Heterozygous antithrombin deficiency improves in vivo haemostasis in factor VIII-deficient mice

Summary

Decreased levels of factor VIII (FVIII) limit the amount of thrombin generated at the site of injury, but not the rate that thrombin is neutralised by antithrombin (AT). We hypothesised that FVIII-deficient mice with heterozygous AT deficiency will demonstrate increased thrombin generation and therefore less in vivo bleeding compared to FVIII-deficient mice with normal AT levels. Therefore, we performed tail bleeding experiments in wild-type (WT), heterozygous AT deficient (AT^+/-) mice, FVIII-deficient (FVIII^-/-) mice, and FVIII-deficient mice with heterozygous AT deficiency (FVIII^-/-/AT^+/-). Amount of bleeding was assessed by measuring absorbance of haemoglobin released from lysed red blood cells collected after tail transection. In addition, we measured thrombin generation, activated partial thromboplastin time (aPTT), and AT activity in plasma from the different mice groups. Tail bleeding was significantly reduced in FVIII^-/-/AT^+/- mice compared to FVIII^-/- mice. On the other hand, there was no difference in tail bleeding between AT^+/- and wild-type mice. Thrombin generation was dependent on the mice geno-type, and increased in the following order: FVIII^-/- < FVIII^-/-/AT^+/- < WT < AT^+/-. The aPTT was not influenced by reduced AT activity (i.e. AT^+/- genotype), but was significantly prolonged in FVIII^-/- and FVIII^-/-/AT^+/- mice. Using FVIII-deficient mice as an in vivo murine model of reduced thrombin generation, we demonstrated that moderately reduced AT levels increase thrombin generation and decrease bleeding after traumatic tail vessel injury. In agreement with congenital thrombotic conditions, our data elucidate that bleeding phenotypes can be modulated by the balance between procoagulant and anticoagulant proteins.

Keywords

Antithrombin - thrombin generation - coagulation factors - haemophilia

• References

• 1 Tanaka KA, Key NS, Levy JH. Blood coagulation: hemostasis and thrombin regulation. Anesth Analg 2009; 108: 1433-1446.
• 2 Baugh RJ, Broze Jr. GJ, Krishnaswamy S. Regulation of extrinsic pathway factor Xa formation by tissue factor pathway inhibitor. J Biol Chem 1998; 273: 4378-4386.
• 3 Lu G, Broze Jr. GJ, Krishnaswamy S. Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III. J Biol Chem 2004; 279: 17241-17249.
• 4 Jesty J, Beltrami E. Positive feedbacks of coagulation: their role in threshold regulation. Arterioscler Thromb Vasc Biol 2005; 25: 2463-2469.
• 5 Mackman N. The role of tissue factor and factor VIIa in hemostasis. Anesth Analg 2009; 108: 1447-1452.
• 6 Wolberg AS, Allen GA, Monroe DM. et al. High dose factor VIIa improves clot structure and stability in a model of haemophilia B. Br J Haematol 2005; 131: 645-655.
• 7 Szlam F, Taketomi T, Sheppard CA. et al. Antithrombin affects hemostatic response to recombinant activated factor VII in factor VIII deficient plasma. Anesth Analg 2008; 106: 719-724.
• 8 Bolliger D, Szlam F, Molinaro RJ. et al. Finding the optimal concentration range for fibrinogen replacement after severe haemodilution: an in vitro model. Br J Anaesth 2009; 102: 793-799.
• 9 Cvirn G, Gallistl S, Leschnik B. et al. Low tissue factor pathway inhibitor (TFPI) together with low antithrombin allows sufficient thrombin generation in neonates. J Thromb Haemost 2003; 01: 263-268.
• 10 Fritsch P, Cvirn G, Cimenti C. et al. Thrombin generation in factor VIII-depleted neonatal plasma: nearly normal because of physiologically low antithrombin and tissue factor pathway inhibitor. J Thromb Haemost 2006; 04: 1071-1077.
• 11 Bi L, Sarkar R, Naas T. et al. Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies. Blood 1996; 88: 3446-3450.
• 12 Ishiguro K, Kojima T, Kadomatsu K. et al. Complete antithrombin deficiency in mice results in embryonic lethality. J Clin Invest 2000; 106: 873-878.
• 13 Sambrano GR, Weiss EJ, Zheng YW. et al. Role of thrombin signalling in platelets in haemostasis and thrombosis. Nature 2001; 413: 74-78.
• 14 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33: 4-15.
• 15 Tanaka KA, Szlam F, Vinten-Johansen J. et al. Effects of antithrombin and heparin cofactor II levels on anticoagulation with Intimatan. Thromb Haemost 2005; 94: 808-813.
• 16 Meeks SL, Healey JF, Parker ET. et al. Non-classical anti-factor VIII C2 domain antibodies are pathogenic in a murine in vivo bleeding model. J Thromb Haemost 2009; 07: 658-664.
• 17 Ovlisen K, Kristensen AT, Valentino LA. et al. Hemostatic effect of recombinant factor VIIa, NN1731 and recombinant factor VIII on needle-induced joint bleeding in hemophilia A mice. J Thromb Haemost 2008; 06: 969-975.
• 18 Schlachterman A, Schuettrumpf J, Liu JH. et al. Factor V Leiden improves in vivo hemostasis in murine hemophilia models. J Thromb Haemost 2005; 03: 2730-2737.
• 19 Tsakiris DA, Scudder L, Hodivala-Dilke K. et al. Hemostasis in the mouse (Mus musculus): a review. Thromb Haemost 1999; 81: 177-188.
• 20 De Nanteuil G, Gloanec P, Beguin S. et al. Low molecular weight activated protein C inhibitors as a potential treatment for hemophilic disorders. J Med Chem 2006; 49: 5047-5050.
• 21 van ’t Veer C, Golden NJ, Kalafatis M. et al. An in vitro analysis of the combination of hemophilia A and factor V (LEIDEN). Blood 1997; 90: 3067-3072.
• 22 Franchini M, Montagnana M, Targher G. et al. Interpatient phenotypic inconsistency in severe congenital hemophilia: a systematic review of the role of inherited thrombophilia. Semin Thromb Hemost 2009; 35: 307-312.
• 23 van Dijk K, van der Bom JG, Fischer K. et al. Do prothrombotic factors influence clinical phenotype of severe haemophilia? A review of the literature. Thromb Haemost 2004; 92: 305-310.
• 24 Mosnier LO, Bouma BN. Regulation of fibrinolysis by thrombin activatable fibrinolysis inhibitor, an unstable carboxypeptidase B that unites the pathways of coagulation and fibrinolysis. Arterioscler Thromb Vasc Biol 2006; 26: 2445-2453.
• 25 Kitchens CS. To bleed or not to bleed?. Is that the question for the PTT? J Thromb Haemost 2005; 03: 2607-2611.
• 26 Mann KG. Thrombin formation. Chest 2003; 124: 4S-10S.
• 27 Beltran-Miranda CP, Khan A, Jaloma-Cruz AR. et al. Thrombin generation and phenotypic correlation in haemophilia A. Haemophilia 2005; 11: 326-334.
• 28 Al Dieri R, Peyvandi F, Santagostino E. et al. The thrombogram in rare inherited coagulation disorders: its relation to clinical bleeding. Thromb Haemost 2002; 88: 576-582.
• 29 Hemker HC, Al Dieri R, De Smedt E. et al. Thrombin generation, a function test of the haemostatic-thrombotic system. Thromb Haemost 2006; 96: 553-561.
• 30 Tanaka KA, Szlam F, Rusconi CP. et al. In-vitro evaluation of anti-factor IXa ap-tamer on thrombin generation, clotting time, and viscoelastometry. Thromb Haemost 2009; 101: 827-833.
• 31 Rodgers GM. Role of antithrombin concentrate in treatment of hereditary anti-thrombin deficiency. An update. Thromb Haemost 2009; 101: 806-812.
• 32 Kawanaka H, Akahoshi T, Kinjo N. et al. Impact of antithrombin III concentrates on portal vein thrombosis after splenectomy in patients with liver cirrhosis and hypersplenism. Ann Surg 2010; 251: 76-83.
• 33 Kempton CL, Escobar MA, Roberts HR. Hemophilia care in the 21st century. Clin Adv Hematol Oncol 2004; 02: 733-740.
• 34 Menache D, Grossman BJ, Jackson CM. Antithrombin III: physiology, deficiency, and replacement therapy. Transfusion 1992; 32: 580-588.
• 35 Shetty S, Vora S, Kulkarni B. et al. Contribution of natural anticoagulant and fibrinolytic factors in modulating the clinical severity of haemophilia patients. Br J Haematol 2007; 138: 541-544.
• 36 Schulman S, Eelde A, Holmstrom M. et al. Validation of a composite score for clinical severity of hemophilia. J Thromb Haemost 2008; 06: 1113-1121.
• 37 Yanada M, Kojima T, Ishiguro K. et al. Impact of antithrombin deficiency in thrombogenesis: lipopolysaccharide and stress-induced thrombus formation in heterozygous antithrombin-deficient mice. Blood 2002; 99: 2455-2458.
• 38 Rapaport SI, Toneff T, Rimon A. et al. The effect of immunodepletion of anti-thrombin III on the response of rabbits to Russell’s viper venom-induced activation of factor X. Arterioscler Thromb Vasc Biol 1997; 17: 409-416.
• 39 Broze Jr. GJ, Yin ZF, Lasky N. A tail vein bleeding time model and delayed bleeding in hemophiliac mice. Thromb Haemost 2001; 85: 747-748.
• 40 Kahn ML, Zheng YW, Huang W. et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-694.