Antithrombin is as effective as heparin and hirudin to prevent formation of microvascular thrombosis in a murine model

Summary

A recently published post-hoc analysis of a trial using high-dose antithrombin (AT) in septic patients (KyberSept) revealed significant reduction of lethality when no concomitant heparin was administered, whereas patients with the combination of heparin and AT did not benefit in terms of survival. Therefore, it seems feasible to study the capability of AT in prevention of microvascular thrombus formation to avoid concomitant application of heparin and AT. Using fluorescence microscopy and a light/dyeinjury mouse ear model, the kinetics of thrombus formation were analyzed quantitatively in vivo upon single iv bolus of saline (control), heparin (100 IU/kg), hirudin (1 mg/kg) or AT (25, 50, 100 or 250 IU/kg) (N=7 animals per group each). In controls, light/dye-injury induced complete thrombotic occlusion in all arterioles and venules studied. Heparin and hirudin prevented thrombotic vessel occlusion in 62% and 43% of arterioles and 11% and 28% of venules. AT-250 was found to be more effective than heparin and hirudin, because thrombus formation was completely banned in all arterioles and venules. AT-100 and AT-50 were also capable of significantly blocking thrombus formation in both arterioles and venules. In blood vessels, which finally clogged, the time for development of complete vessel occlusion was delayed after heparin, hirudin and AT-25, but in particular after AT-50 and AT-100. In conclusion, AT-mediated antithrombotic activity has been characterized in a model of phototoxicity-induced microvascular thrombosis formation, demonstrating thatAT delays and prevents thrombus formation in arterioles and venules at least comparably effective as heparin and hirudin.

Keywords

Intravital fluorescence microscopy - hairless mouse - ear model - light/dye injury - occlusion rate

• References

• 1 Wenzel RP. Treating sepsis. N Engl J Med 2002; 347: 966-7.
• 2 Angus DC, Wax RS. Epidemiology of sepsis: an update. Crit Care Med 2001; 29: S109-S116.
• 3 American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992; 20: 864-74.
• 4 ten Cate H. Pathophysiology of disseminated intravascular coagulation in sepsis. Crit Care Med 2000; 28: S9-S11.
• 5 Abraham E, Reinhart K, Svoboda P. et al. Assessment of the safety of recombinant tissue factor pathway inhibitor in patients with severe sepsis: a multicenter, randomized, placebo-controlled, single-blind, dose escalation study. Crit Care Med 2001; 29: 2081-9.
• 6 Levi M, de Jonge E, van der Poll T. New treatment strategies for disseminated intravascular coagulation based on current understanding of the pathophysiology. Ann Med 2004; 36: 41-9.
• 7 Dempfle CE. Coagulopathy of sepsis. Thromb Haemost 2004; 91: 213-24.
• 8 Amaral A, Opal SM, Vincent JL. Coagulation in sepsis. Intensive Care Med 2004; 30: 1032-40.
• 9 Fourrier F, Jourdain M, Tournois A. et al. Coagulation inhibitor substitution during sepsis. Intensive Care Med 1995; 21: S264-S268.
• 10 Mackman N. Regulation of tissue factor gene expression in human monocytic and endothelial cells. Haemostasis 1996; 26: 17-9.
• 11 Roemisch J, Gray E, Hoffmann JN. et al. Antithrombin: a new look at the actions of a serine protease inhibitor. Blood Coagul Fibrinolysis 2002; 13: 657-70.
• 12 Mammen EF. Antithrombin: its physiological importance and role in DIC. Semin Thromb Hemost 1998; 24: 19-25.
• 13 Triantaphyllopoulos DC. Effects of human antithrombin III on mortality and blood coagulation induced in rabbits by endotoxin. Thromb Haemost 1984; 51: 232-5.
• 14 Dickneite G. Antithrombin III in animal models of sepsis and organ failure. Semin Thromb Haemost 1998; 24: 61-9.
• 15 Emerson Jr TE, Fournel MA, Leach WJ. et al. Protection against disseminated intravascular coagulation and death by antithrombin-III in the Escherichia coli endotoxemic rat. Circ Shock 1987; 21: 1-13.
• 16 Hoffmann JN, Vollmar B, Inthorn D. et al. Antithrombin reduces leukocyte adhesion during chronic endotoxemia by modulation of the cyclooxygenase pathway. Am J Physiol Cell Physiol 2000; 279: C98-C107.
• 17 Iba T, Kidokoro A, Fukunaga M. et al. Antithrombin modulates the leukocyte-endothelial cell interaction in the staphylococcal enterotoxin B-challenged mouse. J Trauma 2003; 55: 546-50.
• 18 Horie S, Ishii H, Kazama M. Heparin-like glycosaminoglycan is a receptor for antithrombin III-dependent but not for thrombin-dependent prostacyclin production in human endothelial cells. Thromb Res 1990; 59: 895-904.
• 19 Uchiba M, Okajima K, Murakami K. et al. Attenuation of endotoxin-induced pulmonary vascular injury by antithrombin III. Am J Physiol 1996; 270: L921-930.
• 20 Hoffmann JN, Vollmar B, Romisch J. et al. Antithrombin effects on endotoxin-induced microcirculatory disorders are mediated mainly by its interaction with microvascular endothelium. Crit Care Med 2002; 30: 218-25.
• 21 Hoffmann JN, Vollmar B, Laschke MW. et al. Adverse effect of heparin on antithrombin action during endotoxemia: microhemodynamic and cellular mechanisms. Thromb Haemost 2002; 88: 242-52.
• 22 Warren BL, Eid A, Singer P. et al. and the KyberSept Trial Study GroupCaring for the critically ill patient. High-dose antithrombin III in severe sepsis: a randomized controlled trial. J Am Med Assoc 2001; 286: 1869-78.
• 23 Dellinger RP, Carlet JM, Masur H. et al. and the Surviving Sepsis Campaign Management Guidelines Committee. Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock. Crit Care Med 2004; 32: 858-73.
• 24 Roesken F, Ruecker M, Vollmar B. et al. A new model for quantitative in vivo microscopic analysis of thrombus formation and vascular recanalisation: The ear of the hairless (hr/hr) mouse. Thromb Haemost 1997; 78: 1408-14.
• 25 Fiebig E, Ley K, Arfors KE. Rapid leukocyte accumulation by “spontaneous” rolling and adhesion in the exteriorized rabbit mesentery. Int J Microcirc Clin Exp 1991; 10: 127-44.
• 26 Valenzeno DP. Photomodification of biological membranes with emphasis on singlet oxygen mechanisms. Photochem Photobiol 1987; 46: 147-60.
• 27 Osterlund M, Owenius R, Carlsson K. et al. Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa. Biochemistry 2001; 40: 9324-8.
• 28 Engelmann B, Luther T, Mueller I. Intravascular tissuefactor pathway- a model for rapid initiation of coagulation within the blood vessel. Thromb Haemost 2003; 89: 3-8.
• 29 Inamo J, Belougne E, Doutremepuich C. Importance of photo activation of rose bengal for platelet activation in experimental models of photochemically induced thrombosis. Thromb Res 1996; 83: 229-35.
• 30 Roesken F, Vollmar B, Ruecker M. et al. In vivo analysis of antithrombotic effectiveness of recombinant hirudin on microvascular thrombus formation and recanalization. J Vasc Surg 1998; 28: 498-505.
• 31 Vargas OHein, von Heymann C, Lipps M. et al. Hirudin versus heparin for anticoagulation in continuous renal replacement therapy. Intensive Care Med 2001; 27: 673-9.
• 32 Broeders MA, Tangelder GJ, Slaaf DW. et al. Endogenous nitric oxide and prostaglandins synergistically counteract thromboembolism in arterioles but not in venules. Arterioscler Thromb Vasc Biol 2001; 21: 163-9.
• 33 Harada N, Okajima K, Uchiba M. et al. Antithrombin reduces ischemia/reperfusion-induced liver injury in rats by activation of cyclooxygenase-1. Thromb Haemost 2004; 92: 550-8.
• 34 Nowak G, Bucha E, Goock T. et al. Pharmacology of r-hirudin in renal impairment. Thromb Res 1992; 66: 707-15.
• 35 Estes JW. Clinical pharmacokinetics of heparin. Clin Pharmacokinet 1980; 05: 204-20.
• 36 Knot EA, de Jong E, ten Cate JW. et al. Antithrombin III: biodistribution in healthy volunteers. Thromb Haemost 1987; 58: 1008-11.
• 37 Lu W, Mant T, Levy JH. et al. Pharmacokinetics of recombinant transgenic antithrombin in volunteers. Anesth Analg 2000; 90: 531-4.
• 38 Okajima K. Antithrombin prevents endotoxin-induced pulmonary vascular injury by inhibiting leukocyte activation. Blood Coagul Fibrinolysis 1998; 9 (Suppl. 02) S25-37.
• 39 Isobe H, Okajima K, Uchiba M. et al. Antithrombin prevents endotoxin-induced hypotension by inhibiting the induction of nitric oxide synthase in rats. Blood 2002; 99: 1638-45.
• 40 Mizutani A, Okajima K, Uchiba M. et al. Antithrombin reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation through promotion of prostacyclin production. Blood 2003; 101: 3029-36.
• 41 Hoffmann JN, Vollmar B, Inthorn D. et al. The thrombin antagonist hirudin fails to inhibit endotoxin-induced leukocyte/endothelial cell interaction and microvascular perfusion failure. Shock 2000; 05: 528-34.
• 42 Hoffmann JN, Wiedermann CJ, Juers M. et al. and the KyberSept investigators. Benefit/risk profile of high-dose antithrombin in patients with severe sepsis treated with and without concomitant heparin. Thromb Haemost 2006; 95: 850-6.