Factor VII and single-chain plasminogen activator-activating protease - Activation and autoactivation of the proenzyme

Structural and biological characteristics of a recently described plasma se rine protease, which displayed factor VII as well as pro-urokinase-activati ng properties in vitro, indicated a dual role for this factor VII-activatin g protease (FSAP) in hemostasis. Only the active protease (two-chain FSAP) has been isolated from plasma and from a prothrombin complex concentrate, w hereas activators of the proenzyme have not been identified so far. After p urification of the FSAP proenzyme from cryo-poor plasma by adsorption to an immobilized mAb and subsequent ion-exchange chromatography, activation to generate two-chain FSAP was followed by a direct chromogenic assay as well as by the ability of two-chain FSAP to activate prourokinase. purified sing le-chain FSAP underwent autoactivation leading to the typical protease two- chain pattern and subsequent degradation products, as demonstrated by Weste rn-blotting analysis using a site-specific mAb. This autoactivation was sig nificantly enhanced in the presence of heparin, whereas Ca2+ ions stabilize d single-chain FSAP (the proenzyme) resulting in slower autoactivation kine tics. Correspondingly, the heparin-augmented reaction, which was associated with autodegradation particularly of the protease domain, was slowed down by co-incubation with Ca2+. Of the other proteases and cofactors tested, on ly urokinase (uPA) was able to generate the typical two-chain FSAP pattern. Studies with different forms of uPA suggest that the catalytic activity of pro-urokinase/uPA is needed to activate single-chain FSAP, indicating that it is the only hemostatic protease that can act as a physiological activat or of FSAP.