Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are the central mediators of thrombosis, with their selective activation induced by different agonists in spatially distinct regions of the thrombus. Platelet extracellular vesicles (pEVs) are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. It is currently unknown if physiological platelet agonists can influence the pEV proteome. In the current study we used physiological platelet agonists of varying potencies which represent the microenvironments that platelets experience during thrombus formation: adenosine diphosphate (ADP), collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV release. Proteomic profiling revealed that pEVs in comparison to parental platelets were impacted in response to various activating stimuli. Furthermore, we found that various protein classes including those related to coagulation (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen, CD9, GPV, and EMILIN1) are attributed to platelet EVs following agonist stimulation. This occurs in the absence of de novo protein synthesis, indicating that protein packaging into pEVs is a specific and active process. This study provides new information on the biogenesis and proteome landscape of pEVs impacted and conserved by physiological agonist stimuli.