Presence Of Tissue Factor Pathway Inhibitor Research Articles

Partial rescue of naturally occurring active site factor X variants through decreased inhibition by tissue factor pathway inhibitor and antithrombin

BackgroundActivated coagulation factor X (FXa) is the serine protease component of prothrombinase, the physiological activator of prothrombin. Factor X Nottingham (A404T) and Taunton (R405G) are two naturally occurring mutations, identified in families with a bleeding phenotype. ObjectiveTo characterize these FX variants functionally. MethodsThe activity and inhibition of recombinant FX variants were quantified in plasma‐based and pure component assays. ResultsThe prothrombin times in FX‐depleted plasma supplemented with FX Nottingham and Taunton were greatly increased compared to that of wild‐type (WT) FX. Kinetic investigations of activated variants in the prothrombinase complex showed kcat/Km reduced ~50‐fold and ~5‐fold, respectively, explaining the prolonged prothrombin time (PT). The substituted residues are located in the protease domain Na+‐binding loop, important for the activity of FXa, as well as its inhibition. Both FXa Nottingham and Taunton showed reduced affinity for Na+. Plasma‐based thrombin generation assays triggered with 1 pmol/L tissue factor (TF) demonstrated only small differences in activities compared to WT FX, but large reductions at 10 pmol/L TF. Severely reduced inhibition of both FXa Nottingham and Taunton by tissue factor pathway inhibitor (TFPI) and antithrombin (AT), was shown in pure‐component FXa inhibition assays. Factor Xa Nottingham and Taunton produced higher amounts of thrombin than WT FXa in pure‐component prothrombinase assays in the presence of TFPI and AT, explaining the results from the plasma‐based assay. ConclusionsFactor X Nottingham and Taunton both display decreased proteolytic activity. However, their reduced activity in plasma triggered by low TF can be rescued by decreased inhibition by the natural FXa inhibitors, TFPI and AT.

Mechanism of Tissue Factor (TF) Enhancing Factor (F)VIII Activity on FXa Generation in Initial Phase of Coagulation and Interaction Between TF and FVIII C2 Domain

In the cell-based coagulation model, factor (F)VIIa complex with tissue factor (TF) initiates the blood coagulation by generating FXa as the extrinsic tenase complex and activates FIX which composes the intrinsic tenase complex. We demonstrated that FVIIa/TF directly activated FVIII in an early coagulation phase (Soeda, JTH, 2010), and TF enhanced the intrinsic tenase activity via possible interaction with FVIIIa (Ogiwara, ASH, 2010). In this study, we clarified the enhancing mechanism of intrinsic tenase activity in the TF-related up-regulation of FVIII, and identified the TF-interactive region on FVIII.To explore the enhancing mechanism of TF for FVIII regulation, we performed the FXa generation assay with various amounts of FVIII or thrombin-mediated FVIIIa, constant FIXa (1 nM), FX (300 nM) and phospholipid vesicles (PL; 20 µM) in the presence of recombinant lipidated TF (rTF, Innovin®). The Km value for FVIII in the presence of rTF was ~2.4-fold lower than that its absence (Km; 5.6±0.5, 13.3±3.7 nM, respectively, p<0.05). Similarly, the Km for FVIIIa in the presence of TF was ~1.5-fold lower than that in its absence (Km; 6.3±0.6 nM, 9.7±1.6 nM, respectively, p<0.05), supporting that the presence of TF could promote the FXa-catalyzed activation of FVIII and FVIIIa-dependent generation of intrinsic FXa. To further evaluate the effect of TF on FVIII-dependent FXa generation, the FXa generation assay with FVIIa/TF-activated FVIIIa (FVIIIa-VIIa/TF) was also performed. The initial velocity on FXa generation with FVIIIa-VIIa/TF was 22.6 nM/min. However, the initial velocity on FXa generation with FVIIIa-VIIa/TF by addition of FVIIa-inhibitor (E-76), not to generate FVIIa/TF-dependent FXa, was 3.4 nM/min, and that with FVIII alone was 0.05 nM/min. In addition, the initial velocity with FVIIa/TF alone was 10.4 nM/min. These findings supported that the TF increased FXa generation greater than the additive effect of FVIII-dependent and FVIIa/TF-dependent FXa generation in early initiation phase of coagulation prior to thrombin generation. Since tissue factor pathway inhibitor (TFPI) is present in physiological circulating whole blood, a similar experiment on FXa generation assay was repeated under the presence of TFPI, estimated to be present at 0.5 nM in the pre-coagulant state or at 15 nM in the coagulant state in circulating blood. The initial velocity on FXa generation with FVIIIa-VIIa/TF was reduced by the presence of 0.5 or 15 nM TFPI (17.9 and 12.6 nM/min, respectively). By contrast, the initial velocity on FVIIIa-VIIa/TF-dependent FXa generation with addition of FVIIa inhibitor was little reduced by 0.5 nM TFPI, whilst was reduced by 15 nM TFPI (3.5 and 2.5 nM/min, respectively). These findings supported that the TFPI possibly didn’t inhibit TF on the enhanced intrinsic tenase on association with FVIII in the pre-coagulant state.We further reported that TF enabled FXa to activate FVIII, irrespective of von Willebrand factor (VWF), and the direct association of rTF and non-lipidated TF with FVIII (Furukawa, ISTH, 2015). Since TF is transmembrane protein, however, we performed a surface plasmon resonance (SPR)-based assay (BIAcore®) and solid phase-based ELISA to identify the interactive region(s) on FVIII to recombinant soluble TF (sTF; Altor BioScience), a portion of TF outside of the PL membrane. An SPR-based assay revealed the direct binding of intact FVIII, LCh (a3-A3C1C2, A3C1C2) subunit, C2 domain to immobilized sTF (Kd; 2.3±0.6, 5.8±1.0, 10.5±3.5, 11.8±0.5 nM, respectively). The intact HCh, A1 or A2 domain to sTF failed to bind, however. A non-equilibrium ELISA also revealed that sTF bound to immobilized C2 domain with moderate affinity (Kdapp; 16.9±2.2 nM), and the interaction was dependent on ionic strength and Ca2+. In addition, the presence of VWF significantly competitively inhibited the C2 and sTF binding by ~90% (IC50; 5.7 µg/ml) at the maximal concentration employed, suggesting that the C2 domain-TF interaction could activate FVIII by FXa even in the presence of VWF.We concluded that it might be possible that TF enhanced the FVIII-mediated FXa generation by not only FVIIa but also FXa, additionally this enhancing mechanism might not be suppressed by TFPI in the initiation phase of coagulation. Furthermore, TF might function to FVIII activation, irrespective of presence of VWF, by the binding to C2 domain through the competition with VWF. DisclosuresNogami:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Shima:Sysmex Corporation: Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

The Effect Of Physiological Metal Ions On The Procoagulant Enzyme-Cofactor Complexes Of The Extrinsic Pathway Of Coagulation

IntroductionThe rapid coagulation response to vascular injury is mediated by the formation of three enzyme cofactor complexes (extrinsic tenase, intrinsic tenase and prothrombinase) on membrane surfaces. A common structural feature of these proteases is their GLA domains, each of which requires the binding of divalent metal ions at multiple sites to achieve the conformation necessary for optimal membrane and cofactor binding. Both Ca2+ and Mg2+ ions have been reported to bind to GLA domain sites. However almost all studies kinetically characterizing these complexes have been done in the presence of Ca2+ (2-5 mM) as the sole metal ion, despite the relatively equivalent availability in plasma of both free Ca2+ (∼1.1 mM) and Mg2+ (∼0.6 mM) (Ca2+/Mg2+). A recent study has systematically examined the effects of various Ca2+ concentrations with and without Mg2+ on the membrane binding of activated protein C (APC) and FVIIa and enzymatic activity of APC and the extrinsic tenase complex which were enhanced in Ca2+/Mg2+ relative to Ca2+ alone (Vadivel, K., et al, 2013 JMB). In the current study we compare the effects of plasma levels of Ca2+ and Mg2+ versus Ca2+alone on the catalytic performances of the extrinsic tenase, intrinsic tenase and prothrombinase complexes individually and collectively. MethodsAll experiments were conducted in Hepes buffered saline pH 7.4 containing 0.1% PEG and either 2 mM Ca2+ or 1.1 mM Ca2+/0.6 mM Mg2+ (Ca2+/Mg2+). In closed system experiments, enzyme-cofactor complexes were assembled on phospholipid vesicles composed of a 3:1 ratio of synthetic phosphatidylcholine and phosphatidylserine (PCPS), and zymogen activation monitored via sampling into assay mixtures containing the appropriate chromogenic substrate. In open system experiments complexes were preassembled on PCPS coated capillaries, the zymogen delivered in the flowing phase and the extent of zymogen activation monitored in the effluent as described previously (Haynes, LM., et al, 2011 Biophys J). The combined interaction of the procoagulant enzyme cofactor complexes under both metal ion conditions was studied in a synthetic coagulation proteome monitoring thrombin (IIa) generation as previously described (van't Veer, C., and Mann, KG, 1997 JBC). ResultsExtrinsic tenase The extrinsic tenase complex had an approximately two-fold higher rate of FXa generation in the presence of Ca2+/Mg2+ (1.78 ±0.05 pM/s) versus Ca2+ alone (0.88 ± 0.02 pM/s) (N=3, p<0.001). Experiments were also conducted in the absence or presence of tissue factor pathway inhibitor (TFPI); in the presence of TFPI and Ca2+/Mg2+ ions the extrinsic tenase complex was three times as catalytically active compared to TFPI and Ca2+ only (N=3, p<0.05). Direct FXa inhibition by TFPI was not significantly different between the Ca2+ and Ca2+/Mg2+ containing buffers. Intrinsic tenase In closed system experiments the intrinsic tenase complex showed impaired FXa generation in the presence of Ca2+/Mg2+ (29.3 ± 3.4 pM/s) compared to Ca2+ alone (51.7 ± 3.0 pM/s) (N=3, p<0.001). Thrombin activation of FVIII was not statistically different between both buffers, however the presence of Ca2+/Mg2+ resulted in a more rapid loss of cofactor activity over Ca2+ alone. Prothrombinase Under flow, IIa generation measured from prothrombinase was not statistically different between both buffers when measured at five different shear rates (100-1000s-1, N≥2). Plasma proteome In the synthetic coagulation proteome (N=4) the presence of Ca2+/Mg2+ led to an average 31% increase in maximum IIa levels compared to Ca2+alone and an average decrease of 1 minute to reach maximum levels. ConclusionsIn summary, the presence of plasma levels of Ca2+ and Mg2+, compared to Ca2+ alone, enhances the initiation phase of the extrinsic pathway of coagulation by enhancing the rate of FXa generation from the extrinsic tenase complex while also impairing the TFPI inhibition of the extrinsic tenase complex. These procoagulant effects are potentially abrogated by the suppressive effects of Ca2+/Mg2+ on the intrinsic tenase complex, potentially caused by the increased rate of spontaneous inactivation of FVIIIa. These results highlight the importance of complementing the assessments of individual enzyme complex systems with studies of the larger complex systems in which they function to identify the net physiologic impact of metal ions that target various components in the coagulation cascade. Disclosures:Mann:Haematologic Technologies, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Expression of tissue factor pathway inhibitor (TFPI) in human breast and colon cancer tissue

Activation of blood coagulation, a phenomenon frequently observed in breast and colon cancer patients, contributes to tumour progression. The principal initiator of blood coagulation activation in cancer patients is tissue factor (TF), while tissue factor pathway inhibitor (TFPI) is the main inhibitor of the TF-dependent pathway of blood coagulation. Previous immunohistochemical studies revealed no expression of TFPI in human cancer cells. The aim of the study was to evaluate the expression of TFPI protein and mRNA in breast and colon cancer tissues. A total of 108 cancer tissues (from primary tumours and metastatic lymph nodes) were obtained from 87 patients during surgical treatment. Immunohistochemical studies using a polyclonal anti-TFPI antibody were performed including a semiquantitative analysis. The in situ hybridisation method employed single-stranded DNA oligonucleotide (probe sequence: 5'Biotin-CCACCATACTTGAAACGTTCACACT-Biotin3') directed against TFPI mRNA. Strong or medium expression of TFPI protein was observed in cancer cell bodies in all breast cancers and in most (39/66 cases) colon cancers examined. Weaker expression of TFPI was detected in cancer cells localised in lymph node metastatic foci of breast cancer. Endothelial cells were also TFPI-positive. TFPI mRNA was demonstrated in all cases of breast and in approximately 80% cases of colon cancer cells. TFPI mRNA and protein are present in association with colon and breast cancer cells, suggesting that the protein may play a role in cancer biology. The presence of TFPI in association with breast cancer cells localised in regional lymph nodes may indicate its role in lymphatic spread.

Tissue factor pathway inhibitor can interact with platelets

Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. However, there is no report on interaction between TFPI and platelets other than that by Tsuji, who found that whole blood anticoagulated with TFPI exhibited remarkable decrease in platelet count. Our study revealed that washed platelets suspended in modified Tyrode's buffer (8 mM CaCl 2) containing TFPI exhibit platelet aggregation. However, platelets aggregation was observed without TFPI, but its increase and intensity were slow and weak, compared to that in the presence of TFPI. This aggregation was inhibited by anti-CD41 (anti-GPIIb) antibody. This finding suggested that TFPI promotes platelet aggregation.

Kinetics of the Inhibition of Tissue Factor-Factor Vila by Tissue Factor Pathway Inhibitor

Tissue factor-factor VIIa catalysed activation of factor IX is inhibited by the complex of tissue factor pathway inhibitor (TFPI) and factor Xa. At present, no information is available as to what extent the kinetics of complex formation between TFPI and factor Xa during factor X activation contribute to the overall rate of inactivation of the factor X converting complex. We have determined the kinetic parameters of the individual reactions, i.e. factor X activation, formation of the TFPI-factor Xa complex, and inactivation of tissue factor-factor VIIa by the TFPI-factor Xa complex. We modelled the overall reaction by assuming a two-step reaction: factor Xa generated by tissue factor-factor VIIa forms a reversible complex with TFPI and in the second step this complex forms a reversible quaternary complex with tissue factor-factor VIIa. The validity of the model was demonstrated by analysis of factor Xa generation curves in the presence of TFPI. Independently determined constants for factor X activation (kcat = 12 s-1, Km = 70 nM) and inhibition of tissue factor-factor VIIa by TFPI-factor Xa complex (rate constant of inhibition of 1.1 x 10(8) M-1S-1) were used. The association rate constant of the formation of the TFPI-factor Xa complex was estimated by fitting the model to the data.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated Factor X and Thrombin Formation Triggered by Tissue Factor on Endothelial Cell Matrix in a Flow Model: Effect of the Tissue Factor Pathway Inhibitor

The procoagulant subcellular matrix of stimulated endothelial cells that contains tissue factor (TF) was used to investigate the mechanism by which TF pathway inhibitor (TFPI) inhibits thrombin formation initiated by TF/factor VIIa (FVIIa) under flow conditions. Purified coagulation factors VII, X, and V and prothrombin were perfused at a wall shear rate of 100 s-1 through a flow chamber containing a coverslip covered with matrix of cultured human umbilical vein endothelial cells. This resulted in a TF- and FVII-dependent FXa and thrombin generation as measured in the effluent at the outlet of the system. Inhibition of this TF/FVIIa-triggered thrombin formation by TFPI purified from plasma was dependent on the amount of TF present on the endothelial cell matrix. The rate of prothrombinase assembly and steady-state levels of thrombin formation were decreased by TFPI. Because persistent albeit decreased steady-state levels of thrombin formation occurred in the presence of TFPI, we conclude that plasma-TFPI does not inhibit FXa present in the prothrombinase complex. The addition of FIX and FVIII to perfusates containing FVII and FX increased the FXa generation on endothelial matrices, and counteracted the inhibition of thrombin formation on endothelial cell matrices by TFPI. Our data provide further evidence for the hypothesis that the rapid inactivation of TF/FVIIa by TFPI in combination with the absence of either FVIII or FIX causes the bleeding tendency of patients with hemophilia A or B.

Activated factor X and thrombin formation triggered by tissue factor on endothelial cell matrix in a flow model: effect of the tissue factor pathway inhibitor

The procoagulant subcellular matrix of stimulated endothelial cells that contains tissue factor (TF) was used to investigate the mechanism by which TF pathway inhibitor (TFPI) inhibits thrombin formation initiated by TF/factor VIIa (FVIIa) under flow conditions. Purified coagulation factors VII, X, and V and prothrombin were perfused at a wall shear rate of 100 s-1 through a flow chamber containing a coverslip covered with matrix of cultured human umbilical vein endothelial cells. This resulted in a TF- and FVII-dependent FXa and thrombin generation as measured in the effluent at the outlet of the system. Inhibition of this TF/FVIIa-triggered thrombin formation by TFPI purified from plasma was dependent on the amount of TF present on the endothelial cell matrix. The rate of prothrombinase assembly and steady-state levels of thrombin formation were decreased by TFPI. Because persistent albeit decreased steady-state levels of thrombin formation occurred in the presence of TFPI, we conclude that plasma- TFPI does not inhibit FXa present in the prothrombinase complex. The addition of FIX and FVIII to perfusates containing FVII and FX increased the FXa generation on endothelial matrices, and counteracted the inhibition of thrombin formation on endothelial cell matrices by TFPI. Our data provide further evidence for the hypothesis that the rapid inactivation of TF/FVIIa by TFPI in combination with the absence of either FVIII or FIX causes the bleeding tendency of patients with hemophilia A or B.