Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), is found in plasma and platelets. PAI-1 circulates in complex with vitronectin (Vn), an interaction that stabilizes PAI-1 in its active conform. In this study, we examined the binding of platelet-derived Vn and PAI-1 to the surface of isolated platelets. Flow cytometry indicate that, like P-selectin, PAI-1, and Vn are found on the surface of thrombin-or calcium ionophore-activated platelets and platelet microparticles. The binding of PAI-1 to the activated platelet surface is Vn-dependent. Vn mediates the binding of PAI-1 to platelet surfaces through a high affinity (K d of 80 nM) binding interaction with the NH 2 terminus of vimentin, and this Vn-binding domain is expressed on the surface of activated platelets and platelet microparticles. Immunological and functional assays indicate that only ؊5% of the total PAI-1 in platelet releasates is functionally active, and it co-precipitates with Vn, and the vimentin-enriched cytoskeleton fraction of activated platelet debris. The remaining platelet PAI-1 is inactive, and does not associate with the cytoskeletal debris of activated platelets. Confocal microscopic analysis of platelet-rich plasma clots confirm the co-localization of PAI-1 with Vn and vimentin on the surface of activated platelets, and platelet microparticles. These findings suggest that platelet vimentin may regulate fibrinolysis in plasma and thrombi by binding platelet-derived Vn⅐PAI-1 complexes.

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...

Study objectives were to determine, in children with systemic lupus erythematosus (SLE), (1) the association of antiphosholipid antibody (APLA) subtypes with thrombotic events (TEs) and (2) the predictive value of persistent versus transient antibodies for TEs. This is a cohort study of 58 SLE children in whom lupus anticoagulants (LAs), anticardiolipin antibodies (ACLAs), anti-␤ 2 -glycoprotein-I (anti-␤ 2 -GPI), and antiprothrombin (anti-PT) were assessed on at least 2 occasions (more than 3 months apart). Antibodies were classified as persistent