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

Joesph R. Wiencek; Mahesheema Na; Jamila Hirbawi; Michael Kalafatis

doi:10.1074/jbc.M113.462374

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

From the ^‡Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115,
the ^§Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195,
the ^¶Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio 44115, and
the ^‖Taussig Cancer Center, Cleveland Clinic, Cleveland, Ohio 44195

↵2 To whom all correspondence should be addressed: Cleveland State University, Department of Chemistry, 2351 Euclid Avenue, Science and Research Center SR370, Cleveland OH 44115. Tel.: 216-687-2460; Fax: 216-687-9298; E-mail: m.kalafatis{at}csuohio.edu.

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 (Arg⁷⁰⁹/Arg¹⁰¹⁸/Arg¹⁵⁴⁵) 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 nm), fVa^ΔB9 (0.4 nm), and fV^ΔB9/Q3 (0.7 nm) were similar to the affinity of fVa^WT for fXa (0.3 nm). 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.

Footnotes

↵1 Supported in part by Doctoral Dissertation Research Expense Award and Fellowship Program (DDREAFP) at Cleveland State University (to JRW).
↵* This work was supported, in whole or in part, by National Institutes of Health Grants R01 HL-73343 from the NHLBI (to M. K.) by a Third frontier grant from the Ohio Department of Development, and T32 HL 007914. This work was also supported by Funds from the Center for Gene Regulation in Health and Disease. A preliminary report of this work was presented at the 52nd Annual Meeting of the American Society of Hematology, December 4–7, 2010, Orlando, FL. (2010) Blood 116, 2221a.