Submitted May 18, 2009
Accepted June 10, 2009
Protamine sulphate downregulates thrombin generation by inhibiting factor V activation
Fionnuala Ni Ainle, Roger J.S. Preston, P. Vincent Jenkins, Hendrik J. Nel, Jennifer A. Johnson, Owen P. Smith, Barry White, Padraic G. Fallon, and James S. O'Donnell*
Haemostasis Research Group, Institute of Molecular Medicine, St James's Hospital, Trinity College Dublin, Dublin, Ireland
Inflammation and Immunity Research Group, Institute of Molecular Medicine, St James's Hospital, Trinity College Dublin, Dublin, Ireland
Department of Haematology, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
National Centre for Hereditary Coagulation Disorders, St James's Hospital, Dublin, Ireland
* Corresponding author; email: jodonne{at}tcd.ie.
Protamine sulphate is a positively-charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly following cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect have not been defined. In vivo administration of pharmacological doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail bleeding time (from 120 to 199 sec). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both PT and APTT. Protamine also markedly attenuated tissue-factor initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41±7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of FV activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.
