Vitronectin functions as a cofactor for rapid inhibition of activated protein C by plasminogen activator inhibitor-1 - Implications for the mechanism of profibrinolytic action of activated protein C

Activated protein C (APC) is a natural anticoagulant in plasma that down-regulates the coagulation cascade by degrading factors Va and VIIIa. In addition to its anticoagulant function, APC is also known to possess a profibrinolytic property. This property of APC has been attributed to its ability to neutralize PAI-1, thereby increasing the concentration of tissue plasminogen activator in plasma leading to up-regulation of the fibrinolytic cascade. This hypothesis, however, has not been well established, since the concentration of PAI-1 in plasma is low, and its reactivity with APC is very slow in a purified system. Here we demonstrate that vitronectin enhances the reactivity of PAI-1 with APC similar to 300-fold making PAI-1 the most efficient inhibitor of APC thus far reported (k(2) = 1.8 x 10(5) M-1 s(-1)). We further show that PAI-1 inhibition of the Glu(192) --> Gln mutant of APC is enhanced similar to 40-fold, independent of vitronectin, suggesting that vitronectin partially overcomes the inhibitory interaction of PAI-1 with Glu(192). Additionally, we show that PAI-1 inhibition of the Lys(37)-Lys(38)-Lys(39) --> Pro-Gln-Glu mutant of APC is severely impaired, suggesting that, similar to tissue plasminogen activator, the basic 39-loop of APC plays a critical role in the reaction. Together, these results suggest that vitronectin functions as a cofactor to promote the profibrinolytic activity of APC.