Platelets are central to blood clotting via their rapid response to stimuli such as thrombin activation. Their activation induces the secretion of proteins that promote platelet aggregation and tissue inflammation. However, detailed analysis of the secreted platelet proteome is hampered by platelets’ tendency to pre-activate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the proteome released from carefully isolated and in vitro thrombin-activated primary platelets from healthy donors. More than 1,300 proteins were detected using a data-dependent acquisition strategy and HCD-based peptide fragmentation. Quantitative comparison of resting and thrombin-treated platelet releasate showed 202 proteins were significantly increased by thrombin stimulation, including several novel proteins involved in immune system activation. This analysis included complementary quantification of the platelet lysates identifying >3,800 proteins to provide anti-correlation analysis for the secreted proteins and a resource for comparing platelet protein abundance. Sub-maximal thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory-pathway proteins. Using an open-search strategy, we performed unbiased scanning for post-translational modifications within the releasate proteins, which highlighted O-glycosylation as being a major component. Subsequent O-glycome analysis of the same releasate fraction highlighted a bias to either O-fucosylated, O-glucosylated, or sialylated core 1 mucin structures. To validate these glycosylation sites and structures, we performed both targeted and untargeted EThcD fragmentation of our releasate digests. For the first time, we detected O-fucosylation on previously uncharacterised sites in the abundant platelet protein multimerin (MMRN1) likely mediated by POFUT1. We hypothesise that fucosylation of MMRN1 at these sites is required for the protein’s function in platelet aggregation. The comprehensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms in platelet function and thrombosis.