GMP-140 binding to neutrophils is inhibited by sulfated glycans.

GMP-140 is a 140-kDa granule membrane glycoprotein localized to the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells. Expression of GMP-140 on the activated cell surface has been shown to mediate the adhesion of thrombin-activated platelets to neutrophils and monocytes and the transient adhesion of neutrophils to endothelium. In contrast, fluid-phase GMP-140 strongly inhibits the CD18-dependent adhesion of tumor necrosis factor alpha-activated neutrophils to endothelium suggesting that GMP-140 can also serve an anti-adhesive function. In the present report, it is demonstrated that fluid-phase GMP-140 which exists predominantly as a tetramer binds to a single class of high affinity receptor on neutrophils and HL60 cells. Binding of 125I-labeled GMP-140 to neutrophils and HL60 cells and the rosetting of neutrophils and HL60 cells by thrombin-activated platelets were inhibited by EDTA, excess unlabeled fluid-phase GMP-140, Fab fragments of an affinity-purified rabbit anti-GMP-140 antibody, and by the murine anti-GMP-140 monoclonal antibody, AK 4. Both neutrophil and HL60 GMP-140 binding and platelet rosetting were strongly inhibited by heparin, fucoidin, and dextran sulfate 500,000, were partially inhibited by dextran sulfate 5,000 and lambda- and kappa-carrageenan, but were not inhibited by chondroitins 4- and 6-sulfate. Since this sulfated glycan specificity is identical to that previously reported by us for GMP-140, the present results suggest that the sulfated glycan binding site and the neutrophil receptor binding site on GMP-140 are either identical or proximal.     

GMP-140 binds to a glycoprotein receptor on human neutrophils: evidence for a lectin-like interaction

GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism.     

The selectin GMP-140 binds to sialylated, fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells

Granule membrane protein-140 (GMP-140) is an inducible receptor for myeloid leukocytes on activated platelets and endothelium. Like other selectins, GMP-140 recognizes specific oligosaccharide ligands. However, prior data on the nature of these ligands are contradictory. We investigated the structural features required for ligand interaction with GMP-140 using purified GMP-140, cells naturally expressing specific oligosaccharides, and cells expressing cloned glycosyltransferases. Like the related selectin endothelial leukocyte adhesion molecule-1 (ELAM-1), GMP-140 recognizes alpha(2-3)sialylated, alpha(1-3)fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells, including the sequence Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNac beta-R (sialyl Lewis x). Recognition requires sialic acid, because cells expressing large amounts of Lewis x, but not sialyl Lewis x, do not interact with GMP-140. Although sialyl Lewis x is expressed by both myeloid HL-60 cells and CHO cells transfected with an alpha 1-3/4 fucosyltransferase, GMP-140 binds with significantly higher affinity to HL-60 cells. Thus, the sialyl Lewis x tetrasaccharide may require additional structural modifications or specific presentations in order for leukocytes in flowing blood to interact rapidly and with high affinity to GMP-140 on activated platelets or endothelium.     

Downregulation of GMP-140 (CD62 or PADGEM) expression on platelets by N,N-dimethyl and N,N,N-trimethyl derivatives of sphingosine.

GMP-140 (CD62 or PADGEM), a member of the selectin family, is a membrane glycoprotein in secretory granules of platelets and endothelial cells. When these cells are activated by agonists such as thrombin or AMP, GMP-140 is rapidly redistributed to the cell surface. The carbohydrate epitope defined by GMP-140 was identified as sialosyl-Le(x) (as for ELAM-1), which may play an essential role in adhesion of leukocytes or tumor cells on endothelial cells, through aggregation with platelets. Redistribution of GMP-140 from alpha-granules of platelets to the cell surface, induced by thrombin and PMA, was strongly inhibited by preincubation of platelets with N,N-dimethylsphingosine (DMS) or N,N,N-trimethylsphingosine (TMS) at 10-20 microM concentration for a brief period (5 min). Inhibition of GMP-140 redistribution to the cell surface by DMS or TMS was also detected by a cell adhesion assay using HL60 cells, which highly express sialosyl-Le(x); i.e., HL60 cells adhered on platelets activated by thrombin or PMA but not on platelets which were briefly preincubated with DMS or TMS followed by activation. The inhibitory effect of DMS or TMS on GMP-140 redistribution is not due to cytotoxicity, since the TMS-treated platelets were fully capable of aggregating in the presence of ristocetin. Sphingosine (SPN) and protein kinase C inhibitors such as H-7 and calphostin C showed weaker inhibitory activity than DMS and TMS. Our results indicate that both DMS and TMS could be useful reagents to inhibit cell surface expression of crucial selectins which promote adhesion of Le(x-) or sialosyl-Le(x)-expressing cells with platelets and endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of GMP-140, an inducible granule membrane protein of platelets and endothelium.

GMP-140 is an integral membrane glycoprotein with an apparent Mr of 140,000 located in secretory granules of human platelets and endothelial cells. When these cells are stimulated, the protein is rapidly redistributed to the plasma membrane; therefore, monoclonal antibodies to GMP-140 are useful markers of activated platelets and endothelium. GMP-140 is cysteine-rich and heavily glycosylated. The cDNA-derived amino acid sequence indicates that it contains a number of modular domains that are likely to fold independently. Beginning at the N-terminus, these comprise a "lectin" domain, an "EGF" domain, nine tandem consensus repeats similar to those in complement-binding proteins, a transmembrane domain, and a cytoplasmic tail. Some cDNAs also predict variant forms of GMP-140, including a putative soluble form lacking the transmembrane domain that appears to arise from alternative splicing of mRNA. The domain organization of GMP-140 is strikingly similar to two other vascular cell surface structures: ELAM-1, a cytokine-inducible endothelial cell receptor that binds neutrophils, and a lymphocyte-homing receptor that mediates the adherence of lymphocytes to high endothelial venules of peripheral lymph nodes. These "selectins" constitute a new gene family of receptors with related structure and potentially related function.     

Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation

GMP-140 is an integral membrane glycoprotein found in secretory granules of platelets and endothelial cells. After cellular activation, it is rapidly redistributed to the plasma membrane. The cDNA-derived primary structure of GMP-140 predicts a cysteine-rich protein with multiple domains, including a "lectin" region, an "EGF" domain, nine tandem consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. Some cDNAs also predict a soluble protein with a deleted transmembrane segment. The domain organization of GMP-140 is similar to that of ELAM-1, a cytokine-inducible endothelial cell receptor that binds neutrophils. This similarity suggests that GMP-140 belongs to a new family of inducible receptors with related structure and function on vascular cells.     

P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities.

P-selectin on platelets and endothelial cells and E-selectin on endothelial cells are leukocyte receptors that recognize lineage-specific carbohydrates on neutrophils and monocytes. The proposed ligands for these receptors contain the Le(x) core and sialic acid. Since other investigators have shown that both E-selectin and P-selectin bind to sialylated Le(x), we evaluated whether E-selectin and P-selectin recognize the same counter-receptor on leukocytes. The interaction of HL60 cells with Chinese hamster ovary (CHO) cells expressing P-selectin or E-selectin was studied. To determine whether a protein component is required in addition to sialyl Le(x) for either P-selectin or E-selectin recognition, HL60 cells or neutrophils were digested with proteases, including chymotrypsin, elastase, proteinase Glu-C, ficin, papain, or thermolysin. Cells treated with these proteases bound E-selectin but not P-selectin. Fucosidase or neuraminidase treatment of HL60 cells markedly decreased binding to both E-selectin- and P-selectin-expressing CHO cells. Growth of HL60 cells in tunicamycin inhibited the ability of these cells to support P-selectin-mediated binding and, to a lesser extent, E-selectin-mediated binding. Purified P-selectin inhibited CHO:P-selectin binding to HL60 cells, but incompletely inhibited CHO:E-selectin binding to HL60 cells. However, purified soluble E-selectin inhibited CHO:P-selectin and CHO:E-selectin binding to HL60 cells equivalently and completely. COS cells, unable to bind to E-selectin or P-selectin, bound E-selectin but not P-selectin upon transfection with alpha-1,3-fucosyltransferase or alpha-1,3/1,4-fucosyltransferase. Similarly, LEC 11 cells expressing sialyl Le(x) bound E-selectin- but not P-selectin-expressing CHO cells. Sambucus nigra lectin, specific for the sialyl-2,6 beta Gal/GalNAc linkage, inhibited P-selectin but not E-selectin binding to HL60 cells. Although sialic acid and Le(x) are components of the P-selectin ligand and the E-selectin ligand, these results indicate that the ligands are related, having overlapping specificities, but are structurally distinct. A protein component containing sialyl Le(x) in proximity to sialyl-2,6 beta Gal structures on the P-selectin ligand may contribute to its specificity for P-selectin.     

Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells

E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150- kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160- kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca(2+)- dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide: N- glycosidase F inhibited cell binding to P- and E-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E- and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N- linked carbohydrates and is common for both endothelial selectins.     

Structural and biosynthetic studies of the granule membrane protein, GMP-140, from human platelets and endothelial cells

GMP-140 is an integral membrane glycoprotein of apparent Mr = 140,000 located in secretory storage granules of platelets and vascular endothelial cells. When these cells are activated, GMP-140 redistributes from the membrane of the granules to the plasma membrane. To gain insight into the potential function of GMP-140, we examined aspects of its structure and biosynthesis. The amino acid composition of platelet GMP-140 revealed elevated numbers of cystinyl (6.1%), prolinyl (7.2%), and tryptophanyl (2.1%) residues. GMP-140 contained 28.8% carbohydrate by weight, distributed among N-acetylneuraminic acid, neutral sugar, and N-acetylglucosamine residues. Enzymatic removal of N-linked oligosaccarides reduced the protein's apparent Mr by more than 50,000. The biosynthesis of GMP-140 in HEL cells, which share biochemical features with megakaryocytes, was studied by pulse-chase labeling with [35S]cysteine followed by immunoprecipitation. HEL cells synthesized a heterogeneous GMP-140 precursor of 98-125 kDa which converted to a mature 140-kDa form within 40-60 min. Removal of high mannose oligosaccarides by endo-beta-N-acetylglucosaminidase H treatment reduced the apparent Mr of the precursor but not the mature protein. Tunicamycin-treated HEL cells synthesized three to four precursors of 80-92 kDa, suggesting the possibility of heterogeneity of GMP-140 at the protein level. Exposure of activated platelets to proteases followed by Western blotting indicated that most of the mass of GMP-140 was located on the extracytoplasmic side of the membrane. Our studies indicate that GMP-140 is a cysteine-rich, heavily glycosylated protein with a large extracytoplasmic domain. These features are compatible with a receptor function for the molecule when it is exposed on the surface of activated platelets and endothelial cells.     

PADGEM-dependent adhesion of platelets to monocytes and neutrophils is mediated by a lineage-specific carbohydrate, LNF III (CD15).

PADGEM (platelet activation-dependent granule-external membrane protein) is a leukocyte receptor of activated platelets that mediates cellular adhesion of platelets to neutrophils and monocytes. To identify the natural ligand on neutrophils and monocytes that interacts with PADGEM, we have evaluated anti-leukocyte antibodies for their ability to block leukocyte-PADGEM binding. Only anti-CD15 antibodies were able to inhibit the binding of neutrophils, monocytes, HL60 cells, and U937 cells to platelets. Anti-CD15 antibodies inhibited the binding of U937 cells to PADGEM-expressing COS cells and to purified PADGEM incorporated into phospholipid vesicles. The CD15 antigen, lacto-N-fucopentaose III (Gal beta 1----4[Fuc alpha 1----3]NAcGlc beta 1----3Gal-beta 1----4Glc), inhibited the interaction of neutrophils or HL60 cells with platelets, whereas lacto-N-fucopentaose I did not; lacto-N-fucopentaose II demonstrated minimal inhibition. Lacto-N-fucopentaose III, and to a lesser extent lacto-N-fucopentaose II, but not lacto-N-fucopentaose I, inhibited the interaction of HL60 cells with COS cells transfected with PADGEM cDNA. CD15, lacto-N-fucopentaose III or Lex, is a component of the PADGEM ligand on neutrophils and monocytes.     

CD62/P-selectin recognition of myeloid and tumor cell sulfatides.

CD62, also called PADGEM protein, GMP-140, or P-selectin, is a granule membrane protein of endothelial cells and platelets that is mobilized to the plasma membrane following exposure to mediators such as thrombin, histamine, complement components, or peroxides. Data presented to date suggest that one ligand of CD62 includes CD15 (Lewis x determinant) and sialic acid. We show here that sulfatides, heterogeneous 3-sulfated galactosyl ceramides, are an apparently unrelated ligand of CD62. Sulfatides are expressed on the plasma membrane of, and are excreted by, granulocytes, and constitute the principal ligand for CD62 on the plasma membrane of some tumor cells. CD62 binds to sulfatides adsorbed to plastic as avidly as it binds to myeloid or tumor cells. We find that granulocytes excrete sulfatides at a rate predicted to allow them to be rapidly released from CD62 once they have exited the bloodstream.     

Two sites (23–30, 76–90) on rat P-selectin mediate thrombin activated platelet-neutrophil interactions

The lectin-like domain of P-selectin, an adhesive receptor (also known as PADGEM, GMP-140 or CD62) is implicated in platelet or endothelial cell interactions with leukocytes. The aim of this study was to characterize the lectin-like domain of rat P-selectin by the use of synthetic peptides. The lectin and EGF-like domains of rat P-selectin were cloned in our laboratory and shown to present very strong homologies to its human counterpart. Peptides corresponding with the lectin-like domain of P-selectin were tested for their ability to inhibit thrombin-activated platelets rosetting to neutrophils. Peptides 23–30 (A) and 76–90 (C), but not peptide 51–61 (B), inhibited thrombin activated rat platelets interactions with rat neutrophils (A = 33%, C = 46%, P < 0.05). Using a combination of peptides (A + B = 35%, P = 0.008 and A + C = 62%, P < 0.001), we observe different degrees of inhibition of platelets binding to neutrophils. The IC₅₀ of peptides A+C was O.llmM. LYP-20, an anti-human P-selectin monoclonal antibody, was also observed to inhibit thrombin-activated rat platelets binding to rat neutrophils in a very significant manner (57% of inhibition, P < 0.001). Moreover, heparin inhibited thrombin-stimulated platelet/neutrophils rosetting (36% of inhibition, P < 0.01). These results show the importance of two sites (23–30 and 76–90) on the lectin-like domain of P-selectin in mediating platelet-neutrophil interactions in rats. Such peptides may be potent in vivo inhibitors of cell-cell interactions involving P-selectin.     

GMP-140: a receptor for neutrophils and monocytes on activated platelets and endothelium.

GMP-140 is a membrane glycoprotein located in secretory granules of platelets and endothelium. When these cells are activated by agonists such as thrombin, GMP-140 is rapidly translocated to the plasma membrane. GMP-140, along with ELAM-1 and the peripheral lymph node homing receptor, defines the selectin family of structurally related molecules that regulate interactions of leukocytes with the blood vessel wall. Each of these molecules contains an N-terminal lectin-like domain, followed by an EGF-like region, a series of consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. The genomic structures of the selectins suggest that they arose by duplication and modification of exons encoding specific structural domains. GMP-140 is a receptor for neutrophils and monocytes when it is expressed on activated platelets and endothelium. This property facilitates rapid adhesion of leukocytes to endothelium at regions of tissue injury as well as platelet-leukocyte interactions at sites of inflammation and hemorrhage. Like other leukocyte adhesion molecules, GMP-140 may also participate in pathologic inflammation, thrombosis, and tumor metastasis. Confirmation of such pathologic roles may lead to design of new drugs that block adhesive receptor function in human disease.     

Inhibition of selectin-dependent tumor cell adhesion to endothelial cells and platelets by blocking O-glycosylation of these cells.

Expression of sialosyl-Le(x) (SLe(x)) and sialosyl-Le(a) (SLe(a)) on tumor cell lines HL60, Colo205, and U937 was greatly suppressed by application of benzyl-alpha-GalNAc for inhibition of O-linked carbohydrate chain extension, which resulted in reduced adhesion of tumor cells to activated endothelial cells or platelets mediated by ELAM-1 (E-selectin) or GMP-140 (P-selectin). Inhibitors or modifiers of N-glycosylation had no effect on expression of SLe(x) or SLe(a) in these tumor cells. These findings suggest the possibility that targeting of O-glycosylation inhibitors or modifiers to tumor cells may effectively suppress metastatic potential.     

PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes

PADGEM (platelet activation dependent granule-external membrane protein) is an integral membrane protein of the alpha granules of platelets and Weibel-Palade bodies of endothelial cells that is expressed on the plasma membrane upon cell activation and granule secretion. Activated platelets, but not resting platelets, bind to neutrophils, monocytes, HL60 cells, and U937 cells. This interaction is inhibited by anti-PADGEM antibodies, PADGEM, and EDTA; anti-GPIIb-IIIa, anti-thrombospondin, anti-GPIV, and thrombospondin produce no effect. Neutrophils and U937 cells, in contrast to Jurkatt cells, contain PADGEM recognition sites, as shown by binding of PADGEM contained in phospholipid vesicles. These results indicate that PADGEM mediates adhesion of activated platelets to monocytes and neutrophils. Therefore, PADGEM shares not only structural but also functional homology with ELAM-1 and MEL-14, members of a new family of vascular cell adhesion molecules.     

Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils

The initial step in extravasation of neutrophils (polymorphonuclear leukocytes [PMNs]) to the extravascular space is adherence to the endothelium. We examined the effect of oxidants on this process by treating human endothelial cells with H2O2, t-butylhydroperoxide, or menadione. This resulted in a surface adhesive for PMN between 1 and 4 h after exposure. The oxidants needed to be present only for a brief period at the initiation of the assay. Adhesion was an endothelial cell-dependent process that did not require an active response from the PMN. The adhesive molecule was not platelet-activating factor, which mediates PMN adherence when endothelial cells are briefly exposed to higher concentrations of H2O2 (Lewis, M. S., R. E. Whatley, P. Cain, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 1988. J. Clin. Invest. 82:2045-2055), nor was it ELAM-1, an adhesive glycoprotein induced by cytokines. Oxidant-induced adhesion did not require protein synthesis, was inhibited by antioxidants, and, when peroxides were the oxidants, was inhibited by intracellular iron chelators. Granule membrane protein-140 (GMP-140) is a membrane-associated glycoprotein that can be translocated from its intracellular storage pool to the surface of endothelial cells where it acts as a ligand for PMN adhesion (Geng, J.-G., M. P. Bevilacqua, K. L. Moore, T. M. McIntyre, S. M. Prescott, J. M. Kim, G. A. Bliss, G. A. Zimmerman, and R. P. McEver. 1990. Nature (Lond). 343:757-760). We found that endothelial cells exposed to oxidants expressed GMP-140 on their surface, and that an mAb against GMP-140 or solubilized GMP-140 completely blocked PMN adherence to oxidant-treated endothelial cells. Thus, exposure of endothelial cells to oxygen radicals induces the prolonged expression of GMP-140 on the cell surface, which results in enhanced PMN adherence.     

Inhibition of P-selectin-mediated cell adhesion by a sulfated derivative of sialic acid

P-selectin, a carbohydrate-binding cell adhesion molecule expressed on activated endothelial cells and platelets, plays a key role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. It simultaneously recognizes a sialic acid-containing carbohydrate chain and the sulfated tyrosine residues of a specific counter-receptor expressed on the leukocyte surface. We examined the inhibitory effects of a synthetic sulfated derivative of sialic acid (NMSO3) on P-selectin-mediated cell adhesion and found the following: (1) P-selectin/IgG chimera bound to immobilized NMSO3. (2) The binding of P-selectin/IgG chimera to purified P-selectin glycoprotein ligand-1 was inhibited by soluble NMSO3. (3) The adhesion of HL60 cells to P-selectin-expressing CHO cells was inhibited by NMSO3. (4) NMSO3 inhibited P-selectin-induced tumor necrosis factor-alpha production in monocytes and activated platelet-induced generation of reactive oxygen species in neutrophils. In conclusion, NMSO3 acts as a specific inhibitor for P-selectin-mediated cell adhesion and for adhesion-dependent leukocyte activation.     

Demonstration that a lectin-like receptor (gp90MEL) directly mediates adhesion of lymphocytes to high endothelial venules of lymph nodes

Lymphocyte migration from the blood into most secondary lymphoid organs is initiated by a highly selective adhesive interaction with the endothelium of specialized blood vessels known as high endothelial venules (HEV). The propensity of lymphocytes to migrate to particular lymphoid organs is known as lymphocyte homing, and the receptors on lymphocytes that dictate interactions with HEV at particular anatomical sites are designated "homing receptors". Based upon antibody blockade experiments and cell-type distribution studies, a prominent candidate for the peripheral lymph node homing receptor in mouse is the approximately 90-kD cell surface glycoprotein (gp90MEL) recognized by the monoclonal antibody MEL-14. Previous work, including sequencing of a cDNA encoding for this molecule, supports the possibility that gp90MEL is a calcium-dependent lectin-like receptor. Here, we show that immunoaffinity-purified gp90MEL interacts in a sugar-inhibitable manner with sites on peripheral lymph node HEV and prevents attachment of lymphocytes. Lymphocyte attachment to HEV in Peyer's patches, a gut-associated lymphoid organ, is not affected by gp90MEL. The results demonstrate that gp90MEL, as a lectin-like receptor, directly bridges lymphocytes to the endothelium.     

Topographic distribution of a granule membrane protein (GMP-140) that is expressed on the platelet surface after activation: an immunogold-surface replica study

Monoclonal and polyclonal antibodies have been developed that recognize a 140 kD glycoprotein on the plasma membrane of activated, but not unstimulated, platelets. This glycoprotein is found in resting platelets as an alpha-granule membrane protein and has therefore been named GMP-140. After thrombin stimulation, alpha-granules fuse with the surface-connected canalicular system and GMP-140 is redistributed to the plasma membrane. In the present study, we immunolabeled unstimulated and activated human platelets and analyzed the distribution of GMP-140 over broad expanses of the plasma membrane using surface replication techniques. Fixed platelets were allowed to settle onto poly-L-lysine-coated coverslips and immunolabeled with polyclonal anti-GMP-140, followed by protein A gold. After critical-point drying, rotary-shadowed surface replicas were made. GMP-140 was not present on the surfaces of unstimulated platelets, but thrombin stimulation resulted in the massive expression of GMP-140 on the cell surface, with the immunogold label monodispersed. In contrast, we recently found that GPIIb-IIIa, the fibrinogen receptor, is monodispersed on unstimulated platelets and clustered on activated platelets. Although GMP-140's hemostatic function is unknown, its monodispersed surface pattern implies significant differences form GPIIb-IIIa with respect to ligand binding and/or cytoskeletal interaction.