Cardiovascular diseases are today the most common welfare diseases and are the primary cause of death in the industrialized society. Platelets are nowadays considered to have an important function in not only haemostasis, but also in inflammatory processes as key players of the innate immune system. Toll like receptors (TLRs), expressed on the platelet surface, recognize pathogen associated molecular patterns (PAMPs) and trigger immune responses. Pam3CSK4, a synthetic lipopeptide, acts as a ligand for TLR2/1 and thereby activates platelets. Pathogens are known to adhere to human tissues, e.g. atherosclerotic plaques, and form biofilms that causes a continuous activation of the immune system. This study aims to investigate how adsorbed Pam3CSK4, resembling a pathogen surface, affects the adhesion and activation of platelets, and to clarify the role of adenine nucleotides in this process. Moreover, to study the release of one pro-inflammatory cytokine upon stimulation with Pam3CSK4.Methods
To examine activation and granule secretion due to Pam3CSK4 stimulation, Pam3CSK4 was immobilized onto hydrophobic surfaces. Platelets were pre-incubated with the P2X1- (ATP) antagonist MRS2159, the P2Y1- (ADP) antagonist MRS2179, the P2Y12- (ADP) antagonist Clopidogrel or the phospholipase C- (PLC) inhibitor U73122 before addition to Pam3CSK4 coated surfaces. Cytokine-measurements were performed with high sensitivity-ELISA for interleukin-7.Results
A significant decrease in cell number measured as cells/mm2 was found when platelets were pre-incubated with MRS2159, indicating that platelet activation by Pam3CSK4-coated surfaces is ATP dependent. Treatment with MRS2179 instead led to a significant increase in cells/mm2. Inhibition of the PLC signalling pathway did not alter the platelet response. Preliminary results indicate that inhibition of both the P2Y1-receptor (with MRS2179) and the P2Y12-receptor (with Clopidogrel) has an antagonizing effect on cell adhesion and spreading. Pam3CSK4-stimulation time- and dose-dependently increased the secretion of the pro-inflammatory cytokine interleukin-7.Conclusion
TLR2/1-mediated adhesion of platelets is dependent on ATP-release while inhibition of P2Y1-receptors enhances platelet P2Y12-responses. These results further clarify TLR2/1-induced platelet activation and strengthen the role of the platelets as an active player in sensing bacterial infections.