Background: Platelet-derived extracellular vesicles (PEVs) are small vesicles released by activated platelets that are gaining growing interest in the field of vascular biology. The mode of platelet activation is a critical determinant of PEVs release, phenotype and function. However, only very limited information is available concerning the impact of the platelet purification procedure on PEVs release. Methods: Washed or isolated platelets were separated by differential centrifugations. For washed platelets, the platelet pellet was washed by resuspension in PIPES buffer and finally resuspended in HEPES buffer. Isolated platelets were obtained by directly resuspending the platelet pellet in HEPES, skipping the washing steps in PIPES buffer. PEVs release was induced in washed or isolated platelets by stimulation with different agonist and analysed by Nanoparticle Tracking Analysis. Results: Isolated platelets showed a higher release of PEVs upon adenosine diphosphate (ADP) stimulation compared to washed platelets, whereas PEVs released upon stimulation with strong agonists (thrombin, collagen, A23187, U46619) were similar in the two groups. This different responsiveness to ADP was also observed as a higher \alpha-granules release and protein kinase C activation in isolated platelets compared to washed ones. Residual plasma contamination appeared to be essential for the ability of platelets to release PEVs in response to ADP. Conclusions: In conclusion, our study strongly suggests that procedure adopted for platelets preparation is a critical determinant of PEVs release upon ADP stimulation.