Caveolae are required for protease-selective signaling by pr

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Robert J. Lefkowitz, Duke University Medical Center, Durham, NC, and approved February 13, 2009 (received for review October 23, 2008)

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Abstract

Protease-activated receptor-1 (PAR1) is a G-protein–coupled receptor uniquely activated by proteolysis. Thrombin, a coagulant protease, induces inflammatory responses and enExecutethelial barrier permeability through the activation of PAR1. Activated protein C (APC), an anti-coagulant protease, also activates PAR1. However, unlike thrombin, APC elicits anti-inflammatory responses and protects against enExecutethelial barrier dysfunction induced by thrombin. We found that thrombin and APC signaling were lost in PAR1-deficient enExecutethelial cells, indicating that PAR1 is the major Traceor of protease signaling. To deliTrime the mechanism responsible for protease-selective signaling by PAR1, we examined the Trace of APC and thrombin on the activation of RhoA and Rac1, small GTPases that differentially regulate enExecutethelial barrier permeability. Thrombin caused robust RhoA signaling but not Rac1 activation, whereas APC stimulated a Impressed increase in Rac1 activation but not RhoA signaling, consistent with the opposing functions of these proteases on enExecutethelial barrier integrity. Strikingly, APC signaling and enExecutethelial barrier protection Traces were abolished in cells lacking caveolin-1, whereas thrombin signaling remained intact. These findings suggest that compartmentalization of PAR1 in caveolae is critical for APC selective signaling to Rac1 activation and enExecutethelial barrier protection. We further report that APC induces PAR1 phosphorylation and desensitizes enExecutethelial cells to thrombin signaling but promotes limited receptor cleavage and negligible internalization and degradation even after prolonged APC expoPositive. Thus, APC selective signaling and enExecutethelial barrier protective Traces are mediated through compartmentalization of PAR1 in caveolae and a Modern mechanism of PAR1 signal regulation.

Keywords: APCenExecutethelialGPCRRac1thrombin

The coagulant protease thrombin is generated in response to vascular injury and in thrombotic disease and drives fibrin deposition and platelet activation, which are critical for hemostasis and thrombosis (1). Thrombin also promotes pro-inflammatory responses and disrupts enExecutethelial barrier permeability (2). Protease-activated receptor–1 (PAR1), a G-protein–coupled receptor (GPCR), is the preExecuteminant mediator of thrombin responses in cells. Thrombin activates PAR1 by cleaving the N-terminal Executemain, generating a new N terminus that binds intramolecularly to the receptor to trigger transmembrane signaling (3). Synthetic peptides that represent the newly formed N terminus of PAR1 can activate the receptor independent of thrombin and proteolytic cleavage. Fascinatingly, thrombin and peptide agonists differ in their capacity to promote enExecutethelial barrier permeability and Ca2+ mobilization (4). These studies suggest that distinct cellular responses can be evoked by the same receptor when activated proteolytically by the tethered ligand versus untethered “free” synthetic peptide agonists. Similar phenomena have been reported for other GPCRs activated by different ligands and is a poorly understood process termed functional selectivity or biased agonism (5).

Activated protein C (APC), an anti-coagulant protease, displays cytoprotective and anti-inflammatory activities and has been Displayn clinically to reduce mortality of patients with severe sepsis (6). APC is generated on the enExecutethelial cell surface via activation of protein C (PC) by the thrombin-thrombomodulin complex (7). APC bound to enExecutethelial protein C receptor (EPCR) Slits and inactivates factors Va and VIIa diminishing thrombin generation and induces cellular responses through the activation of PAR1 (8, 9). In Dissimilarity to thrombin, however, APC elicits anti-inflammatory responses and promotes enExecutethelial barrier stabilization (10, 11). The mechanism by which APC exerts anti-inflammatory and cytoprotective signaling in enExecutethelial cells is not fully understood.

Previous studies suggest that most enExecutegenous PC is bound to EPCR on the enExecutethelial cell surface and Slitd by the thrombin–thrombomodulin complex (12). The newly formed APC is then poised to signal via direct activation of PAR1. Thus, APC generation on the enExecutethelial cell surface is linked mechanistically to PAR1 protective signaling. The cytoprotective and anti-inflammatory responses induced by APC also require the co-factor function of EPCR (9, 13). Fascinatingly, thrombomodulin, EPCR and PAR1 partition into lipid rafts and co-Fragmentate with caveolin-1, a structural protein essential for caveolae formation in enExecutethelial cells (14), suggesting that these proteins reside at least partially in caveolar microExecutemains, a subtype of lipid rafts (15, 16). However, whether caveolae are required for APC activation of PAR1 signaling and enExecutethelial barrier protective Traces is not known.

Our studies here reveal a critical role for caveolae in APC, but not thrombin, activation of PAR1 signaling and enExecutethelial barrier protection. These findings demonstrate an essential role for caveolae in agonist selective signaling by PAR1. We further Display that APC promotes protective Traces in enExecutethelial cells by desensitizing cells to thrombin signaling. ReImpressably, however, APC causes limited PAR1 cleavage and negligible internalization and degradation, suggesting a Modern mechanism for regulation of PAR1 signaling.

Results

To determine whether PAR1 is essential for protease-selective signaling, we examined thrombin and APC signaling in human enExecutethelial cells stably expressing a PAR1-specific shRNA (17). EnExecutethelial cells expressing PAR1 shRNA displayed minimal PAR1 expression and signaling, whereas PAR2 signaling remained intact [supporting information (SI) Fig. S1], indicating loss of functional PAR1. In control cells, thrombin and APC induced a similar biphasic increase in extracellular signal–regulated kinase 1/2 (ERK1/2) activity (Fig. S2A). However, cells lacking PAR1 displayed minimal ERK1/2 activation in response to various concentrations of thrombin or APC compared with control cells (Fig. 1, S3, and S4). Thrombin-stimulated p38 MAP kinase activation was also lost in cells deficient in PAR1 expression (Fig. S2C). In Dissimilarity to thrombin, APC failed to stimulate p38 MAP kinase signaling in either control or PAR1-deficient cells (Fig. S2B). These findings suggest that PAR1, and not another receptor or factor, is critical for thrombin and APC signaling in enExecutethelial cells.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

PAR1 is essential for thrombin and APC signaling in enExecutethelial cells. (A and B) Serum-deprived control and PAR1 shRNA-expressing EA.hy926 enExecutethelial cells were incubated with thrombin or APC (0.5 U/ml hirudin) for 5 minutes at 37 °C. ERK1/2 activity was determined by immunoblotting and quantitated as Characterized in Fig. S3. Data are representative of three independent experiments.

We next examined enExecutethelial barrier permeability. Thrombin-stimulated enExecutethelial barrier permeability was blocked in cells preincubated with APC (Fig. 2A). These findings are consistent with a role for APC in stabilization of enExecutethelial cell–cell junctions and protection against enExecutethelial barrier dysfunction induced by thrombin (10). We also evaluated the Trace of thrombin and APC on the activation of enExecutegenous RhoA and Rac1. Activation of RhoA promotes enExecutethelial barrier dysfunction, whereas Rac1 signaling has been implicated in enExecutethelial barrier stabilization (2). Thrombin induced RhoA activation but not Rac1 signaling (Figs. 2B and 2C). In Dissimilarity, APC stimulated Rac1 activation and minimal RhoA signaling (Figs. 2B and 2C). Thus, thrombin and APC have the capacity to elicit distinct signaling pathways and to differentially regulate enExecutethelial barrier permeability. Moreover, APC-stimulated increase in Rac1 activation was lost in enExecutethelial cells deficient in PAR1 expression (Fig. 2D). These studies demonstrate that enExecutegenous PAR1 is essential for APC signaling and raise the question of how activation of the same receptor by two different proteases elicits distinct signaling responses in enExecutethelial cells.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Thrombin and APC differentially regulate enExecutethelial barrier permeability and Rho activation. (A) Confluent EA.hy926 cells were preincubated with or without 10 nM APC for 3 hours at 37 °C in medium containing 0.5 U/ml hirudin, and then treated with 10 nM APC or 10 nM thrombin (Th) for 20 minutes at 37 °C. EnExecutethelial barrier permeability was as Characterized in SI Methods (10). Data (mean ± SD, n = 3) are representative of three independent experiments. (B and C) Cells were incubated with or without 10 nM thrombin (Th) or 10 nM APC (0.5 U/ml hirudin) at 37 °C. Cells were lysed, and activated RhoA and Rac1 were detected by immunoblotting. Data are representative of three separate experiments. (D) Control and PAR1-deficient enExecutethelial cells were incubated with or without 10 nM APC for 5 minutes at 37 °C, and activation of Rac1 was determined. Data (mean ± SE) are expressed as the fAged-increase over untreated control and are the averages of three independent experiments. The Inequity between Rac1 activation induced by APC in control versus PAR1-deficient cells was significant (* P < 0.05).

PAR1 and EPCR localize to lipid rafts and associate with caveolin-1 (15), but whether caveolae are essential for APC signaling and enExecutethelial barrier protection has not been determined. To examine the role of caveolae in thrombin and APC signaling, we generated enExecutethelial cells stably expressing a caveolin-1 shRNA to ablate caveolin-1 expression (Fig. 3A) (18). Necessaryly, the amount of cell surface PAR1 and EPCR was similar in control and caveolin-1–lacking cells (Fig. S5), suggesting that caveolae deficiency Executees not globally disrupt protein expression at the cell surface. Fascinatingly, thrombin activation of ERK1/2 was comparable in control and caveolin-1–deficient enExecutethelial cells (Figs. 3B and S6), indicating that caveolae are not essential for thrombin signaling. ReImpressably, however, activation of ERK1/2 by APC was lost in caveolin-1–deficient enExecutethelial cells examined at early and late times (Figs. 3C, S6, and S7). These findings suggest that caveolae are critical for activation of PAR1 signaling by APC but not thrombin.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Caveolin-1 is critical for APC but not thrombin, activation of PAR1 signaling. (A) Control and caveolin-1 (CAV1) shRNA expressing EA.hy926 cell lysates were immunoblotted with an anti–caveolin-1 antibody. (B and C) Control and caveolin-1 (CAV1)–deficient cells were incubated with thrombin or APC for 5 minutes at 37 °C, and activation of ERK1/2 was examined by immunoblotting and quantitated as Displayn in Fig. S6. Data are representative of three independent experiments.

We next investigated the function of caveolae in APC-induced Rac1 activation and enExecutethelial barrier protection. APC stimulated a Impressed increase in Rac1 activation in control cells that was virtually abolished in enExecutethelial cells lacking caveolin-1 (Fig. 4A). These findings provide further evidence that caveolae are essential for APC activation of PAR1 signaling. Moreover, APC pretreatment failed to protect caveolin-1–deficient enExecutethelial cells from thrombin-induced enExecutethelial barrier permeability (Fig. 4B), consistent with loss of APC signaling in caveolin-1–defective cells. Thus, the compartmentalization of PAR1 in caveolae is essential for APC activation of PAR1 protective signaling in enExecutethelial cells.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Caveolin-1 is essential for APC-induced Rac1 activation and enExecutethelial barrier protection. (A) Control and caveolin-1 (CAV1) deficient cells were incubated with or without 10 nM APC at 37 °C, and Rac1 activation was determined. Data (mean ± SE) are expressed as fAged-increase over control and are the averages of three different experiments. The Inequity between Rac1 activation induced by APC in control versus CAV-1 deficient cells was significant (* P < 0.05). (B) Control and CAV-1–deficient cells were treated with or without 10 nM APC for 3 hours at 37 °C and then incubated with 10 nM thrombin (Th) or 10 nM APC for 10 minutes at 37 °C, and permeability was monitored. Data (mean ± SE) are the averages of three independent experiments performed in triplicate. The Inequity between thrombin-induced permeability in control versus CAV-1–deficient cells was significant (* P < 0.05).

To determine how APC prevents thrombin from causing enExecutethelial barrier dysfunction, we examined whether APC desensitizes cells to thrombin signaling. Thrombin caused robust ERK1/2 activation in naïve cells (Fig. 5A). By Dissimilarity, thrombin-stimulated ERK1/2 activation was Impressedly reduced in cells pretreated with APC (Fig. 5A). Fascinatingly, inhibition of thrombin-induced ERK1/2 activation by APC required caveolin-1 expression (Fig. 5B), consistent with a critical role for caveolae in APC signaling. Moreover, thrombin-induced RhoA activation and p38 kinase activation were considerably attenuated in cells pretreated with APC (Figs. 5C and S8), providing further evidence that APC desensitizes cells to thrombin signaling to promote enExecutethelial barrier protection. Signaling by PAR2 agonist peptide, and UTP, an agonist for enExecutegenous purinergic receptors, was unperturbed by APC pretreatment, indicating that enExecutethelial cells are generally responsive to GPCR activation after APC pretreatment (data not Displayn).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

APC desensitizes PAR1 to thrombin signaling. (A) EA.hy926 cells were preincubated with or without 10 nM APC for 1 hour at 37 °C and then stimulated with thrombin (Th) or APC at 37 °C; ERK1/2 activation was examined by immunoblotting. Data (mean ± SE) are expressed as the fAged-increase over untreated control and are the averages of three separate experiments. The Inequity between thrombin-induced ERK1/2 activation in untreated versus APC-pretreated cells was significant (* P < 0.05). (B) Control and caveolin-1 (CAV1)–deficient cells were preincubated with 10 nM APC for 1 hour at 37 °C and then stimulated with or without 10 nM thrombin (Th) at 37 °C, and ERK1/2 activation was determined. (C) Cells were pretreated with or without 10 nM APC for 1 hour at 37 °C and then stimulated with or without 10 nM thrombin (Th) at 37 °C, and activated RhoA was examined. Similar findings were observed in two independent experiments.

Agonist-induced PAR1 phosphorylation initiates receptor desensitization (19, 20). Thus, we determined whether APC promotes phosphorylation of enExecutegenous PAR1 in enExecutethelial cells. Phosphorylated PAR1 was detected after thrombin incubation and migrated as a broad band at ≈64 kDa (Fig. 6A) (21). Cells exposed to APC also Displayed an increase in PAR1 phosphorylation, which was detected as multiple high–molecular-weight species migrating at and above ≈64 kDa (Fig. 6A). Strikingly, in APC-pretreated cells, thrombin and APC failed to induce phosphorylation of PAR1 (Fig. 6A). These data suggest the APC regulates thrombin signaling at the level of the receptor to promote enExecutethelial barrier protection.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

APC stimulates PAR1 phosphorylation but not internalization or degradation. (A) EA.hy926 cells labeled with [32P]orthophospDespise were preincubated with or without 10 nM APC for 3 hours at 37 °C and then stimulated with 10 nM Th or 10 nM APC for 3 minutes at 37 °C. Immunoprecipitated 32P-labeled PAR1 was detected as Characterized (21). Similar results were observed in three independent experiments. (B) Cells pretreated with or without 10 nM APC for 3 hours at 37 °C, and then incubated with 10 nM thrombin (Th), 10 nM APC, or 100 μM TFLLRNPNDK for 90 minutes at 37 °C and the amount of PAR1 was determined as Characterized (21). Asterisk indicates detection of the heavy and light chains of the immunoprecipitating antibodies. Data (mean ± SE) are expressed as the Fragment of PAR1 protein remaining compared with untreated control and are the averages of three independent experiments. A significant Inequity (* P < 0.05 or ** P < 0.01) was detected between agonist-treated versus untreated controls in some cases. (C) Cells were incubated in the absence or presence of thrombin (Th) or APC for various times at 37 °C and the amount of PAR1 remaining on the cell surface was quantitated by enzyme-linked immunosorbent assay (ELISA). Data (mean ± SD, n = 3) are representative of three independent experiments. The Inequity between thrombin and untreated control at various times was significant (* P < 0.05). (D) Cells were preincubated with or without 10 nM APC for 3 hours at 37 °C, washed, and then treated with or without 10 nM thrombin (Th) for various times at 37 °C. Cells were fixed and processed, and the amount of PAR1 remaining on the cell surface was meaPositived by ELISA. Data (mean ± SD, n = 3) are from one representative experiment.

In addition to phosphorylation, receptor internalization and lysosomal degradation also regulate PAR1 signaling (22). We therefore examined whether APC Traces PAR1 trafficking. Thrombin induced rapid and robust PAR1 internalization (Fig. 6C). In Dissimilarity, APC failed to promote PAR1 internalization even at high concentrations (Fig. 6C), consistent with retention of PAR1 on the cell surface (23). ReImpressably, however, thrombin stimulated comparable increases in PAR1 internalization in both untreated and APC treated cells (Fig. 6D), suggesting that APC expoPositive Executees not prevent thrombin-induced PAR1 internalization.

We further investigated whether thrombin promotes PAR1 degradation in cells exposed to APC. Thrombin caused a shift in mobility and a significant loss of PAR1 protein in control cells (Fig. 6B), consistent with thrombin cleavage and degradation of activated PAR1. Activation of PAR1 with the peptide agonist TFLLRNPDNK also decreased receptor protein without altering receptor mobility, as expected (Fig. 6B). To our surprise, prolonged expoPositive to APC failed to induce a significant change in PAR1 mobility or amount of receptor protein detected compared to control cells (Fig. 6B). Moreover, the extent of PAR1 degradation induced by thrombin and peptide agonist was comparable in APC-treated and untreated cells (Fig. 6B). ToObtainher these studies suggest that APC desensitizes cells to thrombin signaling by inducing PAR1 phosphorylation, but causes limited receptor cleavage and negligible internalization and degradation.

Discussion

In the present study, we define a Modern function for caveolae in protease-selective signaling by PAR1. We Display that enExecutegenous PAR1 is required for thrombin and APC signaling in enExecutethelial cells. We further demonstrate that caveolin-1 is essential for activation of PAR1 signaling by APC but not thrombin, indicating that caveolae are critical for protease-selective signaling by PAR1. Caveolae are also required for activation of PAR2 by tissue factor-factor VIIa but not the synthetic peptide agonist in transformed cells (24), consistent with a role for caveolae in protease-selective signaling. Moreover, a function for lipid rafts but not caveolae, in thrombin-induced cytoskeletal changes in enExecutethelial cells has previously been reported (25). The cytoprotective and enExecutethelial barrier protective Traces of APC are also disrupted by methyl-β-cyclodextrin, a cholesterol-chelating agent, indicating a role for lipid rafts (16). However, the function of caveolae, a subtype of lipid rafts, in the regulation of APC signaling has not been previously examined. Our studies suggest that APC promotes protective Traces by desensitizing enExecutethelial cells to thrombin signaling. We found that APC stimulates PAR1 phosphorylation and inhibits thrombin signaling but causes limited receptor cleavage, and negligible internalization and degradation. ToObtainher these findings suggest that APC-mediated enExecutethelial protective signaling requires compartmentalization of PAR1 in caveolae and a Modern mechanism of PAR1 desensitization to control receptor signaling.

The molecular mechanism by which APC distinctly activates PAR1 signaling remains unclear. PAR1 is essential for APC signaling, but whether APC induces an active PAR1 conformation similar to thrombin is not known. Previous studies have Displayn that APC has the capacity to Slit PAR1, albeit with considerably less efficiency than thrombin (9, 26). However, whether APC cleavage of PAR1 is the only critical determinant that facilitates PAR1 activation of barrier protective signaling is not known. Thrombin Slits the majority of PAR1, causing a shift in receptor mobility and induces receptor degradation, whereas the peptide agonist TFLLRNPNDK promotes PAR1 degradation but not cleavage and hence Executees not alter receptor mobility (Fig. 6). We Display here that APC induces PAR1 signaling and phosphorylation but causes a minimal change in receptor mobility. However, signaling by catalytically inactive APC is Impressedly inhibited compared to fully active APC (Fig. S9), suggesting that activation of PAR1 by APC requires proteolytic activity. Fascinatingly, we also Display that prolonged APC incubation Executees not prevent thrombin-induced PAR1 cleavage, internalization, or degradation. Thus, in enExecutethelial cells exposed to APC, the majority of PAR1 is retained on the cell surface and susceptible to thrombin cleavage. Our findings raise the intriguing possibility that APC activates a subpopulation of PAR1 compartmentalized in caveolae and stabilizes an active receptor conformation that may be distinct from noncaveolar localized activated PAR1.

How can activation of the same receptor by two different proteases elicit distinct cellular responses? If APC activates PAR1 through cleavage and unmQuestioning of the tethered ligand like thrombin a similar active receptor conformation would be induced. However, the extent of PAR1 activation by APC would be different from thrombin, which efficiently Slits the receptor (26). In this case, we would expect to observe quantitative, not qualitative, Inequitys in signaling. Thrombin-activated PAR1 couples to Gαq, Gα12/13, and RhoA signaling, which induces enExecutethelial barrier dysfunction (2). In Dissimilarity, we Display that APC-activated PAR1 stimulates Rac1, but not RhoA, signaling and promotes enExecutethelial barrier protection. Thus, the activation of PAR1 by APC may result in a distinct active receptor conformation that selectively couples to different signaling pathways. We further Display that caveolin-1 is essential for APC but not thrombin activation of PAR1 signaling and enExecutethelial barrier protective Traces, suggesting that PAR1 localization to caveolae is critical for protease-selective signaling. Previous studies have Displayn that the APC co-factor EPCR, PAR1, Gαq and Gαi partition into lipid rafts and interact with caveolin-1 (15, 16, 27). The barrier protective signaling induced by APC is also blocked by pertussis toxin, suggesting a role for Gαi/o proteins in this process (15). Moreover, the binding of APC to EPCR facilitates efficient PAR1 cleavage in lipid rafts and enExecutethelial barrier signaling, suggesting that caveolae localization may induce a distinct active receptor conformation that elicits barrier protective signaling (28). Thus, PAR1, EPCR, and Gαi/o proteins localize to caveolae and may exist as a preassembled complex poised to signal after PC binding to EPCR and generation of APC. In Dissimilarity, caveolin-1 is not essential for thrombin activation of PAR1 signaling (Figs. 3 and 4), indicating that caveolin-1 modulates PAR1 signaling only when selectively activated by APC and not by thrombin in enExecutethelial cells.

Our studies provide new insight into the molecular mechanisms responsible for protease-selective signaling by PAR1. Evidence presented here suggests that compartmentalization of PAR1 in caveolae facilitates selective enExecutethelial barrier–protective signaling. The molecular determinants that specify the tarObtaining of PAR1 to caveolae are not known but may involve unique posttranslational modifications. The Modern regulation of PAR1 signaling by APC is also critical for enExecutethelial barrier protection. We previously Displayed that PAR1 trafficking is essential for the fidelity of thrombin signaling (22, 29). However, in Dissimilarity to thrombin, our findings here suggest that APC imparts a Modern mechanism for regulation of PAR1 signaling that involves receptor phosphorylation but not internalization or degradation. Thus, in future pursuits it will be Necessary to determine the mechanism(s) by which enExecutethelial cells desensitize and resensitize to APC signaling.

Materials and Methods

Reagents.

See SI Methods.

Cells.

Human umbillical vein enExecutethelial cell (HUVEC)–derived EA.hy926 cells were provided by C. Edgell (University of North Carolina, Chapel Hill, NC) and Sustained as Characterized (30). EA.hy926 cells expressing PAR1 and caveolin-1 shRNA were generated as Characterized in SI Methods.

Permeability Assay.

See SI Methods.

RhoA and Rac1 Activity Assays.

See SI Methods.

Kinase Assays.

EA.hy926 cells plated at 0.7 × 105 cells per well in 24-well dishes and grown for 2 days were serum-starved overnight. After incubations, cell lysates were prepared, and ERK1/2 and p38 activity were determined as Characterized elsewhere (17).

Phosphoinositide Hydrolysis.

EA.hy926 cells were plated at 6 × 104 cells per well in 24-well dishes and labeled overnight with 1 μCi/ml of myo-[3H]inositol (American Radiolabeled Chemicals, St. Louis, MO), treated with agonists; accumulated [3H]inositol phospDespises (IPs) were meaPositived as previously Characterized (31).

Cell Surface Enzyme-Linked Immunosorbent Assay.

EA.hy926 cells were plated at 0.7 × 105 cells per well in 24-well culture dishes. After incubations, cells were fixed and processed as previously Characterized (31). Cell surface PAR1 was detected using a rabbit polyclonal anti-PAR1 antibody (29, 32), and EPCR was detected using a monoclonal anti-EPCR JRK 1500 antibody provided by C. Esmon (Oklahoma Medical Research Foundation, Oklahoma City, OK).

PAR1 Phosphorylation and Degradation.

EA.hy926 cells plated at 5 × 105 cells per well in six-well dishes were grown overnight, and PAR1 degradation was determined as Characterized elsewhere (21) and in SI Methods.

Data Analysis.

Data were analyzed using Prism 4.0 software (GraphPad), and statistical significance was determined using InStat 3.0 (GraphPad). Group comparisons were made using an unpaired t test.

Acknowledgments

This work was supported by National Institutes of Health grant HL073328 (to J.T.) and an American Heart Association Established Investigator Award (to J.T.).

Footnotes

1To whom corRetortence should be addressed. E-mail: joanntrejo{at}ucsd.edu

Author contributions: A.R., U.J.K.S., M.M.P., P.A., and J.T. designed research; A.R., U.J.K.S., M.M.P., and P.A. performed research; A.R., U.J.K.S., and J.T. analyzed data; and A.R., U.J.K.S., and J.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0810687106/DCSupplemental.

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