Tissue Factor Pathway Inhibitor-α Research Articles

Platelet tissue factor pathway inhibitor-α dampens cardiac thrombosis and associated fibrosis in mice

BackgroundTissue factor pathway inhibitor (TFPI) is the primary inhibitor of events initiating the blood coagulation pathway. Tfpi−/− mice die during embryonic development. The absence of protease–activated receptor (PAR) 4, the major thrombin receptor on mouse platelets, rescues Tfpi−/−mice to adulthood. Among the 3 TFPI isoforms in mice, TFPIα is the only isoform within platelets (pltTFPIα) and the only isoform that inhibits prothrombinase, the enzymatic complex that converts prothrombin to thrombin. ObjectivesTo determine biological functions of pltTFPIα. MethodsTfpi−/−/Par4−/− mice were irradiated and transplanted with bone marrow from mice lacking or containing pltTFPIα. Thus, PAR4 expression was restored in the recipient mice, which differed selectively by the presence or absence of pltTFPIα and lacked other forms of TFPI. ResultsRecipient mice lacking pltTFPIα had reduced survival over the 200-day posttransplant period. Necropsy revealed radiation injury associated with large intraventricular platelet-rich thrombi, whereas other organs were not affected. Thrombi were associated with fibrotic presentations, including increased collagen deposition, periostin–positive activated fibroblasts, myofibroblasts, and macrophage infiltrates. Recipient mice containing pltTFPIα showed evidence of radiation injury but lacked heart pathology. ConclusionsTfpi−/−/Par4−/− mice develop severe cardiac fibrosis following irradiation and transplantation with bone marrow lacking pltTFPIα. This pathology is markedly reduced when the mice are transplanted with bone marrow containing pltTFPIα. Thus, in this model system pltTFPIα has an important physiological role in dampening pathological responses mediated by activated platelets within the heart tissue.

Development of a Plasma-Based Assay to Measure the Susceptibility of Factor V to Inhibition by the C-Terminus of TFPIα.

Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPIα, while overexpression of FV splicing variants with increased affinity for TFPIα (FV-short) causes bleeding. This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus. FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPIα C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr). The TFPIr (0.25-0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28-0.31) to FVL heterozygotes (0.35, 95% CI 0.34-0.37) and homozygotes (0.39, 95% CI 0.37-0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-shortAmsterdam had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPIα. We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPIα C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.

Factor V Has Anticoagulant Activity in Plasma in the Presence of TFPIα: Difference between FV1 and FV2.

Activated factor V (FVa) is a potent procoagulant cofactor in the prothrombinase complex, whereas its precursor factor V (FV) stimulates the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-α (TFPIα), presumably by promoting TFPIα binding to phospholipids. Plasma FV comprises two glycosylation isoforms (FV1 and FV2) with low and high phospholipid-binding affinity, respectively. The FV1/FV2 ratio is increased in carriers of the FV R2 haplotype. This article demonstrates the TFPIα-cofactor function of FV in plasma and compares FV1 and FV2. Thrombin generation at low TF concentration was measured in FV-depleted plasma reconstituted with 0 to 100% FV, FV1 or FV2, and in 122 individuals genotyped for the R2 haplotype. The TFPIα-cofactor activities of FV1 and FV2 were also investigated in a model system of TFPIα-mediated FXa inhibition. In the FV titration, thrombin generation first increased (up to 5% FV) and then progressively decreased at higher FV concentrations. This anticoagulant effect of FV, which was also observed with FV2 but not with FV1, was largely abolished by anti-TFPIα antibodies, suggesting that it reflects TFPIα-cofactor activity of FV. In the model system of TFPIα-mediated FXa inhibition, FV2 was a more potent TFPIα-cofactor than FV1, in line with their respective phospholipid affinities. Accordingly, FV R2 carriers had higher thrombin generation than non-carriers, even after correction for demographics and plasma levels of coagulation factors and inhibitors. FV (and particularly its FV2 isoform) contributes to the TFPIα-dependent down-regulation of thrombin generation in plasma triggered with low TF.

Suppressive Role of Tissue Factor Pathway Inhibitor-α in Platelet-Dependent Fibrin Formation under Flow Is Restricted to Low Procoagulant Strength.

Tissue factor pathway inhibitor-alpha (TFPI-α) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI-α thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI-α regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-α in plasma is a function of the local procoagulant strength-defined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI-α enhanced fibrin formation, extending from a platelet thrombus, under flow only at <2 pM coated TF. This enhancement was paralleled by an increased thrombin generation. For mouse plasma, genetic deficiency in TFPI enhanced fibrin formation under flow also at 0 pM TF microspots. On the other hand, using blood from haemophilia A or B patients, TFPI-α antagonism markedly enhanced fibrin formation at microspots with up to 100 pM coated TF. We conclude that, under flow, TFPI-α is capable to antagonize fibrin formation in a manner dependent on and restricted by local TF/factor VIIa and factor Xa activities.

Novel insights into the regulation of coagulation by factor V isoforms, tissue factor pathway inhibitorα, and protein S

Factor V (FV) is a regulator of both pro- and anticoagulant pathways. It circulates as a single-chain procofactor, which is activated by thrombin or FXa to FVa that serves as cofactor for FXa in prothrombin activation. The cofactor function of FVa is regulated by activated protein C (APC) and protein S. FV can also function as an anticoagulant APC cofactor in the inhibition of FVIIIa in the membrane-bound tenase complex (FIXa/FVIIIa). In recent years, it has become clear that FV also functions in multiple ways in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. Of particular importance is a FV splice variant (FV-Short) that serves as a carrier and cofactor to TFPIα in the inhibition of FXa. FV-Short is generated through alternative splicing of exon 13 that encodes the large activation B domain. A highly negatively charged binding site for TFPIα is exposed in the C-terminus of the FV-Short B domain, which binds the positively charged C-terminus of TFPIα, thus keeping TFPIα in circulation. The binding of TFPIα to FV-Short is also instrumental in localizing the inhibitor to the surface of negatively charged phospholipids, where TFPIα inhibits FXa in process that is stimulated by protein S. Plasma FV activation intermediates and partially proteolyzed platelet FV similarly bind TFPIα with high affinity and regulate formation of prothrombinase. The novel insights gained into the interaction between FV isoforms, TFPIα, and protein S have opened a new avenue for research about the mechanisms of coagulation regulation and also for future development of therapeutics aimed at modulating coagulation.

The C‐terminus of tissue factor pathway inhibitor‐α inhibits factor V activation by protecting the Arg1545 cleavage site

Background Factor V (FV) is a carrier and a cofactor of the anticoagulant protein tissue factor pathway inhibitor-α (TFPIα), whose basic C-terminus binds to an acidic region in the B-domain of FV. Proteolysis of FV at Arg709 , Arg1018 and Arg1545 by activated FX (FXa) or thrombin removes the B-domain, and converts FV into a procoagulant cofactor (activated FV [FVa]) of FXa in the prothrombinase complex. However, retention of the acidic region in partially activated FV makes prothrombinase activity susceptible to inhibition by TFPIα. Objective/Methods To investigate the effect of the TFPIα C-terminal peptide (TFPIα C-term) on thrombin generation in plasma and on FV activation in model systems. Results TFPIα C-term inhibited tissue factor-initiated and FXa-initiated thrombin generation in a dose-dependent manner. Failure to inhibit thrombin generation in FV-depleted plasma reconstituted with FVa indicated that the peptide effect was mediated by the acidic region of FV, and was localized at the level of FV activation and/or prothrombinase. In model systems, TFPIα C-term inhibited both FV activation and prothrombinase activity. Western blot analysis showed that the peptide impaired cleavage at Arg1545 by both thrombin and FXa. The inhibition was stronger for FV-short, which binds TFPIα with higher affinity. Similar results were obtained with full-length TFPIα. Conclusions Cleavage of FV at Arg1545 , which abolishes the anticoagulant properties of FV and commits FV to the procoagulant pathway, is inhibited by binding of the TFPIα C-terminus to the FV acidic region. Possible targets of this new anticoagulant function of TFPIα are low-abundance FV(a) species retaining the acidic region.

The C-Terminus of Tfpiα Inhibits Factor V Activation By Protecting the Arg1545 Cleavage Site

Coagulation factor V (FV) is the inactive precursor of FVa, which acts as an essential cofactor of factor Xa (FXa) in the prothrombinase complex. FV is maintained in the inactive state by the interaction between a basic and acidic region in the B-domain. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) is highly homologous to the FV basic region and also binds to the acidic region of FV. In fact, a large fraction of plasma TFPIα circulates in complex with FV. Thanks to this interaction, FV acts as a cofactor of TFPIα in the inhibition of FXa and TFPIα inhibits prothrombinase complexes containing forms of FVa that retain the acidic region. However, when FV is activated through cleavage at Arg709, Arg1018 and Arg1545 by FXa or thrombin, it loses its anticoagulant properties and becomes a strong procoagulant. Recently, a FV splicing variant (FV-short) that lacks the basic region and binds TFPIα with high affinity has been described. FV-short is present in all individuals and represents ~5% of all plasma FV.To gain more insight in the functional implications of the FV-TFPIα interaction, we studied the effects of a peptide identical to the TFPIα C-terminus (TFPIα C-term) on thrombin generation in plasma and on FV activation in model systems. All major findings were confirmed with full-length TFPIα.TFPIα C-term (0-5 µM) prolonged the lag time and decreased the peak height of tissue factor- and FXa-triggered thrombin generation in a dose-dependent manner. These effects were more pronounced at low procoagulant stimuli and in the presence of plasma TFPIα. TFPIα C-term also inhibited thrombin generation in FV-depleted plasma reconstituted with FV, but not in FV-depleted plasma reconstituted with FVa, suggesting an effect on FV activation and/or prothrombinase. In model systems, TFPIα C-term inhibited the activation of purified FV by FXa and thrombin in a dose-dependent manner. This could be due to inhibition of FV proteolysis and/or to inhibition of prothrombinase in the assay used to quantify FVa activity. Therefore, FV activation was also followed by SDS-PAGE and Western blotting. This showed that TFPIα C-term (1 µM) interferes with FV proteolysis by both FXa and thrombin by selectively impairing cleavage of FV at Arg1545, which is located close to the FV acidic region (residues 1493-1537). The effect of TFPIα C-term on FV activation by thrombin was 3-fold stronger for FV-short than for full-length FV, in line with their respective affinities for the TFPIα C-terminus. Full-length TFPIα (10 nM) also inhibited FV cleavage at Arg1545 and delayed FV activation by thrombin. Its effect was also more pronounced on FV-short than on FV.In summary, binding of the TFPIα C-terminus to the acidic region of FV inhibits FV activation by FXa or thrombin by blocking access to the Arg1545 cleavage site. Since cleavage at this site marks the transition of FV from an anticoagulant form (TFPIα-cofactor) to a procoagulant form (FXa-cofactor), this may represent an important new anticoagulant function of TFPIα. The main target of this anticoagulant mechanism is presently unclear, but it is unlikely to be intact FV, whose plasma concentration is 100-fold higher than the TFPIα concentration. More likely candidates are low-abundance FV species that lack the basic region but retain the acidic region, and therefore bind TFPIα with high affinity, such as FV-short, early FV activation intermediates and/or platelet FV.Supported by grant nr. 2014-1 from the Dutch Thrombosis Foundation. DisclosuresNo relevant conflicts of interest to declare.

Tissue factor‐independent inhibition of thrombin generation by tissue factor pathway inhibitor‐α

Tissue factor pathway inhibitor-α (TFPIα) inhibits factor Xa by forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. TF-FVIIa forms a quaternary complex with TFPIα and FXa, which shuts off the initiation of coagulation via the extrinsic pathway. To investigate whether direct inhibition of FXa by TFPIα independently of TF plays a role in downregulating coagulation. Inhibition of FXa by TFPIα in plasma was determined by measuring thrombin generation triggered with FXa, the FX activator from Russell's viper venom (RVV-X), FXIa, or FIXa. TF-independent anticoagulant activities of TFPIα and its cofactor, protein S, were quantified: (i) after neutralization of TFPIα and protein S with anti-TFPI or anti-protein S antibodies; and (ii) in TFPI-depleted or protein S-depleted plasmas supplemented with varying amounts of TFPIα or protein S. Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation in plasma triggered with RVV-X, FXa, FIXa, or FXIa. Anti-TFPI and anti-protein S antibodies decreased the lag time and increased the peak height of thrombin generation to the same extent, indicating that inhibition of FXa by TFPIα requires the presence of protein S. TFPIα and protein S titrations in TFPI-depleted or protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF also revealed TF-independent anticoagulant activity of TFPIα, which was completely dependent on the presence of protein S. Direct inhibition of FXa by TFPIα contributes to the downregulation of coagulation.

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.

Coagulation factor VA2440G causes east Texas bleeding disorder via TFPIα

The autosomal dominantly inherited east Texas bleeding disorder is linked to an A2440G variant in exon 13 of the F5 gene. Affected individuals have normal levels of coagulation factor V (FV) activity, but demonstrate inhibition of global coagulation tests. We demonstrated that the A2440G mutation causes upregulation of an alternatively spliced F5 transcript that results in an in-frame deletion of 702 amino acids of the large activation fragment, the B domain. The approximately 250-kDa FV isoform (FV-short), which can be fully activated by thrombin, is present in all A2440G carriers' plasma (n = 16). FV-short inhibits coagulation through an indirect mechanism by forming a complex with tissue factor pathway inhibitor-α (TFPIα), resulting in an approximately 10-fold increase in plasma TFPIα, suggesting that the TFPIα:FV-short complexes are retained in circulation. The TFPIα:FV-short complexes efficiently inhibit thrombin generation of both intrinsic and extrinsic coagulation pathways. These data demonstrate that the east Texas bleeding disorder-associated F5(A2440G) leads to the formation of the TFPIα:FV-short complex, which inhibits activation and propagation of coagulation.

Factor V, tissue factor pathway inhibitor, and east Texas bleeding disorder

In a report reading like a fascinating detective story, Vincent and colleagues crack the mysterious case of east Texas bleeding disorder. They show that affected individuals have a mutation in exon 13 of the coagulation F5 gene that causes increased expression of an alternatively spliced transcript, which encodes a previously unrecognized factor V (FV) isoform they call FV-short. This FV isoform lacks a large portion of the B domain of FV, which is normally released upon the proteolytic activation of FV by thrombin and binds tightly to the coagulation regulator tissue factor pathway inhibitor-α (TFPIα). This interaction leads to an approximately 10-fold increase in the level of TFPIα circulating in plasma and a resultant anticoagulant effect that produces a hemorrhagic diathesis.

The Kunitz-3 domain of TFPI-α is required for protein S–dependent enhancement of factor Xa inhibition

AbstractProtein S (PS) enhances the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-α (TFPI-α) in the presence of Ca2+ and phospholipids. Altered forms of recombinant TFPI-α were used to determine the structures within TFPI-α that may be involved in this PS-dependent effect. Wild-type TFPI-α (TFPIWT), TFPI-α lacking the K3 domain (TFPI-ΔK3), and TFPI-α containing a single amino acid change at the putative P1 residue of K3 (R199L, TFPIK3P1) produced equivalent FXa inhibition in the absence of PS, whereas the response in FXa inhibition produced by PS was reduced with TFPIK3P1 (EC50 61.8 ± 13.4nM vs 8.0 ± 0.4nM for TFPIWT) and not detectable with TFPI-ΔK3. Ligand blotting and surface plasmon resonance experiments demonstrated that FXa bound TFPIWT and TFPI-ΔK3 but not the isolated K3 domain, whereas PS bound TFPIWT and the K3 domain but not TFPI-ΔK3. Addition of TFPIWT, TFPIK3P1, or TFPI-ΔK3 produced comparable prolongation of FXa-induced coagulation in PS-deficient plasma, but the anticoagulant effect of TFPIWT was substantially greater than that of TFPIK3P1 > TFPI-ΔK3 in normal plasma and PS-deficient plasma reconstituted with PS. We conclude that the PS-mediated enhancement of FXa inhibition by TFPI-α involves an interaction between PS and TFPI-α, which requires the K3 domain of TFPI-α.

Role for the Kunitz-3 Domain of Tissue Factor Pathway Inhibitor-α in Cell Surface Binding

Tissue factor pathway inhibitor (TFPI)-alpha, a key regulator of tissue factor-induced coagulation, contains 3 tandem Kunitz-type inhibitory domains. Kunitz-1 binds and inhibits factor VIIa in the factor VIIa/tissue factor complex, and Kunitz-2 binds and inhibits factor Xa. The role of the Kunitz-3 domain of TFPI-alpha, however, has remained an enigma. To determine the structures within TFPI-alpha involved in its binding to cell surface, altered forms of TFPI-alpha were expressed in C127 (mouse mammary) cells: C-terminal truncated forms TFPI-alpha (252), TFPI-alpha (242), and TFPI-alpha (181), which also lacks the third Kunitz domain (K3); TFPI-alpha (desK3), which lacks only the K3 domain; and TFPI-alpha (R199L), in which the putative P1 site in K3 is changed from arginine to leucine. By flow cytometry (fluorescence-activated cell sorting), the altered forms 252, 242, and R199L showed significantly reduced binding, whereas the forms 181 and desK3 completely failed to bind to the cell surface. Transient expression of WT-, desK3-, and K3/K2-TFPI-alpha (in which K3 is replaced with K2) in another cell line (b-end3, mouse endothelial) produced comparable results. Exogenously added C-terminal truncated and R199L forms of TFPI-alpha bound poorly and desK3 did not bind at all to the surface of ECV304 cells in which TFPI-alpha expression had been "knocked down" by RNA interference. Optimal cell binding of endogenously expressed TFPI-alpha requires its K3 and C-terminal domains, and within the K3 domain, the P1 (R199) residue plays an important role. Thus, one role of the K3 domain involves the cell surface localization of TFPI-alpha.