Protamine sulphate is a positively-charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, protamine also possesses intrinsic anticoagulant properties. Furthermore, administration of excess protamine in the neutralization of UFH is associated with increased bleeding, particularly following cardiothoracic surgery. In this study we have investigated the molecular mechanisms underlying the anticoagulant properties of protamine. In pooled normal plasma, we observed a dose-dependent prolongation of both PT and APTT assays with increasing protamine concentrations (0–30μg/ml). The anticoagulant effects of protamine in normal plasma were also examined using a tissue factor-initiated thrombin generation assay. 30μg/ml protamine resulted in a two-fold prolongation of lag-time, a two-fold reduction in peak thrombin generation, and a 41±17% (p=0.047) decrease in endogenous thrombin potential (ETP). In heparinised plasma (0.3U/ml), addition of increasing protamine concentration initially reversed the anticoagulant effect of heparin, resulting in a progressive increase in ETP to that of normal plasma. However, further increases in protamine resulted in a dose-dependent reduction in ETP to a minimum of 61±16%. Recent studies have shown that platelet factor 4 (PF4), another cationic protein used to reverse heparin, can bind to the anionic Gla domain of protein C, thereby enhancing APC generation (up to 25-fold). Consequently, we investigated potential interaction(s) between protamine and the protein C anticoagulant pathway. As expected, in normal plasma, APC (0–20nM) caused a concentration-dependent prolongation in APTT to 180±8%, and a parallel reduction in ETP. However in the presence of 30μg/ml protamine, the effects of APC on both the APTT and ETP were markedly enhanced compared to the effect of either substance alone. As APC down-regulates thrombin generation by inactivating FVa and FVIIIa, we used a phospholipid-dependent FVa proteolysis assay to elucidate the mechanism responsible for the synergistic interaction between APC and protamine. The ability of APC to reduce FVa cofactor activity in this assay (in the presence or absence of protein S) was not significantly affected by the presence of protamine. However, a potent synergistic anticoagulant interaction between APC and protamine was also observed in plasma from patients with homozygous FV Leiden, suggesting protamine enhances APC cleavage of FVa at position Arg-306. To determine whether protamine influences the rate of FVIIIa proteolysis by APC, we expressed and purified an APC-resistant FVIII variant (R336Q/R562Q). FVIII-deficient plasma was spiked with physiological concentrations of wild type or variant FVIII, and the anticoagulant effects of protamine ± APC studied using plasma thrombin generation assays. Similar synergistic anticoagulant effects of APC in combination with protamine were observed for both wildtype and variant FVIII. To assess whether protamine also influences procoagulant processes, the rate of factor V activation by thrombin was analysed by SDS-PAGE. In the presence of protamine (30μg/ml), FVa generation was significantly reduced. In addition to inhibiting the rate of FVa generation, we also observed that protamine significantly impaired the functional activity of the prothrombinase complex in a concentration dependent manner. In contrast, cationic polybrene had no significant effect on either rate of FVa generation or prothrombinase complex activity. In conclusion, we have shown a novel and profound anticoagulant synergy between protamine sulphate and APC. Moreover, we demonstrate that this synergistic effect is mediated by independent effects on FVa generation and proteolysis respectively. These novel findings provide further insights into the molecular mechanism underlying the anticoagulant effect of excess protamine in human plasma.

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