Abstract
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) approximately 3.5 micrometer) 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-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.
Highlights
To determine whether fibrin-associated vitronectin regulates PAI-1 distribution in clots, plasma clots were processed for dual-labeling immunofluorescence and confocal scanning laser microscopic image analysis to examine the distribution of plasma vitronectin and PAI-1 on fibrin fibrils (Fig. 1)
The studies presented in this report provide the first evidence that vitronectin plays a critical role in the regulation of PAI-1 binding to fibrin
The importance of vitronectin as the mediator of fibrinassociated PAI-1 activity is underscored by the finding that PAI-1-mediated inhibition of clot lysis is vitronectin-dependent in both purified fibrin and plasma clots
Summary
Previous studies have determined that the addition of 0.5 nM vitronectin to 375 nM fibrinogen prior to clotting resulted in the highest ratio of fibrin-bound versus free vitronectin.3 To block nonspecific binding, 100-l aliquots of PBS containing 3% bovine serum albumin and 0.05% Tween-20 (blocking buffer) were added to wells for 2 h at 37 °C. Lysis—To examine the influence of fibrin-associated vitronectin on PAI-1 activity, we monitored the t-PA-mediated lysis of 125I-fibrinogen-labeled plasma clots (Fig. 2).
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