Caveolin-1 Enhances Tissue Factor Pathway Inhibitor Exposure and Function on the Cell Surface

Abstract

Tissue factor pathway inhibitor (TFPI) blocks tissue factor-factor VIIa (TF-FVIIa) activation of factors X and IX through the formation of the TF-FVIIa-FXa-TFPI complex. Most TFPI in vivo associates with caveolae in endothelial cells (EC). The mechanism of this association and the anticoagulant role of caveolar TFPI are not yet known. Here we show that expression of caveolin-1 (Cav-1) in 293 cells keeps TFPI exposed on the plasmalemma surface, decreases the membrane lateral mobility of TFPI, and increases the TFPI-dependent inhibition of TF-FVIIa. Caveolae-associated TFPI supports the co-localization of the quaternary complex with caveolae. To investigate the significance of these observations for EC we used RNA interference to deplete the cells of Cav-1. Functional assays and fluorescence microscopy revealed that the inhibitory properties of TFPI were diminished in EC lacking Cav-1, apparently through deficient assembly of the quaternary complex. These findings demonstrate that caveolae regulate the inhibition by cell-bound TFPI of the active protease production by the extrinsic pathway of coagulation.Tissue factor (TF) 1 is a transmembrane protein that triggers blood coagulation in vivo. Assembly of TF with factor VIIa (FVIIa) on cell surfaces initiates limited proteolysis of factors IX and X (FX), leading to thrombin generation. TF elicits thrombogenic responses in septicemia, cancer, and atherosclerosis (1-4), promotes metastasis, angiogenesis, and intima hyperplasia after arterial injury (5, 6), and acts as signaling receptor upon binding of FVIIa (7).Tissue factor pathway inhibitor (TFPI) is the endogenous regulator of TF-FVIIa activity. TFPI is a Kunitz-type protease inhibitor, which binds FVIIa via Kunitz-1 and FXa via Kunitz-2. Formation of the TF-FVIIa-FXa-TFPI complex provides sustained repression of the TF pathway (8). TFPI was first described as a soluble plasma protein that binds through its C terminus to yet uncharacterized anionic sites on the cell surface. Nevertheless, the majority of TFPI circulating in plasma is C-terminal truncated and bound to plasma lipoproteins, and as such, has significantly less inhibitory activity than the full-length TFPI (9).TFPI in vivo is mainly produced by endothelial cells (EC), has an intact C terminus, and associates with the plasma membrane through mechanisms that are not fully identified (3, 10 -13). Heparin releases a portion of the full-length, functionally active cell-associated TFPI, either from cell surface-binding sites or from intracellular stores (14, 15). However, the bulk of heparin-resistant cellular TFPI is released by phosphatidylinositol-phospholipase C in vitro, which indicates that most cellular TFPI associates with the cell surface via a glycosylphosphatidylinositol link (10 -12, 16, 17). Furthermore, endogenous TFPI in resting endothelium (10), monocytes (18), and the ECV304 cell line (11, 16) partitions in low-density fractions insoluble in cold detergent (lipid rafts). In monocytes and ECV304 endogenous TFPI inhibits efficiently FVIIa-TF...

Extracellular protein disulfide isomerase regulates coagulation on endothelial cells through modulation of phosphatidylserine exposure

“…33 ECs were treated for 30 minutes with anti-PDI, control IgG (10 g/mL), or human recombinant PDI (hPDI; 100nM) and/or ionomycin (5M) for 10 minutes, before the addition of FVIIa (10nM) and excess substrate FX (100nM). Aliquots were taken during a 30-minute incubation at 37°C, FXa generation was quenched with ethylenediaminetetraacetic acid, and the amount of FXa was measured in a chromogenic assay.…”

“…Aliquots were taken during a 30-minute incubation at 37°C, FXa generation was quenched with ethylenediaminetetraacetic acid, and the amount of FXa was measured in a chromogenic assay. 33 For prothrombinase assays, monolayers were treated and then incubated with FVa (50nM), FXa (5nM), and substrate prothrombin (0.5M) in HBS/Ca. The reaction was stopped after 10 minutes with ethylenediaminetetraacetic acid, and the amount of thrombin was measured with Spectrozyme TH.…”

“…33,35 Images were collected with a Nikon C1 confocal system on a Nikon TE2000U microscope (Nikon Instruments) as described, 33 using a PlanApo 60ϫ NA 1.2 water immersion objective lens and/or a PlanApo 20ϫ NA 0.7 dry objective lens. The acquisition software was EZ-C1 Version 3.6 (Nikon).…”

“…Western blot was performed essentially as described, 33 using chemiluminescent detection (GE Healthcare). Densitometric analysis was performed with ImageJ software 1.42b (NIH).…”

“…We have previously reported the use of this cell model for analysis of TF pathway inhibitor-dependent regulation of TF procoagulant function. 33 Characterization of TF expression and activity in this cellular model is detailed in supplemental data and supplemental Figure 1. Cells were washed twice with HEPES-buffered saline (HBS; 10mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid, 137mM NaCl, 40mM KCl, 20mM glucose, and 0.5% bovine serum albumin, pH 7.4) before use.…”