Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells.
The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.
Mimuro
J
J
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Loskutoff
D J
DJ
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010447:Peptide Hydrolases; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors*; D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D006023:Glycoproteins; D015849:Plasminogen Inactivators; D010447:Peptide Hydrolases; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator