Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity*

Abstract

Background: Factor V is activated to factor Va to interact with factor Xa.

Results: Elimination of nine amino acids from the B domain results in binding of unactivated factor V to factor Xa.

Conclusion: Amino acids 1000–1008 of factor V prevent unwanted prothrombinase assembly.

Significance: A short peptide sequence from the B region is a regulatory domain for the generation of factor Va procoagulant activity.

Abstract

Single chain factor V (fV) circulates as an M _r 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000–1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg ⁷⁰⁹/Arg ¹⁰¹⁸/Arg ¹⁵⁴⁵) mutated to glutamine (fV ^Q3), a mutant fV molecule with region 1000–1008 deleted (fV ^ΔB9), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV ^ΔB9/Q3). The recombinant molecules along with wild type fV (fV ^WT) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV ^Q3 was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, K _{D (app)} values for fV ^ΔB9 (0.9 n m), fVa ^ΔB9 (0.4 n m), and fV ^ΔB9/Q3 (0.7 n m) were similar to the affinity of fVa ^WT for fXa (0.3 n m). Two-stage clotting assays revealed that although fV ^Q3 was deficient in its clotting activity, fV ^ΔB9/Q3 had clotting activity comparable with fVa ^WT. The k _cat value of prothrombinase assembled with fV ^ΔB9/Q3 was minimally affected, whereas the K _m value of the reaction was increased 57-fold compared with the K _m value obtained with prothrombinase assembled with fVa ^WT. These findings strongly suggest that amino acid region 1000–1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.