Destabilization of the Factor V B-Domain Results in Procofactor Activation.

A hallmark of hemostasis is that proteins involved in the formation of a blood clot remain in a quiescent state and are only activated following an appropriate stimulus. Blood coagulation factor V (FV), which is structurally homologous to FVIII, cannot function in the prothrombinase complex and is thus considered a procofactor. Thrombin catalyzes the conversion of FV to FVa following three cleavages (Arg⁷⁰⁹, Arg¹⁰¹⁸, and Arg¹⁵⁴⁵) releasing a large heavily glycosylated central B-domain (836 amino acids). Explanations as to how bond cleavage or B-domain release facilitates the transition to the active species are incomplete. Recent studies using a partial B-domainless form of FV (FV-810 des^811–1491) support a model in which removal of B-domain sequences from FV rather than specific proteolysis underlies the mechanism by which cofactor function is realized. This single-chain derivative is functionally equivalent to FVa suggesting that the deleted B-domain sequences somehow suppress cofactor activity. To investigate this further, we have expressed and purified several single-chain derivatives of FV that vary in B-domain length from 155 to 497 residues. Functional activity assays as well as direct binding fluorescent measurements revealed that elimination of most of the C-terminal half of the B-domain (residues 1034–1491; 458 out of 836 a.a. deleted) had no influence on maintaining the procofactor state. However, deletion of sequences from the N-terminal half of the B-domain resulted in derivatives with cofactor-like properties. Using progressively finer deletion variants we were able to demonstrate that either a B-domain length of at least 378 amino acids or specific sequences contained within residues 902–1033 is sufficient to suppress cofactor activity. To examine these possibilities, we constructed additional FV variants in which a B-domain length of 378 amino acids was maintained, but specific portions of 902–1033 were exchanged with FVIII B-domain. Using the FV-1033 derivative (residues 1034–1491 of B-domain deleted) as a scaffold, three constructs were prepared, s-131, s-104, and s-46, representing 131, 104, and 46 amino acids from the FV B-domain exchanged with FVIII B-domain. In activity assays and direct binding measurements, each of these variants had properties consistent with the cofactor-like form indicating that a length of ~375 residues is not sufficient to maintain the procofactor state. These findings demonstrate for the first time that there are indeed specific FV-B domain sequences between 902–1033 that directly or indirectly stabilize the procofactor state. Remarkably, simply replacing these sequences in FV-1033 resulted in activation of the proteins in the absence of proteolysis. These observations change existing ideas about FV activation and provide insight into specific regions of the B-domain that assist in preserving the procofactor state.