Effects of aprotinin on coagulation and fibrinolysis enzymes

Large quantities of aprotinin, the tight-binding inhibitor of trypsin and kallikrein, are used with success to reduce bleeding, e.g. in cardiac pulmonary surgery. The effect has been attributed to inhibition of fibrinolysis, which immediately seems plausible since aprotinin is a known effective inhibitor of plasmin. However, fast kinetic studies reveal that the rate of the plasmin-aprotinin reaction may be too slow to compete effectively with alpha(2)-antiplasmin, even when present at subphysiological concentrations. Fibrinolysis experiments in microtiter plates confirm that the effect of aprotinin on plasmin may be negligible in the presence of alpha(2)-antiplasmin. Therefore, the effects of high concentrations of aprotinin on a number of coagulation and fibrinolysis enzymes previously assumed not to be affected by the inhibitor were also investigated using steady state kinetic-rate methods. It was found that aprotinin is a multi-functional inhibitor which inhibits urokinase, activated protein C (APC) and thrombin sufficiently well, if present in mu M-concentrations, to significantly affect their activity. A kinetic analysis showed simple competitive inhibition in all these cases, corresponding with binding of aprotinin to the active site of the enzyme and thus a reduction in the amount of enzyme available for substrate interaction. However, Factor Xa, Factor VIIa and tissue-plasminogen activator were not affected. All in all, the results indicate that the effects of high concentrations of aprotinin may also stem from enzymes other than plasmin. Known kinetic data on various physiological inhibitors show that the effects of aprotinin on urokinase and APC may be more important than has been hitherto anticipated.