Additive effects of anticoagulants: recombinant human activated protein C and heparin or melagatran, in tissue factor-activated umbilical-cord plasma

Summary

Severe sepsis in children or adults may cause a life-threatening coagulopathy, with widespread consumption of activated protein C (APC); recombinant human APC (rhAPC) is a promising candidate anticoagulant treatment. We investigated the effects of rhAPC and other anticoagulants on coagulation triggered by adding small quantities of lipidated tissue factor to human umbilical-cord plasma in vitro. rhAPC, unfractionated heparin (UH),and melagatran (a direct thrombin inhibitor) were studied individually, and in combinations of rhAPC with either UH or melagatran. rhAPC alone dose-dependently prolonged the activated partial-thromboplastin time (aPTT) but not the prothrombin time (PT), and dose-dependently suppressed two indices of thrombin generation, namely prothrombin fragment F 1.2 (F 1.2) generation and thrombin–antithrombin (TAT) complex formation. UH alone dose-dependently prolonged the aPTT but not the PT, while melagatran alone dose-dependently prolonged both the aPTT and the PT. Adding either UH or melagatran dose-dependently augmented the capacity of rhAPC to suppress F 1.2 generation (with addition of UH showing a greater effect) and TAT formation (with addition of melagatran showing a greater effect). Both the capacity of UH to prolong the aPTT and the capacity of melagatran to prolong the aPTT and the PT were augmented by adding rhAPC. In our in-vitro study, adding either UH or melagatran augmented the capacity of rhAPC to suppress thrombin generation in human umbilical-cord plasma, with the anticoagulant effect of melagatran being more predictable than that of UH. Hence, combining rhAPC with melagatran might be a valuable therapeutic option in patients with severe sepsis.

Keywords

Anticoagulation - heparin - melagatran - recombinant human activated protein C - thrombin

• References

• 1 Bernard GR, Vincent J-L, Laterre P-F. et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699-709.
• 2 Barton P, Kalil AC, Nadel S. et al. Safety, pharmacokinetics, and pharmacodynamics of drotrecogin alfa (activated) in children with severe sepsis. Pediatrics 2004; 113: 7-17.
• 3 Rawicz M, Sitkowska B, Rudzinska I. et al. Recombinant human activated protein C for severe sepsis in a neonate. Med Sci Monit 2002; 8: CS90-CS94.
• 4 Sajan I, Da-Silva SS, Dellinger RP. Drotrecogin alfa (activated) in an infant with gram-negative septic shock. J Intensive Care Med 2004; 19: 51-5.
• 5 Suzuki K, Stenflo J, Dahlback B. et al. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914-20.
• 6 Fulcher CA, Gardiner JE, Griffin JH. et al. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 63: 486-9.
• 7 Dahlbäck B, Villoutreix BO. Molecular recognition in the protein C anticoagulant pathway. J Thromb Haemost 2003; 1: 1525-34.
• 8 Scully MF, Toh CH, Hoogendoorn H. et al. Activation of protein C and its distribution between its inhibitors, protein C inhibitor, alpha 1-antitrypsin and alpha 2-macroglobulin, in patients with disseminated intravascular coagulation. Thromb Haemost 1993; 69: 448-53.
• 9 Cvirn G, Gallistl S, Muntean W. Alpha-2-macroglobulin inhibits the anticoagulant action of activated protein C in cord and adult plasma. Haemostasis 2001; 31: 1-11.
• 10 Petäjä J, Fernández JA, Gruber A. et al. Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C. J Clin Invest 1997; 99: 2655-63.
• 11 van 't Veer C, Golden NJ, Kalafatis M. et al. Inhibitory mechanism of the protein C pathway on tissue factor-induced thrombin generation. Synergistic effect in combination with tissue factor pathway inhibitor. J Biol Chem 1997; 272: 7983-94.
• 12 Cvirn G, Gallistl S, Rehak T. et al. Elevated thrombin- forming capacity of tissue factor-activated cord compared with adult plasma. J Thromb Haemost 2003; 1: 1785-90.
• 13 Andrew M, Paes B, Milner R. et al. Development of the human coagulation system in the full-term infant. Blood 1987; 70: 165-72.
• 14 Gustafsson D, Antonsson T, Bylund R. et al. Effects of melagatran, a new low-molecular-weight thrombin inhibitor, on thrombin and fibrinolytic enzymes. Thromb Haemost 1998; 79: 110-8.
• 15 Butenas S, van t' Veer C, Mann KG. "Normal” thrombin generation. Blood 1999; 94: 2169-78.
• 16 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-70.
• 17 Andrew M, Paes B, Johnston M. Development of the hemostatic system in the neonate and young infant. Am J Pediatr Hematol Oncol 1990; 12: 95-104.
• 18 Dhainaut J-F, Yan SB, Margolis BD. et al. Drotrecogin alfa (activated) (recombinant human activated protein C) reduces host coagulopathy response in patients with severe sepsis. Thromb Haemost 2003; 90: 642-53.
• 19 Dhainaut J-F, Laterre P-F, Janes JM. et al. Drotrecogin alfa (activated) in the treatment of severe sepsis patients with multiple-organ dysfunction: data from the PROWESS trial. Intensive Care Med 2003; 29: 894-903.
• 20 Fernández JA, Petäjä J, Griffin JH. Dermatan sulfate and LMW heparin enhance the anticoagulant action of activated protein C. Thromb Haemost 1999; 82: 1462-8.
• 21 Cvirn G, Gallistl S, Leschnik B, Muntean W. Low tissue factor pathway inhibitor (TFPI) together with low antithrombin allows sufficient thrombin generation in neonates. J Thromb Haemost 2003; 1: 263-8.
• 22 Cvirn G, Gallistl S, Koestenberger M. et al. The anticoagulant action of recombinant human activated protein C (rhAPC, Drotrecogin alpha activated): comparison between cord and adult plasma. Thromb Haemost 2004; 91: 912-8.
• 23 Schmidt B, Ofosu FA, Mitchell L. et al. Anticoagulant effects of heparin in neonatal plasma. Pediatr Res 1989; 25: 405-8.
• 24 Chan AK, Berry LR, Monagle PT. et al. Decreased concentrations of heparinoids are required to inhibit thrombin generation in plasma from newborns and children compared to plasma from adults due to reduced thrombin potential. Thromb Haemost 2002; 87: 606-13.
• 25 Weitz JI, Hudoba M, Massel D. et al. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-91.
• 26 Gustafsson D, Nyström J-E, Carlsson S. et al. The direct thrombin inhibitor melagatran and its oral prodrug H 376/95: intestinal absorption properties, biochemical and pharmacodynamic effects. Thromb Res 2001; 101: 171-81.
• 27 Hirsh J, O'Donnell M, Weitz JI. New anticoagulants. Blood 2005; 105: 453-63.
• 28 White B, Perry D. Acquired antithrombin deficiency in sepsis. Br J Haematol 2001; 112: 26-31.
• 29 Koestenberger M, Gallistl S, Cvirn G. et al. Anticoagulant action of melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, in umbilical cord and adult plasma: an in vitro examination. Thromb Res 2005; 115: 135-42.
• 30 Koestenberger M, Cvirn G, Gallistl S. et al. Drotrecogin alfa activated (recombinant human activated protein C) in combination with heparin or melagatran: effects on prothrombin time and activated partial thromboplastin time. Blood Coagul Fibrinolysis 2004; 15: 693-7.
• 31 Frassica JJ, Vinagre YM, Maas B. Recombinant human-activated protein C (rhAPC) in childhood sepsis. J Intensive Care Med 2004; 19: 56-7.