The effects of heparin, protamine, and heparin/protamine reversal on platelet function under conditions of arterial shear stress

Platelet dysfunction contributes to blood loss after cardiopulmonary bypass . This study examined the antiplatelet effects of heparin, protamine, and v arying heparin/protamine ratios in an in vivo physiologic model and further elucidated the mechanism of the antiplatelet and anticoagulant effects of protamine. We used the Clot Signature Analyzer (CSA (TM)), a system that an alyzes coagulation in flowing whole blood, to test two aspects of platelet function, with different concentrations of heparin and protamine, under con ditions simulating arterial flow:: collagen-induced thrombus formation (CIT F) under moderate shear and high sheer platelet activation, platelet hemost asis time (PH;T). In addition, platelet aggregometry, celite activated clot ting time (Hepcon (TM) ACT), prothrombin time (PT), and partial thromboplas tin time (PTT) were measured. Both PHT and the CITF were prolonged by hepar in at 20 mug/mL, protamine at 20 and 40 mug/mL, and heparin/ protamine rati os of 1:1 and 1:2, but not at 1:1.5. The Hepcon ACT was prolonged by hepari n 20 mug/mL and protamine alone at 20 and 40 mug/mL, was normal at a ratio of 1:1, and was prolonged at 1:1.5 and 1:2. Protamine 80 mug/mL prolonged t he PT and PTT. Depen dency on thrombin, protein kinase C activation, and no nspecific charge effects were examined. The direct thrombin inhibitor D-phe nylalanyl-L-prolyl-L-arginylchloromethyl ketone prolonged the PHT and ACT, but not the CITF, whereas the polycationic molecules polyarginine and polyl ysine prolonged the CITF, but not the PHT. The effect of protamine on the P TT, but not PT, could be shortened by the addition of excess phospholipid. Therefore, heparin inhibits both high shear collagen-independent and modera te shear collagen-dependent platelet activation; however, the latter is not mediated by its antithrombin activity. Protamine's antithrombin effect may explain its inhibition of platelet activation at high shear stress. Protam ine's nonspecific charge effects are more important for inhibiting moderate shear collagen-induced platelet activation.