Restoring the Procofactor State of Factor Va-like Variants by Complementation with B-domain Peptides |
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| Authors: | Matthew W. Bunce Mettine H. A. Bos Sriram Krishnaswamy Rodney M. Camire |
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| Affiliation: | From the ‡Division of Hematology, The Children''s Hospital of Philadelphia and ;the §Department of Pediatrics, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, Pennsylvania 19104 |
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| Abstract: | Coagulation factor V (FV) circulates as an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domain. Conserved basic and acidic sequences within the B-domain appear to play an important role in keeping FV as an inactive procofactor. Here, we utilized recombinant B-domain fragments to elucidate the mechanism of this FV autoinhibition. We show that a fragment encoding the basic region (BR) of the B-domain binds with high affinity to cofactor-like FV(a) variants that harbor an intact acidic region. Furthermore, the BR inhibits procoagulant function of the variants, thereby restoring the procofactor state. The BR competes with FXa for binding to FV(a), and limited proteolysis of the B-domain, specifically at Arg1545, ablates BR binding to promote high affinity association between FVa and FXa. These results provide new insight into the mechanism by which the B-domain stabilizes FV as an inactive procofactor and reveal how limited proteolysis of FV progressively destabilizes key regulatory regions of the B-domain to produce an active form of the molecule. |
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| Keywords: | Coagulation Factors Enzyme Mechanisms Hemostasis Protein Engineering Recombinant Protein Expression Autoinhibition Factor V Factor Va Prothrombinase |
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