Molecular Basis of Procofactor Activation

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DESCRIPTION (provided by applicant): Activation of precursor proteins by specific proteolysis is a hallmark of blood coagulation. The inactive procofactor protein factor V (FV) cannot participate to any significant degree in its macromolecular enzyme complex. Activity is generated following proteolysis, indicating that the conversion of the procofactor to FVa must result in structural changes that impart cofactor function. While there has been widespread interest in studying this protein, little is known about how specific bond cleavage and B-domain release facilitate this conversion process. The long-term objective of this proposal is to decipher these molecular processes and provide detailed insight into how FV is preserved as an inactive procofactor. This molecular process undoubtedly plays critical regulatory roles, evolved to maintain normal hemostasis since FVa has a tremendous influence on IIa generation. In the first aim, we will define the structural requirements necessary to maintain the FV procofactor state. We hypothesize that there are conserved regions in the B-domain that are instrumental in suppressing cofactor activity, despite the fact that this domain is poorly conserved and varies in length among vertebrates. In the second aim, we will investigate the mechanism by which B-domain sequences preserve the procofactor state. We hypothesize that discrete regions of the B-domain make direct contacts on the heavy and/or light chain thereby masking critical structural determinants. In the third aim, we will exploit the diversity of vertebrate FV B-domains to determine whether a common mode of inhibition is preserved across species. We hypothesize that B-domains from vertebrate species retain common sequences that will preserve the human FV procofactor state. In the final aim, we will examine the influence of FV B- domain sequences on protease susceptibility and examine the biological significance of keeping FV inactive. We hypothesize that the B-domain significantly affects how proteases such as activated protein C and thrombin engage and act on FV. These hypotheses will be tested using biochemical, kinetic, and equilibrium binding approaches employing well characterized recombinant proteins. A complete picture of the molecular events leading to the expression of functional binding sites on FVa will not only shed light on the function of this protein but will also enhance our understanding of the biological relevance of preserving FV as an inactive procofactor. Knowledge gained from this proposal may also reveal previously unrecognized ways to modulate FV/FVa function.