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

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Abstract
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(ab′)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 [Ca2+]‐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(ab′)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(ab′)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(ab′)2 fragments increased significantly the phosphorylation of ERK‐1/2 MAPKs. Furthermore, pretreatment of platelets with SB203580 completely abrogated the aPL‐mediated enhanced aggregation of the platelets. Platelets treated with F(ab′)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(ab′)2 aPL. There were no significant changes in intracellular [Ca2+] 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.