Alarcón M
Thrombosis Research Center, Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Research Center for Aging, Universidad de Talca, 2 Norte 685, Talca, Post code 3460000, Chile.
Heliyon. 2019 Apr 8;5(4):e01486. doi: 10.1016/j.heliyon.2019.e01486. eCollection 2019 Apr.
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.
Experiments were designed to evaluate the generation of pro-thrombogenic microparticles in vitro on platelets via TLR4 activation.
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 LPS (0-9 μg/mL) followed by activation with ADP (1 μM, subaggregant concentration), centrifuged for 60 minutes and analyzed by flow cytometry.
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.
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.
不同细菌感染可能通过产生和分泌多种促炎因子增加血栓形成和动脉粥样硬化风险。人类血小板具有Toll样受体4（TLR4），它是脂多糖（LPS）的主要受体。血小板的激活会产生直径小于1.0微米的血小板衍生微粒（PDMPs）（在循环中含量非常丰富>90%），这些微粒与心血管疾病（CVDs）的发生有关，而心血管疾病是全球主要的死亡原因。
设计实验评估通过TLR4激活在体外血小板上促血栓形成微粒的产生情况。
将健康志愿者的富血小板血浆和洗涤后的血小板孵育以产生PDMPs。产生微粒的最佳来源是洗涤后的血小板。然后将洗涤后的血小板与超纯LPS（0 - 9μg/mL）孵育15分钟，随后用ADP（1μM，亚聚集浓度）激活，离心60分钟并通过流式细胞术分析。
用LPS（9μg/mL）和ADP（1μM）孵育血小板使PDMPs的产生增加了34倍。最后，我们评估了该方案以检测对PDMPs产生的抑制作用，发现洗涤后的血小板与乙酰水杨酸（10μM）孵育，在激活TLR4时PDMPs的产生受到7.7倍的抑制。
建立了一种通过流式细胞术产生和分析PDMPs的新的简便方案。未来它可用于确定PDMPs与不同病理状态的关联。
