Structual Correlates of Cofactor and Protease Function

Children'S Hosp Of Philadelphia, Philadelphia PA

Investigators

Linked publications & trials

Abstract

The nonenzymatic cofactor factor Va (FVa) and the serinc proteinase factor Xa (FXa) are essential components of the enzymatic complex termed prothrombinase, the only known physiological activator of prothrombin. The precursors of these active species cannot participate in the prothrombinase complex to any significant degree, indicating that thc proteolytic conversion of FV to FVa and FX to FXa results in appropriate structural changes that impart substrate and enzyme binding capability. The broad long-term objective of this work is directed towards increasing the understanding of how specific sequences within the procofactor FV and zymogen FX contribute to maintaining their relatively inactive precursor states and how sequences in FVa mediate interactions with prothrombin and thrombin. We will test the hypothesis that alleviation of steric constraints from the B- domain leads to the expression ofmacromolccular binding sites following procofactor cleavage. We will also determine whether acidic sequences within the heavy chain of FV provide a recognition exosite for both thrombin and prothrombin. We hypothesize that these acidic sequences facilitate procofactor activation, but do not assist directly in enhancing rates ofprothrombin activation. Finally, we will assess whether intrarnolecular activation of FX following the zymogen to protcase transition results in the expression of functional binding sites on the protease domain. We hypothesize that stabilization of Asp194 via internal salt bridge formation results in the expression of high affinity FVa and Na*-binding sites rather than removal of steric effects-mediated by the FX activation peptide. Taken together these hypotheses will be tested by generating a series of recombinant FX and FV variants or plasma-derived derivatives and these proteins will be evaluated using a combination of equilibrium binding studies employing fluorescence and thermodynamic techniques, kinetic studies of the proteolytic reactions, and protein structural studies. Understanding the molecular events leading to the expression of structural determinants on FVa and FXa and how these sites influence each other is a key step in the development of pharmacologic agents with high specificity aimed at thrombotic and vascular disease states. We believe this proposal will contribute to our understanding of the molecular processes involved in zymogcn and procofactor transitions, which may ultimately lead to unanticipated ways to modulate the properties FXa and FVa.