Plasminogen activator inhibitor-1 promotes formation of endothelial microparticles with procoagulant potential
Plasminogen activator inhibitor-1 promotes formation of endothelial microparticles with procoagulant potential
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DOI：
10.1161/01.cir.0000033972.90653.af
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发表时间：
2002-10-29
期刊：
影响因子：
37.8
通讯作者：
Goligorsky, MS
中科院分区：
文献类型：
--
作者：
Brodsky, SV;Malinowski, K;Goligorsky, MS
背景-内皮功能障碍正在成为各种高度流行的心血管疾病的共同因素。纤溶酶原激活物抑制物-1(PAI-1)水平升高和促凝血活性升高已被认为是内皮功能障碍的标志。本研究旨在探讨PAI-1的细胞作用以及PAI-1与促凝血状态之间的潜在联系。方法与结果：用PAI-1处理人脐静脉内皮细胞，通过激光共聚焦荧光显微镜、免疫沉淀和Western blotting以及FACS分析分离和鉴定内皮细胞微粒。PAI-1处理降低了uPAR的表达，并与小窝蛋白共定位，同时增加了培养基中uPAR的丰度。流式细胞仪分析显示，PAI-1可迅速增加表达uPAR和α(V)β(3)整合素的内皮微粒数量，且呈剂量依赖关系。这一过程可通过中和抗uPAR抗体来减弱。与野生型小鼠相比，PAI-1基因敲除小鼠的循环内皮细胞微粒数量显著减少；然而，PAI-1缺陷动物对PAI-1的注射反应更明显地增加了微粒数量。PAI-1处理增加了Annexin V染色的微粒数量，这是阴离子磷脂表达的证据。同时伴随着凝血酶的加速生成。结论--数据揭示了PAI-1通过减少磷脂的跨膜不对称性而剂量依赖地促进内皮细胞微粒形成的新作用。这种现象可能是观察到的体外凝血酶生成增加的原因。这些发现可能潜在地将内皮功能障碍的这些特征-PAI-1水平升高和血栓形成的倾向联系起来。
Background-Endothelial dysfunction is emerging as a common denominator for diverse and highly prevalent cardiovascular diseases. Increased level of plasminogen activator inhibitor-1 (PAI-1) and procoagulant activity have been recognized as hallmarks of endothelial dysfunction. This study was aimed at investigating cellular actions of PAI-1 and a potential link between PAI-1 and procoagulant state.Methods and Results-Human umbilical vein endothelial cells treated with PAI-1 were subjected to laser confocal fluorescence microscopy, immunoprecipitation and Western blotting, and FACS analysis for isolation and identification of endothelial microparticles. PAI-1 treatment resulted in a reduced expression of uPAR, its colocalization with caveolin, and the concomitant increase of uPAR abundance in the culture medium. FACS analysis revealed that PAI-1 rapidly and dose-dependently increased the number of endothelial microparticles expressing uPAR and alpha(v)beta(3) integrin. This process was attenuated by pretreatment with neutralizing anti-uPAR antibodies. PAI-1 knockout mice showed a significantly decreased number of circulating endothelial microparticles than wild-type mice; however, PAI-1-deficient animals responded to infusion of PAI-1 with a more pronounced rise in the number of microparticles. PAI-1 treatment increased the number of microparticles stained with Annexin V, evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin.Conclusions-The data disclose a novel effect of PAI-1 to dose-dependently promote formation of endothelial microparticles with the reduced transmembrane asymmetry of phospholipids. This phenomenon may be responsible for the observed increase in in vitro thrombin generation. These findings could potentially link these hallmarks of endothelial dysfunction-elevated levels of PAI-1 and propensity toward thrombosis.