Low molecular weight protamine: A potent but nontoxic antagonist to heparin/low molecular weight protamine

To avoid bleeding complications, protamine is routinely used after cardiova scular surgery to neutralize the anticoagulant function of heparin. However , its clinical use is associated with adverse and sometimes fatal reactions . Based on literature review of the mechanism of heparin neutralization and protamine induced immunologic toxicity, we propose the following hypothesi s: if a chain shortened low molecular weight protamine (LMWP) containing th e heparin neutralizing domain could be derived from native protamine, it co uld be a potent and yet nontoxic heparin antagonist. In this study, we pres ent results to validate this hypothesis. LMWP fragments containing an intac t arginine sequence and an average molecular weigh of approximately 1,100 d altons were successfully prepared by enzymatic digestion of protamine with thermolysin. In vitro studies show that such LMWP fragments completely neut ralized the anticoagulant functions of heparin and LMWH, based on the anti- Xa chromogenic and aPTT clotting time assays. In vivo results reveal that a lthough injection of protamine to mice led to obvious production of anti-pr otamine antibodies, injection of LMWP did not elicit any detectable immunog enic responses. In addition, these LMWP fragments exhibited a markedly redu ced antigenicity and cross-reactivity toward the mice anti-protamine antibo dies.