The tissue factor (TF) pathway of coagulation plays a primary role in hemostasis but the aberrant activation of TF-mediated coagulation leads to thrombus formation, the precipitating event in acute myocardial infarction, unstable angina and ischemic stroke. Tissue factor-mediated coagulation also contributes to the pathogenesis of acute lung injury by generating extravascular fibrin deposition in lung parenchyma. Pleural fibrin deposition is a common complication of pleural inflammation and occurs in a wide variety of pleural diseases. Thus, a proper regulation of TF activity is critical for maintenance of hemostatic balance. Tissue factor pathway inhibitor (TFPI) is the primary inhibitor for TF-mediated coagulation whereas antithrombin (AT) may function as an auxiliary second physiologic regulator. In pleural injury, concentrations of plasminogen activator inhibitor (PAI)-1, the predominant inhibitor of tissue-type and urokinase-type plasminogen activators, were increased about 1000-fold in exudative pleural fluids. In addition to inhibiting uPA and tPA, PAI-1 was also shown to inhibit thrombin and activated protein C. In the present study we investigated whether PAI-1 inhibits FVIIa activity. Free FVIIa or FVIIa complexed with relipidated TF (10 nM) was incubated with PAI-1 (1 μM) ± heparin (10 U/ml) or vitronectin (1 μM) for varying time periods and the extent of FVIIa inactivation was measured in a clotting assay. PAI-1 or PAI-1 + heparin exhibited no significant inhibitory effect on free factor VIIa coagulant activity. PAI-1, combined with equimolar concentration of vitronectin, inhibited free FVIIa activity, albeit very slowly (~15% inhibition in 2 h). However if FVIIa was complexed with relipidated TF, it was inhibited much more rapidly by PAI-1. The time required for 50% inactivation of FVIIa/TF by PAI-1 was about 85 min. Heparin increased the rate of PAI-1 inactivation of FVIIa/TF by about 2-fold whereas vitronectin enhanced PAI-1 inactivation of FVIIa/TF by 4 to 5-fold, inhibiting 50% of the FVIIa coagulant activity in less than 20 min. Heparin or vitronectin alone had no inhibitory effect on FVIIa/TF. To compare the efficiency of PAI-1 and PAI-1/vitronectin with AT/heparin to inhibit FVIIa/TF activity, we determined the loss of FVIIa coagulant activity in the presence of varying concentrations of PAI-1 ± vitronectin (1 μM) or AT ± heparin (10 U/ml). The IC50 values as follow; AT, > 5 μM; PAI-1, 817 nM; AT/heparin, 25 nM; PAI-1/vitronectin, 125 nM. A 1:1 stochiometric complex between PAI-1 and FVIIa, with an apparent molecular weight of 100,000 was detected by SDS-PAGE. In additional studies, we investigated the ability of PAI-1 to inactivate FVIIa bound to TF on lung fibroblasts. PAI-1 inhibited FVIIa bound to cell surface TF, but to a lesser extent than FVIIa bound to relipidated TF. However, in the absence of heparin, PAI-1 and not AT is capable of inactivating FVIIa bound to TF. Interestingly, in contrast to the data observed with FVIIa bound to relipidated TF, heparin or vitronectin failed to accelerate PAI-1 inactivation of FVIIa bound to cell surface TF. At present, it is unclear why heparin and vitronectin failed to enhance the PAI-1 inhibitory effect on FVIIa bound to cell surface TF. Overall the data presented here in show that PAI-1 is capable of modulating FVIIa/TF activity. Further studies are needed evaluate whether PAI-1 inhibition of FVIIa/TF plays an important role in pathophysiology, particularly in lung inflammation.

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