Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae - Regulatory mechanism(s) of the anticoagulant properties of the endothelium
Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae - Regulatory mechanism(s) of the anticoagulant properties of the endothelium
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DOI：
10.1161/01.atv.17.11.2964
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发表时间：
1997-11-01
影响因子：
8.7
通讯作者：
Lupu, F
中科院分区：
文献类型：
--
作者：
Lupu, C;Goodwin, CA;Lupu, F
组织因子途径抑制物（TFPI）是组织因子VIIa复合物促凝血活性的主要下调因子，在培养的人内皮细胞（EC）中以均匀分布于细胞表面和细胞内的明确簇存在。我们在这里证明，通过免疫荧光，TFPI共定位在EC与小窝蛋白，尿激酶型纤溶酶原激活物受体，和鞘糖脂。TFPI和小窝蛋白的双免疫金电镜证实TFPI定位于静息内皮细胞的小窝中。通过Nycodenz的密度梯度超浓缩大鼠肺或EC匀浆后，TFPI高度富集在1.05至1.08 g/mL的密度，连同小窝蛋白和碱性磷酸酶。通过ELISA，在Triton X-100不溶性EC提取物中检测到超过一半的细胞TFPI。TFPI掺入[1-H-3]乙醇胺，并通过磷脂酰肌醇-磷脂酶C从细胞表面裂解，表明TFPI在质膜中的特异性糖基磷脂酰肌醇锚定机制。TFPI的聚集及其在小窝中的定位依赖于膜中胆固醇的存在。激动剂诱导的EC刺激引起TFPI和小窝蛋白在亚细胞水平的分布的显着变化，随后增加的细胞表面相关的抑制活性对组织因子。因子VIIa。我们的研究结果表明，除了在转胞吞作用，potocytosis，细胞表面蛋白水解，和信号转导的调节功能，小窝也发挥了直接的作用，在EC抗凝性能的调节。
Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-H-3]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.