Abstract: The main component of Alzheimer's disease (AD) senile plaques in the brain is amyloid-β peptide (Aβ), a proteolytic fragment of the amyloid precursor protein (APP). Platelets contain both APP and Aβ and much evidence suggests that these cells may represent a useful tool to study both amyloidogenic and nonamyloidogenic pathways of APP processing. It has been demonstrated that platelets activated by physiological agonists, such as thrombin and collagen, specifically secrete Aβ ending at residue 40. To verify whether APP β-processing could be observed also in an in vitro system of highly concentrated platelets, we measured the Aβ released in the incubation media of 5 × 109 platelets/mL by enzyme-linked immunosorbent assay (ELISA). The activation status of platelets was investigated by ultrastructural analysis. We found that Aβ40 levels were significantly higher in incubation media of 5 × 109/mL platelets in comparison with 108/mL platelets (normalized values), while Aβ42 levels were not affected by cell density. The ultrastructural analysis showed platelets at different phases of activation: some platelets were at earlier stage, characterized by granule swelling and dilution, others had granules concentrated in a compact mass in the cell centers within constricted rings of circumferential microtubules (later stage). Normally concentrated cells had the characteristic morphology of resting platelets. Our data suggest that high-density platelets undergo activation likely by increased frequency of platelet–platelet collisions. This, in turn, determines the activation of APP β-processing with consequent release of Aβ40. Investigating the biochemical pathways triggering Aβ secretion in platelets might provide important information for developing tools to modulate this phenomenon in AD brains.