Calpain proteolysis of von Willebrand factor enhances its binding to platelet membrane glycoprotein IIb/IIIa: an explanation for platelet aggregation in thrombotic thrombocytopenic purpura
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We have previously observed calpain activity (calcium-dependent cysteine protease) in sera from patients with acute thrombotic thrombocytopenic purpura (TTP). The calpain activity was not present following recovery and was not detected in other thrombocytopenic disorders. We postulated that this enzyme could participate in the pathogenesis of TTP. Because other investigators have demonstrated abnormalities of von Willebrand factor (vWF) in patients with TTP, we proposed that calpain might interact with vWF in TTP. To challenge this hypothesis, we measured the binding of untreated and calpain-treated vWF to normal and ADP or calpain activated platelets. Untreated vWF bound in a specific and saturable fashion to activated platelets, but only at low (30 microM) calcium concentrations. Von Willebrand factor did not bind to activated platelets at physiological (2 mM) calcium concentrations. Calpain proteolysis of vWF changed the binding characteristics of the vWF so that it had greatly increased binding to both ADP and calpain activated platelets. The calpain-proteolyzed vWF bound to activated platelets at both low and physiological calcium concentrations, and was capable of causing platelet aggregation. The calpain-proteolyzed vWF bound to the activated platelets via glycoproteins IIb/IIIa as demonstrated by inhibition studies using monoclonal antibodies against glycoproteins IIb/IIIa and Ib. It also had a high binding affinity and was capable of inhibiting the binding of radiolabelled fibrinogen to the activated platelets at physiological calcium concentrations. Calpain also proteolyzed fibrinogen, but the calpain altered fibrinogen had normal platelet reactivity. These studies provide further insight into the pathogenesis of the platelet aggregation of thrombotic thrombocytopenic purpura. Calpain proteolyses vWF and can produce the characteristic loss of large multimers seen on sodium dodecyl sulphate (SDS)-agarose gel electrophoresis. The altered vWF is highly reactive with activated platelets and binds to platelet glycoproteins IIb/IIIa and participates in formation of the platelet aggregates that characterize this disease.
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