Hydroxychloroquine Reverses Platelet Activation Induced by Human IgG Antiphospholipid Antibodies

Abstract

SummaryProthrombotic properties of antiphospholipid (aPL) antibodies may be explained in part by their ability to enhance the activation of platelets pre-treated with low doses of ADP or thrombin. The antimalarial drug hydroxychloroquine (HQ) has been used successfully in prevention of postoperative thrombosis and in treatment of patients with SLE or APS. In one study, administration of HQ reversed the thrombogenic properties of aPL in mice. However, the mechanism of action of HQ in preventing thrombosis is not clearly understood. In order to explore this further, the effects of HQ on activation of platelets by aPL in the presence of a thrombin agonist was studied. The changes in the expression of GPIIb/IIIa (CD41a) and GPIIIa (CD61) on platelet membrane by flow cytometry were used as indicators of platelet activation. Citrated whole blood from a healthy donor was treated at room temperature with suboptimal doses of a thrombin agonist receptor peptide (TRAP) and affinity-purified aPL antibodies, in the presence and in the absence of hydroxychloroquine (1 mM). TRAP increased the expression of GPIIb/IIIa and GPIIIa on platelet surface. The treatment of the platelets with the six aPL antibodies in the presence of 12 nMol/ml TRAP further increased the expression of GPIIb/IIIa by 42.3 ± 12.3% and the expression of GPIIIa was further incremented by 46.8 ± 13.5%. The effects of aPL and TRAP on expression of platelet surface markers of activation was completely abrogated by HQ in a dose-dependent fashion and was effective at concentrations of HQ as low as 25 µg/ml (0.0125 mM). This suggests at least one possible mechanism by which HQ may prevent thrombosis. This may have important implications in treatment of thrombosis in APS patients.

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

“…Moreover, aPL have been shown to increase production of TXB 2 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).…”

“…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).…”

“…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 [Ca 2ϩ ] (Figure 1) (13)(14)(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)(8)(9)(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 cPLA 2 on intracellular [Ca 2ϩ ] mobilization and on TXB 2 production in the presence of subactivating doses of thrombin.…”

Inhibition of the thrombogenic and inflammatory properties of antiphospholipid antibodies by fluvastatin in an in vivo animal model

“…Nonetheless, the pathogenic mechanisms of aPL seem to be heterogeneous and far from being completely understood (9). Among the mechanisms suggested to explain the prothrombotic activity of aPL are the direct inhibition of the activated protein C pathway (10), abnormalities in platelet function (11,12), up-regulation of the tissue factor pathway (13), and activation of endothelial cells (ECs) (14).…”

Pathogenic Mechanisms of Thrombosis in Antiphospholipid Syndrome (APS)

“…This assay depends only on primary hemostasis and thus is considered the classic test for the assessment of platelet function. Strong evidence of aPLinduced platelet activation is the enhanced expression of platelet membrane glycoproteins, particularly GPIIb-IIIa (fibrinogen receptor, critical in platelet aggregation) and GPIIIa [ 80 ]. More recently, Pierangeli et al showed that in mice with genetic deletion of GPIIb-IIIa, passive immunization with aPL did not increase clot formation [ 81 ].…”