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.
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 cleaved 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Eaton
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D
Rodriguez
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Vehar
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GA
D000595:Amino Acid Sequence; D001777:Blood Coagulation; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005169:Factor VIII / Q000302:isolation & purification / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011486:Protein C; D013917:Thrombin / Q000378:metabolism*
D006023:Glycoproteins; D046911:Macromolecular Substances; D011486:Protein C; D005169:Factor VIII; D005170:Factor X; D013917:Thrombin; D015951:Factor Xa