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Related Collections Signal Transduction Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 June 2001, Vol. 97, No. 12, pp. 3836-3845 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Role of Fc receptor -chain in platelet glycoprotein Ib-mediated signaling Yi Wu, Katsue Suzuki-Inoue, Kaneo Satoh, Naoki Asazuma, Yutaka Yatomi, Michael C. Berndt, and Yukio Ozaki From the Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Tamaho, Nakakoma, Yamanashi, Japan; and Baker Medical Research Institute, Prahran, Victoria, Australia.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) stimulates tyrosine kinases and subsequent tyrosine phosphorylation events in human platelets. This study found that the combination of vWF and botrocetin, by interacting with GPIb, induced tyrosine phosphorylation of Fc receptor -chain (FcR -chain), Syk, linker for activation of T cells (LAT), and phospholipase C 2 (PLC2). Pretreatment of platelets with 10 µM PP1 completely inhibited these tyrosine phosphorylation events. On GPIb stimulation, Src and Lyn formed a complex with FcR -chain and Syk, suggesting that Src and Lyn are involved in FcR -chain tyrosine phosphorylation and downstream signals. In spite of the PLC2 tyrosine phosphorylation, however, there was no intracellular calcium release and inositol 1,4,5-trisphosphate production. In Brij 35 lysates, FcR -chain was found to constitutively associate with GPIb. The number of GPIb expressed on FcR -chain-deficient platelets was comparable to that of the wild-type, as assessed by flow cytometry. However, tyrosine phosphorylation of Syk, LAT, and PLC2 in response to vWF plus botrocetin was significantly suppressed, suggesting that FcR -chain mediates activation signals related to GPIb. Compared with the aggregation response of wild-type platelets, that of FcR -chain-deficient platelets in response to vWF plus botrocetin was impaired, implying that FcR -chain is required for the full activation of platelets mediated by GPIb. (Blood. 2001;97:3836-3845) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References von Willebrand factor (vWF) is a multimeric protein that mediates platelet adhesion to exposed subendothelium at sites of vascular injury. The adhesive property of vWF is tightly regulated so that plasma vWF does not normally interact with circulating platelets. However, after vWF is activated by binding to damaged vessel walls, it serves to bridge the constituents of subendothelium to glycoprotein Ib (GPIb) on the membrane of circulating platelets.1,2 Although much is known about the molecular basis of the interaction between vWF and GPIb, little has been clarified about the intracellular signal transduction pathway in GPIb-mediated platelet activation. Events related to protein-tyrosine phosphorylation have emerged as important signals mediated by GPIb.3-7 GPIb-mediated platelet activation in response to vWF plus botrocetin, shear stress, or vWF from patients with von Willebrand disease type IIb induces tyrosine phosphorylation of multiple proteins,3-5 suggesting that the binding of vWF to GPIb causes the activation of tyrosine kinases. Studies have shown that the activation of Syk and Src and their association can be induced by GPIb stimulation.6-10 To date, there is no report showing that tyrosine residues within the GPIb molecule can be phosphorylated, nor has GPIb itself intrinsic kinase activity. Thus, how clustering of GPIb induced by vWF mediates the activation of tyrosine kinases such as Src and Syk has become an important issue. Because Syk is activated by engagement of its tandem SH2 domains with phosphorylated tyrosine residues in proteins containing the immunoreceptor tyrosine-based activation motif (ITAM),11 it is of interest whether ITAM-containing transmembrane molecules are also involved in GPIb signaling. Two ITAM-containing proteins have been identified in platelets, the low-affinity receptor for immunoglobulin (Ig) G, FcRIIA, and the Fc receptor -chain (FcR -chain). Of particular note is the finding that FcRIIA is physically associated with GPIb and that it mediates some signaling events.12,13 However, the level of FcRIIA tyrosine phosphorylation induced by GPIb clustering is much lower than that inducible by FcRIIA clustering. Further, murine platelets lacking FcRIIA on their membrane appear to have normal responses to GPIb-related signals. Thus, to what extent FcRIIA contributes to the GPIb signaling pathway remains to be determined. The other ITAM-containing protein, FcR -chain, has been recognized for its role in signaling related to the high-affinity receptors for IgE (FcRI)14 and IgG (FcRI),15 and the low-affinity IgG receptor (FcRIII).16 In platelets, FcR -chain is colocalized with GPVI and is critical for GPVI-mediated platelet activation.17,18 Clustering and activation of GPVI induces tyrosine phosphorylation of FcR -chain ITAM,19 which then facilitates the recruitment of Syk, resulting in its activation.20 Moreover, FcR -chain is required for tyrosine phosphorylation of phospholipase C 2 (PLC2).20 Recently, Falati et al6 reported that alboaggregin A, by interacting with GPIb, induces tyrosine phosphorylation of FcR -chain, PLC2 activation, and Ca++ mobilization in platelets. On the basis of their studies, we sought to further define the role of FcR -chain in the GPIb-related signal transduction pathway.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Materials FcR -chain-deficient C57BL/6 mice and control mice were kindly provided by Dr Toshiyuki Takai (Tohoku University, Japan). The following materials were obtained from the indicated suppliers: monoclonal antibodies (MoAbs) against Lyn and Syk (Wako Pure Chemical Industries, Tokyo, Japan); anti-integrin 1 MoAb, antiphosphotyrosine MoAb, PY 20 (Transduction Laboratories, Lexington, KY); antiphosphotyrosine MoAb, 4G10, anti-Src MoAb, anti-FcR -chain, anti-Lck, anti-LAT (linker for activation of T cells) polyclonal Abs (Upstate Biotechnology, Lake Placid, NY); anti-Fyn MoAb, polyclonal anti-PLC2, anti-Syk, peroxidase-conjugated goat antirabbit IgG Abs (Santa Cruz, CA); control fluorescence-labeled monoclonal rat antimouse antibody (Exalpha Biologicals, Boston, MA); thrombin (Green Cross, Osaka, Japan); collagen (Hormon-Chemie, Munich, Germany); PP1 (Biomol, Plymouth Meeting, PA); Gly-Arg-Gly-Asp-Ser (GRGDS) peptide (Peptide Institute, Osaka, Japan); glutathione-Sepharose 4B and protein A-Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden); peroxidase-conjugated goat antimouse IgG Ab (Cappel, Durham, NC); D-myo-inositol 1,4,5-trisphosphate (IP3) assay kit (Amersham, Buckinghamshire, United Kingdom); bovine serum albumin (BSA), Tween-80, prostaglandin I2, phenylmethylsulfonyl fluoride (PMSF), Na3VO4, Triton X-100, Brij 35 (Sigma, St Louis, MO); and Fura-2 AM (Dojindo Laboratories, Kumamoto, Japan). vWF and botrocetin were purified as described previously.7,21 Nonfunctional anti-GPIb MoAb, WGA3, was provided by Dr M. Handa (Keio University, Tokyo, Japan). Fluorescence-labeled rat antimouse GPIb (p0p4), GPIX (p0p6), and GPIIb/IIIa (JON1) MoAbs were supplied by Dr Bernhard Nieswandt (University of Witten-Herdecke, Wuppertal, Germany). Glutathione S-transferase (GST) fusion protein containing the tandem SH2 domains of Syk was obtained from Dr C.-L. Law (University of Washington, Seattle, WA). Jararaca GPIb-binding protein was donated by Dr Y. Fujimura (Nara Medical University, Nara, Japan). Convulxin was a gift from Dr T. Morita (Meiji Pharmaceutical University, Tokyo, Japan). Preparation and stimulation of human and murine platelets Murine blood was taken by cardiac puncture immediately after death. Human venous blood was obtained from healthy, drug-free volunteers on the day of the experiment, using acid citrate dextrose (120 mM sodium citrate, 110 mM glucose, 80 mM citric acid) as anticoagulant. Washed platelets were prepared as previously described7 and were suspended in modified HEPES-Tyrode buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, 1 mM MgCl2, pH 7.3). Unless otherwise stated, murine platelets were resuspended at a count of 2.0 × 108 cells/mL, and human platelets were adjusted to 1.0 × 109 cells/mL. Platelets were stimulated at 37°C under constant stirring at 1000 rpm in an AG-10 aggregation analyzer (Kowa, Tokyo, Japan). In preliminary experiments, we confirmed that human vWF alone does not induce aggregation of washed murine platelets but that aggregation is induced in the copresence of botrocetin. This finding is in agreement with previous reports that GPIb of murine platelets can interact with human vWF.22,23 Platelet aggregation study Platelet aggregation was monitored by measuring light transmission with the use of an AG-10 aggregation analyzer (Kowa, Tokyo, Japan). The instrument was calibrated with either the washed platelet suspension or platelet-rich plasma (PRP) for zero light transmission and with buffer or platelet-poor plasma (PPP) for 100% transmission, respectively. Aggregation was initiated by addition of agonists under constant stirring at 1000 rpm at 37°C. Platelet aggregation of mice was measured not only by changes in light transmission but also by a particle-counting technique using light scatter.24 Briefly, a diode laser light beam (width: 40 µm; wavelength: 675 nm) was passed through mouse PRP in a cylindrical glass cuvette maintained at 37°C with constant stirring. The light scatter by particles in a limited volume (33 × 65 × 65 µm) was measured with an optical system that minimized multiple light scatter. The signals were digitized to quantitate the number and size of platelet aggregates. Light-scattering method provides a sensitive, in situ continuous measurement of platelet aggregation by counting the number and size of aggregates. On the basis of the level of scattered light intensity, platelet aggregates can be divided into small aggregates (0.2 to 2.0 V/min, consisting of less than 500 platelets) and large aggregates (2.0 to 10 V/min).24 Immunoprecipitation and GST-fusion protein precipitation After the indicated time intervals of activation, platelets were solubilized with an equal volume of 2 × ice-cold lysis buffer (100 mM Tris/HCl, pH 7.4, 5.0 mM EGTA, 2.0 mM PMSF, 2.0 mM Na3VO4, 100 µg/mL leupeptin, and 2% Triton X-100) and kept on ice for 30 minutes. All subsequent steps were performed at 4°C. The lysates were centrifuged at 15 000g for 5 minutes, and the resulting supernatants were precleared by incubation for 30 minutes with protein A-Sepharose for immunoprecipitation experiments or with glutathione-Sepharose for GST-fusion protein precipitation. For immunoprecipitation, the supernatants were then incubated with the appropriate antibodies, followed by the addition of protein A-Sepharose. For GST-fusion protein precipitation, the supernatants were incubated with GST-Syk-SH2 (20 µg/sample), followed by the addition of glutathione-Sepharose beads. The precipitates obtained after centrifugation were washed 3 times in 1 × lysis buffer before the addition of Laemmli sample buffer. As indicated elsewhere, the precipitates with GST-Syk-SH2 were subjected to re-immunoprecipitation in some experiments. Briefly, the precipitated proteins with GST fusion protein bound to glutathione-Sepharose beads were eluted with Glutathione Elution Buffer (10 mM reduced glutathione in 50 mM Tris/HCl, pH 8.0). The eluates were collected, dialyzed against 50 mM Tris/HCl (pH 8.0) containing 1 mM PMSF, 50 µg/mL leupeptin, and 1 mM Na3VO4, and subjected to immunoprecipitation with the indicated antibody. Immunoblotting Precipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. The membranes were blocked with 1% BSA in phosphate-buffered saline (PBS). After extensive washing with PBS containing 0.4% Tween-80, the immunoblots were incubated with the appropriate antibodies for 2 hours at room temperature, or overnight at 4°C. Antibody binding was detected by using peroxidase-conjugated secondary antibodies diluted at 1:7500 and was visualized with the enhanced chemiluminescence reaction reagent. For reprobing with other antibodies, the antibody bound to PVDF membranes was removed with a stripping buffer (2% SDS, 62.5 mM Tris/HCl, pH 6.8, 100 µM 2-mercaptoethanol) at 50°C for 20 minutes. After washing, the membranes were blocked with 1% BSA and reprobed with the indicated antibodies. When indicated, the level of proteins as detected by immunoblotting was quantified by using a PDI420oe scanner (PDI, New York, NY). Flow cytometry Washed murine platelets (8 × 107 cells/mL) in a volume of 25 µL were incubated with fluorescence-labeled rat antimouse GPIb (p0p4), antimouse GPIX (p0p6), or antimouse GPIIb/IIIa (JON1) MoAbs at the final concentration of 10 µg/mL for 15 minutes at 37°C, respectively. After dilution with 400 µL PBS, the samples were analyzed immediately on a FACScan (Becton Dickinson, San Jose, CA). Measurement of IP3 production Platelets were suspended in HEPES-Tyrode buffer at a count of 3 × 109/mL. After stimulation, an equal volume of ice-cold 15% trichloroacetic acid (TCA) was added to the platelet suspension to terminate reactions, and the mixtures were kept on ice for 30 minutes. The mixtures were centrifuged at 4000g for 15 minutes at 4°C, and the resultant supernatant was treated 5 times with 5 mL water-saturated diethyl ether to extract trichloroacetic acid. Residual ether was further removed under vacuum for 1 hour. The samples were neutralized by titration with 0.2 N NaOH, and the production of IP3 was measured with an IP3 assay kit (Amersham) according to the manufacturer's instructions. Measurement of the intracellular Ca++ concentration Measurement of intracellular Ca++ concentration ([Ca++]i) was performed with the use of Ca++-sensitive fluorophore, fura2, as described previously.25 The fura2-loaded platelets were adjusted to a count of 2 × 108/mL, and 200 µM EGTA was added to the platelet suspension to inhibit Ca++ influx, as GPIb-mediated platelet activation appears to induce Ca++ influx.5,26 After stimulation, changes in [Ca++]i were measured with a Hitachi 2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The [Ca++]i values were determined from the ratio of the fura2 fluorescence intensity at 340 nm excitation wavelength to that at 380 nm. Statistical analysis The data are expressed as the mean ± SEM. With the use of Statworks statistical software (Cricket Software, Philadelphia, PA), statistical analysis was done by Student t test with P < .05 taken to indicate significance.     Results Top Abstract Introduction Materials and methods Results Discussion References vWF plus botrocetin stimulates tyrosine phosphorylation of FcR -chain In preliminary experiments, we used 2 commercially available anti-FcR -chain polyclonal antibodies to detect tyrosine phosphorylation of FcR -chain in GPIb-mediated or collagen-induced platelet activation. However, there was little detectable change, if any. These antibodies appeared to be unsuitable for immunoprecipitation. Thus, we sought another way to address this issue. Human platelets have only 2 ITAM-containing proteins, FcRIIA and FcR -chain. Because the tandem SH2 domains of Syk bind specifically to the ITAM-containing proteins, they are often employed to detect the tyrosine-phosphorylated ITAMs.19,20 Thus, we used a GST-fusion protein expressing the tandem SH2 domains of Syk, GST-Syk-SH2. Human platelets were stimulated with 10 µg/mL vWF plus 6 µg/mL botrocetin, or 50 µg/mL collagen, the subsequent platelet lysates were incubated with GST-Syk-SH2, and proteins precipitated by GST-Syk-SH2 were checked for the presence of FcR -chain by using anti-FcR -chain polyclonal antibodies (Figure 1A). Approximately 4 proteins of 13, 11, 8.5, and 7 kd were detected as FcR -chain in platelets stimulated with vWF plus botrocetin [Figure 1Ai, lanes 1-4]. The anti-FcR -chain polyclonal antibodies were thus suitable for Western blotting but not for immunoprecipitation. Immunoblotting with antiphosphotyrosine antibodies revealed that the upper 2 bands (13 and 11 kd) were heavily tyrosine-phosphorylated, indicating that tyrosine phosphorylation of FcR -chain leads to a mobility shift on SDS-PAGE [Figure 1Aii, lanes 1-4]. Because only polyclonal antibodies against FcR -chain are available, whether the lower 2, nonphosphorylated bands correspond to the nonphosphorylated FcR -chain awaits elucidation. The pattern of FcR -chain tyrosine phosphorylation, including those of nonphosphorylated bands, is similar to that in GPVI-stimulated platelets, observed by Tsuji et al.18 Tyrosine phosphorylation of FcR -chain reached its peak 60 seconds after stimulation, and partial dephosphorylation occurred at 300 seconds [Figure 1Aii, lanes 1-4]. Precipitates with GST-Syk-SH2 from collagen-activated platelets showed patterns similar to vWF plus botrocetin [Figure 1Ai,ii, lane 9]. It is established that vWF contains not only a binding site for GPIb, the A1 domain, but also a C1 domain peptide sequence, Arg-Gly-Asp (RGD), which mediates vWF binding to integrin IIb3.27 To rule out the possibility that integrin IIb3 signaling is involved in tyrosine phosphorylation of FcR -chain, the platelets were pretreated with RGDS and EGTA and then stimulated with vWF plus botrocetin. As shown in Figure 1Aii, lanes 5-7, blockage of integrin IIb3 by RGDS and EGTA did not inhibit tyrosine phosphorylation of FcR -chain. We next used jararaca GPIb-binding protein (GPIb-BP) to confirm that FcR -chain tyrosine phosphorylation is mediated by GPIb-vWF interaction. Jararaca GPIb-BP itself induced neither platelet aggregation nor serotonin release from platelets, and it completely inhibited vWF binding to GPIb in the presence of botrocetin or ristocetin.28 In jararaca GPIb-BP-pretreated platelets, tyrosine phosphorylation of FcR -chain was abolished [Figure 1Aii, lane 8]. These findings indicate that GPIb stimulation specifically induces tyrosine phosphorylation of FcR -chain. Falati et al6 also found that both ristocetin plus normal vWF and ristocetin plus RGGS-vWF, a mutant vWF able to bind to GPIb but not to integrin IIb3, induced both tyrosine phosphorylation of FcR -chain and its association with Syk. Our findings are in good accord with their report and confirm that tyrosine phosphorylation of FcR -chain indeed takes place in the GPIb-mediated signal transduction pathway. Recently, FcRIIA, which also contains one ITAM, has been shown to be physically proximal to the GPIb-IX-V complex and functionally related to it.13 In our study, however, tyrosine phosphorylation of FcRIIA was not detectable in the GST-Syk SH2 precipitates (data not shown). View larger version (44K): [in this window] [in a new window]   Figure 1. Stimulation of platelets with vWF plus botrocetin induces tyrosine phosphorylation of FcR -chain. (A) Human platelets were preincubated for 10 minutes at 37°C with a vehicle solution (lanes 1-4, 9), 1 mM RGDS plus 1 mM EGTA (lanes 5-7), or 20 µg/mL jararaca GPIb-BP (lane 8). After stimulation with 6 µg/mL botrocetin plus 10 µg/mL vWF (lanes 1-8) or 50 µg/mL collagen (lane 9) for the indicated time periods, the reactions were terminated by the addition of lysis buffer. The platelet lysates were then precipitated with GST-Syk-SH2. (B) After preincubation with 0.25% dimethyl sulfoxide (DMSO) as control or 10 µM PP1 for 5 minutes at 37°C, the human platelets were activated by the addition of 6 µg/mL botrocetin plus 10 µg/mL vWF for the indicated time frames. The cells were processed for precipitation with GST-Syk-SH2 as described in the legend for A. For A and B, precipitated proteins were then separated by SDS-PAGE (15% polyacrylamide) and immunoblotted with anti-FcR -chain (i) and 4G10 plus PY20 (ii). The numbers below the panels show time in seconds. The data are representative of 3 experiments. The open arrowheads indicate the proteins of 13, 11 kd. The closed arrowheads indicate the proteins of 8.5, 7 kd. wc indicates whole cell lysates. Tyrosine phosphorylation of FcR -chain, in response to the activation of immune receptors in lymphocytes or of GPVI in platelets, is catalyzed by Src family tyrosine kinases.29,30 Because previous reports suggest the involvement of Src in GPIb-mediated platelet activation,7,8 the platelets were pretreated with a specific Src family kinase inhibitor, PP1, to examine whether FcR -chain is also tyrosine-phosphorylated by Src family kinases. PP1 is selective to Src family kinase over Syk by 10 000-fold in both in vitro and functional studies.31,32 As shown in Figure 1B, PP1 at 10 µM completely inhibited tyrosine phosphorylation of FcR -chain, suggesting that Src kinase(s) is responsible for FcR -chain tyrosine phosphorylation, a finding in agreement with that of Falati et al.6 FcR -chain tyrosine-phosphorylated by Src kinases recruits Syk An accumulating body of evidence suggests that tyrosine phosphorylation of FcR -chain catalyzed by Src kinases is required for the membrane recruitment and activation of Syk in signal transduction related to immune receptors and GPVI.11,20 Because we found in a previous report that GPIb mediates tyrosine phosphorylation of Syk and its association with Src,7 we then asked if Syk locates downstream of Src kinase activation and FcR -chain tyrosine phosphorylation in GPIb signaling. As shown in Figure 2A, PP1 pretreatment completely suppressed tyrosine phosphorylation of Syk, suggesting that Src family kinase activation lies upstream of Syk tyrosine phosphorylation. To determine which member(s) of the Src family tyrosine kinases is responsible for Syk activation, the proteins precipitated with anti-Syk MoAb were probed with the antibodies against various Src kinases. As shown in Figure 2B, in platelets stimulated with vWF plus botrocetin, not only Src but also Lyn were found to associate in vivo with Syk, which is also complexed with tyrosine-phosphorylated FcR -chain. Fyn and other kinases of the Src family were not detected (Figure 2B and data not shown). To investigate whether Src and Lyn associate with Syk via the SH2 domain of Syk, the platelet lysates were precipitated with GST-Syk-SH2, instead of anti-Syk MoAb. As shown in Figure 2C, the pattern of association of Src/Lyn, FcR -chain with GST-Syk-SH2 was consistent with their association with Syk (Figure 2B), and the precipitates were also negative for Fyn. Pretreatment of platelets with jararaca GPIb-BP inhibited association of Src, Lyn, FcR -chain with GST-Syk-SH2 (Figure 2C), implying that their association is specifically mediated by GPIb stimulation. Additionally, PP1 pretreatment also inhibited the association of Src/Lyn, FcR -chain with Syk or GST-Syk-SH2. This inhibition was not complete at an early time point; the reason remains elusive. In platelets, Src kinases and FcR -chain are membrane-associated proteins, and this complex formation may serve to recruit Syk to the membrane, thereby resulting in its tyrosine phosphorylation. View larger version (33K): [in this window] [in a new window]   Figure 2. Tyrosine phosphorylation of Syk and its association with FcR -chain, Src, and Lyn are dependent on Src kinase activity. (A) After incubation with 0.25% DMSO as the vehicle control or 10 µM PP1 for 5 minutes at 37°C, human platelets were stimulated with 6 µg/mL botrocetin plus 10 µg/mL vWF for the indicated time periods. The lysates were immunoprecipitated with anti-Syk MoAb. Precipitated proteins were then separated by SDS-PAGE and immunoblotted with 4G10 plus PY20 and anti-Syk MoAb. (B) Precipitated proteins with anti-Syk MoAb obtained in A were separated by SDS-PAGE and analyzed by immunoblotting with anti-FcR -chain, anti-Src, anti-Lyn, and anti-Fyn Abs as indicated in the left of each panel. (C) After pretreatment with 0.25% DMSO (control), 10 µM PP1 or 20 µg/mL jararaca-GPIb-BP, human platelets were stimulated with vWF plus botrocetin. The lysates were precipitated with GST-Syk-SH2, and the precipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-FcR -chain, anti-Src, anti-Lyn, anti-Fyn, and anti-Syk Abs. The data are representative of 3 experiments. The arrows represent the band derived from the heavy chains of IgG. pY indicates phosphotyrosine. To further clarify the mechanism of this complex formation, the lysates were first precipitated with GST-Syk-SH2. The precipitates with GST fusion proteins were eluted with glutathione, then the resultant eluates were subjected to immunoprecipitation with anti-Src or anti-Lyn MoAb. As shown in Figure 3A, FcR -chain was recovered with anti-Src and anti-Lyn immunoprecipitates, suggesting that Src and Lyn bind to FcR -chain. Additionally, Syk was not present in GST-Syk-SH2 precipitates (Figure 2C), suggesting that association of Src/Lyn with FcR -chain is not mediated by Syk associated with FcR -chain. To verify that FcR -chain is required for tyrosine phosphorylation of Syk, we performed similar experiments with FcR -chain knockout mice. As shown in Figure 3B (i and ii), tyrosine phosphorylation of Syk was reduced by approximately 60% in the FcR -chain-deficient platelets. Although FcR -chain plays an important role in Syk activation, there may be an additional signal transduction pathway(s) for Syk activation other than FcR -chain in GPIb-mediated platelet activation. Collectively, these findings suggest that Src and Lyn first bind to FcR -chain or an FcR -chain-associated protein and tyrosine-phosphorylate FcR -chain and that Syk subsequently joins in this complex. View larger version (33K): [in this window] [in a new window]   Figure 3. FcR -chain associated with Src and Lyn is required for tyrosine phosphorylation of Syk. (A) Samples were prepared as described in the legend for Figure 2C. The precipitates with GST-Syk-SH2 from human platelet lysates were eluted with Glutathione Elution Buffer (10 mM glutathione in50 mM Tris/HCl, pH 8.0), and the resultant eluates were subjected to re-immunoprecipitation with anti-Src and anti-Lyn MoAbs, respectively. Precipitated proteins were separated by SDS-PAGE and were analyzed by immunoblotting with anti-FcR -chain (i and ii), anti-Src (i), and anti-Lyn (ii) Abs. (Bi) Washed platelets prepared from wild-type C57BL/6 and FcR -chain-deficient mice were stimulated with 6 µg/mL botrocetin plus 10 µg/mL vWF for 3 minutes. Syk was immunoprecipitated from the lysates, and precipitates were separated by SDS-PAGE and analyzed with 4G10 plus PY20 and anti-Syk immunoblotting. pY indicates phosphotyrosine. (Bii) Level of Syk tyrosine phosphorylation shown in Bi was quantified by densitometry. Columns and error bars represent means ± SEM (n = 3). Association of FcR -chain with GPIb in platelets FcR -chain, which is tyrosine-phosphorylated on GPVI stimulation, binds constitutively to GPVI as a coreceptor.18 Because tyrosine phosphorylation of FcR -chain occurs early in GPIb-mediated platelet activation, it is possible that FcR -chain also locates proximal to GPIb. To determine whether there was a constitutive association between FcR -chain and GPIb, the GPIb immunoprecipitates obtained from Triton X-100 lysates were immunoblotted with anti-FcR -chain antibody. As shown in Figure 4, there was little, if any, FcR -chain associated with GPIb in Triton X-100 lysates. When a weaker detergent, Brij 35, was used instead of Triton X-100, FcR -chain association with GPIb was clearly detected, suggesting that FcR -chain is loosely linked with GPIb. In contrast, integrin 1, another integrate transmembrane protein, could not be recovered in any lysates (Figure 4). There was no significant change in the level of FcR -chain associated with GPIb, irrespective of stimulation. As described above, tyrosine phosphorylated FcR -chain is distinguished from the nonphosphorylated form by its mobility shift. However, no band shift was detectable in GPIb-associated FcR -chain. Although our findings hitherto suggest that Src and/or Lyn tyrosine-phosphorylates FcR -chain with subsequent binding of Syk, we were unable to detect Src/Lyn or Syk in GPIb immunoprecipitates. This discrepancy needs to be addressed in the future. View larger version (33K): [in this window] [in a new window]   Figure 4. FcR -chain is associated with GPIb. After stimulation with 6 µg/mL botrocetin plus 10 µg/mL vWF for the indicated time periods, human platelets were solubilized with an equal volume of lysis buffer containing 2% Brij 35 (vol/vol) or 2% Triton X-100 (vol/vol). GPIb was immunoprecipitated with WGA3, an anti-GPIb MoAb. Precipitated proteins were separated by SDS-PAGE and were analyzed by anti-FcR -chain, anti-integrin 1, and anti-GPIb immunoblotting, respectively. The data are representative of 4 experiments. The numbers below the panels show time in seconds. Tyrosine phosphorylation of PLC2 by botrocetin plus vWF is sensitive to PP1 pretreatment and independent of integrin IIb3 signaling PLC2 is the major PLC isoform expressed in platelets33 and is the only type reported to undergo tyrosine phosphorylation on stimulation.34 Tyrosine phosphorylation of PLC2 has been recently reported to occur in response to alboaggregin A, which presumably interacts with GPIb.6 We confirmed in this study that stimulation of platelets by vWF plus botrocetin caused marked tyrosine phosphorylation of PLC2 in a time-dependent manner. Densitometric analysis revealed that the level of PLC2 tyrosine phosphorylation increased 2-fold at 15 seconds and reached a maximum level at 300 seconds (Figure 5A and data not shown). As shown in Figure 5B, tyrosine phosphorylation of PLC2 was markedly inhibited by jararaca GPIb-BP pretreatment. Moreover, the pretreatment of platelets with RGDS and EGTA did not suppress the level of PLC2 tyrosine phosphorylation (Figure 5A). These findings suggest that tyrosine phosphorylation of PLC2 induced by vWF plus botrocetin is specifically mediated by GPIb and is not dependent on integrin IIb3 signaling. Tyrosine phosphorylation of PLC2 induced by vWF plus botrocetin was completely inhibited by PP1 (Figure 5C), suggesting the contribution of Src family kinases. View larger version (36K): [in this window] [in a new window]   Figure 5. GPIb-vWF interaction induces tyrosine phosphorylation of PLC2, independently of integrin IIb3 signaling. Human platelets were preincubated with a vehicle solution as the control (A-C, left panels), 1 mM RGDS peptide plus 1 mM EGTA (A, right panel), 20 µg/mL jararaca GPIb-BP (B, right panel), or 10 µM PP1 (C, right panel) for 5 minutes and then stimulated with 6 µg/mL botrocetin plus 10 µg/mL vWF for the indicated time periods. After cells were solubilized, PLC2 was isolated by immunoprecipitation with the corresponding Ab. Precipitated proteins were then separated by SDS-PAGE and analyzed by immunoblotting with 4G10 plus PY20. The membranes were reprobed by immunoblotting with anti-PLC2. The data are representative of 3 experiments. The numbers below the panels show time in seconds. pY indicates phosphotyrosine. vWF plus botrocetin stimulates tyrosine phosphorylation of LAT LAT is a 36- to 38-kd transmembrane protein that is found in glycolipid-enriched membrane domains.35 Recent studies have shown that LAT plays an essential role in regulation of PLC2 following stimulation of GPVI in platelets.36 Hence, we asked whether LAT is also involved in the GPIb-mediated signal transduction pathway. As shown in Figure 6A, stimulation with vWF plus botrocetin induced tyrosine phosphorylation of LAT in a time-dependent manner. Densitometric analysis revealed that tyrosine phosphorylation of LAT increased rapidly by 7-fold as early as 15 seconds after stimulation. Because pretreatment with RGDS and EGTA failed to inhibit phosphorylation of LAT (Figure 6A), it is suggested that integrin IIb3 signaling is not involved. The potent inhibition of LAT tyrosine phosphorylation by PP1 suggests that this is also downstream of Src family kinase activation (Figure 6B). View larger version (42K): [in this window] [in a new window]   Figure 6. LAT is tyrosine-phosphorylated in GPIb-mediated platelet activation. Human platelets were preincubated with a vehicle solution as the control (A and B, left panels), 1 mM RGDS peptide plus 1 mM EGTA (A, right panel), or 10 µM PP1 (B, right panel) for 5 minutes and then activated with 6 µg/mL botrocetin plus 10 µg/mL vWF for the indicated time periods. After cell lysis, LAT was isolated by immunoprecipitation with the corresponding Ab. Precipitated proteins were then separated by SDS-PAGE and analyzed by immunoblotting with 4G10 plus PY20. The membranes were reprobed for anti-LAT by immunoblotting. The data are representative of 3 experiments. The numbers below the panels show time in seconds. pY indicates phosphotyrosine. Stimulation with vWF plus botrocetin fails to induce IP3 production and calcium release In platelets, stimulation with collagen or FcRIIA cross-linking induces activation of PLC2, which leads to IP3 production and intracellular calcium mobilization.37,38 Because vWF plus botrocetin also stimulates tyrosine phosphorylation of PLC2, we then asked whether PLC2 is activated in GPIb-mediated platelet activation. Activation of PLC2 can be monitored by measurement of IP3 production and intracellular Ca++ release. As shown in Figure 7A, although collagen induced a rapid and prominent increase in IP3, platelet stimulation with vWF plus botrocetin failed to induce IP3 production. In addition, vWF plus botrocetin did not induce intracellular calcium release, whereas collagen was able to induce calcium release as early as 20 seconds after stimulation (Figure 7B), which is in good correlation with the time course of IP3 production. These findings suggest that PLC2 is not activated in vWF/botrocetin GPIb-mediated platelet activation in vitro despite its tyrosine phosphorylation. View larger version (19K): [in this window] [in a new window]   Figure 7. IP3 production and calcium release are not observed in platelets stimulated with vWF plus botrocetin. (A) IP3: after human platelets were stimulated with 75 µg/mL collagen () or 6 µg/mL botrocetin plus 10 µg/mL vWF () for the indicated time periods, the production of IP3 was measured as described in "Materials and methods." The data are expressed as means ± SEM of 3 separate experiments. (B) Ca++: stimulation of human platelets with 75 µg/mL collagen (i) or 6 µg/mL botrocetin plus 10 µg/mL vWF (ii) was initiated at the time indicated by the arrow. The [Ca++]i elevation in the absence of extracellular calcium was monitored as changes in fura2 fluorescence for 300 seconds. The ordinate represents the ratio of fura2 fluorescence. The results are representative of 3 experiments. GPIb-mediated tyrosine phosphorylation of PLC2 and LAT is absent in murine platelets lacking FcR -chain To determine whether tyrosine phosphorylation of PLC2 and LAT is dependent on FcR -chain, we performed experiments with platelets obtained from FcR -chain knockout mice. The level of tyrosine phosphorylation of PLC2 by vWF plus botrocetin stimulation was dependent on the botrocetin dose in platelets of wild-type C57BL/6 mice (Figure 8A). In platelets lacking FcR -chain, tyrosine phosphorylation of PLC2 on botrocetin plus vWF stimulation was reduced by approximately 80%, and tyrosine phosphorylation of LAT was virtually absent (Figure 8Bi,ii). View larger version (29K): [in this window] [in a new window]   Figure 8. GPIb-stimulated tyrosine phosphorylation of LAT and PLC2 are absent in platelets lacking FcR -chain. (A) Washed platelets from wild-type C57BL/6 mice were stimulated with 10 µg/mL vWF plus 0, 3, or 6 µg/mL botrocetin for 5 minutes. After lysis, platelets were immunoprecipitated with anti-PLC2 Ab. (B) Washed platelets from wild-type C57BL/6 and FcR -chain-deficient mice were stimulated with 6 µg/mL botrocetin plus 10 µg/mL vWF for the indicated time periods. After lysis, PLC2 (i) and LAT (ii) were immunoprecipitated with the corresponding Ab, respectively. The precipitated proteins in A and B were separated by SDS-PAGE and were analyzed by 4G10 plus PY20 immunoblotting. The loaded proteins were confirmed to be at comparable levels by immunoblotting with the corresponding Ab. The data are representative of 3 experiments. pY indicates phosphotyrosine. Impaired large aggregate formation of FcR -chain-deficient platelets Because the findings hitherto point to an important role of FcR -chain in the signal transduction pathway in GPIb-mediated platelet activation, we evaluated the functional role of FcR -chain in GPIb signaling. As detected by the light-transmission method (Figure 9A), platelets lacking FcR -chain failed to respond to convulxin, but they aggregated normally on thrombin stimulation as was the case with the platelets from wild-type C57BL/6 mice. Platelet aggregation in response to vWF plus botrocetin with FcR -chain-deficient mice was partially impaired compared with that of wild-type mice (Figure 9A,B). Platelet aggregation was further evaluated with a particle-counting method using light scattering. This method is unique in that it distinguishes the size of aggregates, based on the intensity of light scattering in PRP. Although the formation of small aggregates with FcR -chain-deficient mice was detected at a level comparable to that of the control, the formation of large aggregates was markedly reduced in FcR -chain-deficient platelets (Figure 9C). View larger version (16K): [in this window] [in a new window]   Figure 9. Formation of large aggregates is suppressed in platelets from FcR -chain-deficient mice. (A) Platelets from wild-type or FcR -chain-deficient mice were challenged with 1 U/mL thrombin (i), 10 ng/mL convulxin (ii), or 6 µg/mL botrocetin (iii), respectively. In the case of thrombin, washed platelets were used instead of platelet-rich plasma (PRP). The aggregation was evaluated for 5 minutes by the light transmission method. The arrows show the time points for the addition of the reagents. (B) PRPs from wild-type () or FcR -chain-deficient mice () were stimulated with increasing amounts of botrocetin for 5 minutes. The maximal light transmission of platelet aggregation induced by 12 µg/mL was set as 100%. The ordinate represents the percentage maximal magnitude of platelet aggregation induced by various concentrations of botrocetin. The data are expressed as means ± SEM (n = 3). (C) The formation of small and large aggregates was assessed by a light-scattering method, as described in "Materials and methods." , platelet aggregate formation from wild-type mice (n = 7); , FcR -chain-deficient mice (n = 5). The data are expressed as means ± SEM. NS indicates not significant; SA, small aggregates; LA, large aggregates. GPIb expression in wild-type and FcR -chain-deficient platelets Because FcR -chain is coexpressed with many receptors, we then asked whether it is also required for the expression of GPIb-IX complex. As shown in Figure 10, flow cytometric studies indicated that the expression of GPIb, IX, or GPIIb/IIIa in FcR -chain-deficient platelets was comparable to those of the wild-type. View larger version (32K): [in this window] [in a new window]   Figure 10. Comparable platelet GPIb-IX expression between FcR -chain-deficient and wild-type mice. Platelets from wild-type (upper panels) or FcR -chain-deficient (lower panels) mice were stained with phycoerythrin-labeled p0p4 (-mouse GPIb), fluorescein isothiocyanate-labeled p0p6 (-mouse GPIX), or phycoerythrin-labeled JON1 (-mouse GPIIb/IIIa), respectively. The fluorescence was analyzed by flow cytometry (solid lines). Gray lines represent the control, the fluorescence of rat -mouse Ab. The results shown are representative of 4 experiments.     Discussion Top Abstract Introduction Materials and methods Results Discussion References The FcR -chain constitutes an integral part of several Fc receptors and is coexpressed with GPVI, forming a platelet collagen receptor.18 On stimulation of this complex, Lyn and Fyn, Src family kinases, are activated and phosphorylate the ITAM of FcR -chain.29,30 Subsequently, Syk binds to the tyrosine-phosphorylated FcR -chain and sends downstream signals, leading to PLC2 activation.20 Recently, Falati et al6 reported that the GPIb-mediated activation signal, elicited by alboaggregin A, induces tyrosine phosphorylation of FcR -chain, a complex formation between Lyn, Fyn, FcR -chain, and Syk, as well as activation of PLC2 with resultant Ca++ release. Their findings imply that the GPIb-mediated signal transduction pathway has several features in common with that of the GPVI collagen receptor. In this report, we sought to define changes in FcR -chain-related signaling molecules in response to GPIb-vWF interaction. By using GST-Syk-SH2, we confirmed that vWF plus botrocetin specifically stimulates tyrosine phosphorylation of FcR -chain, an event completely blocked by jararaca GPIb-BP, a specific GPIb inhibitor. Tyrosine phosphorylation of FcR -chain was also independent of integrin IIb3. Our findings confirm those of Falati et al6 and suggest that the activation signal mediated by GPIb induces FcR -chain tyrosine phosphorylation. Furthermore, we showed that Src/Lyn and FcR -chain associate with Syk by its tandem SH2 domains, suggesting that a complex of Src/Lyn, FcR -chain, and Syk is formed in vivo on GPIb stimulation. This complex formation is specifically mediated by GPIb, as jararaca GPIb-BP significantly abrogated its formation. In the present study, we also found that PP1 significantly inhibits the association of Src/Lyn, FcR -chain with Syk, as well as Syk phosphorylation. This finding suggests that the complex formation is dependent of Src kinase activity and that Syk is downstream of activated Src kinase(s). However, the incomplete inhibition by PP1 implies that there may be an additional mechanism of the complex formation, distinct for Src kinase activation. By re-immunoprecipitation experiments, we demonstrated that Src and Lyn either bind directly to FcR -chain or to an FcR -chain-associated protein. On the basis of these findings, it is most likely that Src/Lyn first phosphorylates the tyrosine residues of FcR -chai