Blood coagulation is initiated following disruption of intact endothelial surfaces that exposes TF to FVIIa in flowing blood. The FVIIa-TF complex then activates FIX and FX to initiate the TF-dependent pathway of blood coagulation. TF-dependent blood coagulation plays a primary role in hemostasis after tissue injury. However, aberrant activation of TF-dependent coagulation contributes to the pathogenesis of diseases characterized by thrombotic events or extravascular fibrin deposition, including acute myocardial infarction and diverse forms of acute lung injury [20
]. Therefore, the proper regulation of TF expression is critical not only for maintenance of hemostatic balance but for preservation of normal organ function and general health. TFPI is the major regulator of FVIIa-TF-induced coagulation [5
]. Although AT also has been shown to inhibit TF-FVIIa activity, the regulation of pathophysiologic TF-dependent coagulation remains unclear at this time [31
]. The data presented herein show that PAI-1, an essential inhibitor of the fibrinolytic system, also inhibits the activity of FVIIa-TF, the key initiator of coagulation.
In addition to inhibiting tPA and uPA, PAI-1 also inhibits other serine proteases involved in blood coagulation and fibrinolysis, such as thrombin, APC and plasmin, albeit with low efficiency [32
]. In the presence of cofactors, either vitronectin and/or heparin, PAI-1 becomes an efficient inhibitor of thrombin [36
] and APC [10
]. However, these cofactors do not convert PAI-1 to a general serine protease inhibitor as no inhibition of FXa by PAI-1 was detected irrespective of the presence or absence of these cofactors [39
]. Thus, the present observation that PAI-1 inhibits FVIIa and that vitronectin enhances this inhibition reflects specific inhibition of FVIIa by PAI-1 and not a generic inhibition of serine proteases by serpins. As observed earlier with AT inhibition of FVIIa [27
], PAI-1 inhibits FVIIa complexed with TF but not free FVIIa. Earlier studies have suggested that TF induces allosteric modifications in FVIIa and stabilizes the active conformation of FVIIa [41
]. Such stabilization may be necessary for PAI-1 to inactivate FVIIa. Earlier studies from our laboratory showed that AT-mediated inhibition of FVIIa-TF was strictly dependent on the presence of heparin [27
]. In contrast to AT, PAI-1 inhibition of FVIIa-TF does not require the presence of heparin. The inhibition of FVIIa complexed with relipidated TF by PAI-1 is improved slightly by the presence of heparin.
The simplest mechanism-based inhibition of proteinase (E) by serpin (I) () includes reversible formation of the Michaelis-like complex (EI) and final inhibitory complex (E-I). While both EI and E-I possess no enzymatic activity only acylated enzyme E-I is stable under conditions of SDS PAGE. Although vitronectin accelerated formation of FVIIa/PAI-1 complex in the presence of either soluble or relipidated TF, the corresponding rate of accumulation of E-I () was always higher with relipidated TF. At present the precise reason for the substantial difference between PAI-1 inhibition of FVIIa bound to soluble TF and relipidated TF is unknown, but a more favorable conformational change in FVIIa bound to relipidated TF could be a possible reason. Vitronectin significantly enhanced kinh
for the reaction of FVIIa bound to relipidated TF with PAI-1 () most likely via increasing the affinity of PAI-1 to FVIIa complexed with relipidated TF (an increase in the rate of formation of EI (k1; )). It is unlikely that liposomes are directly contributes to the cofactor effect of vitronectin since the addition of liposomes to soluble TF did not alter the observed enhancement of the FVIIa inhibition by vitronectin (data not shown). Therefore, overall, our data suggest that in the presence of vitronectin, PAI-1 rapidly forms a catalytically inactive Michaelis-like complex EI with FVIIa bound to relipidated TF, which slowly progresses to the final inhibitory complex E-I (). Such an inhibitory mechanism could explain observed differences in PAI-1 ± vitronectin inhibition of FVIIa-TF in different assays. The loss of FVIIa activity in amidolytic assay () reflects formation of both EI and E-I (). In contrast, the proteolytic activity assays, where sample are diluted many fold, and SDS-PAGE analysis measure the terminal stable inhibitor complex and not the intermediate. Earlier studies established that the active form of PAI-1 is spontaneously converted to inactive latent form with a half-life 0.5-1 h under physiological conditions and that vitronectin binding increases half life of active PAI-1 (see rev[42
]). Vitronectin binding to PAI-1 was also shown to induce a conformational change in the reactive center of PAI-1, thus making it more accessible for the catalytic center of target protease [43
]. However, given that vitronectin primarily affects affinity of PAI-1 to FVIIa bound to relipidated TF, it is likely that exosite interactions between the proteinase and serpin (in complex with vitronectin) outside of the active site govern the observed effects of vitronectin.
Studies performed using a stimulated endothelial and fibroblasts cell model system confirmed that PAI-1 is capable of inhibiting FVIIa bound to TF at the cell surface. However, in contrast to the data obtained with FVIIa bound to relipidated or soluble TF, vitronectin either had a modest (2-fold increase) or no effect on PAI-1 inhibition of FVIIa bound to cell surface TF. While it is unclear why vitronectin has a negligible cofactor effect on PAI-1 inhibition of FVIIa bound to cell surface TF, our results clearly indicate that the effects of vitronectin on serpin mechanism strongly depend on the microenvironment.
The biological significance of PAI-1 inhibition of FVIIa-TF at present is unknown. TFPI is the most relevant inhibitor of FVIIa-TF in vivo
]. TFPI-FXa inhibits FVIIa-TF at about three orders of magnitude faster (0.6 to >1 × 109
] than PAI-1 inhibition of FVIIa-TF. Therefore, it is unlikely that PAI-1, which circulates in plasma at a similar or lower concentration (60 to 80 ng/ml) than TFPI (100 ng/ml), inhibits FVIIa-TF under normal physiological conditions. In general, the physiological relevance of interaction between serine proteases and serpins is determined by a number of criteria, including the (1) presence of the target protease, inhibitors and cofactors in the same compartment, (2) concentration of the reactants in such a compartment, and (3) rate of inhibition [47
]. Expression of both PAI-1 and TF are concurrently elevated in a number of diseases, including atherosclerosis and acute respiratory distress syndrome (ARDS) [14
]. TF, PAI-1, and vitronectin were also found to be localized in atherosclerotic human vessel wall [48
]. Leakage of circulating blood through damaged vessel walls or local synthesis of FVII  in atheroma may lead to formation of FVIIa-TF complexes in atherosclerotic vessel wall [48
]. Similarly, bronchoalveolar lavage and pleural fluids from ARDS and other patients are shown to contain PAI-1, TF and FVIIa [16
;51]. In some disease settings, PAI-1 concentrations are elevated by 100 to 2000-fold, exceeding the concentration of TFPI by 2 to 3-orders of magnitude [13
;52]. Under this scenario, despite PAI-1, compared to TFPI, being a poor inhibitor of FVIIa-TF, the relative efficiency of PAI-1 to TFPI for inhibition FVIIa-TF could reach approximately 0.1 to 1. Thus, the regulation of FVIIa-TF activity by PAI-1 could be a relevant mechanism in the context of atherogenesis, sepsis and diseases characterized by elevated expression of PAI-1. Here it may pertinent to note that PAI-1 may also regulate FVIIa-TF pathway indirectly as the end product of the fibrinolytic system, plasmin, can activate FVII .
In summary, the present study documents for the first time that PAI-1 is an inhibitor of FVIIa-TF. Although PAI-1 inhibits FVIIa-TF with low efficiency, PAI-1 inhibition of the FVIIa-TF pathway of coagulation may have pathophysiological significance in disease conditions such as ARDS or atherogenesis, which are characterized by aberrant fibrin formation and increased expression of both TF and PAI-1.