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Allosteric Regulation of Prothrombin Activation by factor Va

Ali, Mahesheema, na
http://rave.ohiolink.edu/etdc/view?acc_num=csu1462805026

Abstract Details

2016, Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, College of Sciences and Health Professions.
Upon vascular injury, the proteolytic conversion of prothrombin to thrombin occurs in the presence of prothrombinase. Prothrombinase is an enzymatic complex between factor Va (fVa) and factor Xa (fXa) assembled on a membrane surface in the presence of divalent metal ions. Blood coagulation factor V (FV) is a multi-domain protein (A1-A2-B-A3-C1-C2) with no procoagulant activity and circulates in blood as a procofactor. Our investigation demonstrates that fV is activated by thrombin in a kinetically preferred order, by a first cleavage at Arg709, followed by cleavage at Arg1545 to ultimately generate the active cofactor species, fVa. The cofactor binds to membrane surfaces and effectively serves as receptor for membrane-bound fXa. Although membrane-bound fXa alone is capable of activating prothrombin through initial cleavage at Arg271, followed by the cleavage at Arg320 (Prethrombin-2 Pathway); the rate of activation is not physiologically compatible with survival. However, incorporation of fVa into prothrombinase results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation with the order of cleavages reversed, with initial cleavage at Arg320 followed by Arg271 (Meizothrombin Pathway), resulting in physiologically relevant rates of thrombin formation at the place of vascular injury. We have shown that fXa interacts with prothrombin through amino acid region 478-482 in a fVa-dependent manner. We further demonstrate that basic amino acids from exosite I of prothrombin are involved in recognizing fXa within prothrombinase also in a fVa-dependent manner. Finally, we characterized the allosteric interactions of these amino acid residues within prothrombin required for optimum prothrombin activation rates and optimal thrombin function. Our data suggest that amino acids Leu480 and Gln481 allosterically interact with basic amino acids from exosite I on prothrombin, thus modulating the enzymatic activity of fXa within prothrombinase. Our results also provide a logical explanation for the deleterious physiological consequences of natural mutations in proexosite I (Arg382 to Cys) and (Arg388 to Hys) and demonstrate that patients harboring these mutations are impaired in their ability to form a fibrin clot and, thus, are prone to severe bleeding. Overall our data underline the crucial physiological importance of fVa for thrombin generation and clot formation.
Michael Kalafatis, Ph.D. (Advisor)
Edward Plow, Ph.D. (Committee Member)
Anton Komar, Ph.D. (Committee Member)
David Anderson, Ph.D. (Committee Member)
Crystal Weyman, Ph.D. (Committee Member)
Biochemistry
factor Va , factor Xa , Prothrombin, Thrombin

Recommended Citations

Citations

  • Ali, na, M. (2016). Allosteric Regulation of Prothrombin Activation by factor Va [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1462805026

    APA Style (7th edition)

  • Ali, na, Mahesheema. Allosteric Regulation of Prothrombin Activation by factor Va. 2016. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1462805026.

    MLA Style (8th edition)

  • Ali, na, Mahesheema. "Allosteric Regulation of Prothrombin Activation by factor Va." Doctoral dissertation, Cleveland State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1462805026

    Chicago Manual of Style (17th edition)

csu1462805026
377
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This open access ETD is published by Cleveland State University and OhioLINK.