Protamine stimulates platelet aggregation in vitro with activation of the fibrinogen receptor and alpha-granule release, but impairs secondary activation via ADP and thrombin receptors

Heparin and protamine are fundamental in the management of anticoagulation during cardiac surgery. Excess protamine has been associated with increased bleeding. Interaction between protamine and platelet function has been demonstrated but the mechanism remains unclear. We examined the effect of protamine on platelet function in vitro using impedance aggregometry, flow cytometry, and thrombin generation. Platelets were exposed to protamine at final concentrations of 0, 20, 40, and 80 µg/mL, alone or together with adenosine diphosphate (ADP) or thrombin PAR1 receptor-activating peptide (TRAP). We found that in the absence of other activators, protamine (80 µg/mL) increased the proportion of platelets with active fibrinogen receptor (binding of PAC-1) from 3.6% to 97.0% (p < .001) measured with flow cytometry. Impedance aggregometry also increased slightly after exposure to protamine alone. When activated with ADP or TRAP protamine at 80 µg/mL reduced aggregation, from 73.8 ± 29.4 U to 46.9 ± 21.1 U (p < .001) with ADP and from 126.4 ± 16.1 U to 94.9 ± 23.7 U (p < .01) with TRAP. P-selectin exposure (a marker of alpha-granule release) measured by median fluorescence intensity (MFI) increased dose dependently with protamine alone, from 0.76 ± 0.20 (0 µg/mL) to 10.2 ± 3.1 (80 µg/mL), p < .001. Protamine 80 µg/mL by itself resulted in higher MFI (10.16 ± 3.09) than activation with ADP (2.2 ± 0.7, p < .001) or TRAP (5.7 ± 2.6, p < .01) without protamine. When protamine was combined with ADP or TRAP, there was a concentration-dependent increase in the alpha-granule release. In conclusion, protamine interacts with platelets in vitro having both a direct activating effect and impairment of secondary activation of aggregation by other agonists.