Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity

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Abstract
Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80,000 to 210,000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210,000 and 80,000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90,000-180,000 polypeptides are derived from the Mr 210,000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80,000-210,000 proteins and the generation of polypeptides of Mr 73,000, 50,000, and 43,000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90,000-210,000 proteins with the generation of an Mr 45,000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80,000 protein was not degraded by APC. Factor Xa cleaves the Mr 80,000-210,000 factor VIII proteins, resulting in the generation of fragments of Mr 73,000, 67,000, 50,000, 45,000, and 43,000. Factor Xa was found to initially activate and subsequently inactivate factor VIII. Activation by factor Xa correlated with the generation of Mr 73,000, 50,000, and 43,000 polypeptides while inactivation correlated with the cleavage of Mr 73,000 and 50,000 polypeptides to fragments of Mr 67,000 and 45,000, respectively. The cleavage sites in factor VIII of thrombin, factor Xa, and APC were identified by amino acid sequencing of the fragments generated after cleavage of factor VIII by these proteases. Interestingly, factor Xa was found to cleave factor VIII at the same sites as APC and thrombin. This may explain why factor Xa activates as well as inactivates factor VIII.