Abstract 492: Arachidonic Acid Induces Activation of Platelet PF4 and Par-1 mRNA, Which Is Attenuated by Aspirin

Arachidonic acid (AA) is converted to thromboxane A2 via the cyclooxygenase pathway; however its exact mechanism of platelet activation is uncertain. Inhibition of this pathway via aspirin highlights the importance of this pathway in decreasing thrombotic events. In the present study, we investigate the effect of AA on platelet activity indicators (leukocyte- and monocyte-platelet aggregation [LPA, MPA] and reticulated platelets [RP]), as well as the expression (mRNA and protein) of platelet markers PF4 and Par-1, previously well established platelet transcripts with quantitative determinations. To this end, whole blood was incubated with AA (150mM) for 30 min at room temperature in the absence or presence of aspirin (1mM) prior to addition of antibodies for platelet activity indicators, and isolating platelets for mRNA and protein expression. LPA and MPA were significantly increased after AA stimulation in a dose dependent manner, and were inhibited by aspirin treatment. AA significantly increased PF4 and Par-1 protein level as determined by flow cytometry and western blot assays. Pretreatment with aspirin also attenuated this increase in protein levels. Surprisingly, AA stimulation significantly increased thiazole orange staining (a measure of nucleic acids), another marker of increased platelet activity. Importantly, these results suggest that AA-mediated platelet activation produced an overall increase in platelet total RNA content. To confirm these findings, we analyzed the mRNA expression of PF4 and Par-1 by quantitative real time PCR from platelets treated with AA. Interestingly, AA significantly up-regulated the platelet mRNA transcripts of PF4 and Par-1 by 40% to 60%, and pretreatment with aspirin completely attenuated this effect supporting the specificity of the AA effect on platelet RNA. Altogether, these data suggest that platelet mRNA is affected by AA stimulation, which is attenuated by pretreatment with aspirin. However, the mechanisms responsible for the increased mRNA levels and expression of PF4 and Par-1 (processing of pre-RNA to mRNA) require further investigation. Importantly, our findings provide novel insight regarding platelet activation and a better understanding of mediators in the processes of thrombosis and hemostasis.