The effects of supra-normal protein C levels on markers of coagulation, fibrinolysis and inflammation in a human model of endotoxemia

Summary

The protein C pathway serves as a modulating system with both anti-inflammatory and anticoagulant properties and is intimately involved in the pathophysiology of inflammation and sepsis. Treatment with recombinant human activated protein C (rhAPC) can reduce the mortality of severe sepsis. We investigated whether an elevation of plasma protein C levels to supranormal levels by infusion of a protein C zymogen concentrate has an effect on coagulation, protein C activation or inflammation in a human endotoxemia model. Eleven healthy male volunteers were enrolled in a double-blind, placebo-controlled two-way cross-over trial. Ten minutes after infusion of 2ng/kg endotoxin each volunteer received either placebo or a plasmaderived protein C zymogen concentrate (Ceprotin^®, Baxter) (150 U/kg as a slow bolus infusion followed by 30 U/kg/h continuous infusion until 4 hours after LPS-infusion). Protein C antigen and activity increased 4– to 5-fold after infusion of the concentrate. APC was generated during endotoxin-induced inflammation in the placebo (1.6 fold increase) and the protein C period (4.0-fold increase).The increase of APC levels correlated with the TNF-α and IL-6 release in both periods (r=0.65–0.68; p<0.05) and paralleled the protein C antigen and activity levels in the period with supranormal protein C levels. Supra normal protein C levels resulted in slightly, although non-significant, lower tissue factor mRNA expression and thrombin generation (TAT, F1+2). Systemic inflammation (TNF-α, IL-6) was not influenced by protein C zymogen concentrate administration. Infusion of protein C zymogen was safe and no adverse effects occurred. The increase of protein C levels several fold above the normal range resulted in a proportional increase of the APC levels, but had no major anticoagulant, anti-inflammatory or profibrinolytic effects. Low grade endotoxemia itself induces significant protein C activation, which correlates with the TNF release.

Keywords

Endotoxin - protein C - coagulation - inflammation

• References

• 1 Levi M, de Jonge E, van der Poll T. et al Disseminated intravascular coagulation. Thromb Haemost 1999; 82: 695-705.
• 2 Dempfle CE. Coagulopathy of sepsis. Thromb Haemost 2004; 91: 213-24.
• 3 Esmon CT, Taylor Jr. FB, Snow TR. Inflammation and coagulation: linked processes potentially regulated through a common pathway mediated by protein C. Thromb Haemost 1991; 66: 160-5.
• 4 Esmon CT. Role of coagulation inhibitors in inflammation. Thromb Haemost 2001; 86: 51-6.
• 5 Fijnvandraat K, Derkx B, Peters M. et al Coagulation activation and tissue necrosis in meningococcal septic shock: severely reduced protein C levels predict a high mortality. Thromb Haemost 1995; 73: 15-20.
• 6 Fourrier F, Chopin C, Goudemand J. et al Septic shock, multiple organ failure, and disseminated intravascular coagulation. Compared patterns of antithrombin III, protein C, and protein S deficiencies. Chest 1992; 101: 816-23.
• 7 Leclerc F, Hazelzet J, Jude B. et al Protein C and S deficiency in severe infectious purpura of children: a collaborative study of 40 cases. Intensive Care Med 1992; 18: 202-5.
• 8 Mesters RM, Helterbrand J, Utterback BG. et al Prognostic value of protein C concentrations in neutropenic patients at high risk of severe septic complications. Crit Care Med 2000; 28: 2209-16.
• 9 Bernard GR, Vincent JL, Laterre PF. et al Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699-709.
• 10 Faust SN, Levin M, Harrison OB. et al Dysfunction of endothelial protein C activation in severe meningococcal sepsis. N Engl J Med 2001; 345: 408-16.
• 11 Rintala E, Kauppila M, Seppala OP. et al Protein C substitution in sepsis-associated purpura fulminans. Crit Care Med 2000; 28: 2373-8.
• 12 Clarke RC, Johnston JR, Mayne EE. Meningococcal septicaemia: treatment with protein C concentrate. Intensive Care Med 2000; 26: 471-3.
• 13 Ettingshausen CE, Veldmann A, Beeg T. et al Replacement therapy with protein C concentrate in infants and adolescents with meningococcal sepsis and purpura fulminans. Semin Thromb Hemost 1999; 25: 537-41.
• 14 Jilma B, Marsik C, Mayr F. et al Pharmacodynamics of active site-inhibited factor VIIa in endotoxininduced coagulation in humans. Clin Pharmacol Ther 2002; 72: 403-10.
• 15 Jilma B, Blann A, Pernerstorfer T. et al Regulation of adhesion molecules during human endotoxemia. No acute effects of aspirin. Am J Respir Crit Care Med 1999; 159: 857-63.
• 16 Pernerstorfer T, Hollenstein U, Hansen J. et al Heparin blunts endotoxin-induced coagulation activation. Circulation 1999; 100: 2485-90.
• 17 Gruber A, Griffin JH. Direct detection of activated protein C in blood from human subjects. Blood 1992; 79: 2340-8.
• 18 Derhaschnig U, Reiter R, Knobl P. et al Recombinant human activated protein C (rhAPC; drotrecogin alfa [activated]) has minimal effect on markers of coagulation, fibrinolysis, and inflammation in acute human endotoxemia. Blood 2003; 102: 2093-8.
• 19 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-8.
• 20 Stolley PD, Strom BL. Sample size calculations for clinical pharmacology studies. Clin Pharmacol Ther 1986; 39: 489-90.
• 21 van Deventer SJ, Buller HR, ten Cate JW. et al Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood 1990; 76: 2520-6.
• 22 Mayr FB, Spiel A, Leitner J. et al Effects of carbon monoxide inhalation during experimental endotoxemia in humans. Am J Respir Crit Care Med 2005; 171: 354-60.
• 23 Dreyfus M, Masterson M, David M. et al Replacement therapy with a monoclonal antibody purified protein C concentrate in newborns with severe congenital protein C deficiency. Semin Thromb Hemost 1995; 21: 371-81.
• 24 Fourrier F, Leclerc F, Aidan K. et al Combined antithrombin and protein C supplementation in meningococcal purpura fulminans: a pharmacokinetic study. Intensive Care Med 2003; 29: 1081-7.
• 25 Turkington PT, Cathepsin G. a regulator of human vitamin K, dependent clotting factors and inhibitors. Thromb Res 1992; 67: 147-55.
• 26 Samis JA, Garrett M, Manuel RP. et al Human neutrophil elastase activates human factor V but inactivates thrombin-activated human factor V. Blood 1997; 90: 1065-74.
• 27 de Kleijn ED, de Groot R, Hack CE. et al Activation of protein C following infusion of protein C concentrate in children with severe meningococcal sepsis and purpura fulminans: a randomized, double-blinded, placebo-controlled, dose-finding study. Crit Care Med 2003; 31: 1839-47.
• 28 Macias WL, Dhainaut JF, Yan SC. et al Pharmacokinetic-pharmacodynamic analysis of drotrecogin alfa (activated) in patients with severe sepsis. Clin Pharmacol Ther 2002; 72: 391-402.
• 29 Okajima K, Koga S, Kaji M. et al Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects. Thromb Haemost 1990; 63: 48-53.
• 30 Rezaie AR, Cooper ST, Church FC. et al Protein C inhibitor is a potent inhibitor of the thrombin-thrombomodulin complex. J Biol Chem 1995; 270: 25336-9.
• 31 Reinhart K, Karzai W. Anti-tumor necrosis factor therapy in sepsis: update on clinical trials and lessons learned. Crit Care Med 2001; 29: S121-5.
• 32 van der Poll T, Coyle SM, Levi M. et al Effect of a recombinant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans. Blood 1997; 89: 3727-34.
• 33 O'Brien Jr. JM, Abraham E. New approaches to the treatment of sepsis. Clin Chest Med 2003; 24: 521-48v.
• 34 White B, Livingstone W, Murphy C. et al An openlabel study of the role of adjuvant hemostatic support with protein C replacement therapy in purpura fulminans-associated meningococcemia. Blood 2000; 96: 3719-24.
• 35 Alberio L, Lammle B, Esmon CT. Protein C replacement in severe meningococcemia: rationale and clinical experience. Clin Infect Dis 2001; 32: 1338-46.
• 36 Rintala E, Seppala OP, Kotilainen P. et al Protein C in the treatment of coagulopathy in meningococcal disease. Crit Care Med 1998; 26: 965-8.
• 37 Hollenstein U, Homoncik M, Knobl P. et al Acenocoumarol decreases tissue factor-dependent coagulation during systemic inflammation in humans. Clin Pharmacol Ther 2002; 71: 368-74.
• 38 Pernerstorfer T, Hollenstein U, Hansen JB. et al Lepirudin blunts endotoxin-induced coagulation activation. Blood 2000; 95: 1729-34.