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.
