrombin receptor agonist peptide (TRAP) and aPL
expressed enhanced levels of activated glycoprotein IIb/
IIIa, indicating platelet activation (8). In another study,
rabbit aCL were shown to enhance collagen-induced
platelet activation (9). Robbins et al showed that aPL–
␤2-glycoprotein I (␤2GPI) complexes significantly increased production of thromboxane A2 (TXA2), a proaggregatory prostanoid in platelets (10).
Platelets contain family members of the MAPKs,
including ERK-1 (p44 MAPK), ERK-2 (p42 MAPK),
and p38 MAPK (Figure 1). The MAPK p38 is a member
of a family of proline-directed serine/threonine kinases
that is dual-phosphorylated on a threonine and tyrosine
residue, separated by 1 single amino acid (11,12). In
platelets, p38 MAPK is activated by stress, such as heat
and osmotic shock, arsenite, H2O2, ␣-thrombin, collagen, and thromboxane analog (11,12), and is involved in
the phosphorylation of [Ca2⫹]-dependent cytosolic
phospholipase A2 (cPLA2), with subsequent production
of TXB2 (Figure 1). Thrombin has also been shown to
induce phosphorylation of ERK-1/2, involving protein
kinase C (PKC), phospholipase C␤ (PLC␤), and the
intracellular mobilization of [Ca2⫹] (Figure 1) (13–15).
Although several studies have shown that aPL
enhance platelet activation in vitro in the presence of low
doses of agonists (ADP, thrombin, collagen, or TRAP)
(7–10,16), the intracellular events involved in this process
are not understood. To address this question, we examined
the effects of aPL on phosphorylation of p38 MAPK,
ERK-1/2 MAPKs, and cPLA2 on intracellular [Ca2⫹] mobilization and on TXB2 production in the presence of
subactivating doses of thrombin. The effects of the specific
inhibitor for p38 MAPK, SB203580 (4-[4-fluorophenyl]-2[4-methylsulfinylphenyl]-5-[4-pyridyl] 1-imidazole), on
aPL-mediated enhancement of platelet aggregation and on
TXB2 production in the presence of thrombin were also
determined.
PATIENTS AND METHODS
Purification of IgG and preparation of F(abⴕ)2 fragments. IgG from 7 patients with APS (in accordance with the
Sapporo criteria [17]) and from the pooled sera of 10 healthy
INTRACELLULAR EVENTS IN aPL-MEDIATED THROMBOSIS
subjects (as controls) were affinity purified by protein G
(Gamma BindG Type 3; Amersham-Pharmacia, Piscataway,
NJ) by elution with 0.5M acetic acid (pH 3) and neutralization
of the fractions in 1M Tris buffer (pH 9). IgG were subsequently dialyzed in a 0.1M acetate buffer, pH 4.5, for 2 hours
at 4°C, and digestion with pepsin to obtain F(ab⬘)2 fragments
was performed by incubation with pepsin-agarose beads
(Sigma-Aldrich, St. Louis, MO) for 18 hours at 37°C. The
beads were separated by centrifugation for 10 minutes at 4,000
revolutions per minute, and the supernatants were dialyzed
against phosphate buffer, pH 7.4, at 4°C. Intact Fc fragments
and nondigested antibody were removed by protein G–sepharose chromatography. F(ab⬘)2 fragments showed a single
band at ⬃110 kd in nonreduced sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) (utilizing silver staining). IgG fractions and F(ab⬘)2 were passed through a
0.22-␮m filter and were checked for the absence or presence of
endotoxin (lipopolysaccharide [LPS]) by the Limulus amebocyte lysate assay (Amebolysate; ICN Biomedical, Costa Mesa,
CA).
Determination of antibodies. The aCL and antiphosphatidylserine (aPS) antibodies in the IgG fractions were
determined by enzyme-linked immunosorbent assay (ELISA)
as previously described (18), using an anti-human IgG antiserum labeled with alkaline phosphatase (␥-chain specific).
Similarly, binding of the F(ab⬘)2 fragments to cardiolipin was
determined by ELISA, using an anti-human ␬ and ␭ secondary
antibody cocktail (1:1,000 dilution) labeled with alkaline phosphatase (Sigma-Aldrich). Titers of aCL were reported in IgG
phospholipid (GPL) units, and levels of aPS antibodies were
reported in net optical density (OD) units.
Anti-␤2GPI antibodies were detected by ELISA as
previously described, with some modifications, using irradiated
Costar microtiter plates (Corning, Corning, NY) (19). Plates
were coated with 20 ␮g/ml purified ␤2GPI, and blocked with
5% ovalbumin–phosphate buffered saline (PBS) solution.
Anti-human IgG (␥-chain specific) and anti-␬ and anti-␭
secondary antibodies labeled with alkaline phosphatase were
used. Anti-␤2GPI activities were determined at 405 nm, with
results expressed in net OD units. LAC activities of the IgG
and of the F(ab⬘)2 fragments were determined by a modified
activated partial thromboplastin time (APTT) as previously
described (20).
Isolation of platelets. Platelets were obtained from the
blood of adult healthy volunteers who had not received
medication for at least 10 days. Blood was collected in acid–
citrate–dextrose anticoagulant (9/1 volume/volume). Plasmarich platelets (PRPs) were obtained by centrifugation for 20
minutes at 120g at room temperature (21).
Preparation of washed platelets. PRPs were centrifuged
at 800g for 10 minutes at room temperature and resuspended
in HEPES buffer (10 mM HEPES, 140 mM NaCl, 3 mM KCl,
0.5 mM MgCl2, 5 mM NaHCO3, 10 mM dextrose, pH 7.4)
supplemented with 0.4 units/ml apyrase and 1 mM aspirin. The
platelet count was adjusted to 1 ⫻ 109 platelets/ml and these
preparations were used in the immunoblot assays and in the
calcium mobilization experiments.
Preparation of gel-filtered platelets (GFPs). For the
aggregation studies, GFPs were used. Platelets were filtered
through a Sepharose 2B column equilibrated with HEPES
buffer as previously described (21) without inhibitors, and the
2913
platelet count was adjusted to 250 ⫾ 50 ⫻ 106/ml for the
aggregation studies and 300 ⫻ 106 platelets/ml for the TXB2
assay. Platelets were maintained at 37°C in a closed (capped)
tube and analyzed within 2 hours after drawing.
Immunoblot analysis for phosphorylation of p38 and
p44/42 MAPKs. One hundred–microliter aliquots of washed
platelets were incubated in microtubes for 5 minutes at 37°C,
under static conditions, with 50, 100, or 200 ␮g/ml IgG aPL
from APS patients or control IgG, or with 200 ␮g/ml of F(ab⬘)2
aPL from APS patients or control F(ab⬘)2 IgG. Platelets were
then treated with thrombin (0.005 units/ml). Platelets to be
used as controls were treated with PBS and 1 unit/ml thrombin.
Platelets were subsequently lysed with Laemmli buffer
(Bio-Rad, Richmond, CA) and 2-mercaptoethanol, and lysates
were heated at 95°C for 10 minutes and centrifuged for 10
minutes at 11,000g. Samples were subjected to SDS-PAGE in
a 12% gel. After the electrophoresis, proteins were transferred
to nitrocellulose membranes at 10V for 30 minutes in a
semidry transfer cell. The membrane was then blocked and
subsequently incubated with one of the following antibodies:
p38 MAPK (Thr180/Tyr182) 28b10 mouse anti-human monoclonal antibody (Cell Signals Technology, Beverly, MA) or p38
MAPK rabbit polyclonal antibody (Santa Cruz Biotechnology,
Santa Cruz, CA), or with phosphorylated ERK-1/2 (Thr202/
Tyr204) E10 mouse anti-human monoclonal antibody (Cell
Signals Technology) or ERK-1/2 rabbit polyclonal antibody
(Santa Cruz Biotechnology), followed by incubation with secondary antibody/goat anti-mouse or goat anti-rabbit IgG labeled with peroxidase (21) and detection by chemiluminescence (Amplight/BioRad, Hercules, CA). The intensity of the
bands was quantitated by densitometric analysis using Gel Pro
Analyzer software, version 4.5 (Fotodyne, Hartland, WI).
Phosphorylation of cPLA2. One hundred microliters of
purified platelets in a concentration of 1 ⫻ 109 platelets/ml
(prepared as described above) was incubated with 50 ␮l (200
␮g/ml final concentration) of F(ab⬘)2 aPL from APS patients
(n ⫽ 5) or 50 ␮l control F(ab⬘)2 IgG or PBS for 5 minutes, and
then stimulated with 0.005 units/ml thrombin, or treated with
PBS and 1 unit/ml thrombin as a positive control. The reaction
was stopped by the addition of an equal volume of 2⫻
immunoprecipitation buffer (100 mM Tris HCl, pH 7.4, 300
mM NaCl, 2 mM EGTA, 5 ␮g/ml leupeptin, 5 ␮g/ml aprotinin,
2 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 2 mM
NaF, 2% Nonidet P40, 0.5% sodium deoxycholate). The
lysates were cleared by treating them with protein G–
Sepharose for 45 minutes.
The cleared supernatants were incubated with 1 ␮g of
cPLA2 antibody (Santa Cruz Biotechnology) for 2 hours at
4°C. The immune complexes were precipitated by addition of
90 ␮l of protein G–Sepharose for 45 minutes. After brief
centrifugation, immunoprecipitates were washed 3 times with 1
ml of 1⫻ immunoprecipitation buffer and then resuspended
with 25 ␮l of SDS–sample buffer, and subjected to SDS-PAGE
in a 10% gel for 210 minutes. The proteins in the gel were
transferred to a nitrocellulose paper and were immunoblotted
as described previously, using the same cPLA2 antibody (rabbit
polyclonal cPLA2; Santa Cruz Biotechnology). The phosphorylated form of cPLA2 is detected as a slower-moving second
band. Thrombin-induced anti-cPLA2 was detectable after 2
minutes of stimulation (approximately half of the protein was
in the phosphorylated form) (21).
2914
VEGA-OSTERTAG ET AL
Aggregation studies. Aggregation of platelets was performed as follows: a 420-␮l aliquot of GFPs and 1 ␮l of CaCl2
(1 mM final concentration) were placed in the aggregometer
cuvettes at 37°C with 30 ␮l of IgG aPL antibodies from APS
patients (final concentration 200 ␮g/ml) or 30 ␮l of control IgG
(final concentration 200 ␮g/ml) for 5 minutes. In some experiments, platelets were pretreated for 20 minutes with
SB203580 (0.1 and 1 ␮M) or with 1% dimethylsulfoxide (22).
Aggregation was then started in a dual channel aggregometer
(Minigator II; Payton Scientific, Buffalo, NY) and registered in
a linear recorder. Platelet aggregation was evaluated as the
amplitude of the aggregation curves (expressed in millimeters)
after 5 minutes.
TXB2 assay. GFPs were adjusted to a concentration of
3 ⫻ 108 platelets/ml in HEPES buffer and incubated for 5
minutes with (Fab⬘)2 aPL fragments from APS patients or
control F(ab⬘)2 IgG fragments or PBS in the presence or the
absence of 1 ␮M/liter SB203580, and then stimulated with
0.005 units/ml thrombin for 3 minutes. The production of
TXB2 was stopped by the addition of indomethacin (0.1 mM)
in HEPES buffer, and the preparations were centrifuged at
10,000 rpm for 30 seconds at 4°C. The supernatants were
rapidly separated and stored at ⫺20°C until analyzed. TBX2
was analyzed in the supernatants utilizing a commercial enzyme immunoassay system (Amersham-Pharmacia) as indicated by the manufacturer.
Intracellular [Ca 2ⴙ ] mobilization experiments.
Washed platelets were incubated for 20 minutes at 37°C with 5
␮M visible light–excitable [Ca2⫹] indicator Fura Red–
acetoxymethyl ester (AM) and 5 ␮M calcium green–AM
(Molecular Probes, Eugene, OR). Subsequently, platelets were
centrifuged at 800g for 10 minutes at room temperature and
resuspended in HEPES buffer together with 0.4 units/ml
apyrase VII, 0.1 ␮M prostaglandin E1, and 1 mM acetylsalicylic
acid, and standardized at a platelet count of 1 ⫻ 109/ml. One
hundred–microliter aliquots of platelets were then incubated
with 200 ␮g/ml F(ab⬘)2 aPL from APS patients or 200 ␮g/ml
control F(ab⬘)2 IgG or PBS for 5 minutes, and then stimulated
with 0.01 units/ml thrombin. Each experiment included a calibration curve utilizing 0.01, 0.1, 0.5, and 1 units/ml thrombin.
The emission peaks of calcium green and Fura Red
signal intensities were determined at 530/30 nm and 640/40 nm,
respectively (21). Elevations in [Ca2⫹] result in increased
fluorescence intensity of calcium green and decreased fluorescence intensity of Fura Red, at 485/20 nm excitation wavelength. Changes in [Ca2⫹] were evaluated on the basis of a
ratio of signal intensity at the G-channel (calcium green,
sensitive peak at 530 nm) relative to the R-channel (red,
sensitivity peak at 640 nm) (21).
Statistical analysis. Data are presented as the mean ⫾
SD. Student’s t-test was used to compare mean values between
treated platelets and control platelets. P values less than or
equal to 0.05 were considered significant.
RESULTS
Characterization of IgG aPL and F(abⴕ)2 aPL.
All of the IgG aPL samples from the 7 APS patients
Figure 2. Effects of different concentrations of thrombin on phosphorylation of p38 MAPK and ERK-1/2. Washed platelets were
treated with various concentrations of thrombin as indicated (0–5
units/ml thrombin). Phosphorylation of p38 MAPK (P38-P) (A) or
phosphorylation of ERK-1/2 (ERK1/ERK2-P) (B) was detected by
immunoblot in the platelet lysates using specific monoclonal antibodies to the phosphorylated form of the enzymes, as described in Patients
and Methods.
were positive for aCL (⬎100 GPL units) and all of the
IgG samples from the healthy controls were negative for
aCL (⬍10 GPL units), when preparations were tested at
a 200 ␮g/ml protein concentration. The anti-␤2GPI
antibody activities of the IgG aPL and control IgG were
a mean ⫾ SD 0.870 ⫾ 0.218 net OD units and 0.140 ⫾
0.019 net OD units, respectively, when samples were
tested at a 100 ␮g/ml protein concentration. Six of the 7
IgG aPL samples were positive for LAC (APTT ratio of
patient plasma to normal plasma 1.21:1.50). All F(ab⬘)2
aPL samples were positive for binding to cardiolipin
(25–186 GPL units/100 ␮g protein), and the anti-␤2GPI
activities of the F(ab⬘)2 aPL and control F(ab⬘)2 IgG
were a mean ⫾ SD 0.505 ⫾ 0.111 net OD units and
0.068 ⫾ 0.024 net OD units, respectively (significantly
different; P ⫽ 0.0023). Binding to phosphatidylserine
was tested by ELISA and was a mean ⫾ SD 0.506 ⫾
0.112 net OD units for the F(ab⬘)2 aPL preparations and
0.018 ⫾ 0.005 net OD units for control F(ab⬘)2 IgG
(significantly different; P ⫽ 0.0002). All IgG preparations and F(ab⬘)2 fragments tested negative for LPS in
the Limulus amebocyte lysate assay.
Effects of aPL on phosphorylation of p38 MAPK
in platelets. The treatment of washed platelets with
varying concentrations of thrombin (0.005–5 units/ml)
produced a dose-dependent phosphorylation of p38
MAPK (Figure 2A). Treatment of platelets with control
IgG and 0.005 units/ml thrombin produced phosphorylation that was not different from that of platelets
treated with 0.005 units/ml thrombin alone. In contrast,
platelets treated with IgG aPL (n ⫽ 7) at 200 ␮g/ml
protein concentration and 0.005 units/ml thrombin produced a significant increase in phosphorylation of p38
MAPK (among the 7 IgG aPL samples, fold increases of
4.4, 7.8, 7.4, 5.8, 7.5, 6.3, and 5.0, when compared with
INTRACELLULAR EVENTS IN aPL-MEDIATED THROMBOSIS
Figure 3. Effects of IgG antiphospholipid antibodies (IgGaPL) on
phosphorylation of p38 MAPK (P38P). Washed platelets were treated
with 200 ␮g/ml of IgGaPL (n ⫽ 7) or IgG from normal healthy controls
(IgGNHS) and stimulated with 0.005 units/ml thrombin, or treated
with phosphate buffered saline (PBS) alone or with 0.2 units/ml
thrombin alone (A). For comparison, platelets were treated with
IgGaPL (n ⫽ 7) or IgGNHS or PBS alone in the absence of thrombin
(B). A positive control comprising platelets treated with 0.2 units/ml
thrombin alone was also included in the experiments. Phosphorylation
of p38 MAPK was detected by immunoblot using specific monoclonal
antibodies to the phosphorylated form of the enzyme, as described in
Patients and Methods. The results shown are representative of 5
experiments, using platelets from different donors.
platelets treated with control IgG and thrombin) (Figure
3A). A 7.6-fold increase was observed in platelets
treated with 0.2 units/ml of thrombin alone (positive
control).
The effect was dependent on the amount of aPL
used. For example, for sample IgGaPL1, a 4.4-, 3.0-, and
1.9-fold increase in p38 MAPK phosphorylation was
observed with 200, 100, and 50 ␮g/ml protein concentrations, respectively. Treatment of platelets with IgG aPL
alone (n ⫽ 7) (in the absence of thrombin) did not
produce any effect on p38 MAPK phosphorylation
(Figure 3B).
To determine whether the effects produced by
IgG aPL were due to interactions of the antibodies with
platelets either through the Fab fragment or through the
Fc portion of the immunoglobulins, the effects of
F(ab⬘)2 fragments on phosphorylation of p38 MAPK on
platelets were determined by immunoblot. As shown in
Figures 4A and B, 4 of the 5 F(ab⬘)2 aPL preparations
produced a significant increase of phosphorylation
(12.6-, 4.6-, 3.7-, and 12.6-fold increases) when compared with platelets treated with control F(ab⬘)2 IgG and
0.005 units/ml thrombin. No increase in phosphorylation
of p38 MAPK was observed in platelets treated with
control F(ab⬘)2 IgG and thrombin when compared with
platelets treated with thrombin alone.
2915
Effects of aPL on phosphorylation of ERK-1/2.
Treatment of platelets with different concentrations of
thrombin (0.005–5 units/ml thrombin) produced a dosedependent phosphorylation of ERK-1/2 (Figure 2B).
Phosphorylation of ERK-1/2 was observed starting at 0.2
units/ml thrombin. Neither the platelets treated with
F(ab⬘)2 aPL (n ⫽ 5) at 200 ␮g/ml protein concentration
and 0.005 units/ml thrombin nor the platelets treated
with control F(ab⬘)2 IgG and 0.005 units/ml thrombin
induced phosphorylation of ERK-1/2 after 2 minutes of
treatment (Figure 4C).
Effects of a specific inhibitor of p38 MAPK on
aPL-mediated platelet aggregation. To confirm whether
the phosphorylation of p38 MAPK is involved in aPLmediated platelet activation, aggregation studies were
performed in the presence and in the absence of the
specific inhibitor of p38 MAPK, SB203580. As shown in
Figure 5, platelets treated with IgG aPL and 0.005
units/ml thrombin produced an aggregation of 55%,
whereas there was no aggregation in platelets treated
with control IgG and 0.005 units/ml thrombin. Pretreatment of the platelets with 0.1 ␮M or 1 ␮M SB203580
abrogated the aggregatory effects of aPL by 89% and
100%, respectively.
Effects of aPL on phosphorylation of cPLA2.
Treatment of washed platelets with 1 unit/ml thrombin
induced strong phosphorylation of cPLA2 after 2 min-
Figure 4. Effects of F(ab⬘)2 aPL on phosphorylation of p38 MAPK
(p38-P) and ERK-1/2 (ERK1/ERK2-P). Washed human platelets were
incubated with 200 ␮g/ml of F(ab⬘)2 IgGNHS or with F(ab⬘)2 aPL (n ⫽
5) and stimulated with 0.005 units/ml thrombin or with PBS and 0.005
units/ml or 1 unit/ml thrombin. Lysates of the platelets were immunoblotted using specific antibodies for the phosphorylated forms of p38
MAPK (A) and of ERK-1/2 (C) and the unphosphorylated form of p38
MAPK (B). Each blot is representative of the results of 5 experiments,
using platelets from different donors. See Figure 3 for other definitions.
2916
VEGA-OSTERTAG ET AL
Table 1. Effects of F(ab⬘)2 antiphospholipid antibodies (aPL) on
thromboxane B2 (TXB2) production in platelets*
Treatment
0.005 units/ml thrombin
F(ab⬘)2 IgG-NHS ⫹ 0.005 units/ml
thrombin
F(ab⬘)2 aPL ⫹ 0.005 units/ml thrombin
F(ab⬘)2 aPL ⫹ 0.005 units/ml thrombin ⫹
1 ␮M SB203580
Figure 5. Effects of a specific inhibitor of p38 MAPK on aPLmediated platelet aggregation. Gel-filtered platelets were treated with
IgG aPL (200 ␮g/ml) and thrombin (0.005 units/ml) in the presence or
absence of 1 ␮M or 0.1 ␮M SB203580 or with IgG NHS (200 ␮g/ml)
and thrombin (0.005 units/ml). Platelet aggregation was determined in
an aggregometer over a period of 5 minutes, as described in Patients
and Methods. Scale is indicated at the bottom left, with axes indicating
the aggregation percentage per minute. See Figure 3 for definitions.
utes (Figure 6). Four of the 5 F(ab⬘)2 aPL fragments
induced significant phosphorylation of cPLA2 in platelets pretreated with 0.005 units/ml thrombin. Neither
treatment with 0.005 units/ml thrombin nor treatment
with control F(ab⬘)2 IgG and 0.005 units/ml thrombin
induced phosphorylation of cPLA2 (Figure 6).
Figure 6. Effects of IgG antiphospholipid antibodies (aPL) on phosphorylation of [Ca2⫹]-dependent cytosolic phospholipase A2 (cPLA2).
Washed platelets were treated with 200 ␮g/ml of F(ab⬘)2 aPL (n ⫽ 5)
or F(ab⬘)2 IgG from normal healthy subjects (IgGNHS) or phosphate
buffered saline (PBS), and then stimulated with 0.005 units/ml of
thrombin for 2 minutes. Platelets were also treated with PBS and 1
unit/ml thrombin as a positive control. The phosphorylated (cPLA2-P)
and the nonphosphorylated form of the enzyme cPLA2 were detected
in the platelet lysates by immunoprecipitation and immunoblot, as
described in Patients and Methods.
TXB2 production,
mean ⫾ SD pg/ml
230.0 ⫾ 12.5
251.0 ⫾ 12.7
535.0 ⫾ 50.9†
280.3 ⫾ 57.0‡
* Platelets were treated with 0.005 units/ml thrombin and with F(ab⬘)2
aPL (n ⫽ 5) or F(ab⬘)2 IgG from normal healthy subjects (IgG-NHS)
(n ⫽ 5), and TXB2 was measured by enzyme-linked immunosorbent
assay, as described in Patients and Methods. In some experiments,
platelets were pretreated with 1 ␮M SB203580. Experiments were
repeated 4 times.
† P ⬍ 0.0027 versus platelets treated with F(ab⬘)2 IgG-NHS plus
thrombin.
‡ P not significant versus platelets treated with F(ab⬘)2 IgG-NHS plus
thrombin.
Effects of aPL on TXB2 production. The production of TXB2 was significantly increased in platelets
treated with F(ab⬘)2 aPL and thrombin, when compared
with platelets treated with control F(ab⬘)2 IgG and
thrombin (mean ⫾ SD levels of TXB2 535.0 ⫾ 50.9
pg/ml versus 251.0 ⫾ 12.7 pg/ml; P ⫽ 0.0027) or with
thrombin alone (230.0 ⫾ 12.5 pg/ml). The mean values
of TXB2 produced by platelets treated with control
F(ab⬘)2 IgG and those treated with 0.005 units/ml thrombin alone were not statistically significantly different
(Table 1). The effect of F(ab⬘)2 aPL on TXB2 production was completely abrogated by pretreatment of the
platelets with SB203580 (mean ⫾ SD levels of TXB2
280.3 ⫾ 57.0 pg/ml), indicating that the activation of p38
MAPK is involved in aPL-mediated production of TXB2
by platelets (Table 1).
Effects of aPL on intracellular [Ca2ⴙ] mobilization. The treatment of platelets with different concentrations of thrombin (0.01, 0.1, 1, and 5 units thrombin)
produced a significant and dose-dependent increase in
intracellular calcium, with a peak between 2 and 3
minutes of treatment (G-channel to R-channel mean
ratios 2.1, 2.8, 3.2, and 5.7, respectively) (Figure 7).
There was no significant increase in intracellular calcium
concentrations for a period of 5 minutes when platelets
were treated with IgG aPL and 0.01 units/ml thrombin,
as compared with platelets treated with 0.01 units/ml
thrombin alone or platelets treated with control IgG and
thrombin (mean G-channel to R-channel ratios at 3
minutes 2.22, 2.1, and 2.2, respectively) (Figure 7). The
INTRACELLULAR EVENTS IN aPL-MEDIATED THROMBOSIS
Figure 7. Effects of aPL on intracellular calcium ([Ca2⫹]) mobilization. Washed platelets were incubated with 200 ␮g/ml F(ab⬘)2 from
healthy controls or F(ab⬘)2 aPL (n ⫽ 5) for 5 minutes and stimulated
with 0.01 units/ml of thrombin. Changes in [Ca2⫹] were evaluated
based on a mean ratio of the signal intensities of G-channel to
R-channel (G/R) over 1-minute (Min.) intervals of time. A ⫽ platelets
treated with 1 unit/ml thrombin; B ⫽ platelets treated with 0.5 units/ml
thrombin; C ⫽ platelets treated with 0.1 units/ml thrombin; D ⫽
platelets treated with F(ab⬘)2 IgG from healthy controls plus 0.01
units/ml thrombin; E ⫽ platelets treated with F(ab⬘)2 aPL and 0.01
units/ml thrombin; F ⫽ platelets treated with 0.01 units/ml thrombin
alone. See Figure 6 for other definitions.
experiments were run 7 times and the results shown in
Figure 7 correspond to 1 representative experiment.
DISCUSSION
Studies have shown conclusively that aPL are
thrombogenic in in vivo animal models (23–25). The
prothrombotic properties of aPL may be explained in
part by their ability to enhance the activation of platelets. Moreover, aPL have been shown to increase production of TXB2 in the presence of low doses of ADP,
collagen, or thrombin (7,10,26,27). A recent study by our
group showed a significant increase in the expression of
activated glycoprotein IIb/IIIa on platelets treated with
aPL and TRAP (8). Furthermore, aPL-enhanced thrombosis in vivo can be abrogated by infusions of a glycoprotein IIb/IIIa antagonist (1B5) monoclonal antibody
and in ␤3-null (glycoprotein IIb/IIIa–deficient) mice
(Vega-Ostertag M, et al: unpublished observations). In
this study, we confirmed that aPL have a direct effect on
platelet activation in the presence of thrombin, and we
examined the intracellular pathways involved in this
process.
The activation of platelets by thrombin has been
shown to involve more than one pathway, comprising the
p38 MAPK pathway, including the downstream calciumdependent phosphorylation of cPLA2, or the PLC␤
2917
pathway, involving activation of PKC, phosphorylation
of ERK1/ERK2 (Figure 1), and the intracellular mobilization of [Ca2⫹]. In both cases, TXB2 is produced and
platelets are activated (11–15).
In this study, we found that phosphorylation of
p38 MAPK mediates aPL-induced activation of platelets
in vitro. The degree of phosphorylation of this enzyme
varied among the 7 different preparations of aPL (4.4–
7.9-fold increase) and the F(ab⬘)2 aPL fragments (3.7–
12.6-fold increase over the control) used in this study.
This variability is not surprising. Antiphospholipid antibodies are known to be heterogeneous in specificity and
function, and have been shown to bind negatively
charged phospholipids, ␤2GPI, prothrombin, annexin V,
and other proteins of the coagulation cascade. A wide
variety of functions have been attributed to aPL, from
activation of endothelial cells, up-regulation of tissue
factor in monocytes, and platelet activation.
The present data conclusively show that aPL
induce phosphorylation of p38 MAPK after pretreatment with low (subactivating) doses of thrombin. Furthermore, these effects are dependent on the dose of
antibody utilized and are abrogated by pretreatment of
the platelets with the specific inhibitor of the enzyme,
SB203580, as shown in the aggregation studies and in the
TXB2 production experiments.
We also show that aPL up-regulate production of
TXB2 in platelets. This is consistent with the study by
Opara et al that recently demonstrated an increase in
platelet TXB2 production and in aggregation by aCL–
␤2GPI complexes (9,28). Furthermore, in our study, we
show that pretreatment of platelets with SB203580 completely abrogates the production of TXB2 induced by
aPL and low doses of thrombin, and that cPLA2 is
significantly phosphorylated in platelets treated with low
doses of thrombin and F(ab⬘)2 aPL, an event downstream of p38 MAPK activation (Figure 1). Therefore,
the data conclusively show involvement of that enzyme
in aPL-mediated platelet activation. These data are also
consistent with the suggestions by Gonzalez-Buritica et
al, who, in 1988, hypothesized that phospholipase A2
plays a role in platelet activation in patients with aPL
(29).
The ERK-1/2 phosphorylation pathway may also
be initiated in platelets by thrombin (Figure 1) (14,15).
Interestingly, the data from our study show that treatment with aPL and low doses of thrombin does not
induce phosphorylation of ERK-1/2. Initial in vitro
studies performed in transfected cells and in HeLa cells
suggested that p42 MAPK phosphorylates cPLA2 at
2918
Ser505, which lies within a consensus sequence for
MAPK (Pro-Xaa-Ser/Thr-Pro). Subsequently, other investigators reported the concomitant activation of
MAPK and phosphorylation of cPLA2 in stimulated
cells (30–34).
Several recent reports, however, dissociated
cPLA2 phosphorylation from MAPK activation in platelets. One study showed that phosphorylation of ERK-1/2
is not required for phosphorylation of cPLA2 in
thrombin-stimulated platelets (35). Those authors concluded that cPLA2 is the physiologic target of p38
MAPK, and that ERK-1/2 phosphorylation of cPLA2 is
not required for its receptor-mediated activation in
platelets (35). Borsch-Haubold et al have shown no
effect on phosphorylation of cPLA2 or release of thromboxane when the specific inhibitor of PKC, Ro31-8220,
was used in platelets stimulated with thrombin or collagen (12,35–38). Similarly, the same group of authors
showed that inhibition of MAPK kinase using PD98059
did not affect platelet responses to the physiologic
stimuli, thrombin and collagen, indicating a role for p38
MAPK in primary activation of human platelets, independent of ERK-1/2 (35–39).
Altogether, these results are in agreement with
our findings and provide evidence against a role for
ERK-1/2 in primary aPL-mediated platelet aggregation.
However, we do not exclude the possibility that these
MAPKs may play a role in postaggregation events in
platelets mediated by aPL.
We did not find significant changes in intracellular [Ca2⫹] when platelets were treated with F(ab⬘)2 aPL
and low doses of thrombin, supporting the hypothesis
that the PLC␤ pathway and the downstream phosphorylation of ERK-1/2 activation are not involved in aPLmediated platelet activation (see Figure 7). The p38
MAPK–dependent activation of cPLA2 in platelets appears to be dependent on intracellular calcium concentrations. In our studies, although no significant changes
in calcium concentrations were observed when aPL were
added to the system, treatment of platelets with 0.01
units/ml thrombin induced a modest increase in calcium
(as shown in Figure 7). We speculate that the observed
change may have been sufficient to initiate cPLA2
activation, but these observations would need to be
further evaluated to confirm this hypothesis.
The effects of aPL on phosphorylation of p38
MAPK and of cPLA2 and production of TXB2 by
platelets reported in this study are due to the influence
of the F(ab⬘)2 fragment and not to the Fc portion of the
antibody. This finding is consistent with the findings of
the study by Robbins et al, which showed that F(ab⬘)2
VEGA-OSTERTAG ET AL
aCL fragments significantly stimulated TXB2 production
in platelets (10).
The present study did not focus on establishing
the nature of the receptors to which aPL bind on
platelets. Studies have shown that aPL bind only to
platelets previously exposed to low doses of agonists or
to platelets that have been frozen and thawed repeatedly, exposing negatively charged phospholipids (i.e.,
phosphatidylserine) (2). However, the precise nature of
the receptors for aPL in platelets is not known. Opara et
al previously hypothesized that ␤2GPI might mediate
aCL binding to the activated platelet cell surface by
binding with phosphatidylserine, thereby promoting increased platelet activation by the aCL–␤2GPI complexes
(28). A recent study demonstrated that dimeric ␤2GPI
binds to members of the low-density lipoprotein receptor family in platelets and induces increased platelet
adhesion to collagen (40). This effect was increased by
addition of anti-␤2GPI monoclonal antibodies to the
system and was abrogated by inhibition of thromboxane
synthesis.
In summary, our study is the first to show that
aPL-mediated platelet activation occurs selectively
through the p38 MAPK pathway. Upon priming of the
platelets with aPL and low doses of thrombin, cPLA2 is
phosphorylated and TXB2 is produced. PKC and ERK1/2 activation do not seem to be involved in this response. These findings may be important in designing
new approaches to targeted treatment of thrombosis in
APS patients.
ACKNOWLEDGMENT
We are grateful to Dr. Jacob Rand of Albert Einstein
School of Medicine (New York, NY) for his helpful comments
on this work.
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