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Differential Mobilization of Tyrosine Kinases in Human Platelets Stimulated with Thrombin or Thrombin Receptor Agonist Peptide
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
225, 1084–1089 (1996)
1299
Differential Mobilization of Tyrosine Kinases in Human Platelets Stimulated with Thrombin or Thrombin Receptor Agonist Peptide Kaneo Satoh, Yukio Ozaki,1 Naoki Asazuma, Yutaka Yatomi, Qi Ruomei, Kenji Kuroda, Libo Yang, and Shoji Kume Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Yamanashi, Japan Received July 8, 1996 Both thrombin and thrombin receptor agonist peptide (TRAP) activated p72syk and p60c-src with similar magnitudes. Both thrombin and TRAP induced translocation of p60c-src and p54/58lyn to cytoskeleton in an aggregation-dependent manner. Thrombin also induced cytoskeletal association of p72syk, but independent of platelet aggregation. Furthermore, p72syk associated with cytoskeleton underwent marked proteolysis, which was partially dependent upon calpain activation. In contrast, TRAP, even at concentrations as high as 100 m M, did not induce p72syk translocation to cytoskeleton. Our findings suggest that cytoskeletal translocation of p72syk induced by thrombin is governed by a mechanism distinct from those of p60c-src and p54/58lyn translocation. It is also suggested that p72syk translocation induced by thrombin requires additional signal(s) other than that mediated by the recently-cloned thrombin receptor that couples with GTP-binding proteins and interacts with TRAP. q 1996 Academic Press, Inc.
Platelets contain high levels of protein-tyrosine kinase activity, mainly due to p60c-src, p72syk, p54/58lyn and p125FAK (1-3). An accumulating body of evidence suggests that activation of these kinases contributes to agonist-stimulated tyrosine phosphorylation and subsequent processes of platelet activation. Several reports have demonstrated that some tyrosine kinases translocate to the Triton X-100-insoluble (cytoskeleton-rich) fraction upon stimulation with thrombin or other agonists (4-8). While the physiological role of tyrosine kinase translocation to cytoskeleton remains to be elucidated, there is evidence to suggest that it plays a role in cellular retraction of fibrin polymer (9). It also remains to be determined whether all tyrosine kinases associate with cytoskeleton upon platelet activation. Recently, a ligand of the cloned thrombin receptor, thrombin receptor agonist peptide (TRAP), has been found to induce platelet activation (10). Although the overall actions of TRAP are similar to those of thrombin, several studies have suggested that there are qualitative differences (11-13). In this study, we evaluated the effects of TRAP and thrombin on p72syk activity and translocation of tyrosine kinases (p60c-src, p72syk, p54/58lyn) to the Triton X-insoluble fraction in human platelets. MATERIALS AND METHODS c-src
Materials. Anti-p60 monoclonal antibody (MoAb), anti-p72syk MoAb, and anti-p54/58lyn MoAb were obtained from Upstate Biotechnology, NY, USA, and Wako, Osaka, Japan, respectively. Anti-p72syk polyclonal antibody was a generous gift of Dr. Hirohei Yamamura (Kobe University, Japan). Anti-GP I b MoAb , NNKY5-5, was a kind donation from Dr. Shosaku Nomura (Kansai Medical University, Japan). Thrombin receptor agonist peptide (TRAP) and calpeptin were kind gifts of Dr. Junichi Kambayashi (Osaka University, Japan). Preparation of washed platelets. Platelet-rich plasma was incubated with 1 mM acetylsalicylic acid for 30 min to
1 Reprint requests to : Yukio Ozaki, M.D., Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi 409-38, Japan. Fax: 81-552-73-6713.
1084 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Time course of p72syk activation assessed by in vitro kinase assay. After platelets (11 109/ml) were stimulated with 0.5 U/ml thrombin (A) or 20 m M TRAP (B) for the indicated time intervals, they were solubilized with lysis buffer. The Triton X-soluble fraction was immunoprecipitated with anti-p72syk MoAb, and was incubated with [g-32P]ATP for in vitro kinase assay. The proteins were separated by SDS-PAGE and transferred onto membrane. The kinase activity was detected with autoradiography, as detailed in Materials and Methods. The bands for the immunoglobulin heavy chain are indicated by arrowheads. The data are representative of three experiments.
exclude the secondary effects of TXA2 . The platelets were washed and resuspended in a HEPES buffer at a concentration of 1 1 109 cells/ml. Analysis of cytoskeleton-associated tyrosine kinases. Platelets were activated with thrombin or TRAP, and after the indicated periods, reactions were terminated with an equal volume of 21 ice-cold lysis buffer (2% Triton X-100, 100 mM Tris/HCl, pH 7.5, 5 mM EDTA, 2 mM vanadate, 1 mM PMSF , and 100 m g/ml leupeptin). The lysate was sonicated and centrifuged at 16,000 1g for 5 min. The pellet, which was the Triton X-insoluble fraction (cytoskeletonrich fraction), was washed once with 11 lysis buffer, and subjected to Western blotting using specific antibodies against tyrosine kinases. Immunoprecipitation kinase assay. The supernatant was precleared with protein A sepharose twice. The supernatant was then mixed with specific anti-tyrosine kinase monoclonal antibody (MoAb) bound to protein A sepharose. The in vitro kinase assay was performed as previously described (14), with slight modifications. The beads were incubated with kinase reaction buffer (100 mM Hepes/NaOH, pH8.0, 5 mM MnCl2 , 50 mM MgCl2) containing 10 m g acidtreated enolase. The reaction was initiated by the addition of 1.5 m M [g-32P]ATP (15 m Ci). After 10 min at 207C, reactions were stopped by addition of Laemmli buffer. After Western blotting, the radioactivity was quantified with a BAS-2000 Phosphoimager (Fuji Film, Japan).
RESULTS AND DISCUSSION
Changes in tyrosine kinase activity in platelets stimulated with thrombin or TRAP. Changes in tyrosine kinase activity induced by thrombin or TRAP were evaluated on three tyrosine kinases, p72syk, p60c-src, and p54/58lyn. When platelets were activated with thrombin (0.5 U/ ml) under constant stirring, the p72syk activity reached its peak 15 sec to 2 min after stimulation, the level of which was almost 5-fold of the resting value, and subsequently decreased almost to the original level within 5 min (Fig. 1A). TRAP (5Ç40 m M) also induced transient activation of p72syk (Fig. 1B), essentially similar to the effects of thrombin. Thrombin and TRAP also induced transient activation of p60c-src with the maximum activity peaking somewhat later than p72syk (data not shown). The maximum increase in the kinase activity over that of the control remained low (2-fold at most). Thus, we have confirmed previous findings that thrombin induces p72syk and p60c-src activation (14, 15). While Pumiglia et al. have already reported that TRAP induced p60c-src activation (16), we are the first to show that TRAP also activates p72syk. p54/58lyn activation induced by thrombin or TRAP was minimal and was not always reproducible, as assessed by kinase assays. Cytoskeletal association of tyrosine kinases induced by thrombin or TRAP. Cytoskeletal 1085
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FIG. 2. Cytoskeletal association of tyrosine kinases induced by thrombin. Immunoblot analysis of cytoskeleton, probed with anti-p60c-src MoAb (A), anti-p54/58lyn MoAb (B), or anti-p72syk MoAb (C). Platelets (11 109/ml) were suspended in a buffer containing 0.2 mM CaCl2 , and then activated with 0.5 U/ml thrombin for the indicated time intervals. Platelets were lysed with lysis buffer, and Triton X-insoluble fractions were harvested by centrifugation. The pellets were washed once, then solubilized with Laemmli buffer. Cytoskeletal association of tyrosine kinases was analyzed by immunoblotting, as detailed in Materials and Methods. The data are representative of four experiments.
association of tyrosine kinases, p60c-src, p54/58lyn, and p72syk was then evaluated on thrombinor TRAP-induced platelet activation. Western blotting with anti-p60c-src MoAb revealed two bands, 60kDa and 52kDa, which may correspond to a proteolytic product of p60c-src (Fig. 2A). These findings suggest that p60c-src associates with cytoskeleton upon thrombin activation, and part of it undergoes proteolysis, as reported by Oda et al. (4, 17). Western blotting with antip54/58lyn detected two major bands with the molecular weights of 54 and 58 kDa, suggesting no or minimal proteolysis of p54/58lyn (Fig. 2B). The cytoskeletal fractions of thrombin-activated platelets show a small amount of p72syk, if any, at the original molecular weight of 72 kDa, and the antibody reacted strongly with 52 and 48 kDa proteins (Fig. 2C). These findings suggest that the majority of p72syk which associated with cytoskeleton underwent proteolysis. Thrombin, at concentrations as low as 0.1 U/ml, could induce cytoskeletal association of 52 and 48kDa proteins (data not shown). On the other hand, TRAP did not induce cytoskeletal association of p72syk, while the translocation of p60c-src and p54/58lyn occurred in a magnitude similar to thrombin (Fig. 3). TRAP, even at concentrations which induce a full picture of platelet activation including aggregation and p72syk activation, did not induce p72syk translocation, whereas thrombin, at concentrations as low as 0.1 U/ml, induced p72syk translocation. These results suggest that translocation of p72syk requires a yet-unidentified functional thrombin receptor(s), which is distinct from ‘‘the seven-transmembrane thrombin receptor’’. Our notion that a full activation of platelets requires the simultaneous binding of thrombin to at least two different thrombinbinding sites is in agreement with the previous report by Lau et al. (11), who found that TRAP, in contrast to thrombin, is not a full agonist for activation and signal transduction in human platelets. Previous reports (18, 19) have demonstrated that p72syk is susceptible to proteolysis and that 1086
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FIG. 3. Cytoskeletal association of tyrosine kinases induced by TRAP. Immunoblot analysis of cytoskeleton, probed with anti-p60c-src MoAb (A), anti-p54/58lyn MoAb (B), or anti-p72syk MoAb (C). Platelets (11 109/ml) were suspended in a buffer containing 0.2 mM CaCl2 , and then stimulated with 20 m M TRAP for the indicated time intervals. Cytoskeletal fractions were harvested and were analyzed as described in the legend for Fig 2. The data are representative of three experiments.
intracellular Ca2/ elevation facilitates p72syk inactivation, which implies that a Ca2/-activated protease, calpain, is responsible. Alternatively, it is possible that thrombin induced cytoskeletal association of certain proteins, with which anti-p72syk MoAb reacted non-specifically. In order to address this issue, we resolubilized proteins from the cytoskeletal fractions with the use of a high concentration of salts, and performed immunoprecipitation with anti-p72syk MoAb. Western blotting with a monoclonal or polyclonal antibody against p72syk revealed that proteolytic fragments of p72syk were indeed present in platelet cytoskeleton activated with thrombin (Fig. 4A). To further confirm that p72syk undergoes proteolysis upon cytoskeletal association, we performed an experiment in which calpain activation was abolished. Platelets were loaded with 50 m M BAPTA-AM, which should inhibit intracellular Ca2/ elevation, and 100 m M calpeptin, a cell-permeant inhibitor of calpain. That this treatment totally suppressed calpain activation has been previously confirmed by Tsujinaka et al. (20) and Ishii et al. (21). Thrombin increased p72syk activity in platelets thus pretreated to a level similar to the cells not loaded with BAPTA-AM and calpeptin. Cytoskeletal association of p60c-src and p54/58lyn was reduced by 60.9{16.7%, and by 65.9{3.2% (mean{range, nÅ2) respectively. On the other hand, the amount of p72syk at the original molecular weight of 72kDa associated with cytoskeleton was markedly increased (Fig. 4B). The proteolytic fragments at lower molecular weights were still of a considerable amount, suggesting that calpain activation cannot totally explain the p72syk proteolysis and that other mode(s) of proteolysis may be involved. The translocation of p72syk to cytoskeleton has been already reported in porcine platelets activated with thrombin (7), while the effects of TRAP has not been evaluated to date. There appeared to be no detectable degradation of p72syk attached to cytoskeleton in porcine platelets, whereas we found that the majority of p72syk associated with cytoskeleton underwent proteolysis in thrombin-activated human platelets. We assume that this discrepancy is due to differences in species between human and porcine platelets. Since the use of calpain inhibitors revealed 1087
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FIG. 4. Translocation of p72syk to cytoskeleton. (A) Platelets (1 1 109/ml) were incubated with buffer (lane 1) or stimulated with 0.5 U/ml thrombin (lane 2) for 2 min. Cytoskeletal fractions were harvested as described in the legend for Fig. 2. The cytoskeletal fractions were resolubilized with 0.5 M NaCl, and the supernatant was immunoprecipitated with anti-p72syk MoAb. The samples were then analyzed by immunoblotting with a MoAb or polyclonal antibody against p72syk. The data are representative of two experiments. (B) BAPTA- and calpeptin-loaded platelets (1 1 109/ ml) were stimulated with 0.5 U/ml thrombin for the indicated periods. Cytoskeletal fractions were harvested as described in the legend for Fig. 2. Immunoblot analysis of cytoskeleton was performed with anti-p72syk MoAb. The data are representative of two experiments.
cytoskeletal association of p72syk without degradation in our study, and there was no evidence of p72syk degradation in cytosol, it is likely that p72syk degradation occurs after its translocation to cytoskeleton. It is known that thrombin binds to and cleaves GPIb, which is also referred to as the high affinity thrombin binding site (22). g-Thrombin, which lacks the binding to GPIb, activates platelets more slowly than a-thrombin (23). Blocking of GPIb by an anti-GPIb antibody modifies several aspects of platelet activation induced by thrombin (24). In the present study, NNKY5-5, an MoAb against GP I b, up to the concentration of 20 m g/ml had no effect on the p72syk translocation induced by thrombin (data not shown). Although the role of GPIb in facilitating thrombin activation is plausible, whether the interaction between GPIb and thrombin contributes to p72syk translocation remains elusive, because of our failure to block thrombininduced p72syk. ACKNOWLEDGMENTS We gratefully acknowledge the kind gift of reagents by Dr. Hirohei Yamamura and Junichi Kambayashi.
REFERENCES 1. Golden, A., Nemeth, S. P., and Brugge, J. S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 852–856. 2. Ohta, S., Taniguchi, T., Asahi, M., Kato, Y., Nakagawara, G., and Yamamura, H. (1992) Biochem. Biophys. Res. Commun. 185, 1128–1132. 3. Lipfert, L., Haimovich, B., Schaller, M. D., Cobb, B. S., Parsons, J. T., and Brugge, J. S. (1992) J. Cell. Biol. 119, 905–912. 4. Oda, A., Druker, B. J., Smith, M., and Salzman, E. W. (1992) J. Biol. Chem. 267, 20075–20081. 5. Horvath, A. R., Muszbek, L., and Kellie, S. (1992) EMBO. J. 11, 855–861. 6. Dash, D., Aepfelbacher, M., and Siess, W. (1995) FEBS Lett. 363, 231–234. 7. Tohyama, Y., Yanagi, S., Sada, K., and Yamamura, H. (1994) J. Biol. Chem. 269, 32796–32799. 8. Fox, J. E. B., Lipfert, L., Clark, E. A., Reynolds, C. C., Austin, C. D., and Brugge, J. S. (1993) J. Biol. Chem. 268, 25973–25984. 9. Schoenwaelder, S. M., Jackson, S. P., Yuan, Y., Teasdale, M. S., Salem, H. H., and Mitchell, C. A. (1994) J. Biol. Chem. 269, 32479–32487. 1088
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Vu, T-KH., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057–1068. Lau, L. F., Pumiglia, K., Cote, Y. P., and Feinstein, M. B. (1994) Biochem. J. 303, 391–400. Lasne, D., Donato, J., Falet, H., and Rendu, F. (1995) Thromb. Haemost. 74, 1323–1328. Kramer, R. M., Roberts, E. F., Hyslop, P. A., Utterback, B. G., Hui, K. W., and Jakubowski, J. A. (1995) J. Biol. Chem. 270, 14816–14823. Clark, E. A., and Brugge, J. S. (1993) Mol. Cell. Biol. 13, 1863–1871. Taniguchi, T., Kitagawa, H., Yasue, S., Yanagi, S., Sakai, K., Asahi, M., Ohta, S., Takeuchi, F., Nakamura, S., and Yamamura, H. (1993) J. Biol. Chem. 268, 2277–2279. Pumiglia, K. M., and Feinstein, M. B. (1993) Biochem. J. 294, 253–260. Oda, A., Druker, B. J., Ariyoshi, H., Smith, M., and Salzman, E. W. (1993) J. Biol. Chem. 268, 12603–12608. Kobayashi, T., Nakamura, S., Taniguchi, T., and Yamamura, H. (1990) Eur. J. Biochem. 188, 535–540. Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S., and Yamamura, H. (1991) J. Biol. Chem. 266, 15790–15796. Tsujinaka, T., Kajiwara, Y., Kambayashi, J., Sakon, M., Higuchi, N., Tanaka, T., and Mori, T. (1988) Biochem. Biophys. Res. Commun. 153, 1201–1208. Ishii, H., Suzuki, Y., Kuboki, M., Morikawa, M., Inoue, M., and Kazama, M. (1992) Biochem. J. 284, 755–760. Marco, L. D., Mazzucato, M., Masotti, A., Fenton, II, J. W., and Ruggeri, Z. M. (1991) J. Biol. Chem. 266, 23776–23783. Jandrot-Perrus, M., Didry, D., Guillin, M. C., and Nurden, A. T. (1988) Eur. J. Biochem. 174, 359–367. Yamamoto, N., Greco, N. J., Barnard, M. R., Tanoue, K., Yamazaki, H., Jamieson, G. A., and Michelson, A. D. (1991) Blood 177, 1740–1748.
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Differential Mobilization of Tyrosine Kinases in Human Platelets Stimulated with Thrombin or Thrombin Receptor Agonist Peptide Kaneo Satoh, Yukio Ozaki,1 Naoki Asazuma, Yutaka Yatomi, Qi Ruomei, Kenji Kuroda, Libo Yang, and Shoji Kume Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Yamanashi, Japan Received July 8, 1996 Both thrombin and thrombin receptor agonist peptide (TRAP) activated p72syk and p60c-src with similar magnitudes. Both thrombin and TRAP induced translocation of p60c-src and p54/58lyn to cytoskeleton in an aggregation-dependent manner. Thrombin also induced cytoskeletal association of p72syk, but independent of platelet aggregation. Furthermore, p72syk associated with cytoskeleton underwent marked proteolysis, which was partially dependent upon calpain activation. In contrast, TRAP, even at concentrations as high as 100 m M, did not induce p72syk translocation to cytoskeleton. Our findings suggest that cytoskeletal translocation of p72syk induced by thrombin is governed by a mechanism distinct from those of p60c-src and p54/58lyn translocation. It is also suggested that p72syk translocation induced by thrombin requires additional signal(s) other than that mediated by the recently-cloned thrombin receptor that couples with GTP-binding proteins and interacts with TRAP. q 1996 Academic Press, Inc.
Platelets contain high levels of protein-tyrosine kinase activity, mainly due to p60c-src, p72syk, p54/58lyn and p125FAK (1-3). An accumulating body of evidence suggests that activation of these kinases contributes to agonist-stimulated tyrosine phosphorylation and subsequent processes of platelet activation. Several reports have demonstrated that some tyrosine kinases translocate to the Triton X-100-insoluble (cytoskeleton-rich) fraction upon stimulation with thrombin or other agonists (4-8). While the physiological role of tyrosine kinase translocation to cytoskeleton remains to be elucidated, there is evidence to suggest that it plays a role in cellular retraction of fibrin polymer (9). It also remains to be determined whether all tyrosine kinases associate with cytoskeleton upon platelet activation. Recently, a ligand of the cloned thrombin receptor, thrombin receptor agonist peptide (TRAP), has been found to induce platelet activation (10). Although the overall actions of TRAP are similar to those of thrombin, several studies have suggested that there are qualitative differences (11-13). In this study, we evaluated the effects of TRAP and thrombin on p72syk activity and translocation of tyrosine kinases (p60c-src, p72syk, p54/58lyn) to the Triton X-insoluble fraction in human platelets. MATERIALS AND METHODS c-src
Materials. Anti-p60 monoclonal antibody (MoAb), anti-p72syk MoAb, and anti-p54/58lyn MoAb were obtained from Upstate Biotechnology, NY, USA, and Wako, Osaka, Japan, respectively. Anti-p72syk polyclonal antibody was a generous gift of Dr. Hirohei Yamamura (Kobe University, Japan). Anti-GP I b MoAb , NNKY5-5, was a kind donation from Dr. Shosaku Nomura (Kansai Medical University, Japan). Thrombin receptor agonist peptide (TRAP) and calpeptin were kind gifts of Dr. Junichi Kambayashi (Osaka University, Japan). Preparation of washed platelets. Platelet-rich plasma was incubated with 1 mM acetylsalicylic acid for 30 min to
1 Reprint requests to : Yukio Ozaki, M.D., Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi 409-38, Japan. Fax: 81-552-73-6713.
1084 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Time course of p72syk activation assessed by in vitro kinase assay. After platelets (11 109/ml) were stimulated with 0.5 U/ml thrombin (A) or 20 m M TRAP (B) for the indicated time intervals, they were solubilized with lysis buffer. The Triton X-soluble fraction was immunoprecipitated with anti-p72syk MoAb, and was incubated with [g-32P]ATP for in vitro kinase assay. The proteins were separated by SDS-PAGE and transferred onto membrane. The kinase activity was detected with autoradiography, as detailed in Materials and Methods. The bands for the immunoglobulin heavy chain are indicated by arrowheads. The data are representative of three experiments.
exclude the secondary effects of TXA2 . The platelets were washed and resuspended in a HEPES buffer at a concentration of 1 1 109 cells/ml. Analysis of cytoskeleton-associated tyrosine kinases. Platelets were activated with thrombin or TRAP, and after the indicated periods, reactions were terminated with an equal volume of 21 ice-cold lysis buffer (2% Triton X-100, 100 mM Tris/HCl, pH 7.5, 5 mM EDTA, 2 mM vanadate, 1 mM PMSF , and 100 m g/ml leupeptin). The lysate was sonicated and centrifuged at 16,000 1g for 5 min. The pellet, which was the Triton X-insoluble fraction (cytoskeletonrich fraction), was washed once with 11 lysis buffer, and subjected to Western blotting using specific antibodies against tyrosine kinases. Immunoprecipitation kinase assay. The supernatant was precleared with protein A sepharose twice. The supernatant was then mixed with specific anti-tyrosine kinase monoclonal antibody (MoAb) bound to protein A sepharose. The in vitro kinase assay was performed as previously described (14), with slight modifications. The beads were incubated with kinase reaction buffer (100 mM Hepes/NaOH, pH8.0, 5 mM MnCl2 , 50 mM MgCl2) containing 10 m g acidtreated enolase. The reaction was initiated by the addition of 1.5 m M [g-32P]ATP (15 m Ci). After 10 min at 207C, reactions were stopped by addition of Laemmli buffer. After Western blotting, the radioactivity was quantified with a BAS-2000 Phosphoimager (Fuji Film, Japan).
RESULTS AND DISCUSSION
Changes in tyrosine kinase activity in platelets stimulated with thrombin or TRAP. Changes in tyrosine kinase activity induced by thrombin or TRAP were evaluated on three tyrosine kinases, p72syk, p60c-src, and p54/58lyn. When platelets were activated with thrombin (0.5 U/ ml) under constant stirring, the p72syk activity reached its peak 15 sec to 2 min after stimulation, the level of which was almost 5-fold of the resting value, and subsequently decreased almost to the original level within 5 min (Fig. 1A). TRAP (5Ç40 m M) also induced transient activation of p72syk (Fig. 1B), essentially similar to the effects of thrombin. Thrombin and TRAP also induced transient activation of p60c-src with the maximum activity peaking somewhat later than p72syk (data not shown). The maximum increase in the kinase activity over that of the control remained low (2-fold at most). Thus, we have confirmed previous findings that thrombin induces p72syk and p60c-src activation (14, 15). While Pumiglia et al. have already reported that TRAP induced p60c-src activation (16), we are the first to show that TRAP also activates p72syk. p54/58lyn activation induced by thrombin or TRAP was minimal and was not always reproducible, as assessed by kinase assays. Cytoskeletal association of tyrosine kinases induced by thrombin or TRAP. Cytoskeletal 1085
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FIG. 2. Cytoskeletal association of tyrosine kinases induced by thrombin. Immunoblot analysis of cytoskeleton, probed with anti-p60c-src MoAb (A), anti-p54/58lyn MoAb (B), or anti-p72syk MoAb (C). Platelets (11 109/ml) were suspended in a buffer containing 0.2 mM CaCl2 , and then activated with 0.5 U/ml thrombin for the indicated time intervals. Platelets were lysed with lysis buffer, and Triton X-insoluble fractions were harvested by centrifugation. The pellets were washed once, then solubilized with Laemmli buffer. Cytoskeletal association of tyrosine kinases was analyzed by immunoblotting, as detailed in Materials and Methods. The data are representative of four experiments.
association of tyrosine kinases, p60c-src, p54/58lyn, and p72syk was then evaluated on thrombinor TRAP-induced platelet activation. Western blotting with anti-p60c-src MoAb revealed two bands, 60kDa and 52kDa, which may correspond to a proteolytic product of p60c-src (Fig. 2A). These findings suggest that p60c-src associates with cytoskeleton upon thrombin activation, and part of it undergoes proteolysis, as reported by Oda et al. (4, 17). Western blotting with antip54/58lyn detected two major bands with the molecular weights of 54 and 58 kDa, suggesting no or minimal proteolysis of p54/58lyn (Fig. 2B). The cytoskeletal fractions of thrombin-activated platelets show a small amount of p72syk, if any, at the original molecular weight of 72 kDa, and the antibody reacted strongly with 52 and 48 kDa proteins (Fig. 2C). These findings suggest that the majority of p72syk which associated with cytoskeleton underwent proteolysis. Thrombin, at concentrations as low as 0.1 U/ml, could induce cytoskeletal association of 52 and 48kDa proteins (data not shown). On the other hand, TRAP did not induce cytoskeletal association of p72syk, while the translocation of p60c-src and p54/58lyn occurred in a magnitude similar to thrombin (Fig. 3). TRAP, even at concentrations which induce a full picture of platelet activation including aggregation and p72syk activation, did not induce p72syk translocation, whereas thrombin, at concentrations as low as 0.1 U/ml, induced p72syk translocation. These results suggest that translocation of p72syk requires a yet-unidentified functional thrombin receptor(s), which is distinct from ‘‘the seven-transmembrane thrombin receptor’’. Our notion that a full activation of platelets requires the simultaneous binding of thrombin to at least two different thrombinbinding sites is in agreement with the previous report by Lau et al. (11), who found that TRAP, in contrast to thrombin, is not a full agonist for activation and signal transduction in human platelets. Previous reports (18, 19) have demonstrated that p72syk is susceptible to proteolysis and that 1086
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FIG. 3. Cytoskeletal association of tyrosine kinases induced by TRAP. Immunoblot analysis of cytoskeleton, probed with anti-p60c-src MoAb (A), anti-p54/58lyn MoAb (B), or anti-p72syk MoAb (C). Platelets (11 109/ml) were suspended in a buffer containing 0.2 mM CaCl2 , and then stimulated with 20 m M TRAP for the indicated time intervals. Cytoskeletal fractions were harvested and were analyzed as described in the legend for Fig 2. The data are representative of three experiments.
intracellular Ca2/ elevation facilitates p72syk inactivation, which implies that a Ca2/-activated protease, calpain, is responsible. Alternatively, it is possible that thrombin induced cytoskeletal association of certain proteins, with which anti-p72syk MoAb reacted non-specifically. In order to address this issue, we resolubilized proteins from the cytoskeletal fractions with the use of a high concentration of salts, and performed immunoprecipitation with anti-p72syk MoAb. Western blotting with a monoclonal or polyclonal antibody against p72syk revealed that proteolytic fragments of p72syk were indeed present in platelet cytoskeleton activated with thrombin (Fig. 4A). To further confirm that p72syk undergoes proteolysis upon cytoskeletal association, we performed an experiment in which calpain activation was abolished. Platelets were loaded with 50 m M BAPTA-AM, which should inhibit intracellular Ca2/ elevation, and 100 m M calpeptin, a cell-permeant inhibitor of calpain. That this treatment totally suppressed calpain activation has been previously confirmed by Tsujinaka et al. (20) and Ishii et al. (21). Thrombin increased p72syk activity in platelets thus pretreated to a level similar to the cells not loaded with BAPTA-AM and calpeptin. Cytoskeletal association of p60c-src and p54/58lyn was reduced by 60.9{16.7%, and by 65.9{3.2% (mean{range, nÅ2) respectively. On the other hand, the amount of p72syk at the original molecular weight of 72kDa associated with cytoskeleton was markedly increased (Fig. 4B). The proteolytic fragments at lower molecular weights were still of a considerable amount, suggesting that calpain activation cannot totally explain the p72syk proteolysis and that other mode(s) of proteolysis may be involved. The translocation of p72syk to cytoskeleton has been already reported in porcine platelets activated with thrombin (7), while the effects of TRAP has not been evaluated to date. There appeared to be no detectable degradation of p72syk attached to cytoskeleton in porcine platelets, whereas we found that the majority of p72syk associated with cytoskeleton underwent proteolysis in thrombin-activated human platelets. We assume that this discrepancy is due to differences in species between human and porcine platelets. Since the use of calpain inhibitors revealed 1087
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FIG. 4. Translocation of p72syk to cytoskeleton. (A) Platelets (1 1 109/ml) were incubated with buffer (lane 1) or stimulated with 0.5 U/ml thrombin (lane 2) for 2 min. Cytoskeletal fractions were harvested as described in the legend for Fig. 2. The cytoskeletal fractions were resolubilized with 0.5 M NaCl, and the supernatant was immunoprecipitated with anti-p72syk MoAb. The samples were then analyzed by immunoblotting with a MoAb or polyclonal antibody against p72syk. The data are representative of two experiments. (B) BAPTA- and calpeptin-loaded platelets (1 1 109/ ml) were stimulated with 0.5 U/ml thrombin for the indicated periods. Cytoskeletal fractions were harvested as described in the legend for Fig. 2. Immunoblot analysis of cytoskeleton was performed with anti-p72syk MoAb. The data are representative of two experiments.
cytoskeletal association of p72syk without degradation in our study, and there was no evidence of p72syk degradation in cytosol, it is likely that p72syk degradation occurs after its translocation to cytoskeleton. It is known that thrombin binds to and cleaves GPIb, which is also referred to as the high affinity thrombin binding site (22). g-Thrombin, which lacks the binding to GPIb, activates platelets more slowly than a-thrombin (23). Blocking of GPIb by an anti-GPIb antibody modifies several aspects of platelet activation induced by thrombin (24). In the present study, NNKY5-5, an MoAb against GP I b, up to the concentration of 20 m g/ml had no effect on the p72syk translocation induced by thrombin (data not shown). Although the role of GPIb in facilitating thrombin activation is plausible, whether the interaction between GPIb and thrombin contributes to p72syk translocation remains elusive, because of our failure to block thrombininduced p72syk. ACKNOWLEDGMENTS We gratefully acknowledge the kind gift of reagents by Dr. Hirohei Yamamura and Junichi Kambayashi.
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