There is evidence that nanoparticles (NP) can enter the bloodstream following deposition in the lungs, where they may interact with platelets. Polystyrene latex nanoparticles (PLNP) of the same size but with different surface charge-unmodified (umPLNP), aminated (aPLNP), and carboxylated (cPLNP)-were used as model NP to study interactions with human blood and platelets. Both the cPLNP and the aPLNP caused platelet aggregation, whereas the umPLNP did not. Whereas cPLNP caused aggregation by classical upregulation of adhesion receptors, aPLNP did not upregulate adhesion receptors and appeared to act by perturbation of the platelet membrane, revealing anionic phospholipids. Neither oxidative stress generation by particles nor metal contamination was responsible for these effects, which were a result of differential surface derivatization. The study reveals that NP composed of insoluble low-toxicity material are significantly altered in their potency in causing platelet aggregation by altering the surface chemistry. The two surface modifications, aminated and carboxylated, that did cause aggregation did so by different mechanisms. The study highlights the fundamental role of surface chemistry on bioactivity of NP in a platelet activation model.