OPTIMAL HEPARIN-PROTAMINE REVERSAL RATIO

Abstract

Introduction: Some investigations have demonstrated an anti-platelet effect of excess protamine, [1] while others have suggested that the usual doses of protamine fail to reverse the anti-platelet effects of heparin. [2] We have previously reported use of the Clot Signature Analyzer (CSA[registered sign]) to assess the anti-platelet activity of protamine. Here we report use of the CSA[registered sign] to assess the effect of heparin reversed with different protamine doses on platelet function. The CSA[registered sign] tests whole blood shear-induced platelet adhesion (the platelet-induced hemostasis time, PHT), collagen-induced thrombus formation (CITF) and the clotting time (CT) in conditions mimicking physiologic blood flow. Methods: After HIC approval, paired whole blood 3ml samples from 9 volunteers were drawn into syringes containing 240[micro sign]l PBS or 120[micro sign]l heparin 1 mg/ml and 120[micro sign]l protamine 1 mg/ml (HEP/PROT 1/1). All samples were incubated for 90 seconds and assayed in the CSA[registered sign]. A similar analysis was performed comparing 120[micro sign]l heparin 1 mg/ml "reversed" with 180[micro sign]l protamine 1 mg/ml (HEP/PROT 1/1.5) with paired controls and 120[micro sign]l heparin 1 mg/ml "reversed" with 240[micro sign]l protamine 1 mg/ml (HEP/PROT 1/2) compared with 120[micro sign]l heparin 1 mg/ml "reversed" with 180[micro sign]l protamine 1 mg/ml (HEP/PROT 1/1.5). Statistical analysis was with the Wilcoxon signed rank test, significance taken at a P value < 0.05 and values expressed as mean +/- SEM. Results: HEP/PROT 1/1: The PHT, CITF and CT were prolonged significantly by heparin/protamine 1/1 incubation compared with controls (Figure 1, marked *). HEP/PROT 1/1.5: The PHT, CITF were not significantly prolonged with heparin/protamine 1/1.5 incubation compared with controls (Figure 2). Only the CT was significantly prolonged (Figure 2). HEP/PROT 1/1.5 v HEP/PROT 1/2: The PHT and CITF were significantly prolonged by heparin/protamine 1/2 compared to heparin/protamine 1/1.5 (Figure 3).Figure 1Figure 2Figure 3Discussion: 1:1 protamine "reversal" still allows an anti-platelet effect of heparin, as shown by a significantly prolonged PHT, CITF and CT. Increasing the protamine "reversal" dose to 1:1.5 successfully reverses heparin's effect on the PHT and CITF, with CT modestly prolonged, suggesting this approximates the optimal reversal ratio for preservation of in vivo platelet function. Further increase in the heparin/protamine ratio to 1/2 induces an increase in the PHT and CITF, possibly attributable to excess protamine. The mechanism of this protamine effect warrants further investigation. We conclude that protamine at 1.5x the usual reversal dose optimally antagonizes the anti-platelet effects of heparin, while minimizing the intrinsic anti-platelet effects of protamine.