title: Regulation of protease nexin-1 target protease specificity by collagen type IV.
abstract: Recent studies have shown that serine protease inhibitors can be regulated in their activity, specificity, and location by glycoprotein or extracellular matrix (ECM) co-factors. Protease nexin-1 (PN-1) is a member of the serpin superfamily of serine protease inhibitors which can rapidly inhibit thrombin, urokinase, and plasmin. PN-1 binds tightly to and is regulated by the ECM. This interaction accelerates the inhibition of thrombin by PN-1 and blocks urokinase and plasmin inhibition by PN-1. Previous work showed that heparan sulfate proteoglycan is largely responsible for the acceleration of thrombin inhibition by PN-1. Our 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 IV decreased the formation of SDS-stable complexes between urokinase or plasmin and PN-1 without affecting formation of complexes between thrombin and PN-1. The second order rate constant for inhibition of urokinase by PN-1 was markedly decreased with increasing collagen type IV, whereas the second order rate constant for inhibition of thrombin by PN-1 was unaffected by addition of collagen type IV. Other ECM components (collagen type I, vitronectin, fibronectin, and heat-denatured collagen type IV) did not affect complex 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-linked immunosorbent assay; the concentration of PN-1 necessary to obtain 50% saturation of the immobilized collagen type IV binding sites was approximately 15 nM. Collagen type IV was also copurified with PN-1 from fibroblast-conditioned medium. These results demonstrate a novel regulation of serpin specificity in which an ECM co-factor decreased the inhibition of certain proteases by the serpin without affecting the inhibition of its target protease.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of California, Irvine 92717.
authors.forename: F M
authors.initials: FM
authors.lastname: Donovan
authors.forename: P J
authors.initials: PJ
authors.lastname: Vaughan
authors.forename: D D
authors.initials: DD
authors.lastname: Cunningham
mesh_terms: D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D058255:Protease Nexins; D011485:Protein Binding; D011956:Receptors, Cell Surface; D058258:Serpin E2; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D016326:Extracellular Matrix Proteins; D046911:Macromolecular Substances; D058255:Protease Nexins; D011956:Receptors, Cell Surface; C545486:SERPINE2 protein, human; D058258:Serpin E2; D003094:Collagen; D013917:Thrombin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator