Generation of platelet-derived microparticles through the activation of the toll-like receptor 4

INTRODUCTION: Infection from different bacterial may increase the risk of thrombosis and atherosclerosis risk by production and secretion of many proinflammatory factors. Human platelets have toll-like receptor 4 (TLR4), the principal receptor for lipopolysaccharide (LPS). The activation of platelet produces Platelet-derived Microparticles (PDMPs) measuring less than 1.0 micron (that are very abundant in circulation >90%), which are associated with the development of Cardiovascular Diseases (CVDs), the leading cause of death in the world. OBJECTIVES: Experiments were designed to evaluate the generation of pro-thrombogenic microparticles in vitro on platelets via TLR4 activation. METHODS: Platelet-rich plasma and washed platelets from healthy volunteers were incubated for the generation of PDMPs. The best source for the generation of microparticles was washed platelets. Then the washed platelets were incubated for 15 minutes with ultrapure Escherichia coli LPS (0–9 μg/mL) followed by activation with ADP (1 μM, subaggregant concentration), centrifuged for 60 minutes and analyzed by flow cytometry. RESULTS: Incubating platelets with LPS (9 μg/mL) and ADP (1 μM) produced a 34-fold increase in PDMPs generation. Finally, we evaluated this protocol to detect the inhibition of PDMPs generation, washed platelets were incubated with acetylsalicylic acid (10 μM) and an inhibition of 7.7-fold in PDMPs generation for activation of TLR4 was found. CONCLUSION: A new and easy protocol for PDMPs generation and analysis by Flow Cytometry is established. In the future it could be used to determine the association of PDMPs with different pathologies.