Activation and Inactivation of Human Protein C by Plasmin

Summary

Transient procoagulant states resulting in failure of recanalization or rethrombosis of the reperfused artery during thrombolytic therapy might be due to an inhibitory effect of plasmin on the anticoagulant properties of protein C. We therefore studied the effect of plasmin on protein C (PC) and activated protein C (APC) using purified human proteins.

Incubation of 70 nM purified human PC with 40-400 nM human plasmin resulted in rapid activation and subsequent inactivation of PC as measured 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 and more sustained activity, while at higher concentrations, both activation and inactivation were more rapid. Anticoagulant activity appeared 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 activity reached its maximum of only 1.4 nM within 30 s and completely disappeared within 90 s.

Plasmin rapidly destroyed both the anticoagulant and amidolytic activity of purified APC, with second order rate constants of 2.8 × 10⁵ M⁻¹ s⁻¹ and 1.2 × 10⁴ M^{−1 −1}, respectively, for 70 nM APC. The rates of activation and subsequent inactivation were slowed by the presence of CaCl₂. The second order rate constant of inactivation of APC amidolytic activity decreased to 6.6 × 10³ M⁻¹ s⁻¹ in the presence of 5 mM CaCl₂. Proteolytic degradation of both PC and APC corresponding to the loss of amidolytic activity was demonstrated on SDS-PAGE using ¹²⁵I-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 suggest that plasmin modulates the anticoagulant properties of protein C in a way that might be of relevance for the success of fibrinolytic therapy.

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