Calpain Regulation of Cytoskeletal Signaling Complexes in Von Willebrand Factor-stimulated Platelets
1997; Elsevier BV; Volume: 272; Issue: 35 Linguagem: Inglês
10.1074/jbc.272.35.21847
ISSN1083-351X
AutoresYuping Yuan, Sacha M. Dopheide, Chris Ivanidis, Hatem H. Salem, Shaun P. Jackson,
Tópico(s)Blood groups and transfusion
ResumoThe adhesion of platelets to sites of vascular injury is critically dependent on the binding of subendothelial bound von Willebrand factor (vWf) to the platelet surface glycoprotein complexes, GP Ib-V-IX and GP IIb-IIIa (integrin αIIbβ3). There is growing evidence that the binding of vWf to these receptors is not only essential for stable platelet adhesion but is also important for the transduction of activation signals required for changes in platelet morphology, granule secretion, and platelet aggregation. In this study we have investigated signaling events induced by vWf binding to GP Ib-V-IX in both spreading and aggregated platelets. The adhesion of platelets to vWf resulted in dramatic actin filament reorganization, as assessed by immunofluorescence with fluorescein isothiocyanate-conjugated phalloidin, and the cytoskeletal recruitment of various structural proteins (talin and integrin αIIbβ3) and signaling enzymes (pp60c- src, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI 3-kinase), and protein-tyrosine phosphatase (PTP)-1B). Time course experiments in both spreading and aggregated platelets revealed that talin, FAK, and PTP-1B were proteolyzed after translocation to the cytoskeleton. The proteolysis of these proteins was dependent on the presence of extracellular calcium and was specifically inhibited by pretreating platelets with the membrane-permeable calpain inhibitors calpeptin, E64d, and MDL 28,170, but not with the membrane-impermeable inhibitors leupeptin, E64, and calpastatin. The cytoskeletal translocation of signaling enzymes in vWf-stimulated platelets was abolished by pretreating platelets with an anti-GP Ib-V-IX antibody but was unaffected by blocking ligand binding to integrin αIIbβ3. In contrast, calpain activation in vWf-stimulated platelets required ligand binding to both GP Ib-V-IX and integrin αIIbβ3. The activation of calpain in both spreading and aggregated platelets resulted in a substantial decrease in the level of tyrosine phosphorylation of multiple platelet proteins and was associated with a 50–80% reduction in the amount of cytoskeletal associated talin, integrin αIIbβ3, PI 3-kinase, FAK, pp60c- src, and PTP-1B. These studies suggest a potentially important role for calpain in regulating the formation and/or stability of cytoskeletal signaling complexes in vWf-stimulated platelets. Furthermore, they demonstrate distinct roles for GP Ib-V-IX and integrin αIIbβ3 in vWf-induced signal transduction. The adhesion of platelets to sites of vascular injury is critically dependent on the binding of subendothelial bound von Willebrand factor (vWf) to the platelet surface glycoprotein complexes, GP Ib-V-IX and GP IIb-IIIa (integrin αIIbβ3). There is growing evidence that the binding of vWf to these receptors is not only essential for stable platelet adhesion but is also important for the transduction of activation signals required for changes in platelet morphology, granule secretion, and platelet aggregation. In this study we have investigated signaling events induced by vWf binding to GP Ib-V-IX in both spreading and aggregated platelets. The adhesion of platelets to vWf resulted in dramatic actin filament reorganization, as assessed by immunofluorescence with fluorescein isothiocyanate-conjugated phalloidin, and the cytoskeletal recruitment of various structural proteins (talin and integrin αIIbβ3) and signaling enzymes (pp60c- src, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI 3-kinase), and protein-tyrosine phosphatase (PTP)-1B). Time course experiments in both spreading and aggregated platelets revealed that talin, FAK, and PTP-1B were proteolyzed after translocation to the cytoskeleton. The proteolysis of these proteins was dependent on the presence of extracellular calcium and was specifically inhibited by pretreating platelets with the membrane-permeable calpain inhibitors calpeptin, E64d, and MDL 28,170, but not with the membrane-impermeable inhibitors leupeptin, E64, and calpastatin. The cytoskeletal translocation of signaling enzymes in vWf-stimulated platelets was abolished by pretreating platelets with an anti-GP Ib-V-IX antibody but was unaffected by blocking ligand binding to integrin αIIbβ3. In contrast, calpain activation in vWf-stimulated platelets required ligand binding to both GP Ib-V-IX and integrin αIIbβ3. The activation of calpain in both spreading and aggregated platelets resulted in a substantial decrease in the level of tyrosine phosphorylation of multiple platelet proteins and was associated with a 50–80% reduction in the amount of cytoskeletal associated talin, integrin αIIbβ3, PI 3-kinase, FAK, pp60c- src, and PTP-1B. These studies suggest a potentially important role for calpain in regulating the formation and/or stability of cytoskeletal signaling complexes in vWf-stimulated platelets. Furthermore, they demonstrate distinct roles for GP Ib-V-IX and integrin αIIbβ3 in vWf-induced signal transduction. Cell adhesion processes play a fundamental role in inflammation, immunity, embryogenesis, and hemostasis. These adhesion processes are regulated by distinct families of cell surface receptors, including the integrins, immunoglobulin gene family, cadherins, selectins, and the leucine-rich glycoprotein gene family, of which the receptor for vWf, 1The abbreviations used are: vWf, von Willebrand factor; GP, glycoprotein; PI 3-kinase, phosphatidylinositol 3-kinase; PTP, protein-tyrosine phosphatase; RGDS peptide, Arg-Gly-Asp-Ser peptide; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; Me2SO, dimethyl sulfoxide; PBS, phosphate-buffered saline; FAK, focal adhesion kinase. GP Ib-V-IX, is the best characterized member (1Ruggeri Z.M. Prog. Hemost. Thromb. 1991; 10: 35-68PubMed Google Scholar, 2Roth G.J. Blood. 1991; 77: 5-19Crossref PubMed Google Scholar). There is growing evidence that many of these receptors not only mediate cell adhesion but also play an important role in transducing signals that regulate cell morphology, motility, growth, and differentiation (3Rosales C. O'Brien V. Kornberg L. Juliano R. Biochim. Biophys. Acta. 1995; 1242: 77-98PubMed Google Scholar, 4Yamada K.M. Miyamoto S. Curr. Opin. Cell Biol. 1995; 7: 681-689Crossref PubMed Scopus (588) Google Scholar). The molecular basis by which these receptors transmit signals has not been clearly defined, although there is increasing evidence that the interaction between these receptors and the cytoskeleton is critical for signal transduction (4Yamada K.M. Miyamoto S. Curr. Opin. Cell Biol. 1995; 7: 681-689Crossref PubMed Scopus (588) Google Scholar). For example, studies in human platelets and a range of cultured cells have demonstrated that the ligation and clustering of integrins on the cell surface lead to the cytoskeletal recruitment of various structural proteins (talin, vinculin, α-actinin, tensin, paxillin) and signaling molecules (FAK, Src kinases, c-CSK, PI 3-kinase, protein kinase C, phospholipase Cγ, rasGAP, Grb2, SOS, SHP1, PTP-1B, Rho, Ras, Raf, MEK kinases, ERK1, ERK2 and calpain) (5Miyamoto S. Akiyama S.K. Yamada K.M. Science. 1995; 267: 883-885Crossref PubMed Scopus (798) Google Scholar, 6Miyamoto S. Teramoto H. Coso O.A. Gutkind J.S. Burbelo P.D. Akiyama S.K. Yamada K.M. J. Cell Biol. 1995; 131: 791-805Crossref PubMed Scopus (1110) Google Scholar, 7Grondin P. Plantavid M. Sultan C. Breton M. Mauco G. Chap H. J. Biol. Chem. 1991; 266: 15705-15709Abstract Full Text PDF PubMed Google Scholar, 8Fox J.E.B. Lipfert L. Clark E.A. Reynolds C.C. Austin C.D. Brugge J.S. J. Biol. 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There is growing evidence that tyrosine phosphorylation events, mediated by the FAK and Src family kinases, play a major role in recruiting these signaling molecules to the cytoskeleton by providing high affinity binding sites for SH2-containing signaling molecules (5Miyamoto S. Akiyama S.K. Yamada K.M. Science. 1995; 267: 883-885Crossref PubMed Scopus (798) Google Scholar, 6Miyamoto S. Teramoto H. Coso O.A. Gutkind J.S. Burbelo P.D. Akiyama S.K. Yamada K.M. J. Cell Biol. 1995; 131: 791-805Crossref PubMed Scopus (1110) Google Scholar, 15Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar, 16Schaller M.D. Parsons J.T. Curr. Opin. Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (500) Google Scholar, 17Schlaepfer D.D. Hanks S.K. Hunter T. Van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1462) Google Scholar). Although considerable progress has been made in identifying several key proteins involved in regulating the formation of cytoskeletal signaling complexes, there is far less information on cellular proteins negatively regulating these signaling events. A potential candidate protein is the ubiquitous thiol protease, calpain. This enzyme has previously been localized to focal adhesions in cultured cells and in the cytoskeletal fraction of thrombin-stimulated platelets (18Beckerle M.C. Burridge K. DeMartino G.N. Croall D.E. Cell. 1987; 51: 569-577Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 19Fox J.E.B. Santos G. Zuerbig S. Saido T.C. Thromb. Haemostasis. 1995; 72 (abstr.): 987Google Scholar), where it cleaves a number of focal adhesion structural proteins (talin, β3 integrin) (20White G.C. Biochim. Biophys. Acta. 1980; 631: 130-138Crossref PubMed Scopus (28) Google Scholar, 21Fox J.E.B. Reynolds C.C. Phillips D.R. J. Biol. Chem. 1983; 258: 9973-9981Abstract Full Text PDF PubMed Google Scholar, 22Du X. Saido T.C. Tsubuki S. Indig F.E. Williams M.J. Ginsberg M.H. J. Biol. Chem. 1995; 270: 26146-26151Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) and several signaling enzymes (FAK, pp60c- src, PTP-1B, phospholipase Cγ, protein kinase C) (23Cooray P. Yuan Y. Schoenwaelder S.M. Mitchell C.A. Salem H.H. Jackson S.P. Biochem. J. 1996; 318: 41-47Crossref PubMed Scopus (155) Google Scholar, 24Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (287) Google Scholar, 25Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar, 26Banno Y. Nakashima S. Hachiya T. Nozawa Y. J. Biol. Chem. 1995; 270: 4318-4324Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Kishimoto A. Mikawa K. Hashimoto K. Yasuda I. Tanaka S. Tominaga M. Kuroda T. Nishizuka Y. J. Biol. Chem. 1989; 264: 4088-4092Abstract Full Text PDF PubMed Google Scholar). Recent studies in thrombin-stimulated platelets have revealed that calpain cleavage of FAK and pp60c- src results in a reduction in their autokinase activity (23Cooray P. Yuan Y. Schoenwaelder S.M. Mitchell C.A. Salem H.H. Jackson S.P. Biochem. J. 1996; 318: 41-47Crossref PubMed Scopus (155) Google Scholar, 25Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar), whereas PTP-1B cleavage is associated with enhanced phosphatase activity (24Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (287) Google Scholar), raising the possibility that calpain may indirectly regulate the level of protein-tyrosine phosphorylation within the cell (28Ariyoshi H. Oda A. Salzman E.W. Thromb. Vasc. Biol. 1995; 15: 511-514Crossref PubMed Scopus (11) Google Scholar). Furthermore, the cleavage of talin, pp60c- src, and FAK is associated with their subcellular relocation (23Cooray P. Yuan Y. Schoenwaelder S.M. Mitchell C.A. Salem H.H. Jackson S.P. Biochem. J. 1996; 318: 41-47Crossref PubMed Scopus (155) Google Scholar, 25Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar), suggesting a potentially important role for calpain in regulating the stability of integrin signaling complexes. We have recently reported that vWf binding to GP Ib-V-IX induces the cytoskeletal association of pp60c- src and PI 3-kinase in aggregated platelets (29Jackson 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). Given the critical role for this receptor in the initial adhesion of platelets to the site of vascular injury, we have investigated the possibility that vWf binding to GP Ib-V-IX can also induce the formation of cytoskeletal signaling complexes in spreading platelets. Our studies indicate that various structural proteins and signaling enzymes associate with the cytoskeletal fraction of platelets spread on a vWf matrix. The formation of these complexes was associated with calpain activation and the cleavage of talin, FAK, pp60c- src, and PTP-1B. Cleavage of these focal adhesion proteins in spreading and aggregated platelets was associated with a substantial reduction in protein-tyrosine phosphorylation and a 50–80% decrease in the cytoskeletal content of integrin αIIbβ3, talin, FAK, pp60c- src, PTP-1B, and PI 3-kinase. These observations suggest a potentially important role for calpain in modulating the formation and/or stability of cytoskeletal signaling complexes in human platelets. Calpeptin was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Arg-Gly-Asp-Ser (RGDS) peptide, bovine serum albumin, and FITC-conjugated phalloidin were purchased from Sigma. Ristocetin was supplied by Paesel and Lorei Inc. (Germany). All other materials were from sources we have described previously (29Jackson 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,30Schoenwaelder S.M. Jackson S.P. Yuan Y. Teasdale M.S. Salem H.H. Mitchell C.A. J. Biol. Chem. 1994; 269: 32479-32487Abstract Full Text PDF PubMed Google Scholar). Anti-GP IIb monoclonal antibody (mAb) SZ22 and anti-GP IIIa mAb SZ21 were from Immunotech (France). Anti-GP Ib mAb AK2 was a generous donation from Dr. Michael Berndt (Baker Medical Research Institute, Melbourne, Australia). Anti-talin mAb 8d4 and anti-actin mAb were from Sigma. Anti-p85 (p85 subunit of PI-3 kinase) polyclonal antibody was from Upstate Biotechnology Inc. Anti-pp60c- src mAb 327 was a generous donation of Dr. Joan Brugge (University of Pennsylvania). Anti-phosphotyrosine mAb PY20 was from ICN Biochemicals Inc. Anti-FAK mAb and anti-PTP-1B mAb were from Transduction Laboratories. Anti-calpain (80 kDa) polyclonal antibody was kindly donated by Dr. T. C. Saido (Metropolitan Institute of Medical Science, Tokyo, Japan). Anti-mouse and rabbit peroxidase-conjugated IgG were from Silenus Laboratories (Victoria, Australia). Blood was obtained from healthy donors who had not taken any anti-platelet medication in the preceding 2 weeks. Platelets were isolated and washed as described previously (29Jackson 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). For platelet aggregation studies, washed platelets were resuspended in modified Tyrode's buffer (10–50 mm Hepes, pH 7.5, 12 mm NaHCO3, 137 mm NaCl, 2.7m KCl, and 5 mm glucose) and then incubated at 37 °C for 30 min before stimulation. Platelets (3 × 108/ml) were activated using either purified vWf (10–50 μg/ml) in the presence of ristocetin (1 mg/ml) or thrombin (1 unit/ml) in the presence of Ca2+ (1 mm). Aggregation was initiated by stirring the platelet reaction mixtures at 950 rpm at 37 °C, in a four-channel automated platelet analyzer (Kyoto Daiichi, Japan). In some experiments platelets were preincubated for 30 min with either vehicle alone (0.5% Me2SO), calpeptin (50 μg/ml), RGDS (1 mm), anti-GP IIIa mAb SZ21 (5 μg/ml) or anti-GP Ib mAb AK2 (5 μg/ml) before stimulation. In other experiments the GP IIb-IIIa complex was disrupted irreversibly by pretreating platelets with 5 mm EDTA for 90 min at 37 °C, as described previously (29Jackson 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). Glass coverslips were coated with vWf (50 μg/ml) for 2 h at room temperature and then blocked with 0.1% bovine serum albumin for 60 min. Washed platelets (1 × 108/ml) were applied to the coverslips for 60 min in the presence of the platelet inhibitor theophylline (1 mm) to allow platelet adhesion without spreading. The nonadherent platelets were aspirated and the adherent cells washed twice with theophylline-free phosphate-buffered saline (PBS) (20 mmNa2HPO4, 4 mmNaH2PO4, pH 7.4, 150 mm NaCl). Gentle washing was essential to minimize detachment of theophylline-treated platelets from the vWf matrix. After platelets had spread for the indicated times (0, 10, 20, 40, and 60 min), the cells were washed three times with PBS, fixed for 30 min in 3.7% formaldehyde in PBS, then permeabilized with 0.1% Triton X-100 in PBS for 30 min. The permeabilized cells were stained with FITC-conjugated phalloidin (1:1,000 dilution) for 60 min in the dark, then washed three times with PBS. The coverslips were mounted onto glass slides withp-phenylenediamine (1 mg/ml) containing 90% glycerol, 10% PBS (v/v), then visualized with a fluorescence microscope (× 310) (Olympus). To obtain sufficient platelet protein for immunoblot analysis, platelet adhesion studies were performed on polystyrene tissue culture dishes (100 mm) (Nunc). These dishes were coated with vWf and the washed platelets applied to these dishes in a manner identical to that described above. After platelets had spread for the indicated times on these dishes, the cells were washed three times, lysed with 1% Triton X-100 lysis buffer (10 mm Tris, pH 7.2, 1% Triton X-100, 158 mm NaCl, 2 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 5 μm calpain inhibitor I, 1 mm Na3VO4, and 2 mmbenzamidine), then scraped from the dishes with a rubber policeman. Triton X-100-soluble and -insoluble (cytoskeleton) extracts were isolated from the adherent platelets in a manner identical to that used in aggregated platelets (30Schoenwaelder S.M. Jackson S.P. Yuan Y. Teasdale M.S. Salem H.H. Mitchell C.A. J. Biol. Chem. 1994; 269: 32479-32487Abstract Full Text PDF PubMed Google Scholar). The cytoskeletal proteins were extracted from the Triton X-100-insoluble material with radioimmunoprecipitation assay buffer (10 mm Tris, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 158 mm NaCl, 2 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 5 μm calpain inhibitor I, 1 mm Na3VO4 and 2 mmbenzamidine) for 60 min at 4 °C. Radioimmunoprecipitation assay buffer-insoluble material was removed by centrifugation at 15,000 × g for 5 min. Whole cell lysates were prepared by lysing adherent platelets with radioimmunoprecipitation assay buffer for 1 h at 4 °C. Equal quantities of platelet extracts were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions according to the method of Laemmli (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (213238) Google Scholar), then transferred to polyvinylidene difluoride membranes. Western blots were performed as described by Towbin et al. (32Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (46453) Google Scholar), using specific primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies, and finally developed using Enhanced Chemiluminescence (DuPont). When studies on tyrosine phosphorylation of platelet proteins were performed, cells were lysed in an equal volume of Laemmli reducing buffer (50 mm Tris, pH 6.8, 4 mm EDTA, 2 mm Na3VO4, 2 mmphenylmethylsulfonyl fluoride, 4 mm benzamidine, 10% glycerol, 4% SDS, 10% β-mercaptoethanol, 0.002% bromphenol blue) and boiled immediately for 5 min before SDS-polyacrylamide gel electrophoresis. vWf was purified from cryoprecipitate as described by Montgomery and Zimmerman (33Montgomery R. Zimmerman T. J. Clin. Invest. 1978; 61: 1498-1507Crossref PubMed Scopus (79) Google Scholar). Protein concentrations were determined using the Bio-Rad protein assay with bovine serum albumin as a standard. vWf is essential for effective platelet adhesion at sites of vascular injury, especially under conditions of high shear stress. Stable platelet adhesion under high shear requires coordinated binding of immobilized vWf to both the GP Ib-V-IX and GP IIb-IIIa (integrin αIIbβ3) complexes on the platelet surface (34Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). Platelet adhesion is followed by dramatic cytoskeletal reorganization, leading to platelet shape change, spreading, and aggregation. In preliminary studies, we examined the time course for platelet spreading on vWf-coated glass coverslips. Washed platelets (1 × 108/ml) were applied to vWf-coated coverslips in the presence of the platelet activation inhibitor theophylline (1 mm) to allow platelet adhesion without spreading, as described under "Experimental Procedures." Platelet adhesion to vWf under these conditions is potentially reversible, therefore gentle washing conditions were required to minimize the displacement of platelets from the matrix. After removal of theophylline, adherent platelets were allowed to spread for the indicated time points (0, 10, 20, 40, and 60 min), before fixation with formaldehyde, permeabilization with Triton X-100, and staining with FITC-conjugated phalloidin to visualize changes in the actin filament organization. As demonstrated in Fig. 1 A, within 10 min of binding to the vWf matrix, multiple filopodial extensions (1–4 μm in length) projected from the platelet surface. By 20 min, broad lamellipodia sheets began extending between these filopodial projections. These lamellipodia continued to radiate out from the cell center until a confluent platelet monolayer had covered the vWf matrix (60 min). We have reported recently that vWf-induced platelet aggregation is associated with the cytoskeletal translocation of pp60c- src and PI 3-kinase (29Jackson 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). We therefore examined whether the changes in the actin filament network induced by vWf binding to GP Ib-V-IX in spreading platelets was associated with the cytoskeletal recruitment of signaling enzymes. To obtain sufficient amounts of cytoskeletal associated protein for immunoblot analysis, platelet adhesion studies were performed on Petri dishes coated with purified vWf. Washed platelets (3 × 108/ml) were applied to this matrix under conditions identical to those used in Fig. 1 A. At various time points (0, 10, 20, 40, and 60 min) the adherent platelets were lysed with Triton X-100 lysis buffer and cytoskeletal proteins isolated as described under "Experimental Procedures." We confirmed that the Triton X-100-insoluble residues remaining on the matrix were cytoskeletal structures by performing immunofluorescence with FITC-conjugated phalloidin. As demonstrated in Fig. 1 B, these Triton X-100-resistant structures demonstrated an overall similar pattern of immunofluorescence to the permeabilized platelets in Fig. 1 A (60 min), except for the loss of actin filament networks lining the surface membrane. SDS-polyacrylamide gel electrophoresis analysis of the proteins extracted from the matrix revealed a protein pattern similar to that reported previously for cytoskeletal extracts from spreading platelets (35Smith C.M. Burris S.M. Rao G.H.R. White J.G. Blood. 1992; 80: 2774-2780Crossref PubMed Google Scholar), with actin constituting greater than 65% of the total cytoskeletal protein (data not shown). An alternative technique for isolating cytoskeletal proteins in which the Triton X-100-insoluble residue from adherent platelets was extracted with SDS-containing buffers yielded identical results (data not shown). Immunoblot analysis of the cytoskeletal extracts from spreading platelets revealed a time-dependent increase in the cytoskeletal content of integrin αIIbβ3 (GP IIb), talin, pp60c- src, FAK (Fig. 1 C), the p85 subunit of PI 3-kinase, and PTP-1B (data not shown). Several lines of evidence suggest that this increase represents specific recruitment and enrichment of these proteins within the cytoskeleton rather than simply a nonspecific increase caused by changes in the total amount of cytoskeletal protein. First, at each of the time points examined, equal amounts of cytoskeletal protein (30 μg) were used for immunoblot analysis. Second, the increase in these proteins was disproportionate to changes in the cytoskeletal actin content. In fully spread platelets (60 min) the ratio of integrin αIIbβ3, talin, pp60c- src, and FAK to actin was substantially greater (at least 10-fold) than at earlier time points of platelet spreading (10 min) (line graph, Fig. 1 C). Finally, changes in the level of other cytoskeletal associated proteins, such as GP Ib, did not correlate with the increases in these other proteins (data not shown). We noted consistently that the cytoskeletal associated forms of talin, FAK, and PTP-1B underwent proteolytic modification in spreading platelets. Talin was proteolyzed to a 190-kDa fragment, FAK to a 90-kDa fragment, and PTP-1B to a 42-kDa fragment. This pattern of proteolysis is identical to that mediated by calpain in thrombin and ionophore A23187-aggregated platelets (21Fox J.E.B. Reynolds C.C. Phillips D.R. J. Biol. Chem. 1983; 258: 9973-9981Abstract Full Text PDF PubMed Google Scholar,23Cooray P. Yuan Y. Schoenwaelder S.M. Mitchell C.A. Salem H.H. Jackson S.P. Biochem. J. 1996; 318: 41-47Crossref PubMed Scopus (155) Google Scholar, 24Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (287) Google Scholar, 25Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar, 26Banno Y. Nakashima S. Hachiya T. Nozawa Y. J. Biol. Chem. 1995; 270: 4318-4324Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We did not, however, detect pp60c-srcproteolytic fragments in these cytoskeletal extracts even though this kinase has been identified previously as a calpain substrate (25Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar). Previous studies in ionophore A23187-stimulated platelets have demonstrated that the proteolysis of pp60c- src leads to its relocation from the particulate to the soluble fraction of platelets (25Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar). We therefore examined the Triton X-100-soluble extracts from spreading platelets for evidence of pp60c- srcproteolysis. As demonstrated in Fig. 2, proteolytic fragments of pp60c- src, FAK, PTP-1B, and talin were evident in these Triton X-100-soluble extracts. The cleavage of each of these proteins was specifically inhibited by pretreating platelets with the calpain inhibitors calpeptin (Fig. 2) or E64d, or by chelating extracellular calcium with EGTA/Mg2+ (data not shown). Inhibiting calpain under each of these experimental conditions had no inhibitory effect on the rate or extent of platelet spreading (data not shown). All reports to date have indicated that calpain activation by physiological agonists is a postaggregation event critically dependent on fibrinogen binding to integrin αIIbβ3(36Fox J.E.B. Taylor R.G. Taffarel M. Boyles J.K. Groll D.E. J. Cell Biol. 1993; 120: 1501-1507Crossref PubMed Scopus (134) Google Scholar). Several lines of evidence suggest that the calpain activation observed in spreading platelets is unlikely to be caused by the formation of platelet aggregates in our static adhesion assays. First, phase-contrast microscopy of platelets adherent to the vWf matrix on Petri dishes failed to reveal platelet aggregate formation in 15 independent experiments. Furthermore, we have observed consistently that the formation of small platelet aggregates in vWf-stimulated nonstirred platelets is not associated with pp60c- srccleavage. 2Y. Yuan, S. Dopheide, C. Ivanidis, H. H. Salem, and S. P. Jackson, unpublished observations. Second, the platelet adhesion studies were performed in the presence of the platelet inhibitor theophylline, as described under "Experimental Procedures." In the presence of theophylline, platelets adhere to the vWf matrix as single cells but do not spread or aggregate. All nonadherent platelets were washed from the matrix before removing the platelet inhibitor to ensure that no platelet aggregates form on the adherent monolayer. Under these assay conditions, cytoskeletal signal complex formation and calpain activation were identical to that observed in platelets not pretr
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