Identification of functionally important residues in TFPI Kunitz domain 3 required for the enhancement of its activity by protein S.

Protein S is a cofactor for tissue factor pathway inhibitor (TFPI) that critically reduces the inhibition constant for FXa to below the plasma concentration of TFPI. TFPI Kunitz domain 3 is required for this enhancement to occur. To delineate the molecular mechanism underlying enhancement of TFPI function, in the present study, we produced a panel of Kunitz domain 3 variants of TFPI encompassing all 12 surface-exposed charged residues. Thrombin-generation assays in TFPI-depleted plasma identified a novel variant, TFPI E226Q, which exhibited minimal enhancement by protein S. This was confirmed in purified FXa inhibition assays in which no protein S enhancement of TFPI E226Q was detected. Surface plasmon resonance demonstrated concentration-dependent binding of protein S to wild-type TFPI, but almost no binding to TFPI E226Q. We conclude that the TFPI Kunitz domain 3 residue Glu226 is essential for TFPI enhancement by protein S.

Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation. It is synthesized by various cells and its expression level increases in inflammatory environments. Mast cells and neutrophils accumulate at sites of inflammation and vascular disease where they release proteinases as well as chemical mediators of these conditions. In this study, the interactions between TFPI and serine proteinases secreted from human mast cells and neutrophils were examined. TFPI inactivated human lung tryptase, and its inhibitory activity was stronger than that of antithrombin. In contrast, mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate molar ratio of 1:500, resulting in markedly decreased TFPI anticoagulant and anti-(factor Xa) activities. N-terminal amino-acid sequencing and MS analyses of the proteolytic fragments revealed that chymase preferentially cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and Tyr268-Glu269, in that order, resulting in the separation of the three individual Kunitz domains. Neutrophil-derived proteinase 3 also cleaved TFPI, but the reaction was much slower than the chymase reaction. In contrast, alpha-chymotrypsin, which shows similar substrate specificities to those of chymase, resulted in a markedly lower level of TFPI degradation. These data indicate that TFPI is a novel and highly susceptible substrate of chymase. We propose that chymase-mediated proteolysis of TFPI may induce a thrombosis-prone state at inflammatory sites.

Oxidized phospholipids in oxidized low-density lipoprotein reduce the activity of tissue factor pathway inhibitor through association with its carboxy-terminal region.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits the initial reactions of blood coagulation. In this study, we explored the nature of active components that reduce the anticoagulant activity of TFPI in oxidized low-density lipoprotein (ox-LDL). The organic solvent-soluble fraction obtained from ox-LDL was fractionated by normal-phase HPLC. The binding profile of each fraction to TFPI showed a single peak eluting near purified oxidized phospholipid. To explore further the components in oxidized phospholipid that inhibit TFPI activity, we used oxidized phospholipids that mimic the biological activity of ox-LDL. The oxidation products of 1- and/or 2-oleoyl phosphatidylcholine or phosphatidylethanolamine were the most potent inhibitors of TFPI activity, whereas those of arachidonyl phosphatidylcholine possessed only a weak inhibitory effect on the TFPI activity. These oxidized phospholipids mainly associated with the C-terminal basic region of the TFPI molecule. The results indicate that oxidation products of delta-9 unsaturated phospholipids are candidate active components of ox-LDL that impair the function of TFPI through specific association with its C-terminal basic region.

Tissue factor expression in atrial endothelia associated with nonvalvular atrial fibrillation: possible involvement in intracardiac thrombogenesis.

INTRODUCTION: Tissue factor plays a key role in the extrinsic coagulation pathway and is induced by inflammatory cytokines. Atrial myocarditis has been detected recently in some patients with lone atrial fibrillation. Virchow's triad of low blood flow, hypercoagulability, and endothelial dysfunction, enhances thrombus formation. The present study was designed to elucidate the role of endothelial dysfunction in thrombogenesis associated with nonvalvular atrial fibrillation. MATERIAL AND METHODS: We investigated tissue factor expression in the endothelia of left atrial appendages obtained from seven patients with nonvalvular atrial fibrillation and cardiogenic thromboembolism. Tissues were divided into 7-13 sections and compared with control specimens from four patients who died of noncardiac events. Expression of tissue factor, von Willebrand factor and tissue factor pathway inhibitor was evaluated by immunohistochemistry. RESULTS: Histopathologically, inflammatory cells infiltrated the endocardium and all seven patients showed features of persistent myocarditis. Activated T cells [15.3+/-9.4 cells/high power field (HPF, mean+/-S.D.) vs. control 2.2+/-4.4/HPF (P=0.0294)] and a few macrophages [5.1+/-8.4 cells/HPF vs. control 2.4+/-3.5 cells/HPF (P=NS)] infiltrated the endocardium. Tissue factor was overexpressed in the endothelia particularly in tissues containing inflammatory cells and denuded matrix of the endocardium, compared with the control group. Von Willebrand factor, but not tissue factor pathway inhibitor, was also overexpressed in these tissues. CONCLUSION: Tissue factor expression induced by local inflammation is involved in the pathogenesis of thrombosis in patients with nonvalvular atrial fibrillation.

Tissue factor pathway inhibitor induces expression of JUNB and GADD45B mRNAs.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that regulates tissue factor-triggered blood coagulation. It has previously been reported that TFPI inhibits the proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that TFPI may act as more than just a mediator of coagulation through changes in gene expression. By using DNA-array techniques and Northern blot analysis, we here revealed that TFPI transiently induced the mRNA expression of JUNB and GADD45B. The inducible effects were not observed in TFPIdeltaC (lacking the C-terminal basic region) or antithrombin (heparin-binding anticoagulant protease inhibitor). Moreover, the TFPI-induced expression of GADD45B was blocked by receptor-associated protein, which masks the ligand-binding domain of very low density lipoprotein receptor (VLDL-R). In conclusion, this is the first report to show an effect of TFPI on mRNA expression, and suggests that TFPI modulates cellular functions by inducing JUNB and GADD45B expression through binding to VLDL-R.

Anti-inflammatory effects of long-lasting locally-delivered human recombinant tissue factor pathway inhibitor after balloon angioplasty.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) has anti-proliferative and anti-migratory effects on cultured smooth muscle cells (SMC) in addition to its anti-thrombotic activity. Here, we assess how long locally delivered recombinant TFPI (rTFPI) remains detectable at the delivery sites and clarify the main mechanism by which rTFPI blocks neointimal growth in vivo. METHODS: The iliac arteries of 85 Japanese white rabbits were injured using a Cutting Balloon. First, to establish the efficacy of local delivery of rTFPI, 5 groups of 3 rabbits each were examined immediately or 1, 2, 4 or 7 days after delivery. They were treated locally with a total amount of 200 microg of rTFPI given at 40 microg per pulse per minute by means of a Pulse Spray catheter. Thereafter, 34 rabbits which had received 200 microg of rTFPI after cutting angioplasty were compared to the same number of controls regarding thrombosis, inhibition of neointimal proliferation and inflammation. RESULTS: A total of 2.6+/-1.6 ng rTFPI persisted on the injured vessel 4 days after delivery. rTFPI was still present on 48 % of the cut sites 7 days after delivery, despite its short half-life in plasma. Thrombosis in the rTFPI-treated group was significantly reduced compared to the controls. The number of macrophages present within the media and intima was significantly decreased at day 7 after delivery of rTFPI. Furthermore, the number of Ki-67-positive cells 14 days after rTFPI delivery was significantly lower than in controls although there were no significant differences between them after 2 days. CONCLUSIONS: Local delivery of rTFPI decreased the degree of neointimal formation 4 weeks after TFPI delivery because of anti-inflammatory and anti-proliferative effects in addition to, or rather than, via anti-thrombosis.

Oxidized low-density lipoprotein associates strongly with carboxy-terminal domain of tissue factor pathway inhibitor and reduces the catalytic activity of the protein.

Tissue factor pathway inhibitor (TFPI) is a physiological protease inhibitor of the extrinsic blood coagulation pathway. Previously we have shown that TFPI associates quite rapidly with oxidized low-density lipoprotein (ox-LDL), with a reduction of the inhibitory activity on factor X activation. In the present study, it was found, by means of agarose gel electrophoresis, that the pre-incubation of full-length rTFPI with heparin or the carboxy (C)-terminal part (peptide 240-265) of TFPI prevented the association with ox-LDL in a dose-dependent manner. When rTFPI lacking the C-terminal basic part of the molecule (rTFPI-C) was mixed with ox-LDL, only a small amount of rTFPI-C was shifted to the position of ox-LDL on electrophoresis. Further, ox-LDL did not reduce the activity of rTFPI-C. These results indicate that the C-terminal domain of TFPI molecule plays a predominant role in the binding to ox-LDL and the binding through the C-terminal part is essential for the ox-LDL-dependent reduction of the anticoagulant activity of TFPI.