Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids

Summary

In the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.

• References

• 1 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-10370
• 2 Wam-Cramer BJ, Rao LMW, Maki SL, Rapaport SI. Modifications of extrinsic pathway inhibitor (EPI) and factor Xa that affect their ability to interact and inhibit factor Vila/tissue factor: evidence for a two-step model of inhibition. Thromb Haemost 1988; 60: 453-456
• 3 Broze GJ, Warren LA, Novotny WF, Higuchi DA, Girard TJ, Miletich JP. The lipoprotein-associated coagulation inhibitor that inhibits the factor Vlla-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action. Blood 1988; 71: 335-343
• 4 Pedersen AH, Nordfang O, Norris F, Wiberg FC, Christensen PM, Møller KB, Meidahl-Pedersen J, 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: 16786-16793
• 5 Callander NS, Rao LVM, Nordfang O, Sandset PM, Wam-Cramer B, Rapaport SI. Mechanisms of binding of recombinant extrinsic pathway inhibitor (EPI) to cultured cell surfaces. Evidence that rEPI can bind to and inhibit factor Vlla-tissue factor complexes in the absence of factor Xa. J Biol Chem 1992; 267: 876-882
• 6 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: 131-136
• 7 Hamamoto T, Yamamoto Y, Nordfang O, Petersen JGL, Foster DC, Kisiel W. Inhibitory properties of full-length and tmncated tissue factor pathway inhibitor. J Biol Chem 1993; 268: 8704-8710
• 8 Valentin S, Reutlingsperger CPM, Nordfang O, Lindhout T. Inhibition of factor X activation at extracellular matrix of fibroblasts during flow conditions: A comparison between tissue factor pathway inhibitor and inactive factor Vila. Thromb Haemost 1995; 74: 1478-1485
• 9 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 inhibitor domains. J Biol Chem 1988; 263: 6001-6004
• 10 Girard TJ, Warren LA, Novotny WF, Likert KM, Brown SG, Miletich JP, Broze GJ. Functional significance of the Kunitz-type inhibitor domains of lipoprotein-associated coagulation inhibitor. Nature 1989; 338: 518-520
• 11 Wesselschmidt R, Likert K, Huang Z, MacPhail L, Broze GJ. Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin. Blood Coag Fibrinol 1993; 4: 661-669
• 12 Petersen JGL, Meyn G, Rasmussen JS, Christiansen L, Petersen J, Bjøm SE, Jonassen I, Nordfang O. Characterization of human tissue factor pathway inhibitor (TFPI) variants expressed in Saccharomyces cerevisae. J Biol Chem 1993; 268: 13344-13351
• 13 Sandset PM, Abildgaard U, Larsen ML. Heparin induces release of extrinsic coagulation pathway inhibitor (EPI). Thromb Res 1988; 50: 803-813
• 14 Novotny WF, Girard TJ, Miletich JP, Broze GJ. Purification and characterization of lipoprotein-associated coagulation inhibitor from human plasma. J Biol Chem 1989; 264: 18832-18837
• 15 Novotny WF, Girard TF, Miletich JP, Broze GJ. Platelets secrete a coagulation inhibitor functionally and antigenically similar to the lipoprotein-associated coagulation inhibitor. Blood 1988; 71: 2020-2025
• 16 Rapaport SI, Rao LVM. The tissue factor pathway: How it has become a “prima ballerina”. Thromb Haemost 1995; 74: 7-17
• 17 Nordfang O, Bjøm 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: 10371-10376
• 18 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: 388-394
• 19 Schousboe I, Halkier T. Zinc ions promote the binding of factor XII/ factor Xlla to acidic phospholipids but have no effect on the binding of high-Mr kininogen. Eur J Biochem 1991: 309-314
• 20 Bloom JW. The interactions of factors X and IX with phospholipid. Thromb Res 1989; 54: 261-268
• 21 Valentin S, Nordfang O, Bregengard 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: 713-720
• 22 Broze GJ, Lange GW, Duffin KL, MacPhail L. Heterogeneity of plasma tissue factor pathway inhibitor. Blood Coag Fibrinol 1994; 5: 551-559