Modulation of tissue factor-factor VIIa signaling by lipid rafts and caveolae

University of Texas Health Science Center at Tyler, Tyler, Texas, United States
Arteriosclerosis Thrombosis and Vascular Biology (Impact Factor: 5.53). 07/2007; 27(6):1447-55. DOI: 10.1161/ATVBAHA.107.143438
Source: PubMed

ABSTRACT Coagulation factor VIIa (VIIa) binding to its cellular receptor, tissue factor (TF), not only initiates the coagulation cascade but also induces cell signaling by activating G-protein coupled protease-activated receptors. The objective of the present study is to investigate the role of lipid rafts and caveolae in modulating TF-VIIa signaling and coagulant functions.
TF-VIIa coagulant function was measured in factor X activation assay and the signaling function was evaluated in phosphoinositide hydrolysis and IL-8 gene induction. Buoyant density gradient centrifugation and immunofluorescence confocal microscopy were used to determine cellular localization of TF and protease-activated receptor 2. The data show that a substantial fraction of TF and protease-activated receptor 2 resides in lipid rafts/caveolae, and disruption of lipid rafts by cholesterol depletion or modification reduced TF-VIIa-induced cell signaling. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling.
Overall our data show that lipid raft/caveolae play a selective role in modulating the TF-VIIa signaling function without affecting the TF-VIIa coagulant activity.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The family of G protein-coupled receptors (GPCRs) constitutes the largest class of signalling receptors in the human genome, controlling vast physiological responses and are the target of many drugs. After activation, GPCRs are rapidly desensitized by phosphorylation and beta-arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and beta-arrestin-dependent pathway and then recycled back to the cell surface or sorted to lysosomes for degradation. Given the vast number and diversity of GPCRs, different mechanisms are likely to exist to precisely regulate the magnitude, duration and spatial aspects of receptor signalling. The G protein-coupled protease-activated receptors (PARs) provide elegant examples of GPCRs that are regulated by distinct desensitization and endocytic sorting mechanisms, processes that are critically important for the spatial and temporal fidelity of PAR signalling. PARs are irreversibly activated through proteolytic cleavage and transmit cellular responses to extracellular proteases. Activated PAR(1) internalizes through a clathrin- and dynamin-dependent pathway independent of beta-arrestins. Interestingly, PAR(1) is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, beta-arrestins mediate activated PAR(2) internalization and function as scaffolds that promote signalling from endocytic vesicles. Moreover, activated PAR(2) is modified with ubiquitin, which facilitates lysosomal degradation. Activated PARs also adopt distinct active conformations that signal to diverse effectors and are likely regulated by different mechanisms. Thus, the identification of the molecular machinery important for PAR signal regulation will enable the development of new strategies to manipulate receptor signalling and will provide novel targets for the development of drugs.
    British Journal of Pharmacology 05/2010; 160(2):191-203. DOI:10.1111/j.1476-5381.2010.00705.x · 4.99 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Tissue factor (TF), originally discovered to initiate coagulation, is more recently recognized to be involved in other biological processes, such as migration and anti-apoptosis. TF-mediated signaling regulates gene expression and protein synthesis, leading to alterations in cellular behavior. The proteolytic activity of factor VIIa (FVIIa), beta-1 integrin interaction and protease-activated receptor (PAR) activation are some of the key events involved in TF signaling. Post-translational modifications of TF may regulate signaling but this remains elusive. In vivo studies have established that TF signaling severely contributes to processes like angiogenesis, cancer growth and inflammation. This review focuses on the mechanism underlying TF-mediated intracellular signaling with its related physiological and mainly pathological consequences.
    Frontiers in bioscience (Scholar edition) 01/2011; 3:1500-10. DOI:10.2741/240
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background and PurposeTransient Receptor Potential Vanilloid-4 (TRPV4) is a calcium-permeant ion channel that is known to affect vascular function. The ability of TRPV4 to cause a vasoconstriction in blood vessels has not yet been mechanistically examined. Further in neuronal cells, TRPV4 signaling can be potentiated by GPCR activation. Thus we studied the mechanisms underlying the vascular contractile action of TRPV4 and the GPCR mediated potentiation of such vasoconstriction, both of which are as yet unappreciated aspects of TRPV4 function.Experimental ApproachThe mechanisms of TRPV4 dependent regulation of vascular tone in isolated mouse aortae were studied using wire myography. TRPV4 dependent calcium signaling and prostanoid production was studied in cultured human umbilical vein endothelial cells.Key ResultsIn addition to the well-documented vasorelaxation response triggered by TRPV4 activation, we report here a TRPV4 triggered vasoconstriction in the mouse aorta that involves a cyclo-oxygenase generated thromboxane receptor (TP) agonist that acts in a MAP kinase and Src Kinase signaling dependent manner. This constriction is potentiated by activation of the GPCRs for angiotensin (AT1R) or proteinases (PAR1 and PAR2) via transactivation of the Epidermal Growth Factor (EGF) receptor and a process involving protein kinase C (PKC). TRPV4 dependent vascular contraction can be blocked by cyclooxygenase inhibitors or with TP antagonists. Further, TRPV4 activation in HUVECs stimulated thromboxane release as detected by an ELISA.Conclusion and implicationsWe conclude that the GPCR potentiation of TRPV4 action and TRPV4 dependent thromboxane receptor activation are important regulators of vascular function and could be therapeutically targeted in vascular diseases.
    British Journal of Pharmacology 01/2015; 172(10). DOI:10.1111/bph.13072 · 4.99 Impact Factor


Available from