Essentials Cysteine oxidation to sulfenic acid plays a key role in redox regulation and signal transduction. Platelet sulfenylome was studied by quantitative proteomics in pathogen inactivated platelets. One hundred and seventy-four sulfenylated proteins were identified in resting platelets. Pathogen inactivation oxidized integrin βIII, which could activate the mitogen-activated protein kinases pathway. ABSTRACT: Background Cysteine-containing protein modifications are involved in numerous biological processes such redox regulation or signal transduction. During the preparation and storage of platelet concentrates, cell functions and protein regulations are impacted. In spite of several proteomic investigations, the platelet sulfenylome, ie, the proteins containing cysteine residues (R-SH) oxidized to sulfenic acid (R-SOH), has not been characterized. Methods A dimedone-based sulfenic acid tagging and enrichment coupled to a mass spectrometry identification workflow was developed to identify and quantify the sulfenic acid-containing proteins in platelet concentrates treated or not with an amotosalen/ultraviolet A (UVA) pathogen inactivation technique. Results One hundred and seventy-four sulfenylated proteins were identified belonging mainly to the integrin signal pathway and cytoskeletal regulation by Rho GTPase. The impact on pathogen inactivated platelet concentrates was weak compared to untreated ones where three sulfenylated proteins (myosin heavy chain 9, integrin βIII, and transgelin 2) were significantly affected by amotosalen/UVA treatment. Of particular interest, the reported oxidation of cysteine residues in integrin βIII is known to activate the receptor αIIbβIII. Following the pathogen inactivation, it might trigger the phosphorylation of p38MAPK and explain the lesions reported in the literature. Moreover, procaspase activating compound-1 (PAC-1) binding assays on platelet activation showed an increased response to adenosine diphosphate exacerbated by the tagging of proteins with dimedone. This result corroborates the hypothesis of an oxidation-triggered activation of αIIbβIII by the pathogen inactivation treatment. Conclusions The present work completes missing information on the platelet proteome and provides new insights on the effect of pathogen inactivation linked to integrin signaling and cytoskeleton regulation.