Protamine and Coagulopathy After Cardiopulmonary Bypass: May the Perfect Electrostatic Force Be With You

To the Editor: We read with interest the recent narrative review by Stone and Vespe on “heparin rebound”, an enigma which lasted for more than 60 years.1 Based on both historical literature and more recent quantitative clinical studies, the author concluded that “heparin rebound” likely represents coagulopathy other than heparin itself, and residual anti-Xa activity measured as a marker of “heparin rebound” bears hardly any correlation to actual bleeding. In view of relatively low incidences of heparin rebound, an extra dose of protamine may do more harm than good by worsening platelet aggregation and reducing thrombin generation. Platelet factor 4 (PF4) and protamine are both positively charged peptides that bind to and neutralize negative charges on the platelet surface. These charge interactions play a crucial role in regulating thrombus formation and hemostasis in vivo. Eslin and colleagues demonstrated in transgenic mice that PF4 knock-out [PF4(-/-)] causes impaired carotid artery occlusion after a ferric chloride-induced vascular injury.2 Thrombus formation is restored when human PF4 or protamine sulfate is infused into PF4(-/-) mice, but both impede vascular occlusion when infused at a high dose. It was also shown that PF4(-/-) and partial knock-out PF4(+/-) mice become extremely sensitive to heparin, suggesting that heparin impedes thrombus formation via charged interactions with platelet-derived PF4 at the site of vascular injury. A simplified schema shows the interactions among platelet-derived PF4, heparin, and protamine, highlighting the role of electrostatic force in thrombus formation (Figure). It is well known that PF4-heparin complex triggers a high rate of immunization of cardiac surgical patients, and in a subset of patients, immunoglobulin G (IgG) against PF4-heparin causes thrombocytopenia (heparin-induced thrombocytopenia [HIT]). Although less frequently diagnosed and described, protamine-heparin complex can also cause immunization and trigger protamine-induced thrombocytopenia (PIT) due to anti-protamine/heparin IgG antibodies in a subset of cardiovascular patients.3 The authors speculated that anecdotally described “oozing” 30–60 min after protamine administration might be due to “progressive inhibitor” activity of antithrombin (AT) without heparin.4 However, classical “progressive inhibition” of thrombin may not be simply inferred to in vivo thrombin regulation, as the source experiments were conducted in the absence of endothelium. Indeed, Raivio and colleagues 5 reported a more than 300% increase over baseline in activation of protein C (APC) 30 minutes after protamine administration during coronary bypass artery grafting surgery (n=100) using cardiopulmonary bypass (CPB). The mean peak level of APC was 546% (range, 178–1,267%), obtunding thrombin generation through inhibitions of Factor V and Factor VIII. Thrombin supports APC generation when it is bound to endothelial thrombomodulin, and it is ultimately inhibited by antithrombin.6 Interestingly, both PF4 (3-10 µg/ml) and protamine (3.3 µg/ml) can promote APC generation by interacting electrostatically with thrombomodulin and protein C.7 An extra dose of protamine at 30-40 mg can achieve plasma level of 5 µg/ml, and thus one might speculate that late “oozing” results from APC generation rather than heparin rebound. Lastly, the authors suggested the initial protamine dose be tailored to the actual amount of heparin in the blood. The problem is that neither activated clotting time nor heparin management system (HMS; Medtronic, Minneapolis, MN) provides an accurate estimate of heparin levels. The issue of over-dosing of protamine and its consequences after CPB were already reported in 1976 by Guffin and colleagues.8 The authors showed that the standard group received approximately a 1:1 ratio of heparin to protamine, while the intervention group had a 1:0.5 ratio. On average, 203 mg less protamine per patient was administered compared to the control group. Improved coagulation tests and platelet count were reported in the low-dose protamine group with lower chest tube drainage volumes (mean 623 ml vs. 1126 ml in the control during 48h). Platelet transfusion was given to 12 patients (40%) in the control vs. 4 patients (13.3%) in the low-dose group. While “heparin rebound” might have been a valid threat in the days of patients with lower body mass index, and no routine antifibrinolytics, their results showed otherwise. Over-dosing of protamine remains common in today's practice, and our exploration for an optimal heparin/protamine ratio continues: “May the perfect electrostatic force be with you!” 1Stone ME, Vespe MW. Heparin rebound: an in-depth review. J Cardiothorac Vasc Anesth 2022;doi: 10.1053/j.jvca.2022.12.019.2Eslin DE, Zhang C, Samuels KJ, et al. Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin. Blood 2004;104:3173-3180.3Bakchoul T, Jouni R, Warkentin TE. Protamine (heparin)-induced thrombocytopenia: a review of the serological and clinical features associated with anti-protamine/heparin antibodies. J Thromb Haemost 2016;14:1685-1695.4Abildgaard U. Inhibition of the thrombin-fibrinogen reaction by heparin and purified cofactor. Scand J Haematol 1968;5:440-453.5Raivio P, Fernandez JA, Kuitunen A, et al. Activation of protein C and hemodynamic recovery after coronary artery bypass surgery. J Thorac Cardiovasc Surg 2007;133:44-51.6Aritomi M, Watanabe N, Ohishi R, et al. Recombinant human soluble thrombomodulin delivers bounded thrombin to antithrombin III: thrombomodulin associates with free thrombin and is recycled to activate protein C. Thromb Haemost 1993;70:418-422.7Slungaard A, Key NS. Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity. A structure-function analysis. J Biol Chem 1994;269:25549-25556.8Guffin AV, Dunbar RW, Kaplan JA, et al. Successful use of a reduced dose of protamine after cardiopulmonary bypass. Anesth Analg 1976;55:110-113.