We have investigated the binding of factor VIII to platelets in the presence of platelet aggregating agents.
Factor VIII was purified from cryoprecipitates by gel filtration on Sepharose 2B column and was iodinated by lactoperoxydase technique. To washed human platelets (5×108/ml in 10mM Tris, 0.15M NaCl, pH 7.4), 125I-factor VIII (5μg/ml) and an aggregating agent were added. After incubation at 24°C for 3 minutes platelets were separated by centrifugation at 7, 000×g for 1 minute through silicone oil. The radioactivity of the platelet pellets was measured by gamma counter.
Platelets bound 33ng of factor VIII per 108 platelets without addition of aggregating agents. The addition of aggregating agents such as ADP (20μM), collagen (Horm, 4μg/ml) or epinephrine (2μg/ml) did not significantly increase the binding of 125I-factor VIII to platelets. The addition of thrombin (1u/ml) markedly increased 125I-factor VIII binding (126ng/108 platelets). This thrombin-induced binding was concentration dependent (0.1-2.5u/ml) and was enhanced when Ca++ was present in the reaction mixture.
These results suggest that thrombin may play an important role in the early phase of hemostasis through its action upon the interaction between factor VIII and platelets.