Kinetics of activation of variant plasminogens in a noncrosslinked fibrin clot: Abnormal plasmin generation with the plasminogen.Streptokinase complex, urokinase and tissue plasminogen activator

Abstract

A purified clot lysis system (noncrosslinked) was developed to evaluate and study variant plasminogens from patients with a history of thrombophilia. Plasmin generation was studied in the soluble phase and in the clot lysis system with different activators, streptokinase and its derivatives, urokinase and tissue plasminogen activator. Five of the variant plasminogens (Dl, D2, K, S, and F) generated low levels of plasmin in the soluble phase, whereas two (M and Y) generated near normal levels of plasmin. Plasmin generation in the clot lysis system divided the variants into three groups: one variant (Dl) had a very prolonged lysis time or a negligible plasmin generation rate with three direct activators, three variants (D2, K and S) had moderately prolonged lysis times or low plasmin generation rates with these activators, and three variants (F, M, and Y) had normal lysis times and normal plasmin generation rates with these activators. Kinetics of activation parameters with three direct plasminogen activators were determined from a non-linear regression analysis of plots of velocity versus plasminogen concentration and were verified by Lineweaver-Buck plots where possible. With plasminogen.streptokinase, the four variants with prolonged lysis times, had normal apparent Michaelis constants and low catalytic rate constants. Three variants with normal lysis times had high apparent Michaelis constants showing decreased affinity for the plasminogen.streptokinase activator complex, and high catalytic rate constants. With urokinase, the variant with the longest clot lysis time had a high apparent Michaelis constant and a low catalytic rate constant, three variants with prolonged lysis times gave low catalytic rate constants; two of them gave normal apparent Michaelis constants and one gave a high apparent Michaelis constant. Of the three variants with normal lysis times, one gave normal kinetic constants and two had high apparent Michaelis constants with high catalytic rate constants. With tissue plasminogen activator, the variant with the longest clot lysis time gave a high apparent Michaelis constant and a high catalytic rate constant. The three variants with prolonged lysis times gave variable results, one had a slightly higher apparent Michaelis constant with a low catalytic rate constant, the second one had a very low apparent Michaelis constant with a low catalytic rate constant and the third one had a high apparent Michaelis constant with a normal catalytic rate constant. Three variants with normal lysis times had slightly higher Michaelis constants and near normal catalytic rate constants. With the noncrosslinked fibrin substrate, all of the variant plasminogens have lowered catalytic efficiencies with two or three plasminogen activators. The theoretical aspects of these plasmin generation data are discussed.