BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies.

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

Association of steroid receptor coactivator AIB1 with estrogen receptor-alpha in breast cancer cells.

The steroid receptor coactivator AIB1 (amplified in breast cancer-1) is a transcriptional coactivator which has been found to be amplified in breast cancer. We have now investigated the role of the AIB1 protein in breast cancer cell lines. Although detectable levels of AIB1 were present in most cell lines, high levels of AIB1 expression were observed only in the ER-positive cell lines MCF-7 and BT-474 by western blot analysis. Newly developed monoclonal antibodies (mAbs) were used in several assays to show an association between AIBI and estrogen receptor-alpha (ER). AIB1 and ER co-localized to the nucleus of ER positive cell lines as shown by immunofluorescence microscopy, and a functional association of native AIB1 and ER in MCF-7 nuclear extracts was shown by EMSA. Recombinant ER also recruited AIB1 protein from nuclear extracts, shown by EMSA and by precipitation of ER-complex proteins bound to a biotinylated-ERE DNA target. Additionally, anti-AIB1 mAbs were able to immunoprecipitate ER from nuclear extracts of chemically cross-linked cells but not from uncross-linked cells. Both immunoprecipitation and oligonucleotide precipitation studies demonstrated the presence of p300 and CBP as part of the ER transcriptional complex. These results suggest that AIB1 and ER do associate physically in ER-positive breast cancer cell lines. We propose that in AIB1 amplified breast cancers, a heightened AIB1/ER association may play a crucial role in the progression of these tumors.

Endogenously expressed estrogen receptor and coactivator AIB1 interact in MCF-7 human breast cancer cells.

Coactivators are believed to mediate estrogen-induced gene responses via interaction with estrogen receptors (ER). Currently, a major challenge is to determine the importance of each coactivator in a specific cell type and promoter context in response to a particular ligand. The potential of ER to interact with a growing list of coactivators has been shown in a variety of in vitro and gene transfer assays, yet very few data have demonstrated the interaction of endogenous coactivators with ER in intact cells. We report here a ligand-specific interaction of endogenous human ER (hER) and the AIB1 coactivator in MCF-7 human breast cancer cells by using immunoprecipitation analyses. Complexes between endogenously expressed hER and AIB1 were detected in estradiol-treated cells and to a much lesser extent in cells treated with the partial agonist, monohydroxytamoxifen. We were unable to detect an hER-SRC-1 complex in our immunoprecipitations from MCF-7 cells. The in vitro-binding affinity for mouse ER interaction with AIB1 was estimated to be 40-120 nM. We conclude that AIB1 is a major coactivator for hER in MCF-7 human breast cancer cells.

Antibody cross-linking of human CD9 and the high-affinity immunoglobulin E receptor stimulates secretion from transfected rat basophilic leukaemia cells.

Previous studies have shown that antibody cross-linking of the tetraspanin protein CD9 stimulates the degranulation of platelets and eosinophils, although the mechanism of activation is unclear. In this work we transfected human CD9 into the rat basophilic leukaemia (RBL-2H3) cell line and studied the stimulation of secretion from these cells in response to a panel of anti-CD9 antibodies. Intact immunoglobulin G1 (IgG1) antibodies activated transfected cells whereas F(ab')2 fragments of antibody and an intact IgG2a did not. Stimulation of secretion was inhibited by co-incubation with monomer murine immunoglobulin E (IgE) but not with an IgG1 isotype control, indicating that the response involves the endogenous high-affinity IgE receptor (FcepsilonRI). The anti-CD9 antibody activation curve was biphasic, and supraoptimal antibody concentrations stimulated little or no degranulation, indicating that multivalent binding of human CD9 molecules is necessary for the formation of an active complex with rat FcepsilonRI. Immunoprecipitation of FcepsilonRI under mild detergent conditions co-precipitated CD9, suggesting the presence of pre-existing complexes of CD9 and FcepsilonRI that could be activated by antibody cross-linking. These data are further evidence that tetraspanins are involved in FcepsilonRI signalling and may reflect the participation of tetraspanins in the formation of complexes with other membrane proteins that use components of Fc receptors for signal transduction.

GPV is a marker of in vivo platelet activation--study in a rat thrombosis model.

Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G-protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin-antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life. In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans.

CD9 participates in endothelial cell migration during in vitro wound repair.

CD9, a widely expressed membrane protein of the tetraspanin family, has been implicated in diverse functions, such as signal transduction, cell adhesion, and cell motility. We tested the effects of an anti-CD9 monoclonal antibody (ALMA.1) on the migration and proliferation of human vascular endothelial cells (ECs) during repair of an in vitro mechanical wound mimicking angiogenic processes. ALMA.1 induced dose-dependent inhibition of wound repair with a 35+/-1.5% decrease at 20 microg/mL. Only cell migration was affected, because the rate of proliferation of ECs at the lesion margin was not modified and because the inhibition of repair was also observed for nonproliferating irradiated ECs. Monoclonal antibodies against CD63 tetraspanin (H5C6) and control mouse IgG (MOPC-21) were inactive. CD9, one of the most abundant proteins at the surface of ECs, colocalized with beta(1) or beta(3) integrins on EC membranes in double-labeling immunofluorescence experiments with ALMA.1 and an anti-beta(1) (4B4) or anti-beta(3) (SDF.3) monoclonal antibody. Moreover, ALMA.1 and 4B4 had additive inhibitory effects on lesion repair, whereas 4B4 alone also inhibited EC proliferation. In transmembrane Boyden-type assays, ALMA.1 induced dose-dependent inhibition of EC migration toward fibronectin and vitronectin with 45+/-6% and 31+/-10% inhibition, respectively, at 100 microg/mL. 4B4 inhibited migration toward fibronectin at 10 microg/mL but had no effect in the case of vitronectin. Adhesion of ECs to immobilized anti-CD9 monoclonal antibodies induced tyrosine-phosphorylated protein levels similar to those observed during interactions with beta(1) or beta(3) integrins. These results point to the involvement of CD9 in EC adhesion and migration during lesion repair and angiogenesis, probably through cooperation with integrins. As such, CD9 is a potential target to inhibit angiogenesis in metastatic and atherosclerotic processes.

A general approach to the generation of monoclonal antibodies against members of the tetraspanin superfamily using recombinant GST fusion proteins.

Tetraspanins belong to a rapidly growing family of proteins characterized by the presence of four conserved transmembrane segments and are involved in such diverse functions as cellular activation, adhesion, migration and differentiation. In an effort to develop reagents against newly discovered tetraspanins, we have devised a simple method for the screening of monoclonal antibodies (mAbs) using recombinant GST fusion proteins. GST fusion proteins containing the second extracellular domain of different tetraspanins (CD9, CD63, CD53, CD81, A15 or CO-029) were produced separately. Mice were immunized with cells having a high expression of the chosen tetraspanin and the constructs were used to screen hybridomas in a solid phase ELISA. Several clones binding the fusion protein were identified for each construct tested: four anti-CD9 hybridoma clones, four anti-CD63, two anti-CD53, two anti-CD81, three anti-A15 and one anti-CO-029. All the newly developed mAbs recognized the native proteins by flow cytometry, immunofluorescence staining of cells and immunoprecipitation and bound to the denatured proteins on immunoblotting. Use of GST fusion protein constructs in a simple ELISA can facilitate screening for mAbs to members of the tetraspanin family, especially in cases where information is limited to the nucleotide sequence. The mAbs obtained by this strategy should prove to be valuable tools for functional studies of newly discovered tetraspanins.

cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

Production and characterization of monoclonal antibodies to the steroid receptor coactivator AIB1.

The steroid coactivator AIB1 (Ampified In Breast cancer 1) is a member of the SRC-1 family of transactivation coactivators that is amplified in about 7% of breast tumor samples. Hybridomas were established that secreted monoclonal antibodies (MAbs) to a recombinant glutathione-S-transferase (GST) fusion protein expressing the steroid receptor interacting domain of AIB1 (GST-AIB.T1:a.a. 605-1294). Four MAbs from these hybridomas were characterized and designated AX15.1, AX15.2, AX15.3, and AX15.4. The MAbs were shown to be specific to AIB1 and did not cross-react with two similar coactivators SRC-1 and TIF2 as shown in Western blot analysis and enzyme-linked immunoadsorbent assay (ELISA). Western blot analysis using the four MAbs showed specific recognition of AIB1 protein as a 160 kDa band in lysates from cell lines containing AIB1 gene-amplification. The MAbs immunoprecipitated in vitro-translated AIB1 and cellular AIB1 from metabolically labeled cells. The results show that these newly described MAbs are useful in studies of AIB1 function and expression.

Measurement of GPV released by activated platelets using a sensitive immunocapture ELISA--its use to follow platelet storage in transfusion.

Thrombin, the most potent platelet agonist, plays a central role in haemostasis and in the occurrence of thrombotic events. This agonist activates platelets by cleaving the PAR G-protein coupled receptors and by binding to glycoprotein (GP) Ib and also cleaves GPV at the platelet surface to liberate the soluble 69 kDa fragment GPVf1. Monoclonal antibodies (MoAbs) to GPV were developed as tools to study the mechanism of platelet GPV cleavage and measure release of GPV in pathological situations. Specificity of the MoAbs for GPV was confirmed by flow cytometry and immunoprecipitation of proteins from human platelets and Dami megakaryocytic cells. A sensitive immunocapture sandwich ELISA for soluble GPV was developed using two MoAbs recognizing different epitopes of GPV and purified platelet or recombinant GPV as reference protein. This ELISA was employed to determine the mean plasma concentration of GPV in 100 normal individuals (17.3 ng/ml), to demonstrate the dose-dependent release of GPVf1 from washed platelets stimulated with thrombin and to follow the progressive release of GPVf1 during storage of therapeutic platelet concentrates. The present report describes a sensitive GPV ELISA of direct application to survey the processing and storage of platelet concentrates for transfusion and of potential value to monitor platelet activation in thrombotic states.

Selective tetraspan-integrin complexes (CD81/alpha4beta1, CD151/alpha3beta1, CD151/alpha6beta1) under conditions disrupting tetraspan interactions.

The tetraspans are molecules with four transmembrane domains which are engaged in multimolecular complexes (the tetraspan web) containing a subset of beta1 integrins (in particular alpha3beta1, alpha4beta1 and alpha6beta1), MHC antigens and several unidentified molecules. The molecules associated with tetraspans are readily detected after immunoprecipitation performed in mild detergents such as Brij 97 or CHAPS. In this study we show that another classical mild detergent, digitonin, dissociated most of these associated molecules, including integrins, from the tetraspans CD9, CD37, CD53, CD63, CD82, Co-029, Talla-1 and NAG-2. In contrast, reciprocal immunoprecipitations from various cell lines demonstrated that two other tetraspans, CD81 and CD151, formed complexes with integrins not disrupted by digitonin. These complexes were CD81/alpha4beta1, CD151/alpha3beta1 and CD151/alpha6beta1. Furthermore, a new anti-CD151 monoclonal antibody (mAb), TS151r, was shown to have a restricted pattern of expression, inversely related to the sum of the levels of expression of alpha6beta1 and alpha3beta1. This mAb was unable to co-precipitate integrins in digitonin, suggesting that its epitope is blocked by the association with integrins. Indeed, the binding of TS151r to the cell surface was quantitatively diminished following alpha3beta1 overexpression. Altogether, these data suggest that, among tetraspans, CD81 interacts directly with the integrin alpha4beta1, and CD151 interacts directly with integrins alpha3beta1 and alpha6beta1. Because all tetraspan-tetraspan associations are disrupted by digitonin, it is likely that the other tetraspans interact indirectly with integrins, through interactions with CD81 or CD151.

Tetraspanin CD9 is associated with very late-acting integrins in human vascular smooth muscle cells and modulates collagen matrix reorganization.

CD9, a member of the tetraspanin family, and very late-acting (VLA) integrins are known to associate and form functional units on the surface of several cell types. We studied the changes in expression of CD9 and beta1-integrins (CD29, VLA) in human vascular smooth muscle cells (VSMCs) under in vitro culture conditions mimicking proliferative vascular diseases. We also investigated possible interactions between CD9 and VLA integrins in VSMCs. We found that CD9 is highly expressed in VSMCs and is subject to modulation, depending on the proliferative/contractile state of the cells. In the contractile phenotype, the levels of CD9, CD81, another tetraspanin, and CD29 are approximately 50% of those found in the proliferative phenotype. Coimmunoprecipitation experiments showed physical association between CD9 and CD29. CD9 was mainly associated with alpha2 and alpha3-integrins (CD49b and c) and also with alpha5-integrin to a weaker extent. Functionally, the addition of anti-CD9 monoclonal antibodies (MoAbs) doubled the extent of collagen gel contraction mediated by VSMCs, a model for the reorganization of the extracellular collagen matrix occurring in the vessel wall. Anti-CD29 MoAbs inhibited gel contraction, but anti-CD9 MoAbs counteracted this inhibitory effect of anti-CD29 MoAbs. Transfection of human CD9 into Chinese hamster ovary cells more than doubled the extent of Chinese hamster ovary cell-mediated collagen gel contraction (130% stimulation), confirming a role for CD9 in extracellular matrix reorganization. Thus, CD9 seems to be involved in the modulation of VLA integrin-mediated collagen matrix reorganization by VSMCs. These findings suggest that high CD9 expression is associated with a proliferative state of VSMCs. The role of CD9 could be to modulate the function of VLA integrins on the surface of VSMCs.

AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer.

Members of the recently recognized SRC-1 family of transcriptional coactivators interact with steroid hormone receptors to enhance ligand-dependent transcription. AIB1, a member of the SRC-1 family, was cloned during a search on the long arm of chromosome 20 for genes whose expression and copy number were elevated in human breast cancers. AIB1 amplification and overexpression were observed in four of five estrogen receptor-positive breast and ovarian cancer cell lines. Subsequent evaluation of 105 unselected specimens of primary breast cancer found AIB1 amplification in approximately 10 percent and high expression in 64 percent of the primary tumors analyzed. AIB1 protein interacted with estrogen receptors in a ligand-dependent fashion, and transfection of AIB1 resulted in enhancement of estrogen-dependent transcription. These observations identify AIB1 as a nuclear receptor coactivator whose altered expression may contribute to development of steroid-dependent cancers.

Gene cloning of rat and mouse platelet glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage.

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (approximately 1,700 nucleotides) entirely contained in one exon and a single intron (approximately 900 nucleotides) in the 5' untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8-amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2-terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2-terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2-terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.

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

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 alpha-granule membranes. These two antigens also colocalized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from alpha-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.

CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus.

Canine distemper virus (CDV), a lymphotropic and neurotropic negative-stranded RNA virus of the Morbillivirus genus, causes a life-threatening disease in several carnivores, including domestic dogs. To identify the cellular receptor(s) involved in the uptake of CDV by susceptible cells, we isolated a monoclonal antibody (MAb K41) which binds to the cell surface and inhibits the CDV infection of several cell lines from various species. Pretreatment of cells with MAb K41 reduces the number of infectious centers and the size of the syncytia. Using affinity chromatography with MAb K41, we purified from HeLa and Vero cell extracts a 26-kDa protein which contained the amino acid sequence TKDEPQRETLK of human CD9, a member of the tetraspan transmembrane or transmembrane 4 superfamily of cell surface proteins. Transfection of NIH 3T3 or MDBK cells with a CD9 expression plasmid rendered these cells permissive for viral infection and raised virus production by a factor of 10 to 100. The mechanism involved is still unclear, since we were unable to detect direct binding of CDV to CD9 by using immunoprecipitation and a virus overlay protein binding assay. These findings indicate that human CD9 and its homologs in other species are necessary factors for the uptake of CDV by target cells, the formation of syncytia, and the production of progeny virus.

CD63/Pltgp40: a platelet activation antigen identical to the stage-specific, melanoma-associated antigen ME491.

CD63 is a 53-Kd lysosomal membrane glycoprotein that has been identified as a platelet activation molecule. The current study presents evidence that CD63 and Pltgp40, a platelet membrane glycoprotein identified in this laboratory, are the same molecule and that CD63/Pltgp40 is identical to the well-characterized, stage-specific melanoma-associated antigen ME491. Identity of CD63 and Pltgp40 was demonstrated by immunoprecipitation and sequential immunodepletion studies, which showed that the anti-Pltgp40 monoclonal antibody (MoAb) H5C6 and an anti-CD63 MoAb CLB/Gran 12 recognized the same 40- to 55-Kd platelet glycoprotein. In addition, the anti-CD63 MoAb specifically recognized immunoaffinity-purified Pltgp40. Amino acid sequences obtained from NH2-terminal and tryptic fragment peptides of Pltgp40 were used to generate complementary DNA (cDNA) probes using the polymerase chain reaction (PCR) technique. A 386-bp cDNA probe partially encoding CD63/Pltgp40 and a full length cDNA probe were 100% identical to the corresponding sequence of ME491. Antibodies H5C6 and CLB/Gran12 immunoprecipitated a 30- to 60-Kd heterodisperse glycoprotein from G361 melanoma cells, as had previously been reported for antibodies recognizing ME491. These data, taken together with the extensive homology recently reported between ME491, the Schistosoma mansoni membrane antigen SM23, CD37, the tumor-associated antigen CO-029, and the target of an antiproliferative antibody-1, suggest that CD63 is a member of a new family of related molecules.

Characterization of a novel self-associating Mr 40,000 platelet glycoprotein.

A novel platelet glycoprotein has been purified and characterized. This glycoprotein, designated Pltgp40, is an acidic sialylated 40,000-dalton protein that bears both O-linked and N-linked oligosaccharides. Treatment of Pltgp40 with neuraminidase resulted in a 5,000-dalton reduction in its Mr and a 1.5 Unit alkaline shift in the isoelectric point, indicating the presence of a large number of sialic acid residues. A similar size reduction and change in pl were observed after treatment of Pltgp40 with O-glycanase showing that sialic acids are present on O-linked oligosaccharides. Digestion of Pltgp40 with N-glycanase reduced the Mr to approximately 20,000 daltons but did not affect the isoelectric point, suggesting that Pltgp40 contains six to seven nonsialylated N-linked carbohydrate chains. High Mr proteins were observed in affinity purified Pltgp40 and were identified as detergent-stable protein oligomers consisting of multiple 40,000-dalton monomers. Immunodepletion and direct binding studies indicated that Pltgp40 was not equivalent to Ig Fc receptor type II, another 40,000-dalton glycoprotein expressed on platelets. However, Pltgp40 copurified with Fc receptor type II when platelet extracts were loaded onto human IgG affinity columns, raising the possibility that Pltgp40 may associate with Fc receptors or Fc receptor-lg complexes. Amino acid sequence analysis of the N-terminus of Pltgp40 was performed and confirmed that Pltgp40 is a novel platelet glycoprotein. Epitopes on Pltgp40 appear to be widely expressed because monoclonal antibodies against Pltgp40 also reacted with a variety of myeloid, lymphoid, and epithelial cells. Pltgp40 was detected on activated but not resting platelets, indicating that Pltgp40 is a platelet activation marker.