Co-localization of CD9 and GPIIb-IIIa (·IIb ·3 integrin) on activated platelet pseudopods and ·-granule membranes

Summary  CD9 is a 24-kDa membrane glycoprotein expressed on the surface of human platelets and potentially involved in cellular activation and adhesion functions. This protein belongs to a recently delineated family of cell-surface antigens that span the membrane four times, called tetraspans, and found mainly in leucocytes and tumour cells. As a first approach to clarify the function of CD9, we used immunoelectron microscopy to determine the localization of this antigen in human platelets, and compared its distribution with that of the GPIIb-IIIa integrin, the platelet receptor for fibrinogen. Monoclonal antibodies against CD9 (MAb7) and GPIIb-IIIa (HP1-1D) coupled to colloidal gold of different sizes (5 and 15 nm) were incubated with intact platelets in suspension or on ultrathin sections of platelets embedded in LR white. CD9 was found in association with GPIIb-IIIa on the inner face of ·-granule membranes. These two antigens also co-localized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from ·-granules but was associated with lysosomal structures. Flow cytometric analysis of platelet CD9 with a series of monoclonal antibodies revealed an increased expression upon thrombin stimulation, confirming the presence of an intracellular granular pool. The observation that CD9 and GPIIb-IIIa are stored in the same intracellular structures and migrate to the same activation zones after platelet stimulation lends support to previous suggestions of a close association between CD9 and GPIIb-IIIa in human platelets and of a possible involvement of CD9 in adhesive functions of platelets. This revised version was published online in November 2006 with corrections to the Cover Date.     

Co-localization of CD9 and GPIIb-IIIa (·IIb ·3 integrin) on activated platelet pseudopods and ·-granule membranes

Summary CD9 is a 24-kDa membrane glycoprotein expressed on the surface of human platelets and potentially involved in cellular activation and adhesion functions. This protein belongs to a recently delineated family of cell-surface antigens that span the membrane four times, called tetraspans, and found mainly in leucocytes and tumour cells. As a first approach to clarify the function of CD9, we used immunoelectron microscopy to determine the localization of this antigen in human platelets, and compared its distribution with that of the GPIIb-IIIa integrin, the platelet receptor for fibrinogen. Monoclonal antibodies against CD9 (MAb7) and GPIIb-IIIa (HP1-1D) coupled to colloidal gold of different sizes (5 and 15 nm) were incubated with intact platelets in suspension or on ultrathin sections of platelets embedded in LR white. CD9 was found in association with GPIIb-IIIa on the inner face of ·-granule membranes. These two antigens also co-localized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from ·-granules but was associated with lysosomal structures. Flow cytometric analysis of platelet CD9 with a series of monoclonal antibodies revealed an increased expression upon thrombin stimulation, confirming the presence of an intracellular granular pool. The observation that CD9 and GPIIb-IIIa are stored in the same intracellular structures and migrate to the same activation zones after platelet stimulation lends support to previous suggestions of a close association between CD9 and GPIIb-IIIa in human platelets and of a possible involvement of CD9 in adhesive functions of platelets. This revised version was published online in November 2006 with corrections to the Cover Date.     

Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance.

The role of transmembrane 4 superfamily (TM4SF) proteins during muscle cell fusion has not been investigated previously. Here we show that the appearance of TM4SF protein, CD9, and the formation of CD9-beta1 integrin complexes were both regulated in coordination with murine C2C12 myoblast cell differentiation. Also, anti-CD9 and anti-CD81 monoclonal antibodies substantially inhibited and delayed conversion of C2C12 cells to elongated myotubes, without affecting muscle-specific protein expression. Studies of the human myoblast-derived RD sarcoma cell line further demonstrated that TM4SF proteins have a role during muscle cell fusion. Ectopic expression of CD9 caused a four- to eightfold increase in RD cell syncytia formation, whereas anti-CD9 and anti-CD81 antibodies markedly delayed RD syncytia formation. Finally, anti-CD9 and anti-CD81 monoclonal antibodies triggered apoptotic degeneration of C2C12 cell myotubes after they were formed. In summary, TM4SF proteins such as CD9 and CD81 appear to promote muscle cell fusion and support myotube maintenance.     

Complex formation of CD23/surface immunoglobulin and CD23/CD81/MHC class II on an EBV-transformed human B cell line and inferable role of tetraspanin

CD23, a low-affinity IgE receptor, is a type II transmembrane protein having a C-type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co-exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L-KT9 cells (an Epstein-Barr virus (EBV)-transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti-CD23 and anti-Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross-linking of CD23 with anti-CD23 and its second antibody as compared with the case of stimulation by anti-CD23 alone. The cross-linking of CD23 with anti-CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L-KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.     

The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies

Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin. Platelet aggregation induced by this antibody is preceded by and/or accompanied by accelerated phosphatidylinositol turnover, the generation of inositol phosphates and diacylglycerol (DAG), calcium mobilization, and protein phosphorylation. The production of inositol phosphate(s) was measurable within 15 s of either anti-p24/CD9 or thrombin addition. Within 10 s of antibody addition (10 micrograms/ml), the level of DAG was 200% over that of the control and similar to that observed with 2 units/ml thrombin (201% over that of the control). Therefore, as it appears to be true for thrombin, platelet response upon binding of anti-p24/CD9 is primarily mediated by the activation of phospholipase C. When platelets pretreated with aspirin (200 microM) and apyrase (1 mg/ml) were subsequently exposed to anti-p24/CD9, aggregation still occurred. This indicates that neither secreted ADP nor thromboxane generation is required for this aggregation response. Using indo-1 and ratio cytofluorometry, we observed that an increase in platelet cytosolic calcium is a relatively early event and occurs in either the presence or absence of calcium in the external media. Phosphorylation studies of platelet proteins showed that anti-p24/CD9 binding to platelets caused increased phosphorylation of four proteins with apparent molecular masses of 50,000, 47,000, 36,000, and 20,000 daltons. These studies suggest that platelet activation mediated by the surface protein p24/CD9 is mainly through the stimulation of a phospholipase C, the activation of which is responsible for the generation of second messengers inositol trisphosphate and DAG.     

Evidence for an association between CD8 molecules and the T cell receptor complex on cytotoxic T cells

The T cell differentiation molecule CD8 is thought to play an important role in class I major histocompatibility complex-restricted T cell activities but the precise function of this molecule is unknown. To explore this question, we have studied several CD3+, CD8+ class I alloantigen-specific cytotoxic T lymphocyte (CTL) lines and clones. The ability of these CTL to proliferate as well as to lyse specific targets was inhibited by either anti-CD3 or anti-CD8 monoclonal antibodies. Exposure of CTL to relevant but not irrelevant target cells induced the rapid (less than 1 hr) disappearance of approximately 20 to 30% of CD3 and CD8 molecules from the cell surface. The modulation of these molecules became maximal at 6 to 12 hr and recovered thereafter in parallel. Treatment of CTL with anti-CD8 prevented alloantigen-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD8. Incubation of CTL with the combination of anti-CD3 and goat anti-mouse Ig also resulted in modulation of CD8. In contrast, the expression of other CTL surface antigens, such as CD2 (Leu-5, T11) and HLA-DR, was not reduced by any of these manipulations. These results suggest that CD8 molecules are associated with the CD3/antigen receptor complex on the surface of CTL, and may play a direct role in antigen-induced modulation and cross-linking of the T cell receptor.     

Contrasting effects of EWI proteins, integrins, and protein palmitoylation on cell surface CD9 organization

CD9, a tetraspanin protein, makes crucial contributions to sperm egg fusion, other cellular fusions, epidermal growth factor receptor signaling, cell motility, and tumor suppression. Here we characterize a low affinity anti-CD9 antibody, C9BB, which binds preferentially to homoclustered CD9. Using mAb C9BB as a tool, we show that cell surface CD9 homoclustering is promoted by expression of alpha3beta1 and alpha6beta4 integrins and by palmitoylation of the CD9 and beta4 proteins. Conversely, CD9 is shifted toward heteroclusters upon expression of CD9 partner proteins (EWI-2 and EWI-F) or other tetraspanins, or upon ablation of CD9 palmitoylation. Furthermore, unpalmitoylated CD9 showed enhanced EWI-2 association, thereby demonstrating a previously unappreciated role for tetraspanin palmitoylation, and underscoring how depalmitoylation and EWI-2 association may collaborate to shift CD9 from homo- to heteroclusters. In conclusion, we have used a novel molecular probe (mAb C9BB) to demonstrate the existence of multiple types of CD9 complex on the cell surface. A shift from homo- to heteroclustered CD9 may be functionally significant because the latter was especially obvious on malignant epithelial tumor cells. Hence, because of its specialized properties, C9BB may be more useful than other anti-CD9 antibodies for monitoring CD9 during tumor progression.     

Interaction of fibrinogen with its platelet receptor. Differential effects of alpha and gamma chain fibrinogen peptides on the glycoprotein IIb-IIIa complex

The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.     

Activation of Fc gamma RII induces tyrosine phosphorylation of multiple proteins including Fc gamma RII.

Platelets provide a useful system for studying Fc gamma receptor-mediated signaling events because these cells express only a single class of Fc gamma receptors and because platelet aggregation and secretion can be activated through Fc gamma receptor stimulation. We report here that stimulation of platelets by cross-linking antibodies to Fc gamma RII or by treatment with an anti-CD9 monoclonal antibody, which acts through Fc gamma RII, causes an induction of tyrosine phosphorylation of multiple platelet proteins. Although the profile of tyrosine-phosphorylated proteins induced by stimulation of this Fc receptor was similar to that induced by thrombin, an additional 40-kDa phosphorylated protein was also detected. This protein co-migrated with Fc gamma RII and was immunoprecipitated with a monoclonal antibody to Fc gamma RII. In addition, after the cross-linking of Fc gamma RII in HEL cells or in COS-1 cells transfected with Fc gamma RII cDNA, the 40-kDa protein immunoprecipitated with anti-Fc gamma RII was also phosphorylated on tyrosine. These data strongly suggest that Fc gamma RII itself is a substrate for a tyrosine kinase(s) activated when Fc gamma RII is stimulated. Fc gamma RII was phosphorylated by the Src protein in vitro, suggesting that this kinase may be responsible for phosphorylation of Fc gamma RII in vivo. These studies establish that activation of platelets and human erythroleukemia cells through Fc gamma RII and CD9 involves an induction of tyrosine phosphorylation of multiple proteins including Fc gamma RII itself and suggest that these phosphorylation events may be involved in Fc gamma RII-mediated cell signaling.     

Molecular cloning of the CD9 antigen. A new family of cell surface proteins.

The CD9 antigen was described originally as a 24-kDa surface protein of non-T acute lymphoblastic leukemia cells and developing B-lymphocytes. It is also strongly expressed on platelets, among other cells, where it shows the property of mediating platelet activation and aggregation upon binding with mAbs. The primary structure has been elucidated by cloning the cDNA from a lambda gt11 expression vector library constructed with megakaryocytic mRNA. Monoclonal antibodies were used as probes with an APAAP amplification of the signal. The 5' region was further cloned in a lambda gt10 randomly primed cDNA library. The initiation codon was immediately followed by a sequence coding for the tetrapeptide corresponding to the NH2-terminal sequence identified in a microsequencing procedure. Only one species of mRNA was found with an estimated size of 1.4 kilobase. CD9 antigen appears to be a 227-amino acid molecule with four hydrophobic domains and one N-glycosylation site. Sequence and structural comparisons showed extensive similarity of the CD9 antigen with a 237-amino acid molecule described previously as the human melanoma-associated antigen ME491 and a Schistosoma mansoni membrane protein of 218 amino acids. These three proteins identify a new family of cell-surface proteins.     

Complement proteins C5b-9 initiate secretion of platelet storage granules without increased binding of fibrinogen or von Willebrand factor to newly expressed cell surface GPIIb-IIIa

The functional and conformational activation of cell surface glycoproteins IIb-IIIa (GPIIb-IIIa) was probed in platelets stimulated to secrete by complement proteins C5b-9. Gel-filtered human platelets exposed to the purified human C5b-9 proteins exhibited non-lytic secretory release of both alpha- and dense granule storage pools with only a small increase in total binding of 125I-fibrinogen (less than 3000 molecules/cell) to the cell surface. By contrast to ADP- or thrombin-activated platelets, increased 125I-fibrinogen bound to C5b-9 platelets was not inhibited by Arg-Gly-Asp-containing peptides, suggesting that the high affinity membrane receptor for fibrinogen is not expressed under these conditions. C5b-9-stimulated platelets also failed to bind 125I-von Willebrand factor (less than 1 ng/10(8) platelets), confirming that the adhesive protein receptor function of cell surface GPIIb-IIIa is not expressed in these cells. Although specific binding of 125I-fibrinogen or 125I-von Willebrand factor did not significantly increase after C5b-9 assembly, these proteins elicited de novo expression of the GPIIb-IIIa activation-associated epitope recognized by monoclonal antibody PAC-1, and binding of this antibody to C5b-9 platelets was fully competed by Arg-Gly-Asp-containing peptides. These data suggest that the metabolic events which trigger granule secretion after C5b-9 insertion into the plasma membrane cause cell surface GPIIb-IIIa to be expressed in an activation-associated but functionally incompetent conformation.     

Alpha IIb beta 3 integrin dissociation induced by EDTA results in morphological changes of the platelet surface-connected canalicular system with differential location of the two separate subunits

Treatment of human platelets by EDTA (5 mM at 37 degrees C and pH 7.4 for 30 min) induces ultrastructural morphological changes of the surface-connected canalicular system (SCCS). The first consists in dilations of some portions of the channels, whereas the second is represented by collapse of parts of the canaliculi. The collapsed elements of the EDTA treated SCCS are made up of two parallel limiting membranes and a central striated zone. Some of the EDTA treated platelets form microaggregates, the cohesion of which is apparently due to the appearance of pentalaminar interplatelet structures. EDTA treatment is known to induce an irreversible loss of platelet aggregability which is due to irreversible dissociation of the membrane GPIIb-IIIa complexes. In the present study, we looked for involvement of GPIIb-IIIa in the formation of these pentalaminar structures, and were able to demonstrate that the morphological changes described are in fact directly dependent on the EDTA induced dissociation of GPIIb- IIIa complexes. Indeed, we observed that these changes (a) cannot be induced in type I Glanzmann's thrombasthenia, where GPIIb-IIIa complexes are absent, (b) do not appear when human platelets are preincubated with monoclonal anti-GPIIb-IIIa complex-dependent (CD41a) antibodies, which protect the complex from EDTA induced dissociation, (c) appear only at alkaline pH and at 37 degrees C, which corresponds to the range of pH and temperature where EDTA can dissociate GPIIb-IIIa complexes, (d) are accompanied by the disappearance in fluorescence flow cytometry of the heterodimer complex-dependent epitopes, when using anti-CD41a antibodies and (e) do not appear in rat platelets, where GPIIb-IIIa does not dissociate after EDTA treatment. Furthermore, using gold-labeled mAbs concomitantly with the addition of EDTA, we observed that almost only GPIIb was present in the collapsed regions of the canaliculi. Using double labeling studies with polyclonal anti- GPIIb antibodies coupled to 10 nm gold particles and polyclonal anti- GPIIIa antibodies coupled to 20 nm gold particles, we observed that while both 10 and 20 nm particles were present in the dilated portions of the canaliculi almost only the small particles, coupled to the anti- GPIIb antibodies, labeled the collapsed portions of the SCCS. On Lowicryl thin sections, polyclonal antibodies against GPIIb labeled the central striated zone while both GPIIb and GPIIIa were found in the dilated portions of the SCCS. All these observations lead us to suggest that homopolymers of GPIIb could be responsible for "zipping" of the SCCS.     

A common pathway for T lymphocyte activation involving both the CD3-Ti complex and CD2 sheep erythrocyte receptor determinants

T lymphocyte activation with monoclonal antibodies directed against the CD2 (T,p50) sheep red blood cell receptor antigen and against CD3 (T,p19,29) has been investigated. Co-stimulation of purified T lymphocytes with anti-CD3 (SP34) and anti-CD2 (9-1), which detects a unique epitope on the CD2 molecule, results in T cell activation and cell proliferation. Each antibody alone is unable to mediate this effect. Co-stimulation of purified T cells with two different anti-CD2 antibodies, 9-1 and 9.6, which detect two different epitopes on the CD2 molecule, are also mitogenic. In contrast, the combination of anti-CD3 (SP34) and anti-CD2 (9.6) cannot induce T cell activation. These data suggest that the CD2 epitope defined by the 9-1 antibody is functionally important for T cell activation via the CD3/Ti complex. Furthermore, it is demonstrated that anti-CD3 (SP34) induces epitopic modulation of the CD2 molecule, resulting in enhanced expression of the CD2, 9-1 epitope. This epitope modulation of the CD2 (9-1) epitope by anti-CD3 (SP34) occurs instantaneously at 4 degrees C and in the presence of NaN3. The functional interaction between CD3 and CD2 occurs in spite of any evidence of complex formation between these two molecules. These data suggest that the T cell differentiation antigens CD3 and CD2 are jointly involved in antigen-specific T cell activation. The data are consistent with a model for antigen-specific T cell activation involving both the CD3/Ti complex and subsequent activation of the CD2 complex T cell activation by co-stimulation with anti-CD3 (SP34) and anti-CD2 (9-1) is substantially enhanced by the addition of exogenous, purified interleukin 1 (IL 1). These data would suggest that the CD2 complex, as well as the putative IL 1 receptor, are involved in separate and complementary receptor-ligand interactions, resulting in the amplification of antigen-specific T cell responses.     

CD38 is associated with lipid rafts and upon receptor stimulation leads to Akt/protein kinase B and Erk activation in the absence of the CD3-zeta immune receptor tyrosine-based activation motifs.

T lymphocytes can be activated via the T cell receptor (TCR) or by triggering through a number of other cell surface structures, including the CD38 co-receptor molecule. Here, we show that in TCR+ T cells that express a CD3-zeta lacking the cytoplasmic domain, cross-linking with CD38- or CD3-specific monoclonal antibodies induces tyrosine phosphorylation of CD3-epsilon, zeta-associated protein-70, linker for activation of T cells, and Shc. Moreover, in these cells, anti-CD38 or anti-CD3 stimulation leads to protein kinase B/Akt and Erk activation, suggesting that the CD3-zeta-immunoreceptor tyrosine-based activation motifs are not required for CD38 signaling in T cells. Interestingly, in unstimulated T cells, lipid rafts are highly enriched in CD38, including the T cells lacking the cytoplasmic tail of CD3-zeta. Moreover, CD38 clustering by extensive cross-linking with an anti-CD38 monoclonal antibody and a secondary antibody leads to an increased resistance of CD38 to detergent solubilization, suggesting that CD38 is constitutively associated with membrane rafts. Consistent with this, cholesterol depletion with methyl-beta-cyclodextrin substantially reduces CD38-mediated Akt activation while enhancing CD38-mediated Erk activation. CD38/raft association may improve the signaling capabilities of CD38 via formation of protein/lipid domains to which signaling-competent molecules, such as immunoreceptor tyrosine-based activation motif-bearing CD3 molecules and protein-tyrosine kinases, are recruited.     

Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein.

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5'(6')-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1',7(3H)-isobenzofuran]-3'-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab')2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab')2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.     

FcgammaRII tyrosine phosphorylation differs between FcgammaRII cross-linking and platelet-activating anti-platelet monoclonal antibodies.

Using glutathione S-transferase Syk fusion proteins, we evaluated the mode of platelet FcgammaRII tyrosine phosphorylation induced by FcgammaRII cross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 domain of Syk (Syk-N-SH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and the domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bound to tyrosine-phosphorylated FcgammaRII with FcgammaRII cross-linking. In the case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk-NC-SH2 bound to tyrosine-phosphorylated FcgammaRII. Since the SH2 domain is specific for a particular structure containing phosphotyrosine, these findings suggest that only one tyrosine residue in the immunoreceptor tyrosine-based activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that both are phosphorylated with FcgammaRII cross-linking. Synthetic peptides corresponding to the ITAM of human platelet FcgammaRII with the N-terminal tyrosine residue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylated (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-phosphorylated FcgammaRII than C-P, suggesting that the N-terminal tyrosine residue is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, these findings imply that Syk-N-SH2 binds to the phosphorylated C-terminal tyrosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken together, our findings suggest that FcgammaRII-dependent platelet activation without FcgammaRII dimerization, such as with anti-CD9 mAb, is distinct from that induced by FcgammaRII cross-linking.     

Crosslinking of the CD69 molecule enhances S100A9 production in activated neutrophils

Expression of CD69 on neutrophils and generation of anti-CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti-CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint-composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte-macrophage colony stimulating factor (GM-CSF)-activated neutrophils was crosslinked by anti-CD69 monoclonal antibodies, and then intracellular proteins were detected using 2-dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up-regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS-27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS-27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL-1beta production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti-CD69 autoantibodies possibly affect joint-composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA.     

A CD9, alphaIIbbeta3, integrin-associated protein, and GPIb/V/IX complex on the surface of human platelets is influenced by alphaIIbbeta3 conformational states.

A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) receptor alphaIIbbeta3, integrin-associated protein (IAP), and glycoprotein (GP) Ib/V/IX complex was isolated from Chaps-solubilized human platelets. The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), IAP (BRIC126), GPIb (SZ1), GPIX (GR-P), beta3 (AP3) and alphaIIb (C3). Additionally, the association between CD9 and alphaIIbbeta3 was demonstrated by ELISA. In this system, CD9 did not bind to vitronectin receptor (alphavbeta3) suggesting that CD9/alphaIIbbeta3 association was alphaIIb-subunit or alphaIIbbeta3-complex dependent. D3, an alphaIIbbeta3-activating mAb that is also an anti-LIBS (ligand-induced binding site), immunoprecipitated primarily alphaIIbbeta3 with GPIb and IAP. CD9 was not detected in D3 immunoprecipitates. D3 binding induced platelet aggregation via direct alphaIIbbeta3 activation and was upregulated by the alphaIIbbeta3 antagonist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In addition, D3 also inhibited whole blood clot retraction, in contrast to AP3 and C3, suggesting that conformational constraints on alphaIIbbeta3 by D3 binding not only influenced the CD9 complex but also affected alphaIIbbeta3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that alphaIIbbeta3 receptor occupancy was not sufficient to cause complex dissociation. CD9 complex isolation was also independent of platelet activation, although a twofold increase in the quantity of CD9 complex was seen after platelet activation by alpha-thrombin in the presence of CaCl2 compared with that present in EDTA. Stirred platelets showed fibrinogen-mediated aggregation by alpha-thrombin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via alphaIIbbeta3 could be partially responsible for this increase. These findings imply that the platelet CD9 complex is independent of platelet activation although it is dependent upon the conformation state of alphaIIbbeta3.     

Cell surface comodulation of CD4 and T cell receptor by anti-CD4 monoclonal antibody

In our study we have used anti-CD4 mAb to investigate the cell surface association between CD4 and the Ag-specific TCR complex on mature peripheral T cells. Anti-CD4 mAb was administered in vivo and in vitro and its effects on CD4 and CD3 cell surface expression were determined. In vivo, anti-CD4 mAb reduced cell surface expression of its ligand, CD4, and secondarily also reduced cell surface expression of CD3/TCR on CD4+ splenic T cells. In vitro, multivalent cross-linking of CD4 by anti-CD4 mAb and either FcR+ cells or anti-Ig mAb also resulted in decreased surface expression of CD4 and specific comodulation of CD3/TCR. The secondary reduction in cell surface CD3/TCR expression induced by CD4 cross-linking could be pharmacologically disrupted by high doses of PMA, indicating that the comodulation of CD3 with CD4 was dependent upon intracellular mediators, possibly including protein kinase C. These results demonstrate that, in the presence of anti-CD4 mAb, CD4 is functionally associated with the CD3/TCR complex, and that this association is dependent upon the activity of intracellular mediators. Such intracellular mediators might induce the coordinate down-modulation of physically unassociated CD4 and CD3/TCR molecules, or, alternatively, might promote a physical interaction between CD4 and CD3/TCR molecules.     

Cross-linking of alpha and gamma-thrombin to distinct binding sites on human platelets

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross-linking with the membrane-impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross-linking of membrane glycoproteins. The proteins covalently linked to 125I-labelled alpha and gamma-thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 125I-alpha-thrombin was detected in high-molecular-mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr approximately equal to 400,000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78-kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active alpha-thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine-protease complex. The 240-kDa complex was formed with 0.8-100 nM alpha-thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl-inactivated alpha-thrombin. After analysis of Triton-X-100-soluble extracts of cross-linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I-alpha-thrombin. These were a precipitate containing GPIIb-IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240-kDa complex. Significantly, 125I-gamma-thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240-kDa and 78-kDa molecular mass range were not observed. We conclude that several binding sites for alpha-thrombin are present at the platelet surface, and that GPIb is one of them. The studies with gamma-thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.