Platelet dysfunction contributes to blood loss after cardiopulmonary bypass. This study examined the antiplatelet effects of heparin, protamine, and varying heparin/protamine ratios in an in vitro 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 analyzes coagulation in flowing whole blood, to test two aspects of platelet function, with different concentrations of heparin and protamine, under conditions simulating arterial flow: collagen-induced thrombus formation (CITF) under moderate shear and high shear platelet activation, platelet hemostasis time (PHT). In addition, platelet aggregometry, celite activated clotting time (Hepcon(TM) ACT), prothrombin time (PT), and partial thromboplastin time (PTT) were measured. Both PHT and the CITF were prolonged by heparin at 20 microg/mL, protamine at 20 and 40 microg/mL, and heparin/protamine ratios of 1:1 and 1:2, but not at 1:1.5. The Hepcon ACT was prolonged by heparin 20 microg/mL and protamine alone at 20 and 40 microg/mL, was normal at a ratio of 1:1, and was prolonged at 1:1.5 and 1:2. Protamine 80 microg/mL prolonged the PT and PTT. Dependency on thrombin, protein kinase C activation, and nonspecific charge effects were examined. The direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone prolonged the PHT and ACT, but not the CITF, whereas the polycationic molecules polyarginine and polylysine prolonged the CITF, but not the PHT. The effect of protamine on the PTT, but not PT, could be shortened by the addition of excess phospholipid. Therefore, heparin inhibits both high shear collagen-independent and moderate 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. Protamine's nonspecific charge effects are more important for inhibiting moderate shear collagen-induced platelet activation. CONCLUSIONS This study suggests that protamine reversal of heparin's antiplatelet effect occurs within a narrow window because of the direct antiplatelet effects of protamine. Antithrombin effects may explain the inhibition of shear activation of platelets by both heparin and protamine. Nonspecific charge effects of protamine may explain the inhibition of collagen platelet activation in the presence of medium shear.