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• W1517951856 abstract "The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, αIIbβ3. Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, U46619, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress. Blocking the ligand binding function of integrin αIIbβ3, by pretreating platelets with c7E3 Fab, demonstrated the existence of integrin αIIbβ3-dependent and -independent mechanisms regulating RhoA activation. Inhibition of RhoA (C3 exoenzyme) or its downstream effector Rho kinase (Y27632) had no effect on integrin αIIbβ3 activation induced by soluble agonists or adhesive substrates, however, both inhibitors reduced shear-dependent platelet adhesion on immobilized vWf and shear-induced platelet aggregation in suspension. Detailed analysis of the sequential adhesive steps required for stable platelet adhesion on a vWf matrix under shear conditions revealed that RhoA did not regulate platelet tethering to vWf or the initial formation of integrin αIIbβ3 adhesion contacts but played a major role in sustaining stable platelet-matrix interactions. These studies define a critical role for RhoA in regulating the stability of integrin αIIbβ3adhesion contacts under conditions of high shear stress. The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, αIIbβ3. Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, U46619, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress. Blocking the ligand binding function of integrin αIIbβ3, by pretreating platelets with c7E3 Fab, demonstrated the existence of integrin αIIbβ3-dependent and -independent mechanisms regulating RhoA activation. Inhibition of RhoA (C3 exoenzyme) or its downstream effector Rho kinase (Y27632) had no effect on integrin αIIbβ3 activation induced by soluble agonists or adhesive substrates, however, both inhibitors reduced shear-dependent platelet adhesion on immobilized vWf and shear-induced platelet aggregation in suspension. Detailed analysis of the sequential adhesive steps required for stable platelet adhesion on a vWf matrix under shear conditions revealed that RhoA did not regulate platelet tethering to vWf or the initial formation of integrin αIIbβ3 adhesion contacts but played a major role in sustaining stable platelet-matrix interactions. These studies define a critical role for RhoA in regulating the stability of integrin αIIbβ3adhesion contacts under conditions of high shear stress. Platelet adhesion and aggregation at sites of vascular injury is essential for the arrest of bleeding and for subsequent vessel wall repair. The ability of platelets to adhere under conditions of rapid blood flow requires the synergistic contribution of multiple receptor-ligand interactions, foremost of which involves the interaction between von Willebrand factor (vWf) 1The abbreviations used are: vWfvon Willebrand factorGPglycoproteinMLCmyosin light chainHvWfhuman vWfRBCred blood cellsGSTglutathione S-transferaseGEFguanine-nucleotide exchange factorSIPAshear-induced platelet activationAs-vWfasialo-vWfFAfocal adhesionRGDSarginine-glycine-aspartic acid-serineTRAPthrombin receptor agonist peptide1The abbreviations used are: vWfvon Willebrand factorGPglycoproteinMLCmyosin light chainHvWfhuman vWfRBCred blood cellsGSTglutathione S-transferaseGEFguanine-nucleotide exchange factorSIPAshear-induced platelet activationAs-vWfasialo-vWfFAfocal adhesionRGDSarginine-glycine-aspartic acid-serineTRAPthrombin receptor agonist peptide and the two major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin αIIbβ3. The vWf·GP Ib/V/IX interaction is characterized by a rapid association rate that enables efficient platelet tethering to the injured vessel wall, whereas subsequent integrin αIIbβ3engagement of vWf is important for promoting platelet arrest (1.Ruggeri Z.M. J. Clin. Invest. 1997; 99: 559-564Crossref PubMed Scopus (191) Google Scholar). The vWf·GP Ib/V/IX interaction is indispensable for normal platelet function, because it slows platelet movement at the vessel wall thereby enabling receptors with slower intrinsic binding kinetics,i.e. integrins, to engage adhesive ligands. A similar dual-step adhesion mechanism, involving selectins and β2integrins, is employed by leukocytes to adhere to post-capillary venules at sites of inflammation (2.Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1869) Google Scholar, 3.Ley K. Gaehtgens P. Fennie C. Singer M.S. Lasky L.A. Rosen S.D. Blood. 1991; 77: 2553-2555Crossref PubMed Google Scholar, 4.Von Andrian U.H. Chamberas J.D. McEvoy L.M. Bargatze R.F. Arfors K.E. Butcher E.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7538-7542Crossref PubMed Scopus (893) Google Scholar). von Willebrand factor glycoprotein myosin light chain human vWf red blood cells glutathione S-transferase guanine-nucleotide exchange factor shear-induced platelet activation asialo-vWf focal adhesion arginine-glycine-aspartic acid-serine thrombin receptor agonist peptide von Willebrand factor glycoprotein myosin light chain human vWf red blood cells glutathione S-transferase guanine-nucleotide exchange factor shear-induced platelet activation asialo-vWf focal adhesion arginine-glycine-aspartic acid-serine thrombin receptor agonist peptide A key requirement of the adhesion contacts formed by platelets and leukocytes is their ability to resist the detaching effects of rapidly flowing blood. In the case of platelets, the shear forces operating on adhesive bonds can be extremely high, particularly at sites of arterial narrowing where shear forces can exceed 380 dyne/cm2(10,000 s−1). The ability of the GP Ib/V/IX complex to maintain platelet adhesive interactions under such high shear conditions is due to the large number of bonds formed between vWf and GP Ib, the inherent biomechanical stability of the vWf·GP Ib interaction, and anchorage of the receptor complex to the membrane skeleton (1.Ruggeri Z.M. J. Clin. Invest. 1997; 99: 559-564Crossref PubMed Scopus (191) Google Scholar). The factors that regulate the stability of integrin αIIbβ3 adhesion contacts in a shear field remain less clearly defined. It is well established that integrin engagement of adhesive ligands leads to receptor clustering and the formation of firm anchorage points with the actin-based cytoskeleton (5.Shattil S.J. Thromb. Haemost. 1999; 82: 318-325Crossref PubMed Scopus (187) Google Scholar), however, the importance of such post-ligand binding events for sustaining platelet adhesion in a shear field remains unclear. The adhesive function of many integrins is regulated in part by RhoA, a member of the Ras homologous (Rho) family of small GTPases (6.Kaibucki K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (883) Google Scholar). RhoA activation has been extensively studied in a range of cultured cell and is critical for the formation of actin stress fibers and focal adhesions (7.Burridge K. Chrzanowska-Wodnicka M. Zhong C. Trends Cell Biol. 1997; 7: 342-347Abstract Full Text PDF PubMed Scopus (196) Google Scholar). Several RhoA effector proteins have been implicated in the formation of these structures, including p140mDia and Rho kinase (p160ROCK, ROKα). The effect of Rho kinase on focal adhesion formation appears to involve phosphorylation and inactivation of myosin light chain (MLC) phosphatase leading to increased MLC phosphorylation and enhanced myosin II contractility, resulting in tension on actin filaments and their subsequent alignment. Furthermore, RhoA-mediated activation of phosphatidylinositol-4-phosphate 5-kinase induces the formation of phosphatidylinositol (4,5)-bisphosphate, a widely acting phospholipid that promotes actin polymerization and cytoskeletal-membrane attachment (8.Schoenwaelder S.M. Burridge K. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (647) Google Scholar, 9.Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1639) Google Scholar). The culmination of these and other RhoA-mediated signaling events leads to bundling of actin filaments into actin cables or thick stress fibers and the clustering of their associated integrin receptors into focal adhesions (7.Burridge K. Chrzanowska-Wodnicka M. Zhong C. Trends Cell Biol. 1997; 7: 342-347Abstract Full Text PDF PubMed Scopus (196) Google Scholar). These cytoskeletal changes induced by RhoA leads to strong cell attachment points with the extracellular matrix (8.Schoenwaelder S.M. Burridge K. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (647) Google Scholar). The platelet contains relatively high levels of RhoA, along with other RhoA effectors (10.Morii N. Teru-uchi T. Tominaga T. Kumagai N. Kozaki S. Ushikubi F. Narumiya S. J. Biol. Chem. 1992; 267: 20921-20926Abstract Full Text PDF PubMed Google Scholar). However, despite this, very little is known about the mechanisms of RhoA activation or its contribution to integrin-mediated adhesive responses in platelets. Recent studies have demonstrated RhoA activation in response to thrombin (11.Bodie S.L. Ford I. Greaves M. Nixon G.F. Biochem. Biophys. Res. Commun. 2001; 287: 71-76Crossref PubMed Scopus (25) Google Scholar) and the thromboxane mimetic, U46619 (12.Gratacap M.-P. Payrastre B. Nieswandt B. Offermanns S. J. Biol. Chem. 2001; 276: 47906-47913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), with activation of RhoA by the latter agonist reported to occur independent of integrin αIIbβ3 (12.Gratacap M.-P. Payrastre B. Nieswandt B. Offermanns S. J. Biol. Chem. 2001; 276: 47906-47913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The role of RhoA in regulating the adhesive function of integrin αIIbβ3remains controversial (10.Morii N. Teru-uchi T. Tominaga T. Kumagai N. Kozaki S. Ushikubi F. Narumiya S. J. Biol. Chem. 1992; 267: 20921-20926Abstract Full Text PDF PubMed Google Scholar, 13.Leng L. Kashiwagi H. Ren X.-D. Shattil S.J. Blood. 1998; 91: 4206-4215Crossref PubMed Google Scholar, 14.Qi W. Loh E. Vilaire G. Bennett J.S. J. Biol. Chem. 1998; 273: 15271-15278Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 15.Missy K. Plantavid M. Pacaud P. Viala C. Chap H. Payrastre B. Thromb. Haemost. 2001; 85: 514-520Crossref PubMed Scopus (27) Google Scholar, 16.Nishioka H. Horiuchi H. Tabuchi A. Yoshioka A. Shirakawa R. Kita T. Biochem. Biophys. Res. Commun. 2001; 280: 970-975Crossref PubMed Scopus (23) Google Scholar). Although initial reports suggested a potentially important role in regulating integrin αIIbβ3 activation (10.Morii N. Teru-uchi T. Tominaga T. Kumagai N. Kozaki S. Ushikubi F. Narumiya S. J. Biol. Chem. 1992; 267: 20921-20926Abstract Full Text PDF PubMed Google Scholar), more recent studies have not supported these conclusions (13.Leng L. Kashiwagi H. Ren X.-D. Shattil S.J. Blood. 1998; 91: 4206-4215Crossref PubMed Google Scholar, 14.Qi W. Loh E. Vilaire G. Bennett J.S. J. Biol. Chem. 1998; 273: 15271-15278Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 17.Eckly A. Gendrault J.-L. Hechler B. Cazenave J.-P. Gachet C. Thromb. Haemost. 2001; 85: 694-701Crossref PubMed Scopus (55) Google Scholar). In general, these latter findings are in keeping with studies on cultured cells in which changes in the activation status of RhoA do not correlate with alterations in integrin affinity (18.Schwartz M.A. Shattil S.J. Trends Biochem. Sci. 2000; 25: 388-391Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In the current study, we have investigated the role of RhoA in regulating the adhesive function of integrin αIIbβ3. Our studies have demonstrated the existence of integrin αIIbβ3-dependent and -independent mechanisms regulating RhoA activation. Although RhoA activation appears to represent a general feature of platelet activation, our studies do not support an important role for RhoA in regulating the affinity status of integrin αIIbβ3. Rather, we demonstrate an important role for RhoA in regulating the stability of integrin αIIbβ3 adhesion contacts. The ability of RhoA to sustain integrin αIIbβ3·matrix interactions appears critical for efficient platelet adhesion in a shear field. The anti-β3 chimeric Fab fragment of the monoclonal antibody 7E3 (c7E3 Fab-abciximab) was from Eli-Lilly (Centocor, Leiden, The Netherlands). The RhoA monoclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and the horseradish peroxidase-rabbit anti-mouse-IgG was from Jackson Laboratories. TRAP(1–6) (SFLLRN), PAR4 agonist peptide (AYPGKF-NH2), and RGDS peptide were prepared using standard solid phase Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based procedures, on a Rainin PS3 automated synthesizer, according to the method Fields and Noble (19.Fields G.B. Noble R.L. Int. J. Pept. Protein Res. 1990; 35: 161-214Crossref PubMed Scopus (2308) Google Scholar). All other reagents were from sources described previously (20.Jackson S.P. Schoenwaelder S.M. Yuan Y. Rabinowitz I. Salem H.H. Mitchell C.A. J. Biol. Chem. 1994; 269: 27093-27099Abstract Full Text PDF PubMed Google Scholar, 21.Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 22.Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 23.Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kularni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Human von Willebrand factor (HvWf) was purified to homogeneity from plasma cryoprecipitate according to the method of Montgomery and Zimmerman (24.Montgomery R.R. Zimmerman T.S. J. Clin. Invest. 1978; 61: 1498-1507Crossref PubMed Scopus (76) Google Scholar). Asialo vWf was prepared from vWf that had been purified from a human dried Factor VIII Fraction (CSL Ltd., Victoria, Australia). Briefly, reconstituted Factor VIII fraction was applied to a Sepharose CL-6B size-exclusion column, fractions were collected, and vWf was detected by SDS-PAGE and ristocetin cofactor activity. Fractions containing purified vWf were pooled and concentrated by centrifugation. Purified vWf (100 μg/ml) was dialyzed in 0.05 m sodium acetate, 150 mm NaCl, 8 mm CaCl2, pH 6.0, at 4 °C, followed by incubation with 0.1 unit of α2–3,6,8-neuraminidase, Vibrio cholerae(Calbiochem-Novabiochem Corp.) for 3 h at 37οC. De-sialylated vWf was dialyzed in 10 mm Tris-HCl, 150 mm NaCl, pH 7.4, and platelet aggregating activity was confirmed by addition of 20 μg/ml purified protein to citrated platelet-rich plasma. Human fibrinogen was purified from fresh frozen plasma, according to the method of Jakobsen and Koerulf (25.Jakobsen E. Koerulf P. Thromb. Res. 1979; 3: 145-159Abstract Full Text PDF Scopus (97) Google Scholar). Whole blood (anticoagulated with acid-citrate-dextrose) was collected from healthy volunteers who had not received any anti-platelet medication in the preceding 2 weeks. Washed platelets were prepared as previously described (21.Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and resuspended in Tyrode's buffer (10 mmHepes, 12 mm NaHCO3, pH 7.4, 137 mmNaCl, 2.7 mm KCl, 5 mm glucose) containing 1 mm calcium, where indicated. Autologous red blood cells (RBCs) were obtained as described previously (23.Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kularni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). A construct encoding recombinant C3 exoenzyme (a gift from Prof. Keith Burridge, University of North Carolina, Chapel Hill, NC) was expressed as a glutathione S-transferase (GST) fusion protein inEscherichia coli, followed by thrombin-mediated cleavage of the GST tag, as described by Dillon and Feig (26.Dillon S.T. Feig L.A. Methods Enzymol. 1995; 256: 174-184Crossref PubMed Scopus (57) Google Scholar). Purified C3 exoenzyme was dialyzed against Tyrode's buffer overnight at 4 °C. Platelets in platelet washing buffer (pH 6.5) were incubated with purified C3 exoenzyme (100 μg/ml) in the presence of 200 units of hirudin for 4 h at room temperature. Using these conditions, we routinely achieved ∼80% inhibition of RhoA (Fig. 3 A), without significant loss of platelet reactivity (data not shown). The Rho kinase inhibitor Y27632 was prepared as described previously by Uehata and colleagues (63.Uehata, M., Ono, T., Satoh, H., Yamagami, K., and Kawahara, T. (April 17, 2001) U. S. Patent 4997834Google Scholar). Washed platelets resuspended in Tyrode's buffer were incubated with vehicle alone (Me2SO) or Y27632 (20 μm) for 10 min at room temperature. ADP-ribosylation of RhoA in platelet extracts was analyzed using a modified method as described by Leng and colleagues, et al. (13.Leng L. Kashiwagi H. Ren X.-D. Shattil S.J. Blood. 1998; 91: 4206-4215Crossref PubMed Google Scholar). Briefly, platelets incubated with vehicle alone or the indicated concentrations of C3 exoenzyme, were lysed on ice by the addition of an equal volume of lysis buffer (20 mm Hepes, pH 7.4, 145 mmNaCl, 1.5% Triton X-100, 0.8% deoxycholate, 0.2% SDS, 3 mm EGTA, 2 mm MgCl2, 50 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin). Immediately prior to lysis, an equivalent amount of C3 exoenzyme was added to control platelets. In vitroADP-ribosylation was performed immediately at 30 °C for 60 min, by addition of 200 μm GTP, 10 μmNAD+, 10 μg/ml C3 exoenzyme, and 05–2.5 μCi/assay [32P]NAD+. Reactions were terminated by the addition of sample buffer, boiled, and analyzed on SDS-PAGE, followed by autoradiography. The level of GTP-bound RhoA in platelet lysates was measured as described previously (27.Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1350) Google Scholar, 28.Schoenwaelder S.M. Petch L.A. Williamson D. Shen R. Feng G.S. Burridge K. Curr. Biol. 2000; 10: 1523-1526Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Western blots were quantified by densitometry (GelPro software), and RhoA activation expressed as -fold increase over the level of active RhoA in resting platelets, after correcting for protein loading (histograms). Washed platelets were stimulated with the indicated concentrations of agonist, in the presence of calcium (1 mm) and fibrinogen (500 μg/ml). Aggregation initiated by thrombin was performed in the absence of fibrinogen. All aggregations were initiated by stirring the suspensions at 950 rpm for 10 min at 37 °C in a four-channel automated platelet analyzer (Kyoto Daiichi, Japan). The extent of platelet aggregation was defined as the percentage change in optical density as measured by the automated platelet analyzer. Static adhesion assays were performed using a modified method of Yap et al. (23.Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kularni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Briefly, glass coverslips were coated with 10 μg/ml HvWf for 2 h at room temperature, followed by blocking with 10% heat-inactivated human serum for 60 min at room temperature. Platelets were incubated with immobilized matrices for between 30–60 min, unless otherwise indicated, and visualized using phase contrast microscopy. Images were captured, and the surface area of adherent platelets was determined using MCID software (Imaging Research Inc.). The number of C3 exoenzyme-treated adherent platelets and surface area of C3 exoenzyme-treated adherent platelets was expressed as a percentage of vehicle-treated control platelets. Flow assays were performed using glass microcapillary tubes (Microslides, Vitro Dynamics Inc.) coated with 100 μg/ml HvWf, according to the method described by Yap et al. (23.Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kularni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Microcapillary tubes were blocked with 10% heat-inactivated human serum prior to use. Washed platelets reconstituted with RBCs (50% hematocrit) were perfused across coated microcapillary tubes at shear rates of 600 and 1800 s−1. Platelet tethering and stationary adhesion was visualized in real time using differential interference contrast microscopy and the first 2 min of flow video recorded for off-line analysis. In all studies, off-line analyses of platelet tethering and stationary adhesion were performed as described previously (23.Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kularni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Briefly, any cell forming an adhesion contact with immobilized HvWf for greater than 40 ms was scored as a tethered cell. Stationary adhesion was arbitrarily defined as cells not moving more than a single cell diameter over a 10-s period. The duration of stationary platelet adhesion contacts was determined on individual platelets that had formed stationary interactions with immobilized vWf for a period of ≥1 s. Individual cells were analyzed every second for a period up to 30 s, and stationary adhesion was arbitrarily defined as cells not moving more than a single cell diameter over a 1-s period. For analysis of platelet spreading, adherent platelets were washed for 15 min with Tyrode's buffer (1800 s−1), visualized using phase contrast microscopy, and video recorded for off-line analysis. Platelet spreading was analyzed as described for the static adhesion assays. Control orY27632- or C3 exoenzyme-treated platelets, resuspended in Tyrode's buffer, pH 7.5, and containing 1 mm CaCl2, were subjected to shear (5000 s−1) in the presence of 20 μg/ml HvWf for 5 min, using a cone-and-plate viscometer, as described previously (29.Mistry M. Cranmer S.L. Yuan Y. Mangin P. Dopheide S.M. Harper I. Giuliano S. Dunstan D.E. Lanza F. Salem H.H. Jackson S.P. Blood. 2000; 96: 3480-3489Crossref PubMed Google Scholar). Platelets were visualized using inverse phase contrast microscopy (×5 objective), and images were captured using a charge-coupled device camera. Five random fields were captured from each experimental sample, and quantification of single platelets was performed using MCID software. PAC1 immunofluorescence was performed under static and flow conditions, as described previously (23.Yap C.L. Hughan S.C. Cranmer S.L. Nesbitt W.S. Rooney M.M. Giuliano S. Kularni S. Dopheide S.M. Yuan Y. Salem H.H. Jackson S.P. J. Biol. Chem. 2000; 275: 41377-41388Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Platelets were imaged via confocal microscopy, using ×100 oil immersion, and quantitation was performed using Leica software. Significant differences were detected using an unpaired t test with one-way analysis of variance, using Prism software (GraphPad Software for Science, San Diego, CA). Platelets respond to a multitude of activating stimuli in vivo, including soluble agonists generated at the site of vascular injury, adhesive substrates present within the damaged vessel wall, and hemodynamic forces generated by the flow of blood. In an attempt to gain further insight into the functional role of RhoA in platelets, we initially examined whether RhoA activation was restricted to a subset of platelet-activating stimuli or occurred as a general feature of platelet activation. Initially, platelets were stimulated in suspension with a diverse range of soluble agonists, including potent agonists such as thrombin, collagen, and the thromboxane A2 mimetic,U46619, or weaker agonists such as ADP. To monitor RhoA activation directly, we utilized an assay that selectively isolates GTP-bound active RhoA from cell lysates (27.Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1350) Google Scholar) by precipitation with the RhoA binding domain of the downstream effector Rhotekin (GST-RBD), as described under “Experimental Procedures.” Consistent with previous reports (11.Bodie S.L. Ford I. Greaves M. Nixon G.F. Biochem. Biophys. Res. Commun. 2001; 287: 71-76Crossref PubMed Scopus (25) Google Scholar, 12.Gratacap M.-P. Payrastre B. Nieswandt B. Offermanns S. J. Biol. Chem. 2001; 276: 47906-47913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), RhoA activation was induced by thrombin (1 unit/ml) and U46619 (500 nm) (Fig. 1,left). In addition, RhoA activation was also induced by collagen (10 μg/ml) and ADP (25 μm), but with the latter agonist the rate and extent of RhoA activation was substantially less than with the other agonists (Fig. 1 A, and data not shown). In cultured cells, the activation status of RhoA is regulated by soluble growth factors and integrin adhesion receptors (18.Schwartz M.A. Shattil S.J. Trends Biochem. Sci. 2000; 25: 388-391Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 30.Zohn I.M. Campbell S.L. Khosravi-Far R. Rossman K.L. Der C.J. Oncogene. 1998; 17: 1415-1438Crossref PubMed Scopus (319) Google Scholar). However, to date, there is no evidence that platelet integrins are involved in regulating RhoA. To examine the potential involvement of the major platelet integrin αIIbβ3 in this process, platelets were pretreated with the integrin αIIbβ3-blocking antibody c7E3 Fab, prior to the performance of platelet aggregation studies. In thrombin-stimulated platelets, c7E3 Fab-pretreatment resulted in a reduction of ∼70% in RhoA activation (Fig. 1 B), whereas with U46619, collagen, and ADP, integrin αIIbβ3 inhibition resulted in >90% inhibition of RhoA activation. Our findings with respect to U46619 are in direct contrast to those recently reported by Gratacap and colleagues (12.Gratacap M.-P. Payrastre B. Nieswandt B. Offermanns S. J. Biol. Chem. 2001; 276: 47906-47913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) who demonstrated that integrin αIIbβ3 blockade with RGDS peptides had no inhibitory effect on RhoA activation. To investigate potential explanations for this difference, we compared the effects of RGDS peptide and c7E3 Fab on the activation of RhoA induced by U46619 under identical experimental conditions employed by Gratacap et al. (12.Gratacap M.-P. Payrastre B. Nieswandt B. Offermanns S. J. Biol. Chem. 2001; 276: 47906-47913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Consistent with previous findings (12.Gratacap M.-P. Payrastre B. Nieswandt B. Offermanns S. J. Biol. Chem. 2001; 276: 47906-47913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), we did not detect any inhibitory effect of RGDS peptide on RhoA activation induced by U46619, however, c7E3 Fab dramatically inhibited RhoA activation under identical experimental conditions (Fig. 1 C). Findings similar to c7E3 Fab were observed with another potent integrin αIIbβ3 antagonist, aggrastat (data not shown). These findings suggest that RGDS peptide may not completely block ligand binding to integrin αIIbβ3, a finding consistent with previous reports (31.Jones K.L. Hughan S.C. Dopheide S.M. Farndale R.W. Jackson S.P. Jackson D.E. Blood. 2001; 98: 1456-1463Crossref PubMed Scopus (111) Google Scholar). To examine whether RhoA activation represents a general feature of platelet activation, changes in RhoA activity were assessed following platelet adhesion to immobilized substrates and in platelets exposed to high shear stress. As demonstrated in Fig. 2, adhesion and spreading of platelets on either a fibrinogen (100 μg/ml) or human vWf (10 μg/ml) matrix was associated with significant RhoA activation, similar to the levels observed with soluble agonist stimulation. Pretreating platelets with c7E3 Fab prior to adhesion to vWf inhibited RhoA activation by >95% (Fig. 2 B). To examine the effects of high fluid shear stress on RhoA activation, shear-induced platelet activation (SIPA) studies were performed using a cone-and-plate viscometer, as described under “Experimental Procedures.” In control studies, we demonstrated that high shear rates (typically ≥3000 s−1) were required to induce aggregation of washed platelets independent of the addition of exogenous stimuli. Furthermore, aggregation under these experimental conditions was dependent on vWf binding to GPIb and integrin αIIbβ3, because it was completely inhibited by pretreating platelets with blocking antibodies against either receptor (data not shown). As demonstrated in Fig. 2 A, exposure of platelets in suspension to high shear (5000 s−1) in the presence of purified soluble human vWf, induced p" @default.
• W1517951856 created "2016-06-24" @default.
• W1517951856 creator A5021542039 @default.
• W1517951856 creator A5022051551 @default.
• W1517951856 creator A5029168209 @default.
• W1517951856 creator A5032907078 @default.
• W1517951856 creator A5048624587 @default.
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• W1517951856 date "2002-04-01" @default.
• W1517951856 modified "2023-10-16" @default.
• W1517951856 title "RhoA Sustains Integrin αIIbβ3Adhesion Contacts under High Shear" @default.
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