The proposed investigation of molecular mechanisms involved in the regulation of human blood coagulation by factor Va forms the basis for development of a new, independent research program by the candidate. The level of blood coagulation factor Va in vivo is controlled by two opposing proteolytic reaction processes which are essential to normal hemostasis, activation of factor V by thrombin and inactivation of factor Va by activated protein C. Factor Va appears to participate in regulating its own concentration by acting as a cofactor to enhance the rate of protein C activation as well as that of prothrombin activation. A regulatory binding interaction of thrombin with factor Va has been implicated in this process which may represent a critical point in determining the balance between procoagulant and anticoagulant systems. The goal of the proposed studies is to quantitate and characterize thrombin-factor Va interactions and to elucidate mechanisms by which factor Va modulates thrombin reactions. Reaction mechanism hypotheses for the participation of thrombin-factor Va interactions in protein C and prothrombin activation will be evaluated by the use of fluorescence techniques to characterize binding interactions among the proteins and the assembly of phospholipid membrane surface-bound complexes, in conjunction with kinetic studies of the reactions. These studies will seek to define the contributions of specific protein-protein interactions, protein-membrane interactions and conformational changes to the rate enhancements. The resulting quantitative description of the poorly understood but potentially significant regulation of thrombin by human factor Va will provide the foundation for evaluating its role in normal hemostasis and in thrombotic disease. The proposed studies reflect a continuing interest of the candidate in applying physical biochemical approaches to investigate molecular mechanisms of vascular disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Modified Research Career Development Award (K04)
Project #
1K04HL002832-01
Application #
3074564
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1993-04-01
Project End
1998-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Boxrud, P D; Fay, W P; Bock, P E (2000) Streptokinase binds to human plasmin with high affinity, perturbs the plasmin active site, and induces expression of a substrate recognition exosite for plasminogen. J Biol Chem 275:14579-89
Anderson, P J; Nesset, A; Dharmawardana, K R et al. (2000) Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation. J Biol Chem 275:16435-42
Boxrud, P D; Bock, P E (2000) Streptokinase binds preferentially to the extended conformation of plasminogen through lysine binding site and catalytic domain interactions. Biochemistry 39:13974-81
Anderson, P J; Nesset, A; Dharmawardana, K R et al. (2000) Characterization of proexosite I on prothrombin. J Biol Chem 275:16428-34
Dharmawardana, K R; Olson, S T; Bock, P E (1999) Role of regulatory exosite I in binding of thrombin to human factor V, factor Va, factor Va subunits, and activation fragments. J Biol Chem 274:18635-43
Dharmawardana, K R; Bock, P E (1998) Demonstration of exosite I-dependent interactions of thrombin with human factor V and factor Va involving the factor Va heavy chain: analysis by affinity chromatography employing a novel method for active-site-selective immobilization of serine proteinase Biochemistry 37:13143-52
Colwell, N S; Blinder, M A; Tsiang, M et al. (1998) Allosteric effects of a monoclonal antibody against thrombin exosite II. Biochemistry 37:15057-65
Bock, P E; Olson, S T; Bjork, I (1997) Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I. J Biol Chem 272:19837-45
Hogg, P J; Bock, P E (1997) Modulation of thrombin and heparin activities by fibrin. Thromb Haemost 77:424-33
Bock, P E; Day, D E; Verhamme, I M et al. (1996) Analogs of human plasminogen that are labeled with fluorescence probes at the catalytic site of the zymogen. Preparation, characterization, and interaction with streptokinase. J Biol Chem 271:1072-80

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