REGULATION OF PROTEASE NEXIN-1 TARGET PROTEASE SPECIFICITY BY COLLAGEN TYPE-IV

Recent studies have shown that serine protease inhibitors can be regul ated in their activity, specificity, and location by glycoprotein or e xtracellular matrix (ECM) co-factors. Protease nexin-1 (PN-1) is a mem ber of the serpin superfamily of serine protease inhibitors which can rapidly inhibit thrombin, urokinase, and plasmin. PN-1 binds tightly t o and is regulated by the ECM. This interaction accelerates the inhibi tion of thrombin by PN-1 and blocks urokinase and plasmin inhibition b y PN-1. Previous work showed that heparan sulfate proteoglycan is larg ely responsible for the acceleration of thrombin inhibition by PN-1. O ur current studies were directed at identifying ECM component(s) that decreased the ability of PN-1 to inhibit urokinase and plasmin. These studies showed that collagen type TV decreased the formation of SDS st able complexes between urokinase or plasmin and PN-1 without affecting formation of complexes between thrombin and PN-1. The second order ra te constant for inhibition of urokinase by PN-1 was markedly decreased with increasing collagen type IV, whereas the second order rate const ant for inhibition of thrombin by PN-1 was unaffected by addition of c ollagen type IV. Other ECM components (collagen type I, vitronectin, f ibronectin, and heat-denatured collagen type IV) did not affect comple x formation or the rate of inhibition of proteases by PN-1, indicating that these effects were specific to collagen type IV. Binding of PN-1 to immobilized collagen type IV was demonstrated using an enzyme-link ed immunosorbent assay; the concentration of PN-1 necessary to obtain 50% saturation of the immobilized collagen type IV binding sites was a pproximately 15 nM. Collagen type IV was also co-purified with PN-1 fr om fibroblast-conditioned medium. These results demonstrate a novel re gulation of serpin specificity in which an ECM co-factor decreased the inhibition of certain proteases by the serpin without affecting the i nhibition of its target protease.