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We explored the hypothesis that zeta potential altered by polycations affects blood platelet activation and reactivity, the phenomena associated with membrane lipid fluidity and platelet mitochondrial bioenergetics.PAMAM dendrimers generation- and dose-dependently enhanced zeta potential of platelets (from −10.7 mV to −4.3 mV). Increased expressions of activation markers, P-selectin and the active complex αIIbβ3, as well as significantly enhanced fibrinogen binding occurred upon the in vitro incubation of blood platelets in the presence of PAMAMs G3 and G4 (resp. 62.1% and 69.4% vs. 1.4% and 2.7% in control for P-selectin, P < 0.0001). PAMAM dendrimers increased fluidity of platelet membrane lipid bilayer, while they did not affect platelet mitochondria respiration. Increased platelet activation and their responses to agonists in vitro were statistically associated with the revealed alterations in zeta potential.Our results support the hypothesis that polycation-mediated “neutralized” zeta potential may underlie the activating effects of PAMAMs on blood platelets.