Unesterified long chain fatty acids inhibit the binding of single chain urokinase to the urokinase receptor.

The interaction of single chain urokinase with its receptor accelerates plasminogen activator activity on cell surfaces and induces intracellular signalling in several cell types. To date, no physiologic inhibitor of this binding has been identified. We report that the binding of scuPA to its cellular receptor is inhibited by long chain fatty acids such as oleic acid (C18, delta 9) at physiological plasma concentrations. Inhibition of single chain urokinase binding to human trophoblastic cells by long chain fatty acids was dose-dependent and saturable. Fifty percent of the binding was inhibited at an oleic acid concentration of 27 microM, while inhibition was maximal (75%) at 150 microM oleic acid. The inhibitory potency of oleic acid was unaffected by fatty acid free albumin or human plasma. Inhibition of single chain urokinase binding by free fatty acid analogues was critically dependent on chain length (> C14 required for inhibition) and was proportional to the extent of unsaturation. Only the fraction of specific scuPA binding to trophoblasts that was dependent on uPAR was susceptible to inhibition by oleic acid, while binding of scuPA to vitronectin, thombospondin, and the alpha 2-macroglobulin receptor/low-density lipoprotein-related receptor was not. [3H]Oleic acid bound specifically to recombinant soluble uPAR in a 1:1 molar ratio in the presence or absence of plasma and totally blocked its specific binding to a cell line expressing glycosyl phosphatidylinositol-linked single chain urokinase. These results indicate that oleic acid and other unsaturated long chain free fatty acids may serve as physiologic regulators of proteolytic events and intracellular signalling that depend upon the interaction of urokinase with its receptor.

Inhibition of plasminogen activation by polymerized ampicillin.

The polymerized beta-lactam antibiotic ampicillin inhibits the proteolytic activity of human plasmin upon 125I-labeled fibrin clots. The inhibition is dose-dependent, with half-maximal inhibition occurring at 1.25 mM of the polymerized antibiotic. Polymerized ampicillin also inhibits binding of plasmin to fibrin, and 38% inhibition of binding occurs at 10 mM of the antibiotic. Furthermore, polymerized ampicillin inhibits the activation of plasminogen by either urokinase-like plasminogen activator (uPA) or tissue type-plasminogen activator (tPA). At 7.5 mM of polymerized ampicillin, the uPA-mediated plasminogen activation is suppressed by 94%, and half-maximal inhibition is obtained at 0.66 mM. The direct activity of uPA on the chromogenic substrate L-pyroglutamyl-glycyl-L-arginine p-nitroanilide hydrochloride (S-2444) is unaffected by polymerized ampicillin levels of up to 10 mM. The inhibitory effects of the polymerized antibiotic on the activation of plasminogen by both uPA and tPA is totally abolished in presence of fibrin. These interactions may serve as a novel model for ligands that enhance the clot-specificity of thrombolytic agents.