AbstractThymoquinone (TQ) is a nutrient with anticarcinogenic activity that stimulates suicidal death of tumor cells. Moreover, TQ triggers suicidal death of erythrocytes or eryptosis, an effect at least partially due to increase in cytosolic Ca2+activity and ceramide formation. The present experiments explored whether TQ influences apoptosis of blood platelets. Cell membrane scrambling was determined utilizing Annexin V binding to phosphatidylserine exposing platelets, cytosolic Ca2+activity utilizing Fluo 3‐AM fluorescence, caspase activity utilizing immunofluorescence and Western blotting of active caspase‐3 and inactive procaspase‐3, mitochondrial potential utilizing DiOC6fluorescence and ceramide by FACS analysis of ceramide‐binding antibodies. A 30 min exposure to TQ (≥5 µM) was followed by Annexin V binding, paralleled by caspase activation, increase of cytosolic Ca2+activity, mitochondrial depolarization, and ceramide formation. P‐selectin exposure and integrin αIIbβ3activation did not increase in response to TQ. Nominal absence of extracellular Ca2+blunted but did not fully abolish the TQ‐induced activation of caspase‐3. The effects of TQ on platelets are significantly abolished with phosphoinositide‐3 kinase (PI3K) inhibitor wortmannin and G‐protein coupled receptor (GPCR) inhibitor pertussis toxin treatment prior to TQ stimulation. In conclusion, TQ triggers suicidal death of blood platelets in a PI3K‐dependent manner, possibly through a GPCR family receptor; an effect paralleled by increase of cytosolic Ca2+activity, ceramide formation, mitochondrial depolarization, and caspase‐3 activation. J. Cell. Biochem. 112: 3112–3121, 2011. © 2011 Wiley Periodicals, Inc.