Heparin-protamine Complexes and C-reactive Protein Induce Activation of the Classical Complement Pathway: Studies in Patients Undergoing Cardiac Surgery and In Vitro

Summary

The administration of protamine to patients undergoing cardiopulmonary bypass (CPB) to neutralize heparin and to reduce the risk of bleeding, induces activation of the classical complement pathway mainly by heparin-protamine complexes. We investigated whether C-reactive protein (CRP) contributes to protamine-induced complement activation.

In 24 patients during myocardial revascularization, we measured complement, CRP, and complement-CRP complexes, reflecting CRPmediated complement activation in vivo. We also incubated plasma from healthy volunteers and patients with heparin and protamine in vitro to study CRP-mediated complement activation. During CPB, CRP levels remained unchanged while C3 activation products increased. C4 activation occurred after protamine administration. CRP-complement complexes increased at the end of CPB and upon protamine administration. Incubation of plasma with heparin and protamine in vitro generated complement-CRP complexes, which was blocked by phosphorylcholine and stimulated by exogenous CRP. C4d-CRP complex formation after protamine administration correlated clinically with the incidence of postoperative arrhythmia.

Protamine administration during cardiac surgery induces complement activation which in part is CRP-dependent, and correlates with postoperative arrhythmia.

Keywords

C-reactive protein - heparin - protamine - complement - extracorporeal circulation

• References

• 1 Jansen PGM, te HVelthuis, Huybregts MAJM, Paulus R, Bulder ER, van der Spoel HI, Bezemer PD, Slaats EH, Eijsman L, Wildevuur CRH. Reduced complement activation and improved postoperative performance after cardiopulmonary bypass with heparin-coated circuits. J Thorac Cardiovasc Surg 1995; 110: 829-34.
• 2 Chenoweth DE, Cooper SW, Hugli TE, Stewart RW, Blackstone EH, Kirklin JW. Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a anaphylatoxins. N Engl J Med 1981; 304: 497-503.
• 3 Kirklin JK, Westaby S, Blackstone EH, Kirklin JW, Chenoweth DE, Pacifico AD. Complement and the damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1983; 86: 845-57.
• 4 te Velthuis H, Jansen PGM, Hack CE, Eijsman L, Wildevuur CRH. Specific complement inhibition with heparin-coated extracorporeal circuits. Ann Thorac Surg 1996; 61: 1153-7.
• 5 Wildevuur CRH, Jansen PGM, Bezemer PD, Kuik DJ, Eijsman L, Bruins P, De Jong AP, Van Hardevelt FWJ, Biervliet JD, Hasenkam JM. et al. Clinical evaluation of Duraflo^® II heparin treated extracorporeal circulation circuits (2nd version) The European working group on heparin coated extracorporeal circulation circuits. Eur J Cardio-thorac Surg 1997; 11: 616-23.
• 6 Bruins P, te HVelthuis, Yazdanbakhsh AP, Jansen PGM, Van Hardevelt FWJ, de Beaumont EMFH, Wildevuur CRH, Eijsman L, Trouwborst A, Hack CE. Activation of the complement system during and after cardiopulmonary bypass surgery: postsurgery activation involves C-reactive protein and is associated with postoperative arrhythmia. Circulation 1997; 96: 3542-8.
• 7 Siegel J, Rent R, Gewurz H. Interactions of C-reactive protein with the complement system. J Exp Med 1974; 140: 631-47.
• 8 Volanakis JE, Clements WL, Schrohenloher RE. C-reactive protein purification by affinity chromatography and physicochemical characterization. J Immunol Methods 1978; 23: 285-95.
• 9 Volanakis JE, Wirtz KWA. Interaction of C-reactive protein with artificial phosphatidylcholine bilayers. Nature 1979; 281: 155-7.
• 10 Wolbink GJ, Brouwer MC, Buysmann S, ten Berge IJM, Hack CE. CRPmediated activation of complement in vivo: Assessment by measuring circulating complement-CRP complexes. J Immunol 1996; 157: 473-9.
• 11 Teien AM, Lie M, Abildgaard U. Assay of heparin in plasma using a chromogenic substrate for activated factor X. Thromb Res 1976; 08: 413-6.
• 12 Bara L, Mardiguian J, Samama M. In vitro effect on heptest^® of low molecular weight heparin fractions and preparations with various anti-IIa and anti-Xa activities. Thromb Res 1990; 57 (04) 585-92.
• 13 Wolbink GJ, Bollen J, Baars JW, ten Berge RJM, Swaak AJG, Paardekooper J, Hack CE. Application of a monoclonal antibody against a neoepitope on activated C4 in an ELISA for the quantification of complement activation via the classical pathway. J Immunol Methods 1993; 163: 67-76.
• 14 Hack CE, Paardekooper J, Van Milligen F. Demonstration in human plasma of a form of C3 that has the conformation of “C3b-like C3”. J Immunol 1990; 144: 4249-55.
• 15 Hack CE, Paardekooper J, Eerenberg AJM, Navis GO, Nijsten MWN, Thijs LG, Nuijens JH. A modified competitive inhibition radioimmunoassay for the detection of C3a. Use of ¹²⁵I-C3 instead of ¹²⁵I-C3a. J Immunol Methods 1988; 108: 77-84.
• 16 Hack CE, Paardekooper J, Smeenk RJT, Abbink JJ, Eerenberg AJM, Nuijens J. Disruption of the internal thioester bond in the third component of complement (C3) results in the exposure of neodeterminants also present on activation products of C3. An analysis with monoclonal antibodies. J Immunol 1988; 141: 1602-9.
• 17 Rent R, Eisenstein R, Gewurz H. Complement activation by interaction of polyanions and polycations: Heparin-protamine induced consumption of complement. J Immunol 1975; 114 (01) 120-4.
• 18 Kirklin JK, Chenoweth DE, Naftel DC, Blackstone EH, Kirklin JW, Bitran DD, Curd JG, Reves JG, Samuelson PN. Effects of protamine administration after cardiopulmonary bypass on complement, blood elements and the hemodynamic state. Ann Thorac Surg 1986; 41: 193-9.
• 19 Best N, Sinosich MJ, Grunzinskas JG, Fisher M. Complement activation during cardiopulmonary bypass by heparin-protamine interaction. Br J Anaesth 1984; 56: 339-43.
• 20 Griselli M, Herbert J, Hutchinson WL, Taylor KM, Sohail M, Krausz T, Pepys MB. C-reactive Protein and Complement Are Important Mediators of Tissue Damage in Acute Myocardial Infarction. J Exp Med 1999; 190: 1733-40.
• 21 Raasveld MH, Bemelman FJ, Schellekens PT, van Diepen FN, van Dongen Av, Royen EA, Hack CE, ten Berge IJ. Complement activation during OKT3 treatment: a possible explanation for respiratory side effects. Kidney Int 1993; 43: 1140-9.
• 22 van Dam AP, Hack CE. Formation of C3-IgG complexes in serum by aggregated IgG and by non-immunoglobulin activators of complement. Immunol 1987; 61: 105-10.
• 23 Hack CE, Wolbink GJ, Schalkwijk C, Speijer H, Hermens WT, van den Bosch H. A role for secretory phospholipase A2 and C-reactive protein in the removal of injured cells. Immunol Today 1997; 18: 111-5.
• 24 Bruins P, te HVelthuis, Jansen PGM, Eijsman L, Hack CE. Biphasic release of secretory phospholipase A2 during and after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1998; 115 (04) 949-51.
• 25 DiCamelli R, Potempa LA, Siegel J, Suyehira L, Petras K, Gewurz H. Binding reactivity of C-reactive protein for polycations. J Immunol 1980; 125: 1933-8.
• 26 Siegel J, Osmand AP, Wilson MF, Gewurz H. Interactions of C-reactive protein with the complement system. II. C-reactive protein-mediated consumption of complement by poly-L-lysine polymers and other polycations. J Exp Med 1975; 142: 709-21.
• 27 Oz MC, Liao H, Naka Y, Seldomridge A, Becker DN, Michler RE, Smith CR, Rose EA, Stern DM, Pinsky DJ. Ischemia-induced interleukin-8 release after human heart transplantation. A potential role for endothelial cells. Circulation. 1995 92. II428-32.
• 28 Kilgore KS, Friedrichs GS, Homeister JW, Lucchesi BR. The complement system in myocardial ischemia/reperfusion injury. Cardiovasc Res 1994; 28: 437-44.