Plasminogen Activator Inhibitor Type 1 Promotes the Self-association of Vitronectin into Complexes Exhibiting Altered Incorporation into the Extracellular Matrix

Abstract

Serine proteinase inhibitors, including plasminogen activator inhibitor type 1 (PAI-1) and antithrombin, are key regulators of hemostatic processes such as thrombosis and wound healing. Much evidence suggests that PAI-1 can influence such processes, as well as pathological events like tumor metastasis, through its ability to directly regulate binding of blood platelets and cells to extracellular substrata. One way that PAI-1 influences these processes may be mediated through its binding to the plasma protein vitronectin. Binding to PAI-1 results in the incorporation of vitronectin into a higher order complex with a potential for multivalent interactions (Podor, T. J., Shaughnessy, S. G., Blackburn, M. N., and Peterson, C. B. (2000) J. Biol. Chem. 275, 25402-25410). In this study, evidence is provided to support this concept from studies on the effects of PAI-1-induced multimerization on the interactions of vitronectin with matrix components and cell surface receptors. By monitoring complex formation and stability over time using sizeexclusion high performance liquid chromatography, a correlation is made between PAI-1-induced multimerization and enhanced cell/matrix binding properties of vitronectin. This evidence indicates that PAI-1 alters the adhesive functions of vitronectin by converting the protein via the higher order complex to a self-associated, multivalent species that is functionally distinct from the abundant monomeric form found in the circulation.The interactions that occur between cellular receptors and proteins that constitute the extracellular matrix are vital to physiological control of processes like cell adhesion and pericellular proteolysis. Events that alter these interactions can be deleterious, leading to pathological sequelae such as improper wound healing and tumor cell migration or metastasis. For example, components of the humoral response system known as fibrinolysis, which play a role in modulating various cellbinding properties of the extracellular matrix, can be exploited by cancerous cells. By altering the content and activity of proteins related to fibrinolysis, abnormally developing cells can acquire the ability to exit residing tissues and eventually invade and metastasize. An example involves the main regulator of fibrinolysis, PAI-1, 1 in which increased levels of the protein in plasma correlate with the presence of malignant ovarian cancer and higher incidence of disease (1).PAI-1 is a member of the serpin family and represents a key regulatory protein in proteolytic processes responsible for tissue remodeling and tumor metastasis. This property is owed to the fact that PAI-1 is the main inhibitor of both plasminogen activators, uPA and tPA. Interesting features of PAI-1 include its structural lability and the propensity it exhibits to spontaneously adopt a more stable, but inactive conformation. This feature of PAI-1 is unique among the serpin family, with active PAI-1 exhibiting a half-life of ϳ90 min (2). In the body, however, this transition of PAI-1 into an inactive for...

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

“…Integrin Binding Assay-Microtiter plates were coated with 100 l of a 2.5 g/ml solution of the integrin GPIIbIIIa in binding buffer (50 mM Tris, pH 7.4, 0.1 M NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 ) according to the protocol described in a previous study (43). Wells were blocked with 3.5% BSA in binding buffer for 1 h at room temperature.…”

The Solution Structure of the N-terminal Domain of Human Vitronectin

“…However, the binding sites are clearly separate and will not necessarily be mutually exclusive in their binding functions. In this regard, we previously have observed that PAI-1 binds to vitronectin and promotes the assembly of higher-order complexes that become preferentially associated with the ECM, are internalized and degraded together in cell culture, and actually bind integrins more avidly (11). The relative disorder within this C-terminal region of the SMB domain housing the RGD sequence, combined with a closely spaced, but separate binding site for PAI-1, suggests that there may be scenarios that can accommodate simultaneous binding of PAI-1 and integrins.…”

“…Vitronectin binds to PAI-1 with high affinity and stabilizes the inhibitor in its active conformation (8,9). Vitronectin can also associate with PAI-1 and assemble to form higher order complexes (10) that exhibit altered adhesive functions (11). Key to the adhesive functions of vitronectin are its interactions with cell-surface receptors including integrins and uPAR.…”

Discovery and Characterization of Potential Prognostic Biomarkers for Dengue Hemorrhagic Fever

“…First, the oligomer binds to heparin, a known receptor for DENV. 37 Second, it regulates coagulation by forming a bridge between integrin and fibrin to induce platelet aggregation to inhibit plasma leakage. 38,39 Western blot analysis did not identify any single isoform as having a greater association with dengue severity compared with the overall Vtn concentration measured by ELISA.…”