Factor Xa Cleavage of Tissue Factor Pathway Inhibitor Is Associated with Loss of Anticoagulant Activity[*]
15 December 1997
Accepted after resubmission 28 April 1998
27 December 2017 (online)
Tissue 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.
* Supported by Grant 902-26-154 from the Dutch Organization for Scientific Research (NWO)
- 1 Broze GJ, Girard TJ, Novotny WF. Regulation of coagulation by a multi-valent Kunitz-type inhibitor. Biochemistry 1990; 29: 7539-46.
- 2 Rapaport SI. The extrinsic pathway inhibitor: A regulator of tissue factor-dependent blood coagulation. Thromb Haemost 1991; 66: 6-15.
- 3 Broze GJ, Warren LA, Novotny WF, Higuchi DA, Girard JJ, Miletich JP. 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: 335-43.
- 4 Novotny WF, Girard TJ, Miletich JP, Broze GJ. Purification and characterization of the lipoprotein-associated coagulation inhibitor from human plasma. J Biol Chem 1989; 264: 18832-7.
- 5 Wun TC, Kretzmer KK, Girard TJ, Miletich JP, Broze GJ. 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: 6001-4.
- 6 Huang ZF, Wun TC, and Broze GJ. Kinetics of Factor-Xa inhibition by tissue factor pathway inhibitor. J Biol Chem 1993; 268: 26950-5.
- 7 Girard TJ, Warren LA, Novotny WF, Likert KM, Brown SG, Miletich JP, Broze GJ. Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 1989; 338: 518-20.
- 8 Petersen LC, Bjørn SE, Olsen OH, Nordfang O, Norris F, Norris K. Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue factor pathway inhibitor. Eur J Bioch 1996; 235: 310-6.
- 9 Enjyoji K, Miyata T, Kamikubo Y, Kato H. 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: 5725-35.
- 10 Valentin S, Nordfang O, Bregengård C, Wildgoose P. Evidence that the C-terminus of tissue factor pathway inhibitor (TFPI) is essential for its in vitro and in vivo interaction with lipoproteins. Blood Coag Fibrinol 1993; 4: 713-20.
- 11 Nordfang O, Bjørn SE, Valentin S, Nielsen LS, Wildgoose P, Beck TC, Hedner U. The C-terminus of tissue factor pathway inhibitor is essential to its anticoagulant activity. Biochemistry 1991; 30: 10371-6.
- 12 Wesselschmidt R, Likert K, Girard T, Wun TC, Broze GJ. Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa. Blood 1992; 79: 2004-10.
- 13 Lindhout T, Willems G, Blezer R, Hemker HC. Kinetics of the inhibition of human factor Xa by full-length and truncated recombinant tissue factor pathway inhibitor. Biochem J 1994; 297: 131-6.
- 14 Lindhout T, Franssen J, Willems G. Kinetics of the inhibition of tissue factor-factor VIIa by tissue factor pathway inhibitor. Thromb Haemost 1995; 74: 910-5.
- 15 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: 12686-94.
- 16 Valentin S, Schousboe I. Factor Xa enhances the binding of tissue factor pathway inhibitor to acidic phospholipids. Thromb Haemost 1996; 75: 796-800.
- 17 Willems GM, Janssen MP, Salemink I, Wun TC, Lindhout T. Transient high affinity binding of Tissue Factor Pathway Inhibitor-Factor Xa complexes to negatively charged phospholipid membranes. Biochemistry 1998; 37: 3321-8.
- 18 Higuchi DA, Wun TC, Likert KM, Broze GJ. The effect of leukocyte elastase on tissue factor pathway inhibitor. Blood 1992; 79: 1712-9.
- 19 Petersen LC, Bjørn SE, Nordfang O. Effect of leukocyte proteinases on tissue factor pathway inhibitor. Thromb Haemost 1992; 67: 537-41.
- 20 Mertens K, Bertina RM. Pathways in the activation of human coagulation factor X. Biochem J 1980; 185: 647-58.
- 21 Lindhout MJ, Kop-Klaassen BH, Hemker HC. Activation of decarboxy-factor X by a protein from Russell’s viper venom. Purification and partial characterization of activated decarboxyfactor X. Biochim Biophys Acta 1978; 533: 327-41.
- 22 Smith RL. Titration of activated bovine Factor X. J Biol Chem 1973; 248: 2418-23.
- 23 Diaz-Collier JA, Palmier MO, Kretzmer KK, Bishop BF, Combs RG, Obukowicz MG, Frazier RB, Bild GS, Joy WD, Hill SR. et al. Refold and characterization of recombinant tissue factor pathway inhibitor expressed in Escherichia coli . Thromb Haemost 1994; 71: 339-46.
- 24 Pedersen AH, Nordfang O, Norris F, Wiberg FC, Christensen PM, Moeller KB, Meidahl PJ, Beck TC, Norris K, Hedner U, Kisiel W. Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions. J Biol Chem 1990; 265: 16786-93.
- 25 Petersen JG, Meyn G, Rasmussen JS, Petersen J, Bjørn SE, Jonassen I, Christiansen L, Nordfang O. Characterization of human tissue factor pathway inhibitor variants expressed in Saccharomyces cerevisiae . J Biol Chem 1993; 268: 13344-51.
- 26 Brinkmann T, Kahnert H, Prohaska W, Nordfang O, Kleesiek K. Synthesis of tissue factor pathway inhibitor in human synovial cells and chondrocytes makes joints the predilected site of bleeding in haemophiliacs. Eur J Clin Chem Clin Biochem 1994; 32: 313-7.
- 27 Erhardtsen E, Ezban M, Madsen MT, Diness V, Glazer S, Hedner U, Nordfang O. Blocking of tissue factor pathway inhibitor (TFPI) shortens the bleeding time in rabbits with antibody-induced hemophilia-A. Blood Coag Fibrinol 1995; 6: 388-94.
- 28 Rosing J, Bakker HM, Thomassen MCLGD, Hemker HC, Tans G. Characterization of two forms of human factor Va with different cofactor activities. J Biol Chem 1993; 268: 21130-6.
- 29 Laemmli MK. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 1970; 227: 680-5.
- 30 Ohkura N, Enjyoji KI, Kamikubo Y, Kato H. A novel degradation pathway of tissue factor pathway inhibitor: incorporation into fibrin clot and degradation by thrombin. Blood 1997; 90: 1883-92.
- 31 Gebhard W, Hochstrasser K. Inter-α-trypsin inhibitor and its close relatives. In: Protease inhibitors. Barret AJ, Salvesen G. (eds) Elsevier; Amsterdam: 1986. p 389.
- 32 Owen WG, Esmon CT, Jackson GM. The conversion of prothrombin to thrombin I. Characterization of the reaction products formed during the activation of bovine prothrombin. J Biol Chem 1974; 249: 594-605.
- 33 Olds RJ, Lane DA, Caso R, Panico M, Morris HR, Sas G, Dawes J, Thein SL. Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family. Blood 1992; 79: 1206-12.
- 34 Harrison HH, Gordon ED, Nichols WC, Benson MD. Biochemical and clinical characterization of prealbumin CHICAGO: an apparently benign variant of serum prealbumin (transthyretin) discovered with high-resolution two-dimensional electrophoresis. Am J Med Genet 1991; 39: 442-52.
- 35 Kazama Y. The importance of the binding of factor Xa to phospholipids in the inhibitory mechanism of Tissue Factor Pathway Inhibitor: The trans-membrane and cytoplasmic domains of Tissue Factor are not essential for the inhibitory action of Tissue Factor Pathway Inhibitor. Thromb Haemost 1997; 77: 492-7.