Platelet Activating Properties of Murine Monoclonal Antibodies to β2-Glycoprotein I

Abstract

SummaryPreviously developed murine monoclonal antibodies (MAbs) to human β2-glycoprotein I (β2GPI), a plasma protein required for the binding of anti-phospholipid antibodies, were studied for anti-platelet reactivity and influence on platelet function. The six MAbs (IgG1 isotype) tested interacted with both intact and fixed platelets in a β2GPI-dependent manner. Carbamylated β2GPI was still recognized by MAbs but was unable to mediate platelet- antibody binding. MAbs induced aggregation and secretion responses of platelets in platelet-rich plasma (PRP) and whole blood, provided subthreshold concentrations of weak agonists (i.e. ADP or adrenaline) were added. When aggregation in PRP was evaluated by a counting technique instead of turbidometrically, the sole addition of MAbs led to a rapid fall in single platelets. Triggering gel-filtered platelets with MAbs together with β2GPI, but not its carbamylated form, led to platelet activation after a lag time, as monitored by aggregometry, measurements of ATP and β-thromboglobulin secretion and calcium mobilization. F(ab')2 fragments of one of the MAbs failed to activate platelets but inhibited the responses to the whole antibody. This process thus depends on MAbs binding to platelets through both Fab and Fc domains, as confirmed by the suppression of platelet responses upon pretreatment with the anti-FcγRII MAb IV.3. Aggregation and secretion induced by MAbs plus β2GPI did not require exogenous fibrinogen and were variably inhibited in the presence of acetyl salicylic acid, apyrase or Ca2+, depending on the concentrations used for the two proteins. We propose that platelet FcγRII crosslinking that follows a platelet-antibody interaction via the platelet binding protein, β2GPI, may be a new mechanism by which anti-β2 GPI antibodies activate platelets and/or synergize with weak agonists.

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

“…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 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).…”

Role of the Fcγ receptor IIA polymorphism in the antiphospholipid syndrome: An international meta‐analysis

“…This complication seems to be a consequence of platelet activation initiated when platelet Fc␥RIIa are crosslinked by antibodies (5,32). Since the interaction of IgG2-containing antibodies with Fc␥RIIa is allotype-dependent (8), it has been hypothesized that the high-binding H131 allele would be overrepresented among subjects with antibodymediated thrombosis (11,33).…”

Differential Effects of Anti–β 2 -Glycoprotein I Antibodies on Endothelial Cells and on the Manifestations of Experimental Antiphospholipid Syndrome

“…The correlation between the pathogenic effects in the passive transfer experiments and the in vitro adherence assays was not complete. These findings could be explained by the multiple effects of aCL on hemostasis, including activation of platelets, 16 induction of tissue factor, 31 and so on. Furthermore, the effect of the mAbs on the EC seems to be multifactorial and probably is not restricted merely to expression of the three adhesion molecules that were evaluated.…”

“…The role of aCL in conferring the prothrombotic predisposition in APS has been strengthened by in vitro studies showing their platelet- 16,17 and EC- 18,19 activating properties. Additionally, it has been demonstrated that passive transfer 20 or active immunization 21 with aCL of naive mice led to the clinical manifestations of APS.…”