Support of ristocetin-induced platelet aggregation by procoagulant- inactive and plasmin-cleaved forms of human factor VIII/von Willebrand factor

Human factor VIII/von Willebrand factor (fVIII/vWf) was purified to homogeneity as defined by electrophoretic and immunologic criteria and tested for fVIII procoagulant and ristocetin cofactor activities. As little as 0.4 μg/ml of purified fVIII/vWf fully aggregated washed human platelets in the presence of ristocetin. Purified fVIII/vWf, whether thrombin-activated or -inactivated, and fVIII/vWf with its procoagulant activity abolished by a human inhibitor, supported ristocetin-induced platelet aggregation as effectively as native fVIII/vWf. Similarly, purified hemophilic fVIII/vWf protein and fVIII/vWf-like protein isolated from normal serum supported the aggregation of platelets in the presence of ristocetin. FVIII/vWf extensively degraded by human plasmin and exposed to denaturing solvents also supported platelet aggregation in the presence of ristocetin. Plasmin hydrolyzed fVIII/vWf to yield noncovalently bonded fragments that could be separated into two pools by gel filtration in guanidine hydrochloride. After dialysis into dilute neutral buffers, the pool of high molecular weight fragments supported significant ristocetin-induced platelet aggregation and cross-reacted with an antibody to native fVIII/vWf. Hence, unlike fVIII procoagulant activity, the ristocetin cofactor activity and antigenicity of fVIII/vWf are retained, despite changes in the structure and conformation of the molecule. These results suggest caution about the interpretation of procoagulant activity:fVIII/vWf antigen ratios or procoagulant activity:ristocetin cofactor activity ratios in whole plasma, fVIII/vWf concentrates or purified preparations of fVIII/vWf, since small amounts of minimally degraded, procoagulant-inactive fVIII/vWf cound markedly alter such values.