BACKGROUND: Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the presence of antiphospholipid antibodies (aPL) in patients with thromboembolic/-inflammatory events and/or obstetric complications. OBJECTIVES: The aim of this study was to examine whether there are alterations in the platelet lipidome of APS patients in comparison to patients affected by thromboembolism without APS (control) and healthy volunteers. METHODS: We applied quantitative mass spectrometry-based lipidomics to investigate the platelet lipidome of isolated resting and thrombin-stimulated platelets as well as platelet release in patients with APS, controls and healthy volunteers. RESULTS: Lipidomic data revealed an increase in lysophospholipids (LPL) in platelets from APS patients, specifically in lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) species. As LPL are cleavage products generated by phospholipase A (PLA) from the corresponding phospholipid (PL) precursor, LPL/PL ratios may be employed as surrogates for PLA1 and PLA2 activities. The surrogate ratios for PLA2, which participates in the release of arachidonic acid (AA) during platelet activation, were significantly increased in APS in both resting platelets and upon thrombin-induced activation for phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The PC-PLA2 surrogate ratio was found to correlate with serum levels of anti-β2-glycoprotein I and anti-cardiolipin IgG. Finally, receiver operator characteristic (ROC) analysis demonstrated excellent discrimination of patients with APS from controls and healthy volunteers. CONCLUSION: These findings provide substantial evidence that platelet activation is enhanced in APS in vivo, involving the activation of PLA2.