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Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of 125 I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K d ϳ 3.5 M) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1⅐vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.Tissue-type plasminogen activator (t-PA) 1 initiates intravascular fibrinolysis by binding to fibrin, where it activates fibrinbound plasminogen (1-4). The major inhibitor of t-PA, type 1 plasminogen activator inhibitor (PAI-1), circulates in plasma and is released from platelet ␣-granules during blood clotting (5, 6). PAI-1 accumulates in thrombi, rendering them resistant to t-PA-mediated fibrinolysis (7-14). In purified systems, PAI-1 has been shown to bind directly to fibrin, with a K d of 3.7 M (15-18). Consequently, it has been hypothesized that PAI-1 accumulation in thrombi reflects a direct interaction of PAI-1 with fibrin. PAI-1 circulates in plasma (19,20) and platelets in complex with vitronectin (21, 22, 23, 24), a glycoprotein that binds PAI-1 with high affinity (25). The vitronectin interaction with PAI-1 stabilizes the inhibitor in its active conformation (26, 27), induces allosteric changes in vitronectin that expose cryptic epitopes (28, 29), and modulates vitronectin-dependent cell adhesion (25,30,31). Domain mapping studies using proteolysis, synthetic peptides, monoclonal antibodies, and site-directed mutagenesis have identified two discrete sites on vitronectin that may bind and stabilize 33,34). Similar approaches have delineated a single vitronectin-binding site on PAI-1 (35, 36).Recent studies from our laboratories have further characterized the PAI-1-vitronectin interaction. Analytical ultracentrifugation experiments indicate that PAI-1 and native vi...

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We have previously shown that oxidized low-density lipoproteins (oxLDLs) act synergistically with β-glycerophosphate to induce the osteogenic differentiation of primary bovine aortic smooth muscle cells (BASMCs). In the present study, we attempt to resolve the mechanism responsible for this effect by examining the expression of several osteoblast-specific transcription factors. Thus, by culturing BASMCs in the absence or presence of β-glycerophosphate and/or oxLDL, we demonstrate that β-glycerophosphate induces both Runx2 and Osterix (Osx) expression. In contrast, oxLDL has no effect on Runx2 expression but rather it enhances β-glycerophosphate-induced osteoblast differentiation by further up-regulating Osx expression. In an attempt to elucidate the mechanism responsible for this latter effect, we examined the ability of oxLDL to affect Msh homeobox 2 (Msx2) expression. Similar to its effect on Osx expression, oxLDL was found to synergistically enhance β-glycerophosphate-induced Msx2 expression in an extracellular signal-regulated kinase 1 and 2 (Erk 1 and 2)-dependent manner. Furthermore, oxLDL's ability to enhance both β-glycerophosphate-induced Osx expression and alkaline phosphatase activity was prevented when the BASMCs were first transfected with Msx2-specific siRNA. Taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteoblast differentiation and vascular calcification in vivo.