Biomedical subjects

N Hanai

Publications and source records attributed to N Hanai.

At least 55 records · Page 3Linked to original sources

PubMed1996

Dramatically different phenotypes in mouse models of human Tay-Sachs and Sandhoff diseases.

We have generated mouse models of human Tay-Sachs and Sandhoff diseases by targeted disruption of the Hexa (alpha subunit) or Hexb (beta subunit) genes, respectively, encoding lysosomal beta-hexosaminidase A (structure, alpha) and B (structure, beta beta). Both mutant mice accumulate GM2 ganglioside in brain, much more so in Hexb -/- mice, and the latter also accumulate glycolipid GA2. Hexa -/- mice suffer no obvious behavioral or neurological deficit, while Hexb -/- mice develop a fatal neurodegenerative disease, with spasticity, muscle weakness, rigidity, tremor and ataxia. The Hexb -/- but not the Hexa -/- mice have massive depletion of spinal cord axons as an apparent consequence of neuronal storage of GM2. We propose that Hexa -/- mice escape disease through partial catabolism of accumulated GM2 via GA2 (asialo-GM2) through the combined action of sialidase and beta-hexosaminidase B.

Animals↗

PubMed1996

Characterization of cyclophilin 40: highly conserved protein that directly associates with Hsp90.

Cyclophilin 40 (CyP4O) is a recently identified member of the cyclophilin family that may be a component of unactivated steroid receptor complexes. It consists of an N-half portion that is highly homologous to cyclophilin A and has peptidyl prolyl isomerase (PPIase) activity, and a C-half portion that resembles the C-terminal portion of FKBP52 (FK506 binding protein 52), another component of unactivated steroid receptor complexes. To better understand the structure and functional characteristics of this new class of cyclophilin, we have raised monoclonal antibodies against the C-half portion of human CyP4O. Immunostaining with the antibodies showed its preferential localization in cytoplasm. One antibody cross-reacted with a 45 kDa protein in yeast, suggesting high conservation throughout evolution. A CyP4O-associated protein was isolated from rabbit reticulocyte lysate by means of an affinity resin, and was identified as hsp90. The C-half portion of CyP4O was necessary and sufficient for the interaction.

Amino Acid Isomerases↗

PubMed1995

Association between sialyl Lewis(a) expression and tumor progression in melanoma.

Twenty-three primary and 27 metastatic melanoma lesions and 17 pigmented nevi lesions were tested utilizing the immunoperoxidase reaction with anti-sialyl Lewis(a) (sLea) and anti-sLex mAbs.sLea was expressed in 9, 25, and 5 and sLex was expressed in 6, 11, and 2 of these lesions, respectively. Expression of sLea in melanocytic tumors is associated with tumor progression. Serum levels of sLea and sLex were analyzed by a sandwich assay using mAbs in 25 melanoma patients. Only 2 patients at stage 4 showed higher levels of sLea and sLex than did normal control subjects. Moreover, sLea and sLex were expressed in 1 and 2 of 5 human melanoma cell lines, respectively, and expression of sLex and sLex was not modulated by cytokines. These findings suggest that the expression of sLea in melanocytic tumors is correlated with disease progression.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed1995

Tumor-associated glycoantigen, sialyl Lewis(a) as a target for bispecific antibody-directed adoptive tumor immunotherapy.

The KM231 mAb recognizing sialyl Lewis(a) (sLe(a)) epitope of glycoprotein or glycolipid expressed on various human cancers was used to prepare bispecific antibody (BSAb) containing anti-CD3 x anti-sLe(a) mAb. The effect of anti-CD3 x anti-sLe(a) BSAb on the induction of cytotoxicity by activated T cells was investigated. The activated CD3+ T cells expressing CD8 or CD4 were induced from human peripheral blood mononuclear cells by culture with recombinant IL-2 plus immobilized anti-CD3 mAb. The activated CD8+ and CD4+ T cells showed marginal cytotoxicity against tumor cells by themselves. However, addition of anti-CD3 x anti-sLe(a) BSAb resulted in a great augmentation of their cytotoxicity against gastrointestinal tumor cells. The BSAb also triggered IL-2 production of CD4+ helper/killer T cells during lysis of tumor cells. Moreover, the BSAb was demonstrated to have a potent in vivo antitumor activity against human colon cancer implanted in nude mice by combination with CD4+ helper/killer cells. These results demonstrated that sLe(a) antigen might be a good target molecule for BSAb-directed adoptive tumor immunotherapy.

Animals↗

PubMed1995

Antibodies to polysialic acid and its N-propyl derivative: binding properties and interaction with human embryonal brain glycopeptides.

There is no efficient vaccine against group B meningococcal meningitis because of tolerance induced by host tissue polysialic acid cross-reacting with the capsular polysaccharide. The specificities of polysialic acid-antibody interactions were studied using a ligand binding assay. Antibodies 735, 20-1, 2-1B, 2-2B, 5E1, and t5E1 and antibodies against N-propionylated group B meningococcal polysaccharide-tetanus toxoid conjugate (NP-4, 106-6) bound polysialylated human embryonal brain glycopeptides but not control glycopeptides or disialosyllactose, whereas antibodies 109-3 and I-627 were more specific for the N-propionylated polysaccharide. Antiganglioside antibodies (KM538, KM641) did not cross-react with polysialic acid. Human class-switched antibodies 5E1 (IgM) and t5E1 (IgG) reacted identically with all compounds tested and no temperature-dependent differences were observed. All anti-polysialosyl antibodies required a polysaccharide chain of 8-10 residues for binding independent of the immunizing antigen, animal species, or immunoglobulin class. The results suggest careful evaluation of polysialic acid cross-reactivity in vaccine development.

Antibodies, Monoclonal↗

PubMed1994

Induction of cholinergic differentiation with neurite sprouting by de novo biosynthesis and expression of GD3 and b-series gangliosides in Neuro2a cells.

The expression of a single glycosyltransferase, GD3 synthase, caused cholinergic differentiation with neurite sprouting. The cells that expressed GD3 were established from Neuro2a cells by transfection of a mammalian expression vector into which were carried a cDNA encoding GD3 synthase and the blasticidin-S-deaminase gene with a SV40 promoter, followed by selection with blasticidin-S-hydrochloride. The blasticidin-S-hydrochloride-resistant colonies derived from the cells transfected with the cDNA encoding GD3 synthase and the clonal cells (N2a-GD3) were spontaneously sprouting neurites but not those derived from cells transfected with only the vector without the cDNA encoding GD3 synthase (N2a-bsr). GD3 expression by N2a-GD3 was confirmed by immunostaining of the cells using the anti-GD3 monoclonal antibody, KM643. N2a-GD3 expressed not only GD3 but also GQ1b, one of the b-series gangliosides, whereas N2a-bsr did not express these gangliosides. Cell proliferation of N2a-GD3 was greatly reduced, as compared with that of N2a-bsr, and, after several passages, it completely stopped. In addition, N2a-GD3 expressed acetylcholine esterase, indicating that the differentiation of Neuro2a cells was induced by expression of GD3 synthase and subsequent modification of the biosynthesis and expression of gangliosides. These results strongly suggest that the de novo synthesis and expression of GD3 and/or b-series gangliosides induce neurite outgrowth and differentiation of Neuro2a cells. Exogenous GM1 stimulated the neuritogenesis of N2a-bsr but not differentiated N2a-GD3, indicating that the mechanism of neurite sprouting in this system may be overlapped en route with that of exogenous GM1.

Acetylcholinesterase↗

PubMed1994

Monoclonal antibodies distinctively recognizing the subtypes of inositol 1,4,5-trisphosphate receptor: application to the studies on inflammatory cells.

Monoclonal antibodies were raised that specifically recognize the COOH-terminal sequences and the loop sequences between the fifth and the sixth transmembrane spanning regions of human inositol 1,4,5-trisphosphate receptor (IP3R) type 1, 2 and 3. Western blot analysis using Jurkat cells, mouse cerebellum, COS-7 expressing IP3R type 3 cDNA showed that those monoclonal antibodies reacted specifically with each of these three IP3R subtypes and that they do not cross-react. These antibodies could be used for the specific immunoprecipitation of IP3Rs. Using these monoclonal antibodies, the expression profiles of IP3R-subtype proteins were found to be different among inflammatory cells such as macrophages, polymorphonuclear cells, mast cells, eosinophils, splenocytes, thymocytes and megakaryocytic cells. Usually, more than one type of IP3R were expressed in a cell simultaneously. The observation of CMK cells under immunofluorescence confocal microscopy revealed that IP3R type 1 and type 2 are located at different subcellular fractions.

Amino Acid Sequence↗

PubMed1994

Role of O-linked carbohydrate chains on leukocyte cell membranes in platelet-induced leukocyte activation.

We previously reported that activated platelets stimulated neutrophils and monocytes to produce superoxide anion (O2-) through the interaction between P-selectin and its carbohydrate ligand, sialyl Lewis X (sLeX) (Nagata, K., Tsuji, T., Todoroki, N., Katagiri, Y., Tanoue, K., Yamazaki, H., Hanai N., and Irimura, T. (1993) J. Immunol. 151, 3267-3273). In the present study, we investigated the role of cell surface carbohydrate chains of leukocytes in this process. Glycoconjugate-specific hydrolytic enzymes and inhibitors of glycosylation processing were applied. Granulocyte-like differentiated HL-60 (gHL-60) cells released an increased amount of O2- in response to activated platelets in a P-selectin-dependent manner. When HL-60 cells were differentiated in the presence of benzyl-alpha-N-acetylgalactosaminide (Bzl-alpha-GalNAc), an inhibitor of chain elongation of O-linked carbohydrates, the enhanced generation of O2- was abrogated in parallel with decrease in the expression of sLex structure and in the adhesion capacity to activated platelets. In contrast, treatment with swainsonine or 1-deoxymannojirimycin, inhibitors of processing of N-linked carbohydrate chains, did not show such effects. O-Sialoglycoprotease treatment of gHL-60 cells decreased the activated platelet-induced O2- production with concomitant reduction of cell surface sLex expression. Treatment of these cells with N-glycanase did not affect the O2- production. These results strongly suggested that serine/threonine-linked carbohydrate chains containing sLex played an essential role in the P-selectin-mediated leukocyte activation. By Western blotting analysis of gHL-60 cell lysates, we identified two glycoproteins which carried sLex structures and were sensitive to Bzl-alpha-GalNAc treatment.

Blood Platelets↗

PubMed1994

Expression cloning of a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase).

A cDNA encoding a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 using an anti-GD3 antibody and a fluorescence-activated cell sorter. Selection of B-cell line Namalwa cells expressing transfected cDNAs in the presence of anti-GD3 monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topology as found for mammalian glycosyltransferases. The following evidence confirms that the cDNA encodes an alpha-2,8-sialyltransferase, which specifically converts GM3 to GD3. (i) Transfection of pAMo-GD3 into Namalwa KJM-1 cells leads to the appearance of GD3 and a GD3 synthase activity. (ii) Northern blot analysis revealed a correlation between the expression of this gene and GD3 in several cell lines. (iii) The putative COOH-terminal active domain of this cloned enzyme fused with protein A has been purified with IgG-Sepharose beads and has been shown to possess GD3-synthesizing activity, excluding the possibility that the cloned cDNA encodes a transacting factor inducing a GD3 synthase. The deduced primary sequence also contains the "sialyl motif" conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located on chromosome 12.

Amino Acid Sequence↗

PubMed1994

Expression cloning of a novel alpha 1,3-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes.

The sialyl Lewis x (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc-NAc) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium or platelets. In our efforts to identify glycosyltransferases involved in the biosynthesis of those ligands, we achieved expression cloning of a novel human alpha 1,3-fucosyltransferase termed Fuc-TVII from a THP-1 cDNA library by enrichment of the Namalwa cells highly expressing that determinant with a fluorescence-activated cell sorter. Expression of the COOH-terminal catalytic domain of Fuc-TVII showed an alpha 1,3-fucosyltransferase activity for a type II oligosaccharide with a terminal alpha 2,3-linked sialic acid among various acceptors, consistent with that in vivo acceptor specificity. Alignment of the primary sequences of five alpha 1,3-fucosyltransferases and assignment of the chromosomal location of Fuc-TVII gene, together with that acceptor specificity, indicate that Fuc-TVII consists of a unique class of the alpha 1,3-fucosyltransferase family. Determination of the expression levels of these alpha 1,3-fucosyltransferases in various cells revealed that both Fuc-TVII and a myeloid fucosyltransferase Fuc-TIV were significantly expressed in myeloid lineage cells. Fuc-TVII-transfected Namalwa cells exhibited significant binding to E-selectin in contrast to little binding of the Fuc-TIV-transfected cells. These results suggest that Fuc-TVII may participate in the biosynthesis of the selectin ligands.

Antibodies, Monoclonal↗

PubMed1994

Recombinant rat nucleoside diphosphate kinase isoforms (alpha and beta): purification, properties and application to immunological detection of native isoforms in rat tissues.

We previously demonstrated that at least two isoforms of nucleoside diphosphate (NDP) kinase, the products of two different tandemly arrayed genes, are present in rat. To understand the physiological role of each isoform, some biochemical properties of recombinant rat NDP kinase alpha- and beta-isoforms, produced in large amount, were studied. cDNAs of the two isoforms were inserted in an expression vector pET3b and recombinant enzymes were overproduced in Escherichia coli. Their primary structures were different from the native enzymes in that the latter suffer from modification of the NH2-terminal end. The two recombinant isoforms were purified from the cell lysate to apparent homogeneity by ammonium sulfate fractionation followed by three successive column chromatographies. Despite their extreme similarity in the amino-acid sequences, the two showed somewhat different enzymic properties in terms of di- and triphosphate nucleotide substrate specificity. They showed similar mobilities on SDS-PAGE as expected from their calculated molecular weight (alpha-isoform, 17,283 versus beta-isoform, 17,192) but differed in isoelectric point (alpha-isoform, pI 6.7; beta-isoform, pI 7.8) and heat stability. Polyclonal antibody which reacted with both isoforms and alpha-isoform-specific monoclonal antibodies differentially recognized native enzymes from rat tissues after the tissue extracts were separated by isoelectric focusing gel electrophoresis under a denaturation condition. The results showed that the alpha-isoform, though its amount varied from one tissue to another, was the major form in rat tissues examined compared with the beta-isoform which was detectable in brain and testis. There was no preference in their subcellular localization when examined with myelin, synaptosomal supernatant and total homogenate fractions from the rat cerebrum and cerebellum.

Amino Acid Sequence↗

PubMed1994

Chimeric anti-ganglioside GM2 antibody with antitumor activity.

Ganglioside GM2, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin, has been focused on as a target molecule for passive immunotherapy. GM2 is thought to be one of the T-cell-independent antigens and to elicit only IgM antibody responses in rodents and humans. We have previously established two murine anti-GM2 monoclonal antibodies with high specificity and strong binding activity, KM696 and KM697, both of which are of the IgM class. Variable heavy and light chain complementary DNAs of these two murine monoclonal antibodies were cloned and used in the construction of mouse/human IgG1 chimeric antibodies, KM966 and KM967, respectively, in this study. One of the chimeric antibodies, KM966, retained strong and specific reactivity with GM2 and showed the similarity of the binding activity with tumor cell lines to that of the original murine monoclonal antibody. Indirect immunofluorescence staining of tumor cell lines with the chimeric KM966 revealed that the antigen was expressed in substantial amounts on pulmonary tumor cells and leukemia cells as well as neuroectodermal origin tumor cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, respectively, chimeric KM966 was fully effective in killing GM2-expressing tumor cells. In addition, i.v. injection of chimeric KM966 markedly suppressed the establishment of human tumor xenografts in nude mice. Taken together, chimeric KM966 is the first antibody of the human IgG class to ganglioside GM2 and has strong antitumor activity both in vitro and in vivo. It is likely that chimeric KM966 will be a useful agent for passive immunotherapy of human cancer.

Amino Acid Sequence↗

PubMed1994

Immunoglobulin class switch of anti-ganglioside monoclonal antibody from IgM to IgG.

Ganglioside GM2, which is one of the major gangliosides expressed on cell surface of neuroectodermal-origin human tumors, has been focused on as a target molecule for passive immunotherapy. One of the problems in this area was that monoclonal antibodies (mAbs) raised against GM2 were of IgM class even if donors of B cells were varied in mouse, rat or human. We stimulated two kinds of mice hybridomas having membrane-bound anti-GM2 IgM on their surface with GM2 incorporated in synthetic liposomes in the presence of the mouse thymocytes to accelerate the class switch of immunoglobulins (Igs). After the stimulation, protein A-reactive clones were sorted out using a cell sorter. We finally isolated two class switch variants generating mouse IgG3, designated KM796 and KM750, from original hybridomas producing mouse IgM anti-GM2 mAbs, KM696 and KM697, respectively after over 20-time repetitions of the sorting. ELISA with 11 common gangliosides revealed that one of the variant, KM750, retained the same binding specificity to N-acetyl GM2 as that of the parental IgM, KM697. By ELISA using panel of anti-idiotype (Id) mAbs to anti-GM2 mAbs, KM750 was shown to retain the parental KM697 Id. Another variant KM796 almost lost its activity in purification process in acidic condition and changes in Id were suggested. In immunofluorescence assay, KM750 was confirmed to bind to GM2-expressing tumor cell lines. The class switch hybridoma has been stably cultured with the production of the IgG3-class mAb for more than 22 months.

Animals↗

PubMed1994

A new vector for the high level expression of chimeric antibodies in myeloma cells.

We previously reported the expression of a mouse/human chimeric anti-ganglioside GD3 antibody, KM871 (IgG1,kappa) in mouse myeloma SP2/0 cells under the control of the ecotropic Moloney virus long terminal repeat by the co-transfection of chimeric heavy (H) and light (L) chain vectors (Shitara et al. (1993) Cancer Immunol. Immunother.). To establish an efficient and high level expression system for the chimeric antibody, we did comparative study on vector systems and host cells. An improved expression vector, named 'a tandem vector, pChi641HLGM4' was constructed, in which both of the chimeric H and L chain gene transcription units and a dihydrofolate reductase (dhfr) gene transcription unit were inserted. When two kinds of mouse myeloma cell lines, SP2/0 and P3U1, were used as host cells, frequency of the incidence of antibody-producing transfectants was markedly increased by the use of the tandem vector compared with the use of the mixture of each chimeric H vector and L chain vector. To select out appropriate host cells, transfection frequency and antibody production level were compared among SP2/0, P3U1 and rat myeloma YB2/0 cells by transfection of the tandem vector. YB2/0 cell was shown to have the highest potential in both the transfection frequency and the antibody production. Introduction of the tandem vector into YB2/0 cells and the subsequent amplification with 50-200 nM methotrexate gave rise to several clones that stably secreted 70-100 micrograms/10(6) cells per 24 h of the chimeric antibody. This productivity of the antibody is one of the highest levels which have been achieved by other investigators using transfected myeloma cells. Using this system it took only 2-3 months to establish the transfectant clones which stably produced the chimeric antibody.

Animals↗

PubMed1993

Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells.

The surface engagement of high affinity immunoglobulin E receptor (Fc epsilon RI) of rat basophilic leukemia 2H3 (RBL-2H3) cells induced histamine secretion and leukotriene release following activation of the tyrosine kinase Lyn together with phosphatidylinositol 3-kinase (PI3-kinase). Wortmannin inhibited the activity of partially purified PI3-kinase from calf thymus, as well as the PI3-kinase activity in anti-PI3-kinase p85 immunoprecipitates from RBL-2H3 cells, at a concentration as low as 1.0 nM and with IC50 values of 3.0 nM, but did not inhibit PI4-kinase activity. The inhibition of PI3-kinase by wortmannin was irreversible. Wortmannin inhibited both Fc epsilon RI-mediated histamine secretion and leukotriene release up to 80% with IC50 values of 2.0 and 3.0 nM, respectively. Wortmannin inhibited PI3-kinase activity in intact cells up to 80% with an IC50 value of 2.0 nM, which is almost equal to those for PI3-kinase in vitro and for histamine secretion and leukotriene release. With anti-wortmannin antibody, we have shown that wortmannin binds to the 110-kDa protein, but not to PI3-kinase 85-kDa regulatory subunit both in vitro and in whole cells. Furthermore, there was a positive correlation between the potencies of wortmannin derivatives as inhibitors of PI3-kinase and as inhibitors of histamine secretion. Wortmannin had no effect on the activation of the tyrosine kinase Lyn. These results suggest that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion following stimulation of Fc epsilon RI and that wortmannin blocks these responses through direct interaction with the catalytic subunit of this enzyme.

Androstadienes↗

PubMed1993

Expression cloning of a novel Gal beta (1-3/1-4) GlcNAc alpha 2,3-sialyltransferase using lectin resistance selection.

This report describes the isolation of a cDNA encoding a novel human Gal beta (1-3/1-4)GlcNac alpha 2,3-sialyl-transferase involved in the biosynthesis of the sialyl Lewis x determinant (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1-3)GlcNAc). A cDNA library of the human melanoma cell line WM266-4 was constructed in an Epstein-Barr virus-based cloning vector. Selection of the B-cell line Namalwa expressing transfected cDNAs in the presence of the cytotoxic lectin Ricinus communis agglutinin 120 gave a cDNA encoding a protein with type II transmembrane topology, as found for mammalian glycosyltransferases. The use of this lectin, which is specific to galactose residues (especially the Gal beta 1-4GlcNAc structure), originates from our prediction that the modification of the Gal beta 1-4GlcNAc structure (a backbone of the sialyl Lewis x structure) by glycosyltransferases may increase the levels of resistance to this lectin. Comparison of this cDNA sequence with those of three other cloned sialyltransferases revealed two conserved regions shared by all four enzymes. Expression of the COOH-terminal catalytic domain of this protein showed alpha 2,3-sialyltransferase activity with substrate specificity different from that of CMP-N-acetylneuraminate:N-acetyllactosaminide alpha-2,3-sialyltransferase (Gal-beta 1-3(4)GlcNAc alpha 2,3-sialyltransferase, EC 2.4.99.6). Furthermore, expression of this cDNA in Namalwa cells increased the level of sialyl Lewis x antigens. The cloning approach based on lectin resistance may be useful for the isolation of cDNAs encoding other mammalian glycosyltransferases.

Amino Acid Sequence↗

PubMed1993

Expression of sialyl Lewis(x) antigen on human T cells.

Sialyl Lewis(x) (sLe(x)) antigen, which has been introduced as tumor-associated cell surface carbohydrates, was demonstrated to be expressed on a subpopulation of human CD4+ and CD8+ T cells. It was also demonstrated that the majority of gamma delta T cells as well as natural killer cells expressed a high level of sLe(x) antigen. The sLe(x) expression on T cells were up-regulated by activation with various T cell stimulants. The sLe(x)+ CD4+ T cells were enriched in CD4+CD45RO+ memory type of T cells but not in CD4+CD45RA+ T cells. Moreover, sLe(x)+ CD4+ T cells were demonstrated to show higher proliferative responses to T cell stimulants such as CD4+CD45RO+ T cells. These results initially clarified the expression of sLe(x) carbohydrate on human T cells and indicated the important role of sLe(x)-expressing T cells in immune responses.

Antibodies, Monoclonal↗

PubMed1993

Activated platelets induce superoxide anion release by monocytes and neutrophils through P-selectin (CD62).

Activated platelets expressing P-selectin in their surfaces are known to adhere to monocytes and neutrophils. We examined the possibility that the leukocytes are functionally modified by their adhesion to activated platelets. We used human peripheral blood monocytes and neutrophils and measured superoxide anion generation by these cells cultured with platelets. The levels of superoxide anion production were found to be markedly elevated when thrombin-activated platelets were used. The extent of this enhancement was much smaller when leukocytes were cultured with resting platelets than activated platelets. The increase depended on incubation time and platelet concentration. The membranes prepared from activated platelets also induced superoxide anion production, but the culture supernatant of activated platelets did not. The enhanced superoxide anion production was inhibited by anti-P-selectin antibody, anti-sialyl-Lewis X antibody, or a soluble recombinant P-selectin fusion protein. These results indicate that the adhesion of activated platelets to the leukocytes through P-selectin was a crucial step for the activation of leukocyte function, and support the idea that activated platelets are actively involved in inflammation processes.

Base Sequence↗