The present paper described interactions of urinary-type plasminogen activator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA-saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in kcat/Km). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds predominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (Kd,app. of 97 nM). Limited proteolysis of vitronectin followed by ligand-blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49,000), and binding was shown to occur through the N-terminal fragment of u-PA. N-terminal sequence analysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the heparin-binding region of the vitronectin molecule. Plasminogen activator inhibitor type I did not compete with u-PA for binding to vitronectin, suggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasminogen, suggesting these molecules share a common binding site on vitronectin. In addition to in vitro studies, experiments were performed to assess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA-saturated U937 cells to vitronectin was inhibited 66% by excess vitronectin, suggesting that direct binding of u-PA to vitronectin is the mechanism by which u-PA-dependent adhesion of U937 cells to vitronectin is mediated.
There are two major isoenzymes of plasminogen (Pg) in human plasma, designated Pg1 and Pg2. Both Pg forms have an identical primary structure, but differ in their extent of glycosylation. Removal of the oligosaccharide chains alters the normal physiological function of the zymogen and decreases the circulation time of both Pg glycoforms. Recent studies in our laboratory demonstrated that Pg2, with one carbohydrate chain, binds to the surface of U937 monocytoid cells considerably better than Pg1, with two carbohydrate chains, indicating a major role for the carbohydrate chains as determinants for differential binding to the cell surface [Gonzalez-Gronow, Grenett, Fuller & Pizzo (1990) Biochim. Biophys. Acta 1039, 269-276]. In this report we provide evidence that removal of terminal sialic acid from the Thr345-linked oligosaccharide chain of Pg2 is accompanied by the appearance of spontaneous amidolytic and fibrinolytic activity in the single-chain zymogen. Kinetic data demonstrate that asialo-Pg hydrolyses peptide substrates approximately 10% as efficiently as Pm. In addition, the change in carbohydrate content also alters Pg binding to U937 cells. Asialo-Pg binds to U937 cells with a decreased capacity but with a greater affinity than native Pg. Furthermore, asialo-Pg does not compete with native Pg for cell binding. These studies directly demonstrate that the oligosaccharide chains contribute to the heterogeneity observed in the physicochemical and biological properties of Pg1 and Pg2.
Plasminogen, the zymogen form of the serine proteinase plasmin, has been implicated in numerous physiological and pathological processes involving extracellular-matrix remodelling. We have previously demonstrated that the activation of plasminogen catalysed by tissue plasminogen activator is dramatically stimulated in the presence of basement-membrane-specific type IV collagen [Stack, Gonzalez-Gronow & Pizzo (1990) Biochemistry 29, 4966-4970]. The present paper describes the binding of plasminogen to type IV collagen. Plasminogen binds to both the alpha 1(IV) and alpha 2(IV) chains of basement-membrane collagen, with binding to the alpha 2(IV) chain preferentially inhibited by 6-aminohexanoic acid. This binding is specific and saturable, with Kd,app. values of 11.5 and 12.7 nM for collagen and gelatin respectively. Although collagen also binds to immobilized plasminogen, this interaction is unaffected by 6-aminohexanoic acid. Limited elastase proteolysis of plasminogen generated distinct collagen-binding fragments, which were identified as the kringle 1-3 and kringle 4 domains. No binding of collagen to mini-plasminogen was observed. These studies demonstrate a specific interaction between plasminogen and type IV collagen and provide further evidence for regulation of plasminogen activation by protein components of the extracellular matrix.