Phospholipid-bound Tissue Factor Modulates both Thrombin Generation and APC-mediated Factor Va Inactivation

Summary

Hemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.

Keywords

Tissue factor - thrombin generation - factor Va - activated protein C

• References

• 1 Rosenberg RD, Rosenberg JS. Natural anticoagulant mechanisms. J Clin Invest 1984; 74: 1-6.
• 2 Esmon CT, Schwarz HP. An update on clinical and basic aspects of the protein C anticoagulant pathway. Trends in Cardiovasc Med 1995; 5: 141-8.
• 3 Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989; 264: 4743-6.
• 4 Bick RL, Pegram M. Syndromes of hypercoagulability and thrombosis: A review. Sem Thromb Hemost 1994; 20: 109-32.
• 5 Nielsen HK. Pathophysiology of venous thromboembolism. Sem Thromb Hemost 1991; 17 (Suppl. 03) 250-3.
• 6 Esmon CT, Taylor Jr FB, Snow TR. Inflammation and coagulation: Linked processes potentially regulated through a common pathway mediated by protein C. Thromb Hemost 1991; 66: 160-5.
• 7 Camerer E, Kolstø A-B, Prydz H. Cell biology of tissue factor, the principal initiator of blood coagulation. Thromb Res 1996; 81: 1-41.
• 8 Carson SD, Brozna JP. The role of tissue factor in the production of thrombin. Blood Coagulat Fibrinol 1993; 4: 281-92.
• 9 Esmon CT. Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. FASEB J 1995; 9: 946-55.
• 10 Sadler JE, Lentz SR, Sheehan JP, Tsiang M, Wu Q. Structure-function relationships of the thrombin-thrombomodulin interaction. Haemostasis 1993; 23 (Suppl. 01) 183-93.
• 11 Walker FJ, Chavin SI, Fay PJ. Inactivation of factor VIII by activated protein C and protein S. Arch Biochem Biophys 1987; 252: 322-8.
• 12 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 63: 486-9.
• 13 Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of human factor V and human factor Va by activated protein C. J Biol Chem 1994; 269: 31869-80.
• 14 Lu D, Kalafatis M, Mann KG, Long GL. Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V. Blood 1996; 87: 4708-17.
• 15 Dreyfus M, Magny JF, Bridey F, Schwarz HP, Planché C, Dehan M, Tchernia G. Treatment of homozygous protein C deficiency and neonatal purpura fulminans with a purified protein C concentrate. N Engl J Med 1991; 325: 1565-8.
• 16 Tans G, Rosing J, Thomassen MCLDG, Heeb MJ, Zwaal RFA, Griffin JH. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991; 77: 2641-8.
• 17 Brummelhuis HGJ. Preparation of the prothrombin complex. In: Curling JM. (ed): Methods of Plasma Protein Fractionation. New York: Academic Press; 1980: 117.
• 18 Turecek PL, Gritsch H, Richter G, Pichler L, Auer W, Schwarz HP. A complex of prothrombin and activated factor X: A two-component therapeutic for the treatment of coagulopathies, particularly hemophilia with inhibitor. Ann Haematol 1997; 74 (Suppl. 02) A111.
• 19 Sultan Y, Loyer F. In vitro evaluation of factor VIII-bypassing activity of activated prothrombin complex concentrate, prothrombin complex concentrate, and factor VIIa in the plasma of patients with factor VIII inhibitors. Thrombin generation test in the presence of collagen-activated platelets. J Lab Clin Med 1993; 121: 444-52.
• 20 Hope MJ, Bally MB, Webb G, Cullis PR. Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochem Biophys Acta 1985; 812: 55-65.
• 21 Ruf H, Georgalis Y, Grell E. Dynamic laser light scattering to determine size distribution of vesicles. Meth Enzymol 1989; 172: 364-90.
• 22 Pitlick FA, Nemerson Y. Binding of the protein component of tissue factor to phospholipids. Biochemistry 1970; 9: 5105-13.
• 23 Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. The plasma membrane. In: Molecular Biology of the Cell (Chapt 6). New York, London: Garland Publishing Inc; 1989: 276.
• 24 Nicolaes GAF, Tans G, Thomassen MCLGD, Hemker HC, Pabinger I, Varadi K, Schwarz HP, Rosing J. Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor Va^R506Q by activated protein C. J Biol Chem 1995; 270: 21158-66.
• 25 Kessels H, Willems G, Hemker HC. Analysis of thrombin generation in plasma. Comput Biol Med 1994; 24: 277-88.
• 26 Willems GM, Lindhout T, Hermens WT, Hemker HC. Simulation model for thrombin generation in plasma. Haemostasis 1991; 21: 197-207.
• 27 Béguin S, Kessels H, Dol F, Hemker HC. The consumption of antithrombin III during coagulation. Its consequences for the calculation of prothrombinase activity and the standardisation of heparin activity. Thromb Haemost 1992; 68: 136-42.
• 28 Duchemin J, Pittel J-L, Tartary M, Béguin S, Gaussem P, Alhenc-Gelas M, Aiach M. A new assay based on thrombin generation inhibition to detect both protein-C and protein-S deficiencies in plasma. Thromb Haemost 1994; 71: 331-8.
• 29 Hemker HC, Willems GM, Béguin S. A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb Haemost 1986; 56: 9-17.
• 30 Bauer KA, Kass BL, Beeler DL, Rosenberg RD. Detection of protein C activation in humans. J Clin Invest 1984; 74: 2033-41.
• 31 Francis RB, Seyfert U. Rapid amidolytic assay of protein C in whole plasma using an activator from the venom of Agkistrodon contortrix. Am J Clin Pathol 1987; 87: 619-25.
• 32 Gruber A, Griffin JH. Direct detection of activated protein C in blood from human subjects. Blood 1992; 79: 2340-8.
• 33 Ohishi R, Watanabe N, Aritomi M, Gomi K, Kiyota T, Yamamoto S, Ishida T, Maruyama I. Evidence that the protein C activation pathway amplifies the inhibition of thrombin generation by recombinant human thrombomodulin in plasma. Thromb Haemost 1993; 70: 423-6.
• 34 Bakker HM, Tans G, Janssen-Claessen T, Thomassen MCLGD, Hemker HC, Griffin JH, Rosing J. The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C. Eur J Biochem 1992; 208: 171-8.
• 35 Hall AJ, Vos HL, Bertina RM. Lipopolysaccharide induction of tissue factor in THP-1 cells involves Jun protein phosphorylation and nuclear factor kappaB nuclear translocation. J Biol Chem 1999; 274: 376-83.
• 36 Roberts HR, Monroe DM, Oliver JA, Chang JY, Hoffman M. Newer concepts of blood coagulation. Haemophilia 1998; 4: 331-4.
• 37 Morrissey JH, Neuenschwander PF, Huang Q, McCallulm CD, Su B, Johnson AE. Factor VIIa-tissue factor: functional importance of protein-membrane interactions. Thromb Haemost 1997; 78: 112-6.
• 38 Nemerson Y. Analysis of the kinetics of factor X activation by tissue factor-factor VIIa. Haemostasis 1996; 26 (Suppl. 04) 98-101.
• 39 Dahlbäck B. The protein C anticoagulant system: Inherited defects as basis for venous thrombosis. Thromb Res 1995; 77: 1-43.
• 40 Martin DMA, Wiiger MT, Prydz H. Tissue factor and biotechnology. State of the art article. Thromb Res 1998; 90: 1-25.
• 41 Ruf W, Edgington TS. Structural biology of tissue factor, the initiator of thrombogenesis in vivo. FASEB J 1994; 8: 386-90.
• 42 Carmeliet P, Collen D. Molecules in focus; tissue factor. Int J Biochem Cell Biol 1998; 30: 661-7.
• 43 Weiss DJ, Rashid J. The sepsis coagulant Axis: a review. J Vet Intern Med 1998; 12: 317-24.