Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 288, Issue 42, Pages 30151-30160Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.506840
Keywords
Coagulation Factors; Enzyme Mechanisms; Hemostasis; Protein Engineering; Recombinant Protein Expression; Autoinhibition; Factor V; Factor Va; Prothrombinase
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Funding
- National Institutes of Health [R01 HL88010, P01 HL74124]
- NHLBI [T32 HL-07439]
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Background: B-domain fragments of factor V (FV) were used to assess the mechanism by which it is maintained as a procofactor. Results: A basic region fragment binds to FV containing an intact acidic region and inhibits FXa binding. Conclusion: B-domain sequences function as cis- and trans-acting elements to suppress FV(a) procoagulant function. Significance: The results provide mechanistic insight into FV autoinhibition and activation. 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 Arg(1545), 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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