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Role of CD36 in platelet function.

机译：CD36在血小板功能中的作用。

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摘要

CD36 is a Class B scavenger receptor expressed on platelets, erythrocyte precursors, monocytes, microvascular endothelial cells, epithelial cells, adipocytes, and cardiac and skeletal myocytes. It recognizes multiple ligands including thrombospondins, oxidized phospholipids and apoptotic cells, and has been shown to play a role in phagocytosis, angiogenesis and atherosclerosis. The function of CD36 on platelets is incompletely characterized, but our group has recently identified CD36 on platelets as a signaling receptor which can modulate platelet function by binding to ligands such as oxidized LDL. Endothelial cell (EC) derived microparticles (MP) have been identified in the circulation of patients with diseases such as diabetes, anti-phospholipid syndrome and acute coronary syndrome in which patients are prone to arterial thrombosis, and thus platelet activation and aggregation play a pivotal role. Because EC MP express phosphatidyl serine (PS) on their surfaces, a potential CD36 ligand, we hypothesize that MP may bind to platelets via a PS-CD36 interaction and function to transmit an activating signal, thereby promoting a prothrombotic state.; To test this hypothesis, we first isolated EC-derived MP by stimulating human umbilical vein EC with TNFalpha and cyclohexamide according to a previously published protocol. MP were characterized and quantified by flow cytometry and shown to express CD105 and PS. Binding of MP to platelets was detected and quantified by flow cytometry and immunofluorescence microscopy. Platelet activation was assessed by aggregometry and flow cytometry. Washed human platelets (CD105 negative) were incubated with EC-derived MP at a ratio of 1:9 and analyzed by flow cytometry with a fluorescence tagged anti-CD105 dye) positive MP formed rosettes around (Calcein-Green Tagged) platelets. With both the flow cytometry and microscopy assays, platelet-MP association was inhibited by addition of anti-CD36 antibody or by using platelets from CD36 null donors. This inhibition by CD36 antibody was statistically significant (p=0.02). Furthermore, pretreatment of platelets with other CD36 ligands such as oxLDL inhibited MP-platelet association by more than 50%. Next we determined the functional effect of the MP-platelet association. We observed a significant increase in the rate and extent of platelet aggregation to low concentrations (2muM) of ADP and an increase in platelet secretion (measured as surface P-selectin expression) when platelets were incubated with EC-derived MP prior to addition of agonist. This effect was markedly diminished in platelets from CD36 null donors and also inhibited by pre-incubation with anti CD36 antibody. To test the MP-platelet interaction in vivo, carotid arteries were injured by FeCl3 in wild type and CD36 knock out mice. The thrombosed arteries were sectioned and immunostained with an endothelial cell specific antibody to CD105 (red) and a platelet specific antibody to CD61 (green). We reasoned that CD105 staining of thrombi would reflect incorporation of EC-derived MP into the thrombi. We observed significantly more CD105 staining within the thrombi from wild type mice compared to CD36 null mice.; We thus propose a model where CD36 ligands presented to platelets renders them "hyperactive" predisposing patients to pathological thrombosis. It is also possible that CD36 ligands such as EMPs, generated during an acute thrombotic event, could increase the thrombotic response in a CD36 dependent manner by signaling platelets in a positive feedback loop. (Abstract shortened by UMI.)
机译：CD36是在血小板，红细胞前体，单核细胞，微血管内皮细胞，上皮细胞，脂肪细胞以及心脏和骨骼肌细胞上表达的B类清除剂受体。它识别多种配体，包括血小板反应蛋白，氧化的磷脂和凋亡细胞，并已显示在吞噬作用，血管生成和动脉粥样硬化中起作用。 CD36在血小板上的功能尚不完全清楚，但我们小组最近已将血小板上的CD36鉴定为信号受体，可以通过与诸如氧化的LDL等配体结合来调节血小板功能。内皮细胞（EC）衍生的微粒（MP）已在患有糖尿病，抗磷脂综合征和急性冠状动脉综合征等疾病的患者的血液中被发现，其中患者容易发生动脉血栓形成，因此血小板活化和聚集起关键作用角色。因为EC MP在其表面上表达磷脂酰丝氨酸（PS），这是潜在的CD36配体，所以我们假设MP可能通过PS-CD36相互作用与血小板结合并起传递激活信号的作用，从而促进血栓形成前的状态。为了验证这一假设，我们首先根据先前公布的方案，通过用TNFalpha和环己酰胺刺激人的脐静脉EC来分离EC衍生的MP。通过流式细胞仪对MP进行表征和定量，并显示CD105和PS。通过流式细胞仪和免疫荧光显微镜检测并定量MP与血小板的结合。通过凝集测定法和流式细胞术评估血小板活化。将洗涤过的人血小板（CD105阴性）与EC衍生的MP以1：9的比例孵育，并通过流式细胞术用荧光标记的抗CD105染料进行分析，在（钙黄绿素-绿色标记的）血小板周围形成了阳性MP。通过流式细胞术和显微镜测定法，通过添加抗CD36抗体或使用来自CD36无效供体的血小板来抑制血小板-MP缔合。 CD36抗体的这种抑制作用具有统计学意义（p = 0.02）。此外，用其他CD36配体（例如oxLDL）预处理血小板可抑制MP-血小板缔合超过50％。接下来，我们确定了MP-血小板缔合的功能作用。我们观察到血小板在添加激动剂之前与EC衍生的MP孵育时，血小板聚集率和程度降低至低浓度（2μM）的ADP时血小板聚集率和程度显着增加，并且血小板分泌量增加（以表面P-选择素表达衡量）。 。该作用在来自CD36无效供者的血小板中显着减弱，并且还通过与抗CD36抗体预孵育而被抑制。为了测试体内MP血小板的相互作用，在野生型和CD36敲除小鼠中，FeCl3损伤了颈动脉。切开血栓的动脉，并用针对CD105的内皮细胞特异性抗体（红色）和针对CD61的血小板特异性抗体（绿色）进行免疫染色。我们认为血栓的CD105染色将反映出EC衍生的MP掺入血栓中。与无CD36的小鼠相比，我们观察到野生型小鼠的血栓内CD105染色明显更多。因此，我们提出了一种模型，其中向血小板呈递的CD36配体使它们“过度活跃”，使患者容易发生病理性血栓形成。在急性血栓形成事件期间产生的CD36配体（例如EMP）也可能通过在正反馈回路中发信号通知血小板，以CD36依赖性方式增加血栓形成反应。 （摘要由UMI缩短。）

著录项

  • 作者

    Ghosh, Arunima.;

  • 作者单位

    Cleveland State University.;

  • 授予单位 Cleveland State University.;
  • 学科 Biology Cell.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 126 p.
  • 总页数 126
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;
  • 关键词

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