Prothrombinase is composed of the protein cofactor, factor Va, and the enzyme, factor Xa, associated on a cell surface in the presence of divalent metal ions. Incorporation of factor Va into prothrombinase and its interaction with factor Xa results in a 300,000-fold acceleration of the catalytic efficiency of the enzyme as compared to the catalysis of the reaction by factor Xa alone. The procofactor, factor V, does not participate in prothrombinase. Following activation of factor V by thrombin, factor Va is composed of heavy and light chains associated via divalent metal ions. Both chains of the cofactor interact with factor Xa while only the heavy chain of the cofactor binds prothrombin. The factor Va cofactor activity is efficiently down-regulated following proteolysis of the heavy chain by activated protein C (APC) only in the presence of a membrane surface and results in the inability of the cofactor to bind factor Xa. Thus, the positive and negative regulatory processes associated with factor V activation and its inactivation are directly associated with the capability of the cofactor to be incorporated into prothrombinase and to bind factor Xa. The amino acids responsible for the interaction of factor Va with factor Xa and prothrombin remain to be identified. We have data demonstrating that the heavy chain of the cofactor possesses a binding region for factor Xa within amino acid region 323-331, whereas the NH2-terminal portion of the light chain (amino acid residues 1546-1558) also interacts with factor Xa. In addition, we have data suggesting that the COOH-terminal portion of the heavy chain contain an interactive site for prothrombin while previous data have suggested that a binding site for thrombin is located on the B region of the procofactor.
The specific aims of this grant proposal are: (1) to identify and characterize the factor Xa-binding domain(s) on factor Va light chain; (2) to identify and characterize the thrombin and prothrombin-binding domain(s) on the factor V molecule; (3) to test the physiological relevance of our findings by studying the assembly and function of prothrombinase on platelets. To achieve these goals we have designed a series of experiments that are prioritized and integrated with complementary molecular and structural approaches. Characterization of the specific amino acid regions of factor V that are critical for its function will allow for a profound understanding of the macromolecular interactions that control prothrombinase and are required for its assembly, function, and specificity. We have established a system to study phospholipids-driven macromolecular complex formation, which may be a model for the generation of complexes that form extra-and intra-cellularly.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hemostasis and Thrombosis Study Section (HT)
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Link, Rebecca P
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Cleveland State University
Schools of Arts and Sciences
United States
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Wiencek, Joesph R; Na, Mahesheema; Hirbawi, Jamila et al. (2013) Amino acid region 1000-1008 of factor V is a dynamic regulator for the emergence of procoagulant activity. J Biol Chem 288:37026-38
Barhoover, Melissa A; Kalafatis, Michael (2011) Cleavage at both Arg306 and Arg506 is required and sufficient for timely and efficient inactivation of factor Va by activated protein C. Blood Coagul Fibrinolysis 22:317-24
Hirbawi, Jamila; Vaughn, John L; Bukys, Michael A et al. (2010) Contribution of amino acid region 659-663 of Factor Va heavy chain to the activity of factor Xa within prothrombinase . Biochemistry 49:8520-34
Barhoover, Melissa A; Orban, Tivadar; Beck, Daniel O et al. (2008) Contribution of amino acid region 334-335 from factor Va heavy chain to the catalytic efficiency of prothrombinase. Biochemistry 47:6840-50
Hirbawi, Jamila; Bukys, Michael A; Barhoover, Melissa A et al. (2008) Role of the acidic hirudin-like COOH-terminal amino acid region of factor Va heavy chain in the enhanced function of prothrombinase. Biochemistry 47:7963-74
Barhoover, Melissa A; Orban, Tivadar; Bukys, Michael A et al. (2008) Cooperative regulation of the activity of factor Xa within prothrombinase by discrete amino acid regions from factor Va heavy chain. Biochemistry 47:12835-43
Erdogan, E; Bukys, M A; Kalafatis, M (2008) The contribution of amino acid residues 1508-1515 of factor V to light chain generation. J Thromb Haemost 6:118-24
Bukys, Michael A; Orban, Tivadar; Kim, Paul Y et al. (2008) The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase. Thromb Haemost 99:511-22
Erdogan, Evrim; Bukys, Michael A; Orfeo, Thomas et al. (2007) Identification of an inactivating cleavage site for alpha-thrombin on the heavy chain of factor Va. Thromb Haemost 98:998-1006
Bukys, Michael A; Kim, Paul Y; Nesheim, Michael E et al. (2006) A control switch for prothrombinase: characterization of a hirudin-like pentapeptide from the COOH terminus of factor Va heavy chain that regulates the rate and pathway for prothrombin activation. J Biol Chem 281:39194-204

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