The Majority of Type 1 Plasminogen Activator Inhibitor Associated with Cultured Human Endothelial Cells Is Located under the Cells and Is Accessible to Solution-phase Tissue-type Plasminogen Activator

The interactions between exogenously added tissue-type plasminogen activator (t-PA) and the active form of type 1 plasminogen activator inhibitor (PAl-l) produced by and present in cultured human umbilical vein endothelial cells (HUVECs) were investigated. Immunoblotting analysis of the conditioned media obtained from monolayers of HUVECs treated with increasing concentrations of t-PA (~<10/zg/ml) revealed a dose-dependent formation of both t-PA/PAI-1 complexes, and of a 42,000-M, cleaved or modified form of the inhibitor. Immunoradiometric assays indicated that t-PA treatment resulted in a fourfold increase in PAI-1 antigen present in the conditioned media. This increase did not result from the release of PAI-1 from intracellular stores, but rather reflected a t-PAdependent decrease in the PAI-1 content of the Triton X-100 insoluble extracellular matrix (ECM). Although the rate of t-PA-mediated release of PAI-1 was increased by the removal of the monolayer, similar quantities of PAI-I were removed in the presence or absence of the cells. These results suggest that the cells only represent a semipermeable barrier between ECMassociated PAI-1 and exogenous t-PA. Treatment of HUVECs with t-PA (1 /xg/ml, 2 h) to deplete the ECM of PAI-1 did not affect the subsequent rate of PAI-1 production and deposition into the ECM. Immunogold electron microscopy of HUVECs not only confirmed the location of PAI-1 primarily in the region between the culture substratum and the ventral cell surface but failed to demonstrate significant (<1%) PAI-1 on the cell surface. Thus, the majority of PAId associated with cultured HUVEC monolayers is present under the cells in the ECM and is accessible to solution-phase t-PA. p LASMINOGEN activators (PAs) ~ play an important role not only during fibrinolysis but also in a variety of other physiological processes, including tissue repair, macrophage function, ovulation, embryo implantation, neovascularization, and malignant transformation (Astrup, 1975; Reich, 1978; Bachmann and Kruithof, 1984). Precise regulation of PA activity thus constitutes a critical feature of many biological systems. Such control may be at the level of the formation and resolution of fibrin itself (Hoylaerts et al., 1982), at the level of the interactions of PAs with cells (Vassalli et al., 1985), or through the action of specific PA inhibitors (PAIs; Erickson et al., 1985; Sprengers and Kluft, 1987; Loskutoff et al., 1988). Although four molecules with PAI activity have been detected, the endothelial cell-derived inhibitor (PAI-1) (Sprengers and Kluft, 1987; Loskutoff et al., 1988) appears to be 1. Abbreviations used in this paper: CM, conditioned medium; ECM, extracellular matrix; IRMA, immunoradiometric assay; HUVECs, human umbilical vein endothelial cells; PA, plasminogen activator; PAl, plasminogen activator inhibitor; PAId, type 1 plasminogen activator inhibitor; t-PA, tissue-type plasminogen activator. the physiologic inhibitor of tissue-type plasminogen inhibitor (t-PA). PAI-I is a member of the serine proteinase inhibitor (Serpin) superfamily (Carrel and Travis, 1985; Ny et al., 1986, Pannekoek et al., 1986) and differs from other PAls in its stability to SDS (van Mourik et al., 1984; Hekman and Loskutoff, 1985), its electrophoretic mobility (Erickson et al., 1986), and its ability to inhibit single-chain t-PA (Colucci et al., 1986; Hekman and Loskutoff, 1988). In addition, PAI-1 is synthesized as an active molecule but is unstable in solution and rapidly decays into a latent, inactive form at 37°C (Levin, 1986; Kooistra et al., 1986). Active PAI-1 appears to be stabilized through its binding to other proteins (Loskutoff et al., 1988). For example, PAI-1 has been detected in the extracellular matrix (ECM) of a variety of cultured cells (Laiho et al., 1986; Levin and Santell, 1987; Knudsen et al., 1987; Mimuro et al., 1987; Rheinwald et al., 1987; Pollanen et al., 1987; Knudsen and Nachman, 1988) and shown to be active (Levin and Santell, 1987; Mimuro et al., 1987, Knudsen and Nachman, 1988). The binding of PAI-1 to the ECM protects it from the spontaneous inactivation that occurs in solution, increasing its apparent half-life © The Rockefeller University Press, 0021-9525/89/01/15519 $2.00 The Journal of Cell Biology, Volume 110, January 1990 155-163 155 on Jne 6, 2017 D ow nladed fom Published January 1, 1990