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Signaling through Gα13 Switch Region I Is Essential for Protease-activated Receptor 1-mediated Human Platelet Shape Change, Aggregation, and Secretion

2007; Elsevier BV; Volume: 282; Issue: 14 Linguagem: Inglês

10.1074/jbc.m605678200

1083-351X

Jinsheng Huang, Lanlan Dong, Tohru Kozasa, Guy C. Le Breton,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

This study investigated the involvement of Gα13 switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the Gα13 SRI (Myr-G13SRIpep) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G13SRIpep and Myr-G13SRIRandom-pep were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G13SRIpep specifically bound to platelet RhoA guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation in platelets and human embryonic kidney cells. These results suggest a direct interaction of Gα13 SRI with p115RhoGEF and a mechanism for Myr-G13SRIpep inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G13SRIpep specifically inhibited PAR1-stimulated shape change, aggregation, and secretion in a dose-dependent manner but did not inhibit platelet activation induced by either ADP or A23187. It was also found that Myr-G13SRIpep inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G13SRIpep but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G13SRIpep effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of Gα13 SRI with p115RhoGEF is required for G13-mediated RhoA activation in platelets; 2) signaling through the G13 pathway is critical for PAR1-mediated human platelet functional changes and low dose thrombin-induced aggregation; and 3) G13 signaling elicits calcium mobilization in human platelets through a Rho kinase-independent mechanism. This study investigated the involvement of Gα13 switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the Gα13 SRI (Myr-G13SRIpep) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G13SRIpep and Myr-G13SRIRandom-pep were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G13SRIpep specifically bound to platelet RhoA guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation in platelets and human embryonic kidney cells. These results suggest a direct interaction of Gα13 SRI with p115RhoGEF and a mechanism for Myr-G13SRIpep inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G13SRIpep specifically inhibited PAR1-stimulated shape change, aggregation, and secretion in a dose-dependent manner but did not inhibit platelet activation induced by either ADP or A23187. It was also found that Myr-G13SRIpep inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G13SRIpep but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G13SRIpep effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of Gα13 SRI with p115RhoGEF is required for G13-mediated RhoA activation in platelets; 2) signaling through the G13 pathway is critical for PAR1-mediated human platelet functional changes and low dose thrombin-induced aggregation; and 3) G13 signaling elicits calcium mobilization in human platelets through a Rho kinase-independent mechanism. Thrombin and thrombin receptors in platelets play critical roles in heart attack, stroke, and thromboembolic processes (reviewed in Ref. 1Hollenberg M.D. Compton S.J. Pharmacol. Rev. 2002; 54: 203-217Crossref PubMed Scopus (406) Google Scholar). Consequently, investigating the mechanisms of thrombin receptor-mediated signaling pathways is of great importance to developing therapeutic approaches or anti-thrombotic agents (2Brass L.F. Chest. 2003; 124: S18-S25Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) to treat certain cardiovascular diseases.The first thrombin receptor was cloned and sequenced by Coughlin and co-workers in 1991 (3Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2650) Google Scholar). It was found that this receptor is a member of the seven-transmembrane receptor class that signals through different G proteins. Thrombin acts by first cleaving the extracellular N-terminal domain of the receptor, and the new N terminus (SFLLRN) then binds to the second extracellular loop of the receptor protein to cause cellular activation (3Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2650) Google Scholar). To date, four protease-activated receptors (PARs), 2The abbreviations used are: PAR, protease-activated receptor; SRI, switch region I; PRP, platelet-rich plasma; PGI2, prostaglandin I2; TMB, tetramethylbenzidine; BSA, bovine serum albumin; HRP, horseradish peroxidase; HEK, human embryonic kidney; MALDI-TOFMS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis; ELISA, enzyme-linked immunosorbent assay; GPCR, G protein-coupled receptor; Ab, antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Myr, myristoylated; Bio, biotinylated; TRAP, thrombin receptor agonist peptide.2The abbreviations used are: PAR, protease-activated receptor; SRI, switch region I; PRP, platelet-rich plasma; PGI2, prostaglandin I2; TMB, tetramethylbenzidine; BSA, bovine serum albumin; HRP, horseradish peroxidase; HEK, human embryonic kidney; MALDI-TOFMS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis; ELISA, enzyme-linked immunosorbent assay; GPCR, G protein-coupled receptor; Ab, antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Myr, myristoylated; Bio, biotinylated; TRAP, thrombin receptor agonist peptide. i.e. PAR1, PAR2, PAR3, and PAR4, have been cloned, all of which except for PAR2 are identified as receptors for thrombin (4Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Crossref PubMed Scopus (518) Google Scholar). Regarding platelets, there is a species-specific expression of the PAR subtypes. For example, PAR3 and PAR4 (but not PAR1) are expressed in mouse platelets. The functional significance of these receptors was demonstrated by the finding that knock-out of either PAR3 or PAR4 led to impaired thrombin-induced platelet activation (5Hamilton J.R. Cornelissen I. Coughlin S.R. J. Thromb. Haemostasis. 2004; 2: 1538-7933Crossref Scopus (81) Google Scholar). In contrast to mouse, human platelets possess PAR1 and PAR 4 but not PAR 3 receptors. Therefore, in this case, the combination of PAR1 and PAR4 signaling pathways is thought to account for a substantial portion of thrombin-mediated platelet activation (6Kahn M.L. Nakanishi-Matsui M. Shapiro M.J. Ishihara H. Coughlin S.R. J. Clin. Investig. 1999; 103: 879-887Crossref PubMed Scopus (688) Google Scholar).As mentioned previously, PAR signaling pathways involve G protein-initiated effector activation. Furthermore, PARs are thought to couple to different G proteins depending upon the cell type. Our present understanding of PAR-G protein signaling in platelets is primarily based on the collective information obtained from experimental approaches in two separate species, i.e. mouse and human. Specifically, Offermanns et al. (7Offermanns S. Toombs C.F. Hu Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (494) Google Scholar) evaluated the potential role of PAR-Gq signaling using Gq knock-out mice. The results using these mice demonstrated a complete inhibition of thrombin-induced platelet aggregation but not shape change (7Offermanns S. Toombs C.F. Hu Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (494) Google Scholar). Further studies indicated that PARs may also be coupled to Gi (8Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (305) Google Scholar), because thrombin caused a decrease in cAMP levels in the wild-type as well as in the Gq null mice platelets. As an extension of this work, the effects of a conditional G13 knock-out on mouse platelet function were also evaluated (9Moers A. Nieswandt B. Massberg S. Wettschureck N. Gruner S. Konrad I. Schulte V. Aktas B. Gratacap M.P. Simon M.I. Gawaz M. Offermanns S. Nat. Med. 2003; 9: 1418-1422Crossref PubMed Scopus (210) Google Scholar). It was found that the G13 null platelets exhibited diminished thrombin-induced platelet shape change, aggregation, and RhoA activation at low thrombin concentrations but appeared to have a normal thrombin response at higher thrombin concentrations (9Moers A. Nieswandt B. Massberg S. Wettschureck N. Gruner S. Konrad I. Schulte V. Aktas B. Gratacap M.P. Simon M.I. Gawaz M. Offermanns S. Nat. Med. 2003; 9: 1418-1422Crossref PubMed Scopus (210) Google Scholar). Furthermore, knock-out of both Gq and G13 resulted in a complete unresponsiveness of platelets to thrombin (10Moers A. Wettschureck N. Gruner S. Nieswandt B. Offermanns S. J. Biol. Chem. 2004; 279: 45354-45359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Therefore, taken together these results suggest that Gq,G13, and possibly Gi play important roles in the intracellular signaling pathway of thrombin receptor activation in mouse platelets.However, the results in mice cannot necessarily be extrapolated to human platelet activation, because different PAR profiles are found in each case. Regarding human platelets, initial in vitro experiments indicated that thrombin could stimulate both Gαq/11 and Gα12/13 phosphorylation in human platelet membranes, suggesting that these two G protein families may functionally couple to PARs (11Covic L. Gresser A.L. Kuliopulos A. Biochemistry. 2000; 39: 5458-5467Crossref PubMed Scopus (250) Google Scholar). Support for a Gq-mediated pathway for both PAR1 and PAR4 receptors in human platelets was provided by studies using PAR-specific peptides. The results indicated that although both PAR1 and PAR4 receptor activation caused an increase in intraplatelet calcium levels, the kinetics of this calcium mobilization appeared to be different for each receptor type (11Covic L. Gresser A.L. Kuliopulos A. Biochemistry. 2000; 39: 5458-5467Crossref PubMed Scopus (250) Google Scholar). In a subsequent study employing human platelets deficient in Gαq and PLC-β2 (12Vaidyula V.R. Rao A.K. Br. J. Haematol. 2003; 121: 491-496Crossref PubMed Scopus (25) Google Scholar), it was found that the PAR1 and PAR4-induced calcium responses were reduced relative to normal platelets (12Vaidyula V.R. Rao A.K. Br. J. Haematol. 2003; 121: 491-496Crossref PubMed Scopus (25) Google Scholar). Consequently, it appears that both PAR1 and PAR4 have the capacity to signal through Gq and elicit calcium mobilization in human platelets.Other reports have provided indirect evidence linking the human G12/13 signaling pathway to PAR1-mediated platelet function (13Rasmussen U.B. Gachet C. Schlesinger Y. Hanau D. Ohlmann P. Obberghen-Schilling E. Pouyssegur J. Cazenave J.P. Pavirani A. J. Biol. Chem. 1993; 268: 14322-14328Abstract Full Text PDF PubMed Google Scholar, 14Dorsam R.T. Kim S. Jin J. Kunapuli S.P. J. Biol. Chem. 2002; 277: 47588-47595Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 15Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Crossref PubMed Google Scholar, 16Otterdal K. Pedersen T.M. Solum N.O. Thromb. Res. 2001; 103: 411-420Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Thus, it was found that selective activation of PAR1 with the peptide YFLLRNP resulted in platelet shape change (13Rasmussen U.B. Gachet C. Schlesinger Y. Hanau D. Ohlmann P. Obberghen-Schilling E. Pouyssegur J. Cazenave J.P. Pavirani A. J. Biol. Chem. 1993; 268: 14322-14328Abstract Full Text PDF PubMed Google Scholar) that could be blocked by the Rho kinase inhibitor Y-27632 (14Dorsam R.T. Kim S. Jin J. Kunapuli S.P. J. Biol. Chem. 2002; 277: 47588-47595Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 15Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Crossref PubMed Google Scholar). Because RhoA activation has been linked previously to G13 signaling (8Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (305) Google Scholar, 17Paul B.Z. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), these results led the authors to suggest that this shape change response is mediated through the G12/13 signaling pathway (14Dorsam R.T. Kim S. Jin J. Kunapuli S.P. J. Biol. Chem. 2002; 277: 47588-47595Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). It was also reported that in the presence of Gi activation, selective stimulation of G12/13 signaling by low doses of agonists resulted in platelet aggregation and αIIbβ3 integrin activation (14Dorsam R.T. Kim S. Jin J. Kunapuli S.P. J. Biol. Chem. 2002; 277: 47588-47595Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 18Nieswandt B. Schulte V. Zywietz A. Gratacap M.P. Offermanns S. J. Biol. Chem. 2002; 277: 39493-39498Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). These findings were interpreted as evidence that PAR1-mediated G12/13 signaling can contribute to human platelet aggregation if Gi signaling is simultaneously activated. Although recent evidence suggests that human PARs may not directly signal through Gi, such Gi signaling would presumably occur as a consequence of PAR-mediated ADP secretion (19Kim S. Foster C. Lecchi A. Quinton T.M. Prosser D.M. Jin J. Cattaneo M. Kunapuli S.P. Blood. 2002; 99: 3629-3636Crossref PubMed Scopus (147) Google Scholar).These data therefore suggest that human platelet PAR1 primarily signals through Gq and G12/13. Despite these findings, however, the relative contribution of each specific G protein to the overall PAR1-mediated signaling process in human platelets remains unclear, and the requirement for G12/13 signaling in this response is presently unknown. In this study we report the use of a myristoylated peptide representing the SRI of Gα13 (Myr-G13SRIpep) to study PAR1-mediated G13 signaling and function in intact human platelets. Our results provide evidence that PAR1-induced RhoA activation involves the interaction of Gα13 SRI with p115RhoGEF. Platelet functional experiments revealed that Gα13 SRI signaling is required for PAR1-induced functional change in both human platelets and human endothelial cells. Finally, the results identify a Rho kinase-independent G13 signaling pathway involving the mobilization of intraplatelet calcium. Taken together, these data indicate that signaling of Gα13 through SRI is critical for PAR1-induced cellular activation.EXPERIMENTAL PROCEDURESReagents—Myristoylated (Myr) Gα13 SRI peptide (Myr-LLARRPTKGIHEY, Myr-G13SRIpep) and Myr-Gα13SRI random peptide (Myr-LIRPYLHRATKEG, Myr-G13SRIRandom-pep) were synthesized by GenScript Corp. (Piscataway, NJ) or the Research Resource Center, University of Illinois, Chicago; biotinylated (Bio) Gα13 SRI peptide (Bio-LLARRPTKGIHEY, Bio-G13SRIpep), Bio-Gα13SRI random peptide (Bio-LIRPYLHRATKEG, Bio-G13SRIRandom-pep), Myr-Bio-Gα13 SRI peptide (Myr-Bio-KLLARRPTKGIHEY, Myr-Bio-G13SRIpep), Myr-Bio-Gα13SRI random peptide (Myr-Bio-KLIRPYLHRATKEG, Myr-Bio-G13SRIRandom-pep), thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP1), and PAR4-activating peptide (AYPGKF) were synthesized by the Research Resource Center, University of Illinois, Chicago. Luciferin/luciferase reagent (Chrono-lume) was purchased from Chrono-Log (Havertown, PA); HRP-conjugated goat anti-biotin was from Vector Laboratories (Burlingame, CA); HRP-conjugated goat anti-rabbit IgG (H + L) was from Bio-Rad; human embryonic kidney (HEK) cells and human live microvascular endothelial cells were purchased from the American Type Culture Collection (ATCC). Polyvinylidene difluoride membranes were from Millipore Corp. (Bedford, MA). Enhanced chemiluminescence substrate, biotinylation reagent EZ-Link Biotin-LC-PEO-Amine, and the BCA protein assay kit were from Pierce. Bovine serum albumin (BSA), tetramethylbenzidine (TMB), apyrase (grade VII) and Y-27632 were from Sigma. Protein kinase C inhibitor Ro 31-8220 was from Cayman (Ann Arbor, MI). Nunc-Immuno™ plates were from Fisher. 3H-TRAP1 (specific activity 19.55Ci/mm) was custom-labeled by Amersham Biosciences. Fura-2AM was from Molecular Probes (Eugene, OR). Human platelet-rich plasma (PRP, anticoagulated with acid/citrate/dextrose, 2–3 × 108 platelets/ml) were purchased from Life Source Blood Services (Glenview, IL), and outdated platelets were kindly provided by the Hospital Blood Bank, University of Illinois, Chicago.Platelet Membrane Preparation and Solubilization—Solubilized platelet membranes were prepared as described previously (20Manganello J.M. Djellas Y. Borg C. Antonakis K. Le Breton G.C. J. Biol. Chem. 1999; 274: 28003-28010Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Briefly, outdated human platelets were incubated with 3 mm aspirin for 45 min and pelleted by centrifugation at 1,600 × g for 20 min. The platelet pellets were washed with buffer A (25 mm Tris-HCl, 5 mm MgCl2, pH 7.4) containing PGI2 (40 nm), sonicated on ice, and centrifuged at 1,600 × g for 5 min. The supernatant was placed into centrifuge tubes, and the pellets were again resuspended, sonicated, and centrifuged. The supernatants were then combined and centrifuged at 100,000 × g for 30 min. The pellet was resuspended in buffer A plus 10 mm CHAPS, homogenized, and centrifuged again at 100,000 × g for 30 min. The resulting membrane pellet was resuspended in buffer A to yield a final protein concentration of 2–4 mg/ml.Purification of Recombinant p115—Full-length p115RhoGEF cDNA was ligated into BamHI, XhoI sites of pFastBacHTc vector with hexahistidine tag at the N terminus. Baculovirus expressing His6-p115 was generated, and Sf9 cells were infected with the virus for 48 h. Recombinant p115 protein was purified from the cytosolic fractions of infected cells using nickel-nitrilotriacetic acid affinity column with the yield of 0.5 mg from 500 ml of Sf9 cell culture. The protein was ∼90% pure by SDS-PAGE visualization.Dot Blot for Myristoylated Peptides—Human PRP was incubated with 100 μm biotinylated Myr peptides or free biotinylation reagent (as a secondary control) for 5 min. The platelets were then washed three times by centrifugation in Tyrode's buffer containing PGI2 (40 nm), disrupted with lysis buffer (50 mm Tris-HCl containing 100 mm NaCl, 1 mm EDTA, 5 mm MgCl2, 10% glycerol, 50 mm NaF, 1 mm Na3VO4, 1 mm dithiothreitol, 50 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml each of leupeptin and pepstatin, and 0.2% Nonidet P-40, pH 7.5), and sonicated on ice (three 45-s bursts with 15-s pauses). The platelet samples were next loaded on methanol-pretreated polyvinylidene difluoride membranes, blocked by 5% nonfat dry milk, and incubated with HRP-conjugated goat anti-biotin Ab at room temperature for 1 h. After incubation, the membranes were washed three times with Tyrode's buffer and subsequently incubated with HRP substrates. The film was then developed and fixed.Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometric Analysis (MALDI-TOFMS)—MALDI-TOFMS analysis was performed as described previously (21Lee B.S. Krishnanchettiar S. Lateef S.S. Gupta S. Rapid Commun. Mass Spectrom. 2005; 19: 1545-1550Crossref PubMed Scopus (27) Google Scholar). Specifically, human PRP was incubated with 500 μm Myr-G13SRIpep or Myr-G13SRIRandom-pep for 5 min at room temperature. Following incubation, the platelets were washed three times with Tyrode's buffer containing PGI2 (40 nm) and disrupted with lysis buffer and sonicated as described above. The platelet samples were then mixed 1:1 with the matrix solution (10 mg of cyano-4-hydroxycinnamic acid in 1 ml of aqueous solution of 50% acetonitrile containing 0.1% trifluoroacetic acid). Finally, the sample aliquots were spotted onto a MALDI-TOF target and analyzed at 1,764 daltons using a Voyager-DE PRO mass spectrometer (Applied Biosystems) equipped with a 337 nm pulsed nitrogen laser.3H-TRAP1 Binding to Intact Human Platelets—To ensure that the association of 3H-TRAP1 with platelets was not because of uptake of the ligand, displacement studies were employed to measure the extent of the specific binding. In these studies, PRP was initially incubated with 500 μm Myr-G13SRIpep or Myr-G13SRIRandom-pep for 5 min at room temperature. At this point, 40 nm 3H-TRAP1 was added, and the reaction mixture was incubated at 4 °C for 10 min. The reaction was then supplemented with 40 μm unlabeled TRAP1 (to determine specific binding) and incubated for an additional 10 min. Aliquots (1 ml) of the PRP were immediately layered over a silicon oil mixture (75 μl; 81% Dow 550; 19% Dow 200) in a centrifuge tube and centrifuged at 7,000 × g for 1 min to separate the platelets from their plasma (22Feinstein M.B. Michel I. Born G.V.R. J. Lab. Clin. Med. 1974; 84: 926-934Google Scholar, 23Kattelman E.J. Venton D.L. Le Breton G.C. Thromb. Res. 1986; 41: 471-481Abstract Full Text PDF PubMed Scopus (98) Google Scholar). The supernatant was quickly removed by aspiration, and the tube tips containing the platelet pellets were cut and transferred to scintillation vials. One ml of 0.3 n NaOH was added to solubilize the platelets, and 100 μl of 3 n HCl were added to neutralize the reaction mixture. Finally, 10 ml of scintillation fluid were added, and the solution was counted on a Beckman LS 6500 liquid scintillation counter. Under these conditions, specific binding of 3H-TRAP1 was ∼33%.Pulldown Assay for GTP-Rho—RhoA activation was determined as described previously with slight modifications (24Manganello J.M. Huang J.S. Kozasa T. Voyno-Yasenetskaya T.A. Le Breton G.C. J. Biol. Chem. 2003; 278: 124-130Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, 400 μl of PRP were incubated with 500 μm Myr-G13SRIpep, 500 μm Myr-G13SRIRandom-pep, or Me2SO vehicle at room temperature for 5 min and then treated with 25 μm PAR1 agonist (TRAP1) for 1 min. One volume of cold lysis buffer was added, and the samples were incubated on ice for 10 min. The platelet lysates were then centrifuged at 15,000 × g for 1 min, and the supernatant was incubated (4 °C for 1 h) with 20 μg of glutathione S-transferase-Rho binding domain fusion protein-conjugated beads. The beads were washed three times with lysis buffer and subjected to SDS-PAGE. Activated RhoA was detected by Western blot analysis using a polyclonal antibody against RhoA. In separate experiments, 400 μl of HEK cells were incubated with 500 μm Myr-G13SRIpep, 500 μm Myr-G13SRIRandom-pep, or Me2SO vehicle at room temperature for 15 min and then treated with 50 μm TRAP1 for 1 min. After centrifugation, 400 μl of cold lysis buffer was added, and the sample was sonicated on ice. The cell lysates were then centrifuged at 15,000 × g for 1 min, and the supernatant was subjected to the same procedure as described above.Measurement of p115 RhoGEF Binding to G13SRIpep—The design of this experiment was to coat Nunc-Immuno plate wells with G13SRIpep or G13SRIRandom-pep and to measure whether either peptide could capture p115RhoGEF from solubilized human platelets using a sandwich ELISA. However, to establish that equal amounts of G13SRIpep and G13SRIRandom-pep were immobilized to the wells, the following experiment was first conducted. In brief, the wells were coated with various concentrations (0.1 nm to 400 μm) of biotinylated G13SRIpep or biotinylated G13 SRIRandom-pep at 4 °C overnight. The wells were then washed three times with TBS buffer containing 0.03% Tween 20 and blocked with 3% BSA in TBS-Tween buffer. Following incubation at room temperature for 1 h with avidin-HRP, the wells were washed seven times with TBS-Tween buffer, and the TMB substrates were added. When the appropriate color appeared, 50 μl of 1 n HCl were added to stop the reaction, and the absorbance (A) at 450 nm was measured in each sample. A comparison of the A450 values revealed that 0.23 μm G13SRIpep and 12.5 μm G13SRIRandom-pep resulted in equal coating of the wells (Fig. 5A).In the sandwich ELISA experiments, the Nunc-Immuno plate wells were equally coated with either G13SRIpep or G13SRIRandom-pep and blocked as outlined above. After washing three times with TBS-Tween buffer, 100 μg of solubilized platelet membrane protein (20Manganello J.M. Djellas Y. Borg C. Antonakis K. Le Breton G.C. J. Biol. Chem. 1999; 274: 28003-28010Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) were added, and the samples were incubated at room temperature for 2 h. The wells were then supplemented with p115RhoGEF Ab and incubated at room temperature for an additional 1 h. After three washes with TBS-Tween buffer, the HRP-linked secondary Ab was added, and the reaction was incubated at room temperature for an additional 1 h. Finally, TMB substrates were added, and the A450 values were measured.In alternative ELISA experiments, the Nunc-Immuno plate wells were coated with 100 μg/ml recombinant human p115RhoGEF and blocked with 3% BSA. The wells were then supplemented with Bio-G13SRIpep or Bio-G13SRIRandom-pep. After incubation at 37 °C for 4 h, the avidin-HRP was added, and the A450 values were measured as described above.Platelet Shape Change—Human PRP was incubated with Me2SO (vehicle control), Myr-G13SRIpep, or Myr-G13SRIRandom-pep for 5 min, followed by addition of 3 mm EGTA and 10 μm indomethacin. After 2 min of incubation, shape change was induced by adding different agonists and measured by the turbidimetric method (23Kattelman E.J. Venton D.L. Le Breton G.C. Thromb. Res. 1986; 41: 471-481Abstract Full Text PDF PubMed Scopus (98) Google Scholar) using a model 400 aggregometer (Chrono-Log, Havertown, PA).Platelet Aggregation in PRP—Human PRP was incubated with Me2SO vehicle, Myr-G13SRIpep, or Myr-G13SRIRandom-pep at room temperature for 5 min and 10 μm indomethacin for 2 min. Platelet aggregation was induced by adding different agonists, and measured by the turbidimetric method (23Kattelman E.J. Venton D.L. Le Breton G.C. Thromb. Res. 1986; 41: 471-481Abstract Full Text PDF PubMed Scopus (98) Google Scholar) using a model 400 aggregometer (Chrono-Log).Platelet Aggregation in Resuspended Platelets—Human PRP was incubated with 500 μm Myr-G13SRIpep or Myr-G13SRIRandom-pep for 5 min. Following the addition of 40 nm PGI2, the plasma was removed by centrifugation at 500 × g for 3 min. Platelets were resuspended in Tyrode's buffer (supplemented with 1 mm calcium and 0.38% BSA), and the platelet count was adjusted to 3 × 108 platelets/ml. Following addition of 10 μm indomethacin and 0.05 units/ml apyrase, thrombin (0.1 or 0.2 units/ml) was added, and platelet aggregation was measured.Platelet Dense Granule Secretion—Human PRP was pretreated with Me2SO vehicle, 75–250 μm Myr-G13SRIpep, or 250 μm Myr-G13SRIRandom-pep and incubated with luciferin/luciferase for 3 min before stimulation. Platelet dense granule secretion was induced by adding 25 μm TRAP1, and secreted ATP was measured with a Chrono-Log lumi-aggregometer.Platelet Calcium Mobilization—Human PRP was incubated with Fura-2 and 500 μm Myr-G13SRIpep (or 500 μm Myr-G13SRIRandom-pep) for 30 min. The platelets were then supplemented with 10 μm indomethacin, 0.05 units/ml apyrase and sedimented by centrifugation at 500 × g for 3 min. Following resuspension in Tyrode's buffer (supplemented with 1 mm calcium, 0.38% BSA, 10 μm indomethacin, and 0.05 units/ml apyrase), platelet calcium mobilization was induced with 25 μm TRAP1 or 10 μm ADP. In the experiments with Y-27632, the platelets were loaded with Fura-2 as described above. Platelets were then incubated with vehicle or 30 μm Y-27632 for 3 min, and calcium mobilization was induced with 25 μm TRAP1. The samples were excited at 340/380 nm, and the fluorescent emissions were detected at 510 nm using a PTI spectrofluorometer.Stress Fiber Formation and Cell Contraction—Human live microvascular endothelial cells were seeded on coverslips and starved for 24 h after the cells became 100% confluent. The cells were then preincubated with 500 μm Myr-G13SRIRandom-pep, Myr-G13SRIpep, or vehicle (in 6% BSA Hanks' buffer) for 30 min and stimulated with 50 μm TRAP1 or vehicle for 15 min. After fixation with 3.7% formaldehyde and permeabilization with 0.1% Triton X-100, the cells were stained with Alex Fluor 488 phalloidin/4′,6-diamidino-2-phenylindole. Finally, the cells were washed and mounted, and the images were obtained using a confocal microscope (Zeiss LSM 510).Statistical Analysis—Data were analyzed with GraphPad PRISM statistical software (San Diego, CA). Statistical significance is defined as p < 0.05 or p < 0.0001, as indicated.RESULTSCharacterization of Myr-G13SRIpep Uptake by Intact Human Platelets—Our previous studies showed that a peptide representing Gα13 SRI can be useful to study G13 signaling (24Manganello J.M. Huang J.S. Kozasa T. Voyno-Yasenetskaya T.A. Le Breton G.C. J. Biol. Chem. 2003; 278: 124-130Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To further explore G13 signal transduction in intact human platelets, myri