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 affinity-purified 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 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–β2-glycoprotein I (β2GPI) 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, α-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 Cβ (PLCβ), and the intracellular mobilization of [Ca2+] (Figure 1) (13–15).
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; PLCβ = phospholipase Cβ; PLCγ = phospholipase Cγ; DG = diacylglycerol; IP3 = inositol triphosphate; PKC = protein kinase C; cPLA2 = [Ca2+]-dependent cytosolic phospholipase A2; AA = acetylsalicylic acid; TXB2 = thromboxane B2; TXRc = thromboxane receptor.
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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.
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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 β3-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 calcium-dependent phosphorylation of cPLA2, or the PLCβ 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, β2GPI, 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–β2GPI 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 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 PLCβ pathway and the downstream phosphorylation of ERK-1/2 activation are not involved in aPL-mediated 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 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 β2GPI might mediate aCL binding to the activated platelet cell surface by binding with phosphatidylserine, thereby promoting increased platelet activation by the aCL–β2GPI complexes (28). A recent study demonstrated that dimeric β2GPI 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-β2GPI 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 ERK-1/2 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.