ACTIVATION AND INACTIVATION OF HUMAN PROTEIN-C BY PLASMIN

Transient procoagulant states resulting in failure of recanalization o r rethrombosis of the reperfused artery during thrombolytic therapy mi ght be due to an inhibitory effect of plasmin on the anticoagulant pro perties of protein C. We therefore studied the effect of plasmin on pr otein C (PC) and activated protein C (APC) using purified human protei ns. Incubation of 70 nM purified human PC with 40-400 nM human plasmin resulted in rapid activation and subsequent inactivation of PC as mea sured by amidolytic and anticoagulant assays. The rates of activation and inactivation were dependent on the concentration of plasmin. Lower concentrations of plasmin resulted in higher peaks of generated APC a nd more sustained activity, while at higher concentrations, both activ ation and inactivation were more rapid. Anticoagulant activity appeare d more sensitive to inactivation by plasmin than amidolytic activity; e. g., while amidolytic activity reached a maximum of 13.8 nM in 6 min and declined to approximately 6 nM after 30 min, anticoagulant activi ty reached its maximum of only 1.4 nM within 30 s and completely disap peared within 90 s. Plasmin rapidly destroyed bath the anticoagulant a nd amidolytic activity of purified APC, with second order rate constan ts of 2.8 x 10(5) M(-1) s(-1) and 1.2 X 10(4) M(-1) s(-1), respectivel y, for 70 nM APC. The rates of activation and subsequent inactivation were slowed by the presence of CaCl2. The second order rate constant o f inactivation of APC amidolytic activity decreased to 6.6 x 10(3) M(- 1) s(-1) in the presence of 5 mM CaCl2. Proteolytic degradation of bot h PC and APC corresponding to the loss of amidolytic activity was demo nstrated on SDS-PAGE using I-125-labelled proteins. When normal human plasma was incubated with plasmin or streptokinase a substantial loss of PC anticoagulant activity was observed. These results in vitro sugg est that plasmin modulates the anticoagulant properties of protein C i n a way that might be of relevance for the success of fibrinolytic the rapy.