Investigation of Adrenergic Receptor-Mediated Mitochondrial DNA Release from Human platelets

Abstract

Atherosclerosis, a chronic inflammatory disease, is a major cause of deaths associated with cardiovascular diseases. The strong link between atherosclerosis and inflammation has been observed in several studies. The investigation of the inflammation in atherosclerosis has provided insight to the role of immune cells in the development of this disease. Many inflammatory mediators and cells are involved in pathogenesis of atherosclerosis. Beyond pronounced role in thrombosis and haemostasis, platelets also induce inflammation. Several studies indicated that platelets persuade inflammatory response in neighboring ce lls such as endothelial cells and leukocytes. Platelets are crucial in proinflammatory surroundings like atherosclerosis, rheumatoid artlu'itis, allergy and cancer. [n atherosc lerosis, platelets recruit inflammatory cells towards the lesion site and release cytokines and chemokines in excess. Beside these inflammatory mediators, inflammatory response is activated by endogenous damage associated mol ecular patterns (DAMPs). The mitochondrial DNA (mtDNA) is endogenous DANtP that may activate immune responses in case of cell damage and death. Platelets have a small number of mitochondria, that is necessary for platelet functioning. Activated platelets release mitochondria and mtDNA in extracellular milieu. Many studies have demonstrated the increase of mtDNA level in several diseases. The stimulation of certain adrenergic receptors (AR) has been linked with platelet aggregation, but the contribution of AR signaling in platelet mtDNA release remains unexplored. This study was conducted to investigate the role of AR signaling in mtDNA release from platelets after treatment with agonists. For this purpose, we enriched platelets by differential centrifugation of healthy participants' blood. Our data confirm the previously published reports that stimulation with AR agonists triggers strong platelet aggregation within seconds of treatment. There was a significant increase in platelets aggregation response in treated samples as compared to controls. [n order to answer the main question of this work we extracted mtDNA from the cell supernatants after treatment of platelets with AR agonists at different time points. Our results indicate the release of a significant amount of mtDNA from both treated and untreated platelets. However, there was complete absence of nuclear DNA which confirmed the absence of any nucleated cells in our platelet preparations. After confinnation of the DNA presence in our samples, the mtDNA copy Investigation of Adrenergic Receptor-Mediated Mitochondrial DNA Releasefrom number was quantified by absolute qPCR. The released mtDNA showed a tendency to increase slightly in samples treated with phenylephrine (alpha AR agonist) after 6 hours of treatment. However, there was no significant difference in the release of mtDNA between two groups. Similarly, a small rise of mtDNA was observed after 16 hour of phenylephrine treatment. In order to assess the possible mechanism behind mtDNA release from platelet, we estimated the mitochondrial membrane potential (MMP) in treated cells using a combination of Tetramethylrhodamine (TMRM) fluorescent probe and flow cytometry. Our data demonstrate that there was more dye accumulation in unstimulated cells and signals disappeared in platelets treated with AR agonists. To fUliher shed light on this pathway we measured caspase 3 activation by western blotting to determine the stati of cell death signaling. Analysis of Caspase-3 revealed an unexpected cleavage product which is not associated with apoptosis. However previous studies have linked this caspase 3 cleavage with Cal pain-mediated degradation which may be a sign of necrotic mode of cell death. Degradation of tubulin protein in phenylephrine-treated platelets also highlights the probable activation of Cal pain in an AR-dependent manner. However future studies are required to confirm the accelerated mtDNA release from platelet upon AR activation and also to further investigate the possible involvement of Calpains in this process.