Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3602-3612 ◽  
Author(s):  
Ivo M. B. Francischetti ◽  
Jesus G. Valenzuela ◽  
John F. Andersen ◽  
Thomas N. Mather ◽  
José M. C. Ribeiro
Keyword(s):  
Salivary Gland ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Ixodes Scapularis ◽  
Factor Ix ◽  
Hard Tick ◽  
Factor X ◽  
Amidolytic Activity ◽  
Tissue Factor Pathway

Saliva of the hard tick and Lyme disease vector, Ixodes scapularis, has a repertoire of compounds that counteract host defenses. Following sequencing of an I scapularis salivary gland complementary DNA (cDNA) library, a clone with sequence homology to tissue factor pathway inhibitor (TFPI) was identified. This cDNA codes for a mature protein, herein called Ixolaris, with 140 amino acids containing 10 cysteines and 2 Kunitz-like domains. Recombinant Ixolaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor (TF)–induced factor X (FX) activation with an inhibitory concentration of 50% (IC50) in the picomolar range. In nondenaturing gel, Ixolaris interacted stoichiometrically with FX and FXa but not FVIIa. Ixolaris behaves as a fast-and-tight ligand of the exosites of FXa and γ-carboxyglutamic acid domainless FXa (des-Gla-FXa), increasing its amidolytic activity. At high concentration, Ixolaris attenuates the amidolytic activity of FVIIa/TF; however, in the presence of DEGR-FX or DEGR-FXa (but not des-Gla-DEGR-FXa), Ixolaris becomes a tight inhibitor of FVIIa/TF as assessed by recombinant factor IX (BeneFIX) activation assays. This indicates that FX and FXa are scaffolds for Ixolaris in the inhibition of FVIIa/TF and implies that the Gla domain is necessary for FVIIa/TF/Ixolaris/FX(a) complex formation. Additionally, we show that Ixolaris blocks FXa generation by endothelial cells expressing TF. Ixolaris may be a useful tool to study the structural features of FVIIa, FX, and FXa, and an alternative anticoagulant in cardiovascular diseases.

Penthalaris, a novel recombinant five-Kunitz tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick vector of Lyme disease, Ixodes scapularis

2004 ◽  
Vol 91 (05) ◽  
pp. 886-898 ◽  
Author(s):  
Thomas Mather ◽  
José Ribeiro ◽  
Ivo Francischetti
Keyword(s):  
Salivary Gland ◽  
Lyme Disease ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Ixodes Scapularis ◽  
Gel Filtration ◽  
Factor X ◽  
Tick Saliva ◽  
Tissue Factor Pathway

SummaryTick saliva is a rich source of molecules with antiinflammatory, antihemostatic and immunosupressive properties. In this paper, a novel tick salivary gland cDNA with sequence homology to tissue factor pathway inhibitor (TFPI) and coding for a protein called Penthalaris has been characterized from the Lyme disease vector, Ixodes scapularis. Penthalaris is structurally unique and distinct from TFPI or TFPI-like molecules described so far, including Ixolaris, NAPc2, TFPI-1 and TFPI-2. Penthalaris is a 308-amino-acid protein (35 kDa, pI 8.58) with 12 cysteine bridges and 5 tandem Kunitz domains. Recombinant Penthalaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor(TF)-induced factor X (FX) activation with an IC50 of ∼ 100 pM. Penthalaris tightly binds both zymogen FX and enzyme FXa (exosite), but not FVIIa, as demonstrated by column gel-filtration chromatography. At high concentrations, Penthalaris attenuates FVIIa/TF-induced chromogenic substrate (S2288) hydrolysis and FIX activation. In the presence of DEGR-FX or DEGR-FXa, but not des-Gla-DEGR-FXa as scaffolds, tight and stoichiometric inhibition of FVIIa/TF was achieved. In addition, Penthalaris blocks cell surface-mediated FXa generation by monomer (de-encrypted), but not dimer (encrypted) TF in HL-60 cells. Penthalaris may act in concert with Ixolaris and other salivary anti-hemostatics in order to help ticks to successfully feed on blood. Penthalaris is a novel anticoagulant and a tool to study FVIIa/TF-initiated biologic processes.

Proteases in blood clotting

2002 ◽  
Vol 38 ◽  
pp. 95-111 ◽  
Author(s):  
Peter N Walsh ◽  
Syed S Ahmad
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Serine Proteases ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Cell Receptor ◽  
Factor Ix ◽  
Factor X ◽  
Sequence Of Events ◽  
Tissue Factor Pathway

The serine proteases, cofactors and cell-receptor molecules that comprise the haemostatic mechanism are highly conserved modular proteins that have evolved to participate in biochemical reactions in blood coagulation, anticoagulation and fibrinolysis. Blood coagulation is initiated by exposure of tissue factor, which forms a complex with factor VIIa and factor X, which results in the generation of small quantities of thrombin and is rapidly shutdown by the tissue factor pathway inhibitor. The generation of these small quantities of thrombin then activates factor XI, resulting in a sequence of events that lead to the activation of factor IX, factor X and prothrombin. Sufficient thrombin is generated to effect normal haemostasis by converting fibrinogen into fibrin. The anticoagulant pathways that regulate blood coagulation include the protein C anticoagulant mechanism, the serine protease inhibitors in plasma, and the Kunitz-like inhibitors, tissue factor pathway inhibitor and protease nexin 2. Finally, the fibrinolytic mechanism that comprises the activation of plasminogen into plasmin prevents excessive fibrin accumulation by promoting local dissolution of thrombi and promoting wound healing by reestablishment of blood flow.

scholarly journals Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

2013 ◽  
Vol 289 (3) ◽  
pp. 1732-1741 ◽  
Author(s):  
Michael Dockal ◽  
Rudolf Hartmann ◽  
Markus Fries ◽  
M. Christella L. G. D. Thomassen ◽  
Alexandra Heinzmann ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Anticoagulant Activity ◽  
Tight Binding ◽  
Factor Xa ◽  
Factor X ◽  
Intermediate Segment ◽  
Tissue Factor Pathway ◽  
Α Helix

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nm). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nm) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nm). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

Tissue Factor Pathway Inhibitor Inhibits Aortic Smooth Muscle Cell Migration Induced by Tissue Factor/Factor VIIa Complex

1997 ◽  
Vol 78 (03) ◽  
pp. 1138-1141 ◽  
Author(s):  
Yuichiro Sato ◽  
Yujiro Asada ◽  
Kousuke Marutsuka ◽  
Kinta Hatakeyama ◽  
Yuichi Kamikubo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Protease Inhibitor ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Dependent Manner ◽  
Factor X ◽  
Migration Ability ◽  
Tissue Factor Pathway

SummaryTissue factor (TF), a transmembrane glycoprotein, forms a high affinity complex with factor Vll/VIIa (FVIIa) and thereby initiates blood coagulation. Tissue factor pathway inhibitor (TFPI) is an endogenous protease inhibitor of TF/FVIIa-initiated coagulation. We previously reported that TF was a strong chemotactic factor for cultured vascular smooth muscle cells (SMCs). In this study, we examined the contribution of FVIIa and the effect of TFPI to TF-induced cultured SMC migration. TF/FVIIa complex showed a strong migration ability, however, neither TF alone nor FVIIa induced SMC migration. TF/FVIIa treated by a serine protease inhibitor and the complex of TF and inactivated FVIIa (DEGR-FVIIa) did not stimulate SMC migration. Pretreatment with hirudin and the antibodies to a-thrombin and factor X had no effect on TF/FVIIa-induced SMC migration, although a-thrombin and factor Xa also induced SMC migration respectively. TFPI markedly inhibited TF/FVIIa-induced SMC migration in a concentration-dependent manner, but did not affect the SMC migration induced by platelet-derived growth factor (PDGF)-BB, basic fibro blast-growth factor (bFGF), or a-thrombin. These results indicate that the catalytic activity of TF/FVIIa complex is important on SMC migration, and TFPI can reduce SMC migration as well as thrombosis.

Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor

Blood ◽  
2011 ◽  
Vol 117 (20) ◽  
pp. 5514-5522 ◽  
Author(s):  
Emily K. Waters ◽  
Ryan M. Genga ◽  
Michael C. Schwartz ◽  
Jennifer A. Nelson ◽  
Robert G. Schaub ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Intrinsic Pathway ◽  
Extrinsic Pathway ◽  
Tissue Factor Pathway

Abstract Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

scholarly journals Inhibition of Tissue Factor-Factor VIIa-catalyzed Factor X Activation by Factor Xa-Tissue Factor Pathway Inhibitor

1999 ◽  
Vol 274 (40) ◽  
pp. 28225-28232 ◽  
Author(s):  
Irene Salemink ◽  
Jo Franssen ◽  
George M. Willems ◽  
H. Coenraad Hemker ◽  
Theo Lindhout
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor X ◽  
Tissue Factor Pathway ◽  
Factor X Activation

State-of-the-Art Review : Role of Antithrombin and Tissue Factor Pathway Inhibitor in the Control of Thrombosis and Mediation of Heparin Action

1996 ◽  
Vol 2 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Nina Iverson ◽  
Ulrich Abildgaard
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
State Of The Art ◽  
Factor Viia ◽  
Heparin Therapy ◽  
Factor X ◽  
Factor Ixa ◽  
Tissue Factor Pathway ◽  
Blocking Antibodies

Deficiency of any of the two coagulation in hibitors antithrombin (AT) and tissue factor pathway in hibitor (TFPI) lowers the resistance to thrombosis. He reditary deficiency of AT leads to a high risk of throm bosis, which occasionally responds poorly to heparin therapy. Experimental deficiency of TFPI lowers the re sistance to infusion of both tissue factor and endotoxin, both regarding microvascular thrombosis and fatality. Administration of either AT or TFPI protects against mi cro- and macrovascular thrombosis. Injection of heparin and some other glycosaminoglycans releases intima bound TFPI to the blood. Heparin accelerates the inhib itory effects of both inhibitors, in particular the effect of AT. The influence of the two inhibitors on the various anticoagulant reactions have been studied using blocking antibodies. It is suggested that the anticoagulant and an tithrombotic effects of heparin are mainly mediated by the accelerated inactivation of thrombin, factor IXa and factor X by AT, and augmented inactivation of tissue factor-factor VIIa by TFPI released to the blood.

scholarly journals Factor Xa Cleavage of Tissue Factor Pathway Inhibitor Is Associated with Loss of Anticoagulant Activity

1998 ◽  
Vol 80 (08) ◽  
pp. 273-280 ◽  
Author(s):  
Irene Salemink ◽  
Jo Franssen ◽  
George Willems ◽  
Coenraad Hemker ◽  
Anguo Li ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Inhibitory Activity ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Peptide Bond ◽  
Factor X ◽  
Tissue Factor Pathway ◽  
Factor Assay

SummaryTissue factor : factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa : TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa : TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa : TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Arg199-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa : TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa : TFPI towards TF : factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF : factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.

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

1995 ◽  
Vol 74 (03) ◽  
pp. 910-915 ◽  
Author(s):  
Theo Lindhout ◽  
Jo Franssen ◽  
George Willems
Keyword(s):  
Tissue Factor ◽  
Rate Constant ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Full Length ◽  
Factor X ◽  
Tissue Factor Pathway ◽  
Factor X Activation ◽  
Kinetics Of

SummaryTissue factor-factor VIIa catalysed activation of factor X and 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 × 108 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. The rate constants of association of the complex between factor Xa and the variants full length TFPI, TFPI 1-247 and TFPI1-61 were very close to the values determined independently in a kinetic study on the inhibition of factor Xa in the presence of phospholipids, namely 3.4 × 106 M-1s-1, 0.4 × 106 M-1s-1 and 0.3 × 106 M-1s-1, respectively. These results indicate that the factor Xa-dependent inhibition of tissue factor-factor VIIa-catalysed factor X activation by TFPI can be adequately described by the two-step reaction sequence. We found that phospholipids (25 mol % phosphat-idylserine/75 mol % phospatidylcholine) increased the rate constant of association with factor Xa for full length TFPI, but not for the C-ter- minus truncated TFPI. Our results further indicate that optimal inhibition of tissue factor-factor VIIa activity is obtained with full length TFPI because of the higher rate of TFPI-factor Xa complex formation.

Inhibition of Recombinant Human Blood Coagulation Factor VIIa Amidolytic and Proteolytic Activity by Zinc Ions

1991 ◽  
Vol 65 (05) ◽  
pp. 528-534 ◽  
Author(s):  
Anders H Pedersen ◽  
Torben Lund-Hansen ◽  
Yutaka Komiyama ◽  
Lars C Petersen ◽  
Per B Oestergård ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Factor Viia ◽  
Umbilical Vein ◽  
Factor Ix ◽  
Zinc Ions ◽  
Factor X ◽  
Amidolytic Activity ◽  
Carboxyglutamic Acid ◽  
Umbilical Vein Endothelial Cells

SummaryAlthough it is well established that calcium is an essential cofactor in blood coagulation, recent experimental evidence suggests that zinc may also play an important role in hemostasis. In the present study, we have examined the effects of zinc ions on the amidolytic and proteolytic activity of recombinant factor VIIa in the presence of physiological levels of calcium ions. The amidolytic activity of factor VIIa was inhibited half-maximally by 20 ΜM zinc. The amidolytic activity of a derivative of factor VIIa lacking the Γ-carboxyglutamic acid domain was also inhibited half-maximally by 20 ΜM zinc, suggesting that the mechanism of zinc inhibition of factor VIIa amidolytic activity did not involve its Γ-carboxyglutamic acid residues. The amidolytic activity of a complex of recombinant tissue factor and factor VIIa was inhibited half-maximally by 70 ΜM zinc. In contrast to the results obtained with factor VIIa, the amidolytic activities of other human vitamin K-dependent coagulation proteases including factor Xa, thrombin and activated protein C were not appreciably affected by 50-100 ΜM zinc. The proteolytic activation of factor X by a complex of factor VIIa and relipidated tissue factor apoprotein was inhibited half-maximally by 40 ΜM zinc, whereas activation of factor IX in this system was inhibited half-maximally by 70 ΜM zinc ions. Considerably higher levels of zinc (∽100 ΜM) were required to inhibit half-maximally the rate of factor X activation by a complex of factor VIIa and functional tissue factor on the surface of either a human bladder carcinoma cell line, J82, or stimulated human umbilical vein endothelial cells. Activation of factor IX by factor VIIa and tissue factor on the surface of J82 cells was not influenced by zinc. However, the activation rate of factor IX on human umbilical vein endothelial cells was inhibited half-maximally at 100 ΜM zinc. The activation of factor X by factor VIIa in the presence of small unilamellar phospholipid vesicles was inhibited half-maximally by 20 ΜM zinc whereas factor IX activation by factor VIIa was not appreciably influenced by 10-100 ΜM zinc. Our data demonstrate that plasma levels of zinc ions inhibit the amidolytic and proteolytic activities of factor VIIa. The mechanism of this inhibition, as well as its possible physiological relevance, is unknown.

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