Studies of Coagulation and Fibrinolysis
Kim, Paul Ye Sir
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The mechanism and regulation of prothrombinase function in the activation of prothrombin and prethrombin-1 were studied. In addition, the kinetics of plasmin-mediated fibrin degredation were determined. First, the mechanism of prothrombin activation by prothrombinase was investigated using two models – a one-form model which posits a single form of prothrombinase and a two-form model which posits the existence of two, equilibrating forms of prothrombinase each specific for either activation cleavages. In summary, the two-form model fit the data better, as well as predict the initial rates of prothrombin activation better than the one-form model, and thus better represents the mechanism of prothrombinase on prothrombin activation. Second, the differences observed in the activation kinetics of prethrombin-1 by prothrombinase consisting of either human or bovine factor Va (FVa) was investigated. The difference was due to the species origin of the heavy chain. Because the conserved domains show high sequence homology, the carboxy-terminus was studied. The calculated pI of the ten carboxy-terminal residues are 12.5 for human and 4.26 in bovine, suggesting that with bovine FVa, the net negative charge of the ten residues augments the negative charge of DSDYQ, thus facilitating the FVa-prethrombin-1 interaction required for Arg320 cleavage. Third, the kinetics of activated protein C (aPC)-mediated inactivation of FVa in real-time, during prothrombin activation was investigated. A profound protection of FVa from aPC was observed (>100-fold), which was much greater than the individual protection with either factor Xa (3 to 10-fold) or prothrombin (10-fold) alone. The addition of protein S, the cofactor for aPC, resulted in a two-fold increase in the rate constant, but did not ameliorate the protection. Lastly, the kinetics of plasmin-mediated lysis of fibrin clots was investigated. The clot lysis by plasmin showed a low Km (1 to 2uM) suggesting zero-order kinetics. In contrast, the clot formation showed first-order kinetics. These results suggest that as the concentration of fibrinogen increases, especially above the physiologic level, the balance between fibrinolysis and clotting shifts toward the latter, thus providing a biochemical rationale for the increased risk of cardiovascular disease associated with elevated level of fibrinogen.