Tissue Factor Pathway Inhibitor Activity Research Articles

Toll-Like Receptor 9 Signaling Regulates Tissue Factor and Tissue Factor Pathway Inhibitor Expression in Human Endothelial Cells and Coagulation in Mice

Bacterial DNA (CpG DNA) persists in tissues and blood under pathological conditions that are associated with enhanced intravascular coagulation. Toll-like receptor 9 recognizes CpG DNA and elicits innate and adoptive immunity, yet the impact of CpG DNA on coagulation has not been studied. In this study, we investigated the effects of CpG DNA on the expression and activity of tissue factor, a key initiator of coagulation and tissue factor pathway inhibitor in human coronary artery endothelial cells and on coagulation in mice. Controlled in vitro and in vivo studies. University research laboratory. Cultured human coronary artery endothelial cell, wild-type mice, and TLR9-deficient mice. Human coronary artery endothelial cell was challenged with CpG DNA, and tissue factor and tissue factor pathway inhibitor expression and activity were assessed. In mice, the effects of CpG DNA on bleeding time and plasma levels of thrombin-antithrombin complexes and tissue factor were measured. We found that CpG DNA, but not eukaryotic DNA, evoked marked nuclear factor-κB-mediated increases in tissue factor expression at both messenger RNA and protein levels, as well as in tissue factor activity. Conversely, CpG DNA significantly reduced tissue factor pathway inhibitor transcription, secretion, and activity. Inhibition of Toll-like receptor 9 with a telomere-derived Toll-like receptor 9 inhibitory oligonucleotide or transient Toll-like receptor 9 knockdown with small interfering RNA attenuated human coronary artery endothelial cell responses to CpG DNA. In wild-type mice, CpG DNA shortened the bleeding time parallel with dramatic increases in plasma thrombin-antithrombin complex and tissue factor levels. Pretreatment with inhibitory oligonucleotide or anti-tissue factor antibody or genetic deletion of TLR9 prevented these changes, whereas depleting monocytes with clodronate resulted in a modest partial inhibition. Our findings demonstrate that bacterial DNA through Toll-like receptor 9 shifted the balance of tissue factor and tissue factor pathway inhibitor toward procoagulant phenotype in human coronary artery endothelial cells and activated blood coagulation in mice. Our study identifies Toll-like receptor 9 inhibitory oligonucleotides as potential therapeutic agents for the prevention of coagulation in pathologies where bacterial DNA may abundantly be present.

Reactive oxygen species induce a procoagulant state in endothelial cells by inhibiting tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a serine-protease inhibitor, which modulates coagulation tissue factor-dependent (TF). It binds directly and inhibits the TF-FVII/FVIIa complex as well as FXa. Time to reperfusion of acute ischemic myocardium is essential for tissue salvage. However, reperfusion also results in a unique form of myocardial damage, such as contractile dysfunction, decreased coronary flow and altered vascular reactivity. Oxidants and reactive oxygen species (ROS) formation is increased in the post-ischemic heart and is responsible of post-ischemic injury. It has been reported that ROS promote a procoagulant state via TF expression while no data are available on the effect on TFPI. Endothelial cells were incubated with two different ROS generating systems, xanthine (X)/xanthine oxidase (XO) for 5 min, or H2O2 (500 μM) for 24 h. TFPI activity was measured in supernatants by chromogenic assay and TFPI-mRNA analyzed by RT-PCR 2 h after ROS exposure. Unstimulated cells and cells exposed to either X or XO served as controls. Western blot and ligand dot blot was performed to evaluate ROS effect on TFPI structure and binding to FXa. ROS generated by X/XO as well as H2O2 system resulted in decreased TFPI activity compared to unstimulated cells while X or XO alone had no effect. No differences in TFPI mRNA levels versus controls was observed. A significant degradation of TFPI was induced by ROS exposure, resulting in a decreased ability to bind FXa. ROS induce a procoagulant state in endothelial cells by altering TFPI structure, resulting in inhibition of TFPI binding to Factor Xa and loss of activity. This phenomenon might have important consequences during reperfusion of post-ischemic myocardium.

Expression of human tissue factor pathway inhibitor on vascular smooth muscle cells inhibits secretion of macrophage migration inhibitory factor and attenuates atherosclerosis in ApoE-/- mice.

Tissue factor (TF) and coagulation proteases are involved in promoting atherosclerosis, but the molecular and cellular bases for their involvement are unknown. We generated a new strain (ApX4) of apolipoprotein E-deficient mice expressing a membrane-tethered human tissue factor pathway inhibitor fusion protein on smooth muscle actin-positive cells, including vascular smooth muscle cells (SMCs). ApX4 mice developed little atherosclerosis on either a normal chow or high-fat diet. Lipid levels were similar to those in parental ApoE(-/-) mice, and there was no detectable difference in systemic (circulating) tissue factor pathway inhibitor levels or activity. The small lipid-rich lesions that developed had markedly reduced leukocyte infiltrates, and in contrast to ApoE(-/-) mice, SMCs did not express macrophage migratory inhibitory factor (MIF), including at sites distant from atheromatous lesions. Low levels of circulating MIF in ApX4 mice normalized to levels seen in ApoE(-/-) mice after injection of an inhibitory anti-human tissue factor pathway inhibitor antibody, which also led to MIF expression by tissue factor-positive medial SMCs. MIF production by SMCs in ApoE(-/-) mice in vitro and in vivo was shown to be dependent on tissue factor and protease-activated receptor signaling, which were inhibited in ApX4 mice. Our data indicate that tissue factor plays a hitherto unreported role in the generation of MIF by SMCs in atherosclerosis-prone ApoE(-/-) mice, inhibition of which significantly prevents the development of atherosclerosis, through inhibition of leukocyte recruitment. These data significantly enhance our understanding of the pathophysiology of this important pathology and suggest new potential translational strategies to prevent atheroma formation.

Endogenous tissue factor pathway inhibitor has a limited effect on host defence in murine pneumococcal pneumonia.

Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Coagulation and inflammation interact in the host response to infection. Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein that inhibits tissue factor (TF), the main activator of inflammation-induced coagulation. It was the objective of this study to investigate the effect of endogenous TFPI levels on coagulation, inflammation and bacterial growth during S. pneumoniae pneumonia in mice. The effect of low endogenous TFPI levels was studied by administration of a neutralising anti-TFPI antibody to wild-type mice, and by using genetically modified mice expressing low levels of TFPI, due to a genetic deletion of the first Kunitz domain of TFPI (TFPIK1(-/-)) rescued with a human TFPI transgene. Pneumonia was induced by intranasal inoculation with S. pneumoniae and samples were obtained at 6, 24 and 48 hours after infection. Anti-TFPI reduced TFPI activity by ~50 %. Homozygous lowTFPI mice and heterozygous controls had ~10 % and ~50 % of normal TFPI activity, respectively. TFPI levels did not influence bacterial growth or dissemination. Whereas lung pathology was unaffected in all groups, mice with ~10 % (but not with ~50 %) of TFPI levels displayed elevated lung cytokine and chemokine concentrations 24 hours after infection. None of the groups with low TFPI levels showed an altered procoagulant response in lungs or plasma during pneumonia. These data argue against an important role for endogenous TFPI in the antibacterial, inflammatory and procoagulant response during pneumococcal pneumonia.

Thrombin Down-Regulates Tissue Factor Pathway Inhibitor Expression in a PI3K/Nuclear Factor-κB–Dependent Manner in Human Pleural Mesothelial Cells

Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the extrinsic coagulation cascade, and its expression is reported to be relatively stable. Various pathophysiologic agents have been shown to influence TFPI activity by regulating its expression or by modifying the protein. It is not clear how TFPI activity is regulated in normal physiology or in injury. Because thrombin and TFPI are locally elaborated in pleural injury, we sought to determine if thrombin could regulate TFPI in human pleural mesothelial cells (HPMCs). Thrombin significantly decreased TFPI mRNA and protein levels by > 70%. Thrombin-mediated down-regulation of TFPI promoted factor X activation by HPMCs. The ability of thrombin to significantly decrease TFPI mRNA and protein levels was maintained at nanomolar concentrations. Protease-activated receptor (PAR)-1, a mediator of thrombin signaling, is detectable in the mesothelium in human and murine pleural injury. PAR-1 silencing blocked thrombin-mediated decrements of TFPI in HPMCs. Thrombin activates PI3K/Akt and nuclear factor κB (NF-κB) signaling in HPMCs. Inhibition of PI3K (by PX-866) and NF-κB (by SN50) prevented thrombin-mediated TFPI mRNA and protein down-regulation. These are the first studies to demonstrate that thrombin decreases TFPI expression in HPMCs. Our findings demonstrate a novel mechanism by which thrombin regulates TFPI expression in HPMCs and promotes an unrestricted procoagulant response, and suggest that interactions between PI3K and NF-κB signaling pathways are linked in HPMCs and control TFPI expression. These findings raise the possibility that targeting this pathway could limit the ability of the mesothelium to support extravascular fibrin deposition and organization associated with pleural injury.

Tissue factor pathway inhibitor in activated prothrombin complex concentrates (aPCC) moderates the effectiveness of therapy in some severe hemophilia A patients with inhibitor

Some hemophilia A patients who have developed inhibitors are poorly responsive to activated prothrombin complex concentrates (aPCC) after daily dosage, but the mechanism(s) underlying this remain unknown. We examined two representative cases. In case 1, we found that changing to recombinant factor VIIa (rFVIIa) therapy was more effective, and the response to aPCC was restored within ~2 weeks. Tissue factor (TF)-triggered thrombin generation demonstrated a prolonged lag-time and decreased peak thrombin, and this impairment was focused on TF pathway inhibitor (TFPI). Plasma-free TFPI was elevated post-infusion of aPCC, while this was unaffected by rFVIIa. TFPI returned to normal range within 2-3 weeks. Plasmas obtained from patients with poor or good response to aPCC (aPCC-poor or aPCC-good), and good response to rFVIIa (FVIIa-good) demonstrated that free TFPI levels are increased in both aPCC groups, but not in FVIIa-good. TFPI levels pre- and post-infusion in aPCC-poor were significantly higher than those in aPCC-good. Addition of anti-TFPI antibody to the reaction samples demonstrated a greater increase of peak thrombin in aPCC-poor compared to aPCC-good, showing the higher TFPI activity in aPCC-poor. Free TFPI contained in aPCC corresponded to the increasing levels in plasma. In conclusion, TFPI in aPCC attenuated thrombin generation, and the reduced effectiveness of therapy in these circumstances appeared to be related to TFPI activity.

A Phase 2 Randomized, Double-Blind, Placebo-Controlled Trial Demonstrating Reversal Of Rivaroxaban-Induced Anticoagulation In Healthy Subjects By Andexanet Alfa (PRT064445), An Antidote For Fxa Inhibitors

BackgroundDirect factor Xa inhibitors have demonstrated compelling anticoagulant efficacy and/or safety profiles across multiple diverse patient populations. A specific antidote to reverse anticoagulation during episodes of serious uncontrolled bleeding or before urgent/emergent surgery is lacking. Andexanet alfa (proposed INN)(AnXa, PRT064445) is a modified, recombinant human fXa molecule that is catalytically inactive but retains high-affinity binding to direct fXa inhibitors. It thus acts as a decoy to reverse fXa inhibitor-mediated anticoagulation in preclinical and early clinical studies. MethodsThis ongoing Phase 2, double-blind, placebo-controlled study is examining the reversal by AnXa of the anticoagulant activity of rivaroxaban (riva), as well as the pharmacokinetics and safety in healthy subjects. Reversal of riva anticoagulation will be studied with up to 6 different dose cohorts/ regimens of AnXa or placebo in a 6:3 ratio (i.e., 9 subjects per cohort). Riva is administered at an oral dose of 20 mg qd for 6 days and AnXa administered intravenously on Day 6, 3 hours after the last riva dose – the approximate time of maximum riva concentration (mean ± SD: 0.64 ± 0.22 mM, n=18). Pharmacodynamic and safety data are collected through Day 48 with pharmacokinetic data through Day 10. ResultsWe report here available data from the first 2 AnXa dose cohorts (210 mg and 420 mg, n =18). Immediately after completion of the 210 mg and 420 mg doses, anti-fXa activity decreased dose-dependently by 20% and 53%, respectively, from the pre-AnXa level and returned to placebo levels by approximately 2 hours after treatment (Figure). In parallel, the plasma concentrations of unbound riva were decreased by 32% and 51%, respectively, relative to pre-AnXa values. In addition, riva-induced inhibition of thrombin generation and prolongation of both prothrombin time and activated clotting time were also rapidly partially reversed by AnXa in a dose-dependent manner. At 2 minutes after AnXa administration, the molar ratio of AnXa to total plasma riva was 0.8 for the 210 mg dose (1.2 µM/1.6 µM, respectively) and 1.2 for the 420 mg dose (2.6 µM/2.1 µM, respectively). AnXa infusion was not associated with increases in prothrombin fragments F1+2, thrombin-antithrombin, or D-dimer (all values were within normal ranges). As expected, tissue factor pathway inhibitor activity decreased due to its binding to AnXa. AnXa was well tolerated and there were no thrombotic events, serious, or severe adverse events. Adverse events occurring in 1 or more AnXa or placebo recipients included infusion-related reactions (n = 3, all mild in severity) and post-procedural hematoma, headache, or postural dizziness (n = 2 each). [Display omitted] Summary/ConclusionsResults from this ongoing clinical trial demonstrate that AnXa is able to dose-dependently partially reverse the anticoagulant effects of rivaroxaban, as assessed by pharmacodynamic markers, in healthy subjects. These data are consistent with previously reported results with apixaban in that AnXa sequesters rivaroxaban and apixaban in a similar stoichiometric manner. Additional data with higher doses of AnXa will also be presented. AnXa is well-tolerated and a potentially promising, universal antidote for fXa inhibitors. Disclosures:Mark:Portola Pharmaceuticals: Consultancy. Off Label Use: The use of PRT064445 as an antidote for reversal of anticoagulation from direct and indirect fXa inhibitors is investigational. Vandana:Portola Pharmaceuticals: Consultancy. Michael:Portola Pharmaceuticals: Employment, Equity Ownership. Genmin:Portola Pharmaceuticals: Employment, Equity Ownership. Conley:Portola Pharmaceuticals: Employment, Equity Ownership. Stanley:Portola Pharmaceuticals: Employment, Equity Ownership. Castillo:Portola Pharmaceuticals: Employment, Equity Ownership. Hutchaleelaha:Portola Pharmaceuticals: Consultancy. Karbarz:Portola Pharmaceuticals: Employment. Lin:Portola Pharmaceuticals: Employment. Barron:Portola Pharmaceuticals: Employment. Russell:Portola Pharmaceuticals: Employment. Levy:Portola Pharmaceuticals: Employment. Connolly:Portola Pharmaceuticals: Consultancy. Curnutte:Portola Pharmaceuticals: Employment, Equity Ownership.

Tissue Factor Pathway Inhibitor Modulates Fibrin Deposition In The Brain In a Manner That Is Independent Of Thrombomodulin and Activated Protein C

BackgroundFibrin deposition in cerebral vessels contributes to the pathogenesis of stroke and vascular dementia. Under quiescent conditions fibrin deposition is prevented or limited by natural anticoagulants of the endothelium. Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are the two major endothelial anticoagulants and act on different portions of the clotting cascade. TFPI inhibits the initiation of coagulation by inhibiting TF-FVIIa and FXa. TM slows the propagation of coagulation by promoting the activation of protein C (APC), which inactivates FVa and FVIIIa. Both proteins are reported to have low expression in the brain and their function in prevention of cerebral vascular fibrin deposition is unclear. This is emphasized by findings from murine models of decreased TFPI and TM (TMpro/pro) activity. TFPI null embryos have fibrin deposition in the brain and liver, while TMpro/pro mice have fibrin deposition in the lungs, heart, spleen and liver, but the brain is protected from fibrin deposition even following lethal LPS doses. Interestingly, TFPI heterozygosity combined with TMpro/pro results in cerebral vascular fibrin. Here, we report a detailed analysis of TFPI expression in the brain vasculature, the consequences of vascular TFPI deficiency in adult mice, and the capacity of APC to compensate for TFPI deficiency. Methods and ResultsReal time PCR studies demonstrated that TFPI expression in total brain is lower than in other tissues in comparison to the RPL-19 housekeeping gene. However, when the endothelial-specific genes, VE-cadherin or CD31 were used as housekeeping genes, brain TFPI expression was higher than that of lung endothelium (TFPIa: ∼9 fold higher; TFPIb: ∼5-fold higher). In situ hybridization studies revealed that TFPI and TM are expressed on endothelium throughout the brain parenchyma. In addition, TFPI was expressed within the granule layer of the cerebellum. TFPI null embryos succumb to embryonic lethality with necrotic lesions and fibrin deposition in the brain. To determine if increased TM-APC anticoagulant activity would rescue TFPI null embryos, a transgene over expressing human activated protein C (hHPC) was bred into heterozygous TFPI mice, which were then bred to determine if TFPI null mice expressing hHPC survive embryogenesis. However, the hHPC transgene did not rescue TFPI null embryos. Furthermore, TFPI null embryos had similar brain lesions and brain fibrin deposition regardless of the presence of the hHPC transgene. A hematopoietic cell transplant model system was used to examine the consequences of TFPI deficiency in the adult mouse brain. Mice transplanted with hematopoietic cells lacking TFPI had cerebral vascular fibrin deposition that was not observed in mice transplanted with hematopoietic cells producing normal amounts of TFPI. Interestingly, the fibrin was often found at sites of endothelial injury as demonstrated by co-localization with anti-C1qA, which binds to phosphatidylserine on damaged endothelium. ConclusionsTFPI prevents necrotic brain lesions and cerebral fibrin deposition in developing mouse embryos and over expression of hHPC is not sufficient to compensate for the absence of TFPI. Adult mice lacking TFPI in hematopoietic cells have increased amounts of fibrin deposited at sites of cerebral vascular injury despite having normal endothelial TM. These data indicate that the distinct function of TFPI in the cerebral vasculature is not compensated by the anticoagulant activity of TM in regulating cerebral vascular thrombosis. Disclosures:Mast:Novo Nordisk: Honoraria, Research Funding.

TFPI Expresses Anticoagulant Activity Independent Of TF-FVIIa

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting the tissue factor-factor VIIa complex (TF-FVIIa). Inhibition of TF-FVIIa by TFPI is greatly enhanced by FXa. In order to inhibit TF-FVIIa, TFPI first binds to and inhibits Factor Xa (FXa) thus forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. The TFPI-FXa complex subsequently forms a quaternary complex with TF-FVIIa and blocks the initiation of coagulation. Although TFPI can directly inhibit FXa, this inhibition is generally considered to be of limited physiological significance because the plasma TFPI concentration (0.25-0.5 nM) is several orders of magnitude lower than the plasma concentration of other FXa inhibitors e.g. antithrombin (AT = 2.5-5 μM).We investigated whether TFPI expresses anticoagulant activity independent of TF-FVIIa i.e. to test whether direct inhibition of FXa by TFPI contributes to the down-regulation of coagulation.In model systems, the inhibition of FXa by TFPI and AT was determined in the presence of calcium chloride, phospholipids and protein S using a chromogenic substrate to monitor FXa activity. FXa inhibition by TFPI in plasma was determined by measuring thrombin generation curves triggered with FXa, with the FXa activator from Russell's viper venom (RVV-X) and with FXIa or FIXa in normal pooled plasma. These experiments were performed in the presence of a cocktail of anti TF/anti FVIIa antibodies to inhibit any contribution of traces of TF that might be present in plasma to initiate coagulation. The TF-independent contributions of TFPI and/or protein S to the down-regulation of thrombin generation were quantified 1) after neutralization of TFPI and protein S present in plasma with an anti-TFPI antibody cocktail or anti-protein S antibodies and 2) via TFPI and protein S titrations in TFPI- and in protein S-depleted plasma.In model systems containing purified proteins, physiological concentrations TFPI (0.25 nM) appeared to be more effective in FXa inhibition (t1/2 = 2.5 min) than physiological concentrations AT (2.5 μM) which inhibited FXa with t1/2 = 3.2 min. Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation that was triggered in plasma with RVV-X, FXa, FIXa or FXIa. Depending on the trigger and trigger concentration used anti-TFPI and anti-protein S antibodies decreased the lag time and/or increased the peak height of thrombin generation. At high trigger concentrations neither anti-TFPI nor anti-protein S antibodies enhanced thrombin generation. TFPI and protein S titrations in TFPI- and protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF confirmed that both TFPI and protein S express TF-independent anticoagulant activity. TFPI titrations in protein S-depleted plasma triggered with RVV-X or FXIa showed that the TF-independent anticoagulant activity of TFPI was greatly reduced in the absence of protein S. Supplementation of the protein S-depleted plasma with protein S restored the anticoagulant effect of TFPI.We demonstrated that TFPI, despite its low plasma concentration, expresses anticoagulant activity in the absence of TF. Kinetic experiments in model systems at plasma TFPI concentrations show that direct inhibition of FXa by TFPI can account for the TF-independent anticoagulant effect of TFPI on thrombin generation. Blocking the anticoagulant activity of protein S with anti-protein S antibodies also enhances thrombin generation with triggers other than TF. The fact that neutralization of TFPI and of protein S with specific antibodies resulted in a similar stimulation of thrombin generation suggests that TFPI is strongly dependent on the presence of protein S for expression of TF-independent anticoagulant activity in plasma. The ability of TFPI to inhibit thrombin generation initiated via the intrinsic coagulation pathway is an important observation particularly in the light of recent reports which indicate that the intrinsic coagulation pathway plays a relevant role in thrombus formation and in cardiovascular disease. Disclosures:Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment. Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment.

The Potential to Enhance Hemostasis by Inhibiting Tissue Factor Pathway Inhibitor

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factors (TF) X and VIIa. As such, it is a major regulator of the extrinsic clotting pathway. Compounds that block TFPI activity, or “inhibit the inhibitor,” effectively restore hemostasis in animal models of hemophilia. These compounds are thought to act by allowing unfettered TF-mediated thrombin generation through the extrinsic pathway in a manner that bypasses the need for the intrinsic pathway proteins, factor VIII and factor IX. Improved understanding of TFPI tissue expression and function is needed to better recognize how pharmaceutical agents targeting TFPI alter biochemical and physiological mechanisms of blood coagulation. Several advances have been made recently. TFPI is an alternatively spliced protein with two primary isoforms, TFPIα and TFPIβ, which have distinct cellular expression patterns and anticoagulant functions. TFPIα is produced by endothelial cells, and appears to localize within an intracellular granule with release following stimulation with thrombin or heparin. TFPIα is also produced by megakaryocytes and is released from activated platelets. Platelet TFPIα limits clot growth following vascular injury and alters bleeding in a murine hemophilia model. Interestingly, the anticoagulant activity produced by platelet TFPIα appears to be isoform specific and not mediated through inhibition of TF. TFPIβ is made by endothelial cells, localizes to endothelium, and is not present in platelets. TFPIβ is an effective inhibitor of TF-mediated cellular migration and dampens the effects of TF-induced intravascular coagulation in mice. This new knowledge of TFPI isoform expression patterns and anticoagulant activities has important implications for the development of TFPI-blocking pharmaceuticals to treat hemophilia. The recent findings also provide new insights into the biochemical mechanisms that activate blood coagulation pathways and regulate TF-mediated thrombotic and inflammatory disease. Disclosures:Mast:Novo Nordisk: Honoraria, Research Funding.

Protein S Is a Cofactor for Platelet and Endothelial Tissue Factor Pathway Inhibitor-α but Not for Cell Surface–Associated Tissue Factor Pathway Inhibitor

Tissue factor pathway inhibitor (TFPI) is produced in 2 isoforms: TFPIα, a soluble protein in plasma, platelets, and endothelial cells, and TFPIβ, a glycosylphosphatidylinositol-anchored protein on endothelium. Protein S (PS) functions as a cofactor for TFPIα, enhancing the inhibition of factor Xa. However, PS does not alter the inhibition of prothrombinase by TFPIα, and PS interactions with TFPIβ are undescribed. Thus, the physiological role and scope of the PS-TFPI system remain unclear. Here, the cofactor activity of PS toward platelet and endothelial TFPIα and endothelial TFPIβ was quantified. PS enhanced the inhibition of factor Xa by TFPIα from platelets and endothelial cells and stabilized the TFPIα/factor Xa inhibitory complex, delaying thrombin generation by prothrombinase. By contrast, PS did not enhance the inhibitory activity of TFPIβ or a membrane-anchored form of TFPI containing the PS-binding third Kunitz domain (K1K2K3) although PS did function as a cofactor for K1K2K3 enzymatically released from the cell surface. The PS-TFPI anticoagulant system is limited to plasma TFPIα and TFPIα released from platelets and endothelial cells. PS likely functions to localize solution-phase TFPIα to the cell surface, where factor Xa is bound. PS does not alter the activity of membrane-associated TFPI. Because activated platelets release TFPIα and PS, the PS-TFPIα anticoagulant system may act physiologically to dampen thrombin generation at the platelet surface.

Cellular expression and biological activities of alternatively spliced forms of tissue factor pathway inhibitor

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factor-factor VIIa (TF-fVIIa) and factor Xa (fXa). Recent studies revealed distinct cellular expression patterns for TFPIα and TFPIβ and spurred additional experiments to define unique functions for these alternatively spliced TFPI isoforms. TFPIα is produced by endothelial cells, localizes to an intracellular granule, and is released following cellular stimulation with thrombin or heparin. TFPIα also is produced by megakaryocytes and released from activated platelets. Platelet TFPIα limits clot growth following vessel injury and alters bleeding in hemophilia, suggesting that its primary physiological role is modulation of clot development. TFPIβ is made by endothelial cells, localizes to the endothelium surface, and is not in platelets. TFPIβ is an effective inhibitor of TF-mediated cellular migration and may act to dampen the adverse effects of intravascular TF expressed during inflammation. Knowledge of TFPI isoform expression and activity provides new insights into the biochemical regulation of TF-mediated thrombotic and inflammatory disease. Recent findings have therapeutic implications for use of recombinant TFPI to treat severe sepsis in community-acquired pneumonia or to achieve improved engraftment of hematopoietic stem cells, and for development of TFPI-blocking pharmaceuticals to treat hemophilia.

The role of tissue factor pathway inhibitor in atherosclerosis and arterial thrombosis

Tissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor (TF)-mediated coagulation. In atherosclerotic plaques TFPI co-localizes with TF, where it is believed to play an important role in attenuating TF activity. Findings in animal models such as TFPI knockout models and gene transfer models are consistent on the role of TFPI in arterial thrombosis as they reveal an active role for TFPI in attenuating arterial thrombus formation. In addition, ample experimental evidence exists indicating that TFPI has inhibitory effects on both smooth muscle cell migration and proliferation, both which are recognized as important pathological features in atherosclerosis development. Nonetheless, the clinical relevance of these antithrombotic and atheroprotective effects remains unclear. Paradoxically, the majority of clinical studies find increased instead of decreased TFPI antigen and activity levels in atherothrombotic disease, particularly in atherosclerosis and coronary artery disease (CAD). Increased TFPI levels in cardiovascular disease might result from complex interactions with established cardiovascular risk factors, such as hypercholesterolemia, diabetes and smoking. Moreover, it is postulated that increased TFPI levels reflect either the amount of endothelial perturbation and platelet activation, or a compensatory mechanism for the increased procoagulant state observed in cardiovascular disease. In all, the prognostic value of plasma TFPI in cardiovascular disease remains to be established. The current review focuses on TFPI in clinical studies of asymptomatic and symptomatic atherosclerosis, coronary artery disease and ischemic stroke, and discusses potential atheroprotective actions of TFPI.

Plasmin‐dependent proteolysis of tissue factor pathway inhibitor in a mouse model of endotoxemia

The development of a procoagulant state in sepsis, owing to aberrant expression of tissue factor (TF) and a sharp decrease in the level of its major inhibitor, TF pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline in TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (lipopolysaccharide [LPS]) in mice. Plasmin generation and TFPI activity were assayed in the lungs of mice deficient in tissue-type plasminogen (Plg) activator (t-PA) or Plg, at 2 h after LPS or saline injection. The sharp loss of lung-associated TFPI activity at 2 h after LPS challenge paralleled the abrupt increase in plasmin generation. TFPI activity was significantly retained in both t-PA(-/-) and Plg(-/-) mice, which are unable to generate plasmin. The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications.

TFPI resistance related to inherited or acquired protein S deficiency

BackgroundProtein S (PS) is an essential component of the protein C pathway and PS deficiency can explain a poor response to activated protein C. It has recently been shown that PS also acts as a cofactor of Tissue Factor Pathway Inhibitor (TFPI). ObjectivesIn the present study, we investigated whether PS deficiency could be responsible for a poor response to TFPI. Patients/MethodsThirty-one patients with inherited PS deficiency, seven pregnant women and 36 controls were enrolled in the study. We measured the plasma response to added TFPI using a two-step diluted prothrombin time (dPT) assay. The response of the different plasmas to the anticoagulant activity of TFPI was expressed as TFPI Normalised Ratio (TFPI NR). ResultsThe median TFPI NR was statistically significantly lower in patients with inherited PS deficiency (0.5) than in controls (1.0) (p<0.0001). It was statistically significantly lower in patients with type I inherited PS deficiency (0.47) compared to patients with type III inherited PS deficiency (0.58) (p=0.018). In contrast, it did not differ between patients with and without thrombosis. Median TFPI NR values were statistically significantly lower during pregnancy (0.54) than 3months after delivery (0.71) (p=0.016). TFPI NR values correlated well with PS activity values (R2=0.681) whatever the nature of the PS deficiency. ConclusionsOur findings confirm that PS deficiency results in a poor anticoagulant response to TFPI, demonstrating again the cofactor role of PS in TFPI activity.

TLR9 signaling regulates tissue factor and tissue factor pathway inhibitor expression and activity in human coronary artery endothelial cells

Although human endothelial cells recognize and respond to bacterial DNA (CpG DNA), the role of bacterial DNA in procoagulation is not known. We investigated the impact of bacterial DNA on expression and activity of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in human coronary artery endothelial cells (HCAEC). CpG DNA (1–32 μg/ml) markedly induced TF expression between 4 and 8 hours in both protein and mRNA levels and TF activity. CpG DNA markedly enhanced NF‐êB activation, which was blocked by the telomere‐derived TLR9 antagonist oligonucleotide TTAGGG. Consistently, the specific NF‐êB inhibitors BAY 117082 and SN50 prevented CpG DNA‐induced TF transcription and secretion. Conversely, culture of HCAEC with CpG DNA for 24 to 48 hours reduced TFPI transcription, secretion, and activity. Methylation of cytosines in CpG DNA resulted in a complete loss of biological activities. Our results demonstrate that bacterial DNA through TLR9 can alter the balance of TF and TFPI expression and activity in HCAEC, thereby contributing to thrombus formation, the major cause of acute coronary artery disease.(Grant support: CIHR MOP‐97742).

Caveolae optimize tissue factor–Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIβ and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.

Strong Correlation of High Endogenous Plasma Levels of Tissue Factor Pathway Inhibitor (TFPI) with Poor Blood Clotting and Corrective Action of BAX 513 Under FVIII-Inhibited Conditions

Abstract 1201▪▪This icon denotes a clinically relevant abstractTFPI is the major physiological inhibitor of the extrinsic pathway of blood coagulation. Thus, blocking TFPI activity is a possible approach to treat bleeding disorders such as hemophilia A and B. BAX 513, a fucoidan derived from the brown seaweed L. japonica, and other non-anticoagulant sulfated polysaccharides (NASPs) improve coagulation in hemophilic blood and plasma (Liu et al. Thromb Haemost 2006;95:68). This procoagulant activity of NASPs can at least partially be attributed to the inhibition of TFPI. For this study, we predicted that endogenous TFPI levels are associated with decreased hemostatic capacity under hemophilic conditions. We correlated clotting parameters measured by global hemostatic assays with the total and full-length (FL) TFPI levels in human plasma samples in the absence or presence of BAX 513.Tissue factor-triggered coagulation of normal and FVIII-inhibited plasma or blood from 30 healthy subjects was monitored by calibrated automated thrombograph (CAT) assay and rotation thromboelastometry (ROTEM). The experiments were performed with or without factor VIII blockade with a goat anti-human Factor VIII antibody to simulate acquired hemophilia. The total and FL-TFPI plasma levels were quantified by ELISA. We observed longer clotting and clot formation times by ROTEM and decreased thrombin generation in the presence of high TFPI levels in normal plasma (p<0.05). These correlations became substantially stronger when FVIII was inhibited (reaching up to r2= 0.64; p<0.001). In the presence of BAX 513 (0.1–1.2 μg/mL), most plasma samples showed a pro-coagulant response with an at least 50% increase in thrombin peak and a decrease in time to peak. The BAX 513-mediated increase in thrombin generation correlated strongly with the individual FL-TFPI plasma level. The effect was even more apparent when FVIII was inhibited, where the extrinsic pathway is the only driver of thrombin formation.These data indicate that high endogenous TFPI levels critically compromise hemostasis in acquired factor VIII deficiency and that TFPI is a threshold factor for the intrinsic amplification of thrombin formation. As low TFPI levels are associated with increased blood clotting, TFPI inhibition is a rationale treatment approach in patients with hemophilia. Importantly, our findings validate the inhibition of TFPI activity as a mode of action for BAX 513 and related pro-coagulant compounds. Disclosures:Dockal:Baxter Innovations GmbH: Employment. Gorczyca:Medical University of Vienna: Employment. Knappe:Baxter Innovations GmbH: Employment. Palige:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Increased tissue factor pathway inhibitor activity is associated with myocardial infarction in young women: results from the RATIO study.

The tissue factor pathway inhibitor (TFPI)/protein S anticoagulant system is a potent inhibitor of blood coagulation. TFPI and protein S are major determinants of thrombin generation (TG) tests determined at low tissue factor (TF) and at high TF concentrations in the presence of activated protein C (APC). Both TFPI and protein S protect against venous thrombosis, but the importance of the TFPI/protein S system in arterial thrombosis remains unclear. To investigate the influence of the TFPI/protein S anticoagulant system on the risk of myocardial infarction (MI) in young women. The RATIO study is a case-control study in women under 50 years of age, including 205 patients and 638 controls. TFPI and protein S were quantified using ELISA. The TFPI/protein S activity (nTFPIr) and the APC sensitivity ratio (nAPCsr) were determined using TG tests. Odds ratios (ORs) adjusted for putative confounders and corresponding 95% confidence intervals (95% CI) were determined. Women with MI had higher TFPI levels than controls (135.9 ± 40% vs. 124.2 ± 41%), resulting in increased TFPI/protein S activities and increased APC sensitivity. Furthermore, an increased TFPI activity was associated with MI [nTFPIr: adjusted OR Q1 vs. Q4 = 2.1 (95%CI 1.1-4.1)]. Additionally, an increased APC sensitivity was associated with MI [nAPCsr: adjusted OR Q1 vs. Q4 = 1.7 (95% CI 0.9-3.2)] Women with MI had increased TFPI levels compared with controls. Consequently, the TFPI/protein S activity and APC sensitivity are increased in women with MI. Whether this increase in TFPI activity acts as a compensating mechanism for an increased procoagulant state or is a marker of endothelial damage remains to be investigated.

Novel protein ADTRP regulates TFPI expression and function in human endothelial cells in normal conditions and in response to androgen

AbstractThrombosis and cardiovascular disease (CVD) represent major causes of morbidity and mortality. Low androgen correlates with higher incidence of CVD/thrombosis. Tissue Factor Pathway Inhibitor (TFPI) is the major inhibitor of tissue factor-factor VIIa (TF-FVIIa)–dependent FXa generation. Because endothelial cell (EC) dysfunction leading to vascular disease correlates with low EC-associated TFPI, we sought to identify mechanisms that regulate the natural expression of TFPI. Data mining of NCBI's GEO microarrays revealed strong coexpression between TFPI and the uncharacterized protein encoded by C6ORF105, which is predicted to be multispan, palmitoylated and androgen-responsive. We demonstrate that this protein regulates both the native and androgen-enhanced TFPI expression and activity in cultured ECs, and we named it androgen-dependent TFPI-regulating protein (ADTRP). We confirm ADTRP expression and colocalization with TFPI and caveolin-1 in ECs. ADTRP-shRNA reduces, while over-expression of ADTRP enhances, TFPI mRNA and activity and the colocalization of TF-FVIIa–FXa-TFPI with caveolin-1. Imaging and Triton X-114–extraction confirm TFPI and ADTRP association with lipid rafts/caveolae. Dihydrotestosterone up-regulates TFPI and ADTRP expression, and increases FXa inhibition by TFPI in an ADTRP- and caveolin-1-dependent manner. We conclude that the ADTRP-dependent up-regulation of TFPI expression and activity by androgen represents a novel mechanism of increasing the anticoagulant protection of the endothelium.