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Thromb Haemost 2016; 115(03): 580-590
DOI: 10.1160/th15-04-0354
Coagulation and Fibrinolysis
Schattauer GmbH

Role of exosite binding modulators in the inhibition of Fxa by TFPI

Sameera Peraramelli   *
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands
,
Stella Thomassen   *
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands
,
Alexandra Heinzmann
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands
,
Tilman M. Hackeng
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands
,
Rudolf Hartmann
2   Baxalta Innovations GmbH, Vienna, Austria
,
Friedrich Scheiflinger
2   Baxalta Innovations GmbH, Vienna, Austria
,
Michael Dockal
2   Baxalta Innovations GmbH, Vienna, Austria
,
Jan Rosing
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands
› Author Affiliations
Further Information

Publication History

Received: 29 April 2015

Accepted after major revision: 21 October 2015

Publication Date:
20 March 2018 (online)

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Summary

Tissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 μM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2–1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1–150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25–0.5 nM TFPI) inhibit FXa with a t1/2 between 3–15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.

Keywords

Exosites - prothrombin - factor V/Va - factor Xa - TFPI

* These authors contributed equally.


 

  • References

  • 1 Rao LV, Rapaport SI. Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation. Blood 1987; 69: 645-651.
    PubMedGoogle Scholar
  • 2 Broze Jr GJ, Warren LA, Girard JJ. et al. Isolation of the lipoprotein associated coagulation inhibitor produced by HepG2 (human hepatoma) cells using bovine factor Xa affinity chromatography. Thromb Res 1987; 48: 253-259.
    CrossrefPubMedGoogle Scholar
  • 3 Broze Jr GJ, Warren LA, Novotny WF. et al. The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action. Blood 1988; 71: 335-343.
    PubMedGoogle Scholar
  • 4 Wun TC, Kretzmer KK, Girard TJ. et al. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. J Biol Chem 1988; 263: 6001-6004.
    PubMedGoogle Scholar
  • 5 Chang JY, Monroe DM, Oliver JA. et al. TFPIbeta, a second product from the mouse tissue factor pathway inhibitor (TFPI) gene. Thromb Haemost 1999; 81: 45-49.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 6 Zhang J, Piro O, Lu L. et al. Glycosyl phosphatidylinositol anchorage of tissue factor pathway inhibitor. Circulation 2003; 108: 623-627.
    CrossrefPubMedGoogle Scholar
  • 7 Girard TJ, Warren LA, Novotny WF. et al. Functional significance of the Kunitztype inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 1989; 338: 518-520.
    CrossrefPubMedGoogle Scholar
  • 8 Broze Jr GJ, Girard TJ, Novotny WF. Regulation of coagulation by a multivalent Kunitz-type inhibitor. Biochemistry 1990; 29: 7539-7546.
    CrossrefPubMedGoogle Scholar
  • 9 Huang ZF, Wun TC, Broze Jr GJ. Kinetics of factor Xa inhibition by tissue factor pathway inhibitor. J Biol Chem 1993; 268: 26950-26955.
    PubMedGoogle Scholar
  • 10 Morrison JF, Walsh CT. The behavior and significance of slow-binding enzyme inhibitors. Advances in enzymology and related areas of molecular biology 1988; 61: 201-301.
    PubMedGoogle Scholar
  • 11 Ndonwi M, Tuley EA, Broze Jr. GJ. The Kunitz-3 domain of TFPI-alpha is required for protein S-dependent enhancement of factor Xa inhibition. Blood 2010; 116: 1344-1351.
    CrossrefPubMedGoogle Scholar
  • 12 Ahnstrom J, Andersson HM, Hockey V. et al. Identification of functionally important residues in TFPI Kunitz domain 3 required for the enhancement of its activity by protein S. Blood 2012; 120: 5059-5062.
    CrossrefPubMedGoogle Scholar
  • 13 Sandset PM, Abildgaard U, Larsen ML. Heparin induces release of extrinsic coagulation pathway inhibitor (EPI). Thromb Res 1988; 50: 803-813.
    CrossrefPubMedGoogle Scholar
  • 14 Lindahl AK, Abildgaard U, Stokke G. Release of extrinsic pathway inhibitor after heparin injection: increased response in cancer patients. Thromb Res 1990; 59: 651-656.
    CrossrefPubMedGoogle Scholar
  • 15 Ndonwi M, Girard TJ, Broze Jr. GJ. The C-terminus of tissue factor pathway inhibitor alpha is required for its interaction with factors V and Va. J Thromb Haemost 2012; 10: 1944-1946.
    CrossrefPubMedGoogle Scholar
  • 16 Valentin S, Schousboe I. Factor Xa enhances the binding of tissue factor pathway inhibitor to acidic phospholipids. Thromb Haemost 1996; 75: 796-800.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Valentin S, Nordfang O, Bregengard C. et al. Evidence that the C-terminus of tissue factor pathway inhibitor (TFPI) is essential for its in vitro and in vivo interaction with lipoproteins. Blood Coagul Fibrinolysis 1993; 4: 713-720.
    CrossrefPubMedGoogle Scholar
  • 18 Yegneswaran S, Hackeng TM, Dawson PE. et al. The thrombin-sensitive region of protein S mediates phospholipid-dependent interaction with factor Xa. J Biol Chem 2008; 283: 33046-33052.
    CrossrefPubMedGoogle Scholar
  • 19 Bjork I, Olson ST. Antithrombin. A bloody important serpin. Adv Exp Med Biol 1997; 425: 17-33.
    PubMedGoogle Scholar
  • 20 McRae SJ, Stafford AR, Fredenburgh JC. et al. In the presence of phospholipids, glycosaminoglycans potentiate factor Xa-mediated protein C activation by modulating factor Xa activity. Biochemistry 2007; 46: 4195-4203.
    CrossrefPubMedGoogle Scholar
  • 21 Mast AE, Broze Jr. GJ. Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase. Blood 1996; 87: 1845-1850.
    PubMedGoogle Scholar
  • 22 Hackeng TM, Sere KM, Tans G. et al. Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor. Proc Natl Acad Sci USA 2006; 103: 3106-3111.
    CrossrefPubMedGoogle Scholar
  • 23 Franssen J, Salemink I, Willems GM. et al. Prothrombinase is protected from in-activation by tissue factor pathway inhibitor: competition between prothrombin and inhibitor. Biochem J 1997; 323: 33-37.
    CrossrefPubMedGoogle Scholar
  • 24 Castoldi E, Simioni P, Tormene D. et al. Hereditary and acquired protein S deficiencies are associated with low TFPI levels in plasma. J Thromb Haemost 2012; 8: 294-300.
    PubMedGoogle Scholar
  • 25 Duckers C, Simioni P, Spiezia L. et al. Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency. Blood 2008; 112: 3615-3623.
    CrossrefPubMedGoogle Scholar
  • 26 Lindahl AK, Jacobsen PB, Sandset PM. et al. Tissue factor pathway inhibitor with high anticoagulant activity is increased in post-heparin plasma and in plasma from cancer patients. Blood Coagul Fibrinolysis 1991; 2: 713-721.
    CrossrefPubMedGoogle Scholar
  • 27 Nishiyama K, Ogawa H, Yasue H. et al. Heparin-releasable endothelial cell-associated tissue factor pathway inhibitor (TFPI) is increased in the coronary circulation after coronary spasm in patients with coronary spastic angina. Thromb Res 1998; 89: 137-146.
    CrossrefPubMedGoogle Scholar
  • 28 Sere KM, Rosing J, Hackeng TM. Inhibition of thrombin generation by protein S at low procoagulant stimuli: implications for maintenance of the hemostatic balance. Blood 2004; 104: 3624-3630.
    CrossrefPubMedGoogle Scholar
  • 29 Dockal M, Hartmann R, Fries M. et al. Small peptides blocking inhibition of factor Xa and tissue factor-factor VIIa by tissue factor pathway inhibitor (TFPI). J Biol Chem 2014; 289: 1732-1741.
    CrossrefPubMedGoogle Scholar
  • 30 Girard TJ, Broze Jr. GJ. Tissue factor pathway inhibitor. Methods Enzymol 1993; 222: 195-209.
    PubMedGoogle Scholar
  • 31 Esnouf MP, Lloyd PH, Jesty J. A method for the simultaneous isolation of factor X and prothrombin from bovine plasma. Biochem J 1973; 131: 781-789.
    CrossrefPubMedGoogle Scholar
  • 32 Peraramelli S, Suylen DP, Rosing J, Hackeng TM. The Kunitz 1 and Kunitz 3 domains of tissue factor pathway inhibitor are required for efficient inhibition of factor Xa. Thromb Haemost 2012; 108: 266-276.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 Kato H. Tissue factor pathway inhibitor; its structure, function and clinical significance. Pol J Pharmacol 1996; 48: 67-72.
    PubMedGoogle Scholar
  • 34 Rezaie AR. Heparin-binding exosite of factor Xa. Trends in cardiovascular medicine 2000; 10: 333-338.
    CrossrefPubMedGoogle Scholar
  • 35 Abildgaard U. Heparin/low molecular weight heparin and tissue factor pathway inhibitor. Haemostasis 1993; 23 (Suppl. 01) 103-106.
    PubMedGoogle Scholar
  • 36 Reglinska-Matveyev N, Andersson HM, Rezende SM. et al. TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain. Blood 2014; 123: 3979-3987.
    CrossrefPubMedGoogle Scholar
  • 37 Walker FJ. Regulation of vitamin K-dependent protein S. Inactivation by thrombin. J Biol Chem 1984; 259: 10335-10339.
    PubMedGoogle Scholar
  • 38 Broze Jr. GJ, Girard TJ. Factor V, tissue factor pathway inhibitor, and east Texas bleeding disorder. J Clin Invest 2013; 123: 3710-3712.
    CrossrefPubMedGoogle Scholar
  • 39 Wood JP, Bunce MW, Maroney SA. et al. Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation. Proc Natl Acad Sci USA 2013; 110: 17838-17843.
    CrossrefPubMedGoogle Scholar
  • 40 Rosing J, Tans G, Govers-Riemslag JW. et al. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-283.
    PubMedGoogle Scholar
  • 41 Warn-Cramer BJ, Rao LV, Maki SL. et al. Modifications of extrinsic pathway inhibitor (EPI) and factor Xa that affect their ability to interact and to inhibit factor VIIa/tissue factor: evidence for a two-step model of inhibition. Thromb Haemost 1988; 60: 453-456.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 42 Wesselschmidt R, Likert K, Huang Z. et al. Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin. Blood Coagul Fibrinolysis 1993; 4: 661-669.
    CrossrefPubMedGoogle Scholar
  • 43 Lindhout T, Franssen J, Willems G. Kinetics of the inhibition of tissue factor-factor VIIa by tissue factor pathway inhibitor. Thromb Haemost 1995; 74: 910-915.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 44 Willems GM, Janssen MP, Salemink I. et al. Transient high affinity binding of tissue factor pathway inhibitor-factor Xa complexes to negatively charged phos-pholipid membranes. Biochemistry 1998; 37: 3321-3328.
    CrossrefPubMedGoogle Scholar
  • 45 Srivasatava KR, Majumder R, Kane WH. et al. Phosphatidylserine and FVa regulate FXa structure. Biochem J 2014; 459: 229-239.
    CrossrefPubMedGoogle Scholar
  • 46 Enjyoji K, Miyata T, Kamikubo Y. et al. Effect of heparin on the inhibition of factor Xa by tissue factor pathway inhibitor: a segment, Gly212-Phe243, of the third Kunitz domain is a heparin-binding site. Biochemistry 1995; 34: 5725-5735.
    CrossrefPubMedGoogle Scholar
  • 47 Tollefsen DM. Insight into the mechanism of action of heparin cofactor II. Thromb Haemost 1995; 74: 1209-1214.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 48 Walenga JM, Jeske WP, Samama MM. et al. Fondaparinux: a synthetic heparin pentasaccharide as a new antithrombotic agent. Expert Opin Investig Drugs 2002; 11: 397-407.
    CrossrefPubMedGoogle Scholar
  • 49 Jesty J, Wun TC, Lorenz A. Kinetics of the inhibition of factor Xa and the tissue factor-factor VIIa complex by the tissue factor pathway inhibitor in the presence and absence of heparin. Biochemistry 1994; 33: 12686-12694.
    CrossrefPubMedGoogle Scholar
  • 50 Olson ST, Chuang YJ. Heparin activates antithrombin anticoagulant function by generating new interaction sites (exosites) for blood clotting proteinases. Trends in cardiovascular medicine 2002; 12: 331-338.
    CrossrefPubMedGoogle Scholar
  • 51 Vincent LM, Tran S, Livaja R. et al. Coagulation factor V(A2440G) causes east Texas bleeding disorder via TFPIalpha. J Clin Invest 2013; 123: 3777-3787.
    CrossrefPubMedGoogle Scholar
  • 52 Ndonwi M, Broze Jr. G. Protein S enhances the tissue factor pathway inhibitor inhibition of factor Xa but not its inhibition of factor VIIa-tissue factor. J Thromb Haemost 2008; 6: 1044-1046.
    CrossrefPubMedGoogle Scholar
  • 53 Krishnaswamy S. Exosite-driven substrate specificity and function in coagulation. J Thromb Haemost 2005; 3: 54-67.
    CrossrefPubMedGoogle Scholar
  • 54 Krishnaswamy S. The transition of prothrombin to thrombin. J Thromb Hae-most 2013; 11 (Suppl. 01) 265-276.
    CrossrefPubMedGoogle Scholar
  • 55 van Rijn JL, Govers-Riemslag JW, Zwaal RF. et al. Kinetic studies of prothrom-bin activation: effect of factor Va and phospholipids on the formation of the enzyme-substrate complex. Biochemistry 1984; 23: 4557-4564.
    CrossrefPubMedGoogle Scholar