Thrombin activation of factor VIII: the effect of inhibitors.

Abstract

Factor VIII is a large glycoprotein which can be separated into a high molecular weight component which retains factor VIII-related antigens and supports ristocetin-induced platelet agglutination, and a low molecular weight component which has procoagulant activity. It has been suggested that this separation, observed in high ionic strength buffers, might be the consequence of proteolysis by plasma proteins. To consider this possibility, we have examined the interaction of the proteolytic enzyme thrombin with factor VIII. This study, carried out with highly purified materials, has used thrombin-mediated factor VIII proagulant activation as an indicator of this interaction. Several proteolytic inhibitors have been studied to determine their ability to inhibit factor VIII activation by thrombin. Under the current experimental conditions, diisopropylfluorophosphate (DFP) and hirudin inhibited the reaction, while heparin was an effective inhibitor only when plasma proteins were added. Benzamidine inhibited factor VIII activation when used at high concentrations, and phenylmethyl sulphonylfluoride and soybean trypsin inhibitor were found to be ineffective. These results show that DFP and hirudin prevent thrombin alteration of factor VIII and will be useful in purification and characterization studies of the factor VIII molecule.