. Factor V is a protein cofactor that is essential for the prothrombinase complex. This complex consists of factor Va, factor Xa, calcium, and a phospholipid surface. The generation of thrombin is a critical reaction during hemostasis. Thrombin catalyzes a number of important reactions during hemostasis including the activation of platelets, the activation of factors V, VIII and XIII, and the conversion of fibrinogen to fibrin. In addition, thrombin bound to its endothelial receptor thrombomodulin catalyzes activation of protein C which then inactivates factors V and VIII. In order to understand the mechanisms by which the activation of prothrombin is regulated we have undertaken an investigation of the structure and function of the factor V molecule. Initially, we developed methods to isolate human factor V from plasma and studied its proteolytic activation. Subsequently we investigated the role of factor Va as the factor Xa receptor on human platelets. More recently, we have determined the complete primary sequence of the molecule. We have demonstrated that factor V is a multidomain protein with homology to ceruloplasmin and factor VIII. We are currently using site directed mutagenesis to investigate structure function relationships in recombinant factor V expressed in mammalian cells. The goals of the present study are to identify regions of the molecule that constitute the binding sites for phospholipid, cell surface receptors, factor Xa and prothrombin. We will also define the critical proteolytic events required for the activation and inactivation of this protein. We will use both genetic and reverse genetic approaches to define the function of this important protein. Using the factor V cDNA and mammalian cell expression systems we will express recombinant factor V with specific mutations. We will also use PCR to amplify platelet mRNA isolated from patients with factor V deficiency and determine natural mutations that affect factor V function. The results of these experiments will ultimately define the exact molecular interactions involved in the assembly and regulation of the prothrombinase complex. Expression and characterization of chimeric factor V/factor VIII molecules will further enhance understanding of the structure function relationships in this family of related proteins. This knowledge may provide insight into the pathogenesis of thrombotic disorders.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Hematology Subcommittee 2 (HEM)
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Duke University
Internal Medicine/Medicine
Schools of Medicine
United States
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Srivasatava, Kinshuk Raj; Majumder, Rinku; Kane, William H et al. (2014) Phosphatidylserine and FVa regulate FXa structure. Biochem J 459:229-39
Majumder, Rinku; Liang, Xiaoe; Quinn-Allen, Mary Ann et al. (2011) Modulation of prothrombinase assembly and activity by phosphatidylethanolamine. J Biol Chem 286:35535-42
Majumder, Rinku; Quinn-Allen, Mary Ann; Kane, William H et al. (2008) A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex. Blood 112:2795-802
Jeimy, Samira B; Quinn-Allen, Mary Ann; Fuller, Nola et al. (2008) Location of the multimerin 1 binding site in coagulation factor V: an update. Thromb Res 123:352-4
Peng, W; Quinn-Allen, M A; Kane, W H (2005) Mutation of hydrophobic residues in the factor Va C1 and C2 domains blocks membrane-dependent prothrombin activation. J Thromb Haemost 3:351-4
Majumder, Rinku; Quinn-Allen, Mary Ann; Kane, William H et al. (2005) The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex. Biochemistry 44:711-8
Saleh, Mahasen; Peng, Weimin; Quinn-Allen, Mary Ann et al. (2004) The factor V C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis. Thromb Haemost 91:16-27
Peng, Weimin; Quinn-Allen, Mary Ann; Kim, Suhng Wook et al. (2004) Trp2063 and Trp2064 in the factor Va C2 domain are required for high-affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex. Biochemistry 43:4385-93
Hayward, Catherine P M; Fuller, Nola; Zheng, Shilun et al. (2004) Human platelets contain forms of factor V in disulfide-linkage with multimerin. Thromb Haemost 92:1349-57
Jeimy, Samira B; Woram, Rachael A; Fuller, Nola et al. (2004) Identification of the MMRN1 binding region within the C2 domain of human factor V. J Biol Chem 279:51466-71

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