title: Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium.
abstract: 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 [1-3H]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.
authors.affiliation: Thrombosis Research Institute, London, UK. clupu@ceasar.tri-london.ac.uk
authors.forename: C
authors.initials: C
authors.lastname: Lupu
authors.forename: C A
authors.initials: CA
authors.lastname: Goodwin
authors.forename: A D
authors.initials: AD
authors.lastname: Westmuckett
authors.forename: J J
authors.initials: JJ
authors.lastname: Emeis
authors.forename: M F
authors.initials: MF
authors.lastname: Scully
authors.forename: V V
authors.initials: VV
authors.lastname: Kakkar
authors.forename: F
authors.initials: F
authors.lastname: Lupu
mesh_terms: D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002704:Chlorates / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D015942:Factor VIIa / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006595:Hexosamines / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007476:Ionophores / Q000494:pharmacology; D008074:Lipoproteins / Q000032:analysis* / Q000302:isolation & purification; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis*; D016253:Microscopy, Immunoelectron; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D051381:Rats; D012995:Solubility; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D014471:Umbilical Veins; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis
chemical_list: C495540:CAV1 protein, human; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D002704:Chlorates; D017261:Glycosylphosphatidylinositols; D006595:Hexosamines; D007476:Ionophores; D008074:Lipoproteins; D008563:Membrane Lipids; D008565:Membrane Proteins; C051928:lipoprotein-associated coagulation inhibitor; C008322:mannosamine; D000001:Calcimycin; D002784:Cholesterol; D010738:Type C Phospholipases; D015942:Factor VIIa; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium; C032706:sodium chlorate