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PubMed1984

Direct evidence for the transmembrane orientation of the hepatic glycoprotein receptors.

The technique of vectorial labeling has been used to study the orientation of the rat and chicken receptors for asialo- and agalactoglycoproteins in hepatocyte membranes. The membrane-impermeant enzyme lactoperoxidase was used to radioiodinate the outer surfaces of intact cells and endocytic vesicles, as well as both sides of total microsomal membranes. Proteolytically and chemically produced fragments of the receptor polypeptides were analyzed to identify the tyrosine residues modified in each case. The results reveal that each of these receptors is a transmembrane glycoprotein arranged with its NH2 terminus facing the cytoplasm and its COOH terminus, containing the carbohydrate-binding site, exposed at the cell surface. While the primary structures of the chicken and rat receptors are highly homologous in the extracellular portions of the proteins, the cytoplasmic domains show no sequence similarity.

Animals↗
PubMed1983

Platelet membrane glycoprotein I: structure and function. The domain of glycoprotein I involved in the von Willebrand receptor.

The basic structure of platelet membrane glycoprotein I (GPI) and its relation to glycocalicin are now well understood. Glycocalicin is a proteolytic fragment produced by the action of an endogenous Ca2+ activated protease. GPI consists of two glycopeptides, an alpha and a beta chain connected by a disulphide bridge. Glycocalicin is the major part of the GPI alpha chain and can be split by trypsin into a heavily glycosylated trypsin-resistant fragment and a peptide containing at least one intramolecular disulphide bridge and a thrombin binding site. Both the alpha and the beta chains of GPI show hydrophobic properties and are probably integral membrane proteins. The position of the von Willebrand factor binding site within the GPI molecule is still controversial but the bulk of the evidence points to it lying within the non-glycosylated part of the glycocalicin fragment. It is however evident that the GPI beta chain may influence the GPI alpha chain in maintaining the correct conformation of the binding site. The von Willebrand factor binding site and the thrombin binding site appear to be independent but may nevertheless influence one another.

Binding Sites↗
PubMed1983

Molecular defects of platelets in Bernard-Soulier syndrome.

In 1969, it was shown that platelets of patients with Bernard-Soulier (B-S) syndrome exhibited an altered electrophoretic mobility arising from a surface membrane sialic acid deficiency. Subsequent studies showed that a major membrane glycoprotein, GP Ib, was either deleted, reduced in concentration, or structurally modified in B-S platelets. Controversy persists, however, as to the specificity of the defect in platelets of different patients, especially with regard to studies performed on patients in different laboratories. We have now studied platelets from six patients, and during these studies have analyzed the platelet proteins and glycoproteins by single and two-dimensional SDS-PAGE or by crossed immunoelectrophoresis. The surface proteins of the platelets of different patients have been radiolabelled with 3H or 125I. Our studies point to the deletion or severe reduction in concentration of GPIb alpha and Ib beta in the platelets of five patients, detectable but reduced levels of Ib were present in the platelets of the other patient. Studies using 3H-labelled glycoproteins suggest that mol.wt. 82,000 (GP V) and mol.wt. 17,000 glycoproteins are also missing or abnormal. The relationship between these additional defects and the major GP Ib lesion remains to be determined. It should be emphasized that these are membrane abnormalities, no alpha-granule or cyto-plasmic protein deficiencies have been located in B-S platelets.

Blood Coagulation Disorders↗
PubMed1982

Phosphorylation of a membrane receptor for glycoproteins. Possible transmembrane orientation of the chicken hepatic lectin.

The chicken hepatic lectin, a receptor for partially deglycosylated serum glycoproteins, has been identified as a phosphoprotein. Phosphorylation was detected by incorporation of 32P into the protein in cultured hepatocytes and by two-dimensional gel analysis of protein purified from liver tissue. In addition, forms of the receptor containing one, two, and three sialic acid residues have been detected, with the disialylated form predominating. The site of phosphorylation has been identified as Ser7 in the complete amino acid sequence of the receptor (Drickamer, K. (1981) J. Biol. Chem. 256, 5827-5839). The presence of a protein kinase target site near the NH2-terminal of the receptor, a stretch of 25 uncharged, hydrophobic residues in positions 24 through 48, and a site of glycosylation at position 67 suggests that the chicken hepatic lectin is probably a transmembrane protein, oriented with COOH-terminal outside the cell and NH2-terminal in the cytoplasm.

Amino Acid Sequence↗
PubMed1984

Purification of a major sialoglycoprotein (SGP140) on P12/Ichikawa cells and its expression on differentiated HL-60 cells.

SGP140 glycoprotein, a major cell surface sialoglycoprotein with an apparent m.w. of 140,000, was detected on the human T lymphoblastoid cell line P12/Ichikawa by labeling with periodate-tritiated sodium borohydride, followed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Then SGP140 was purified from P12/Ichikawa cells for study of its biochemical character and its distribution in various cell lines. The purification was performed by 0.2% Triton X-100 solubilization from crude membranes, DEAE-Sephacel column chromatography, WGA-agarose column chromatography, Blue-Sepharose 6MB column chromatography, and Sephadex G-150 gel filtration. Antiserum raised against SGP140 was then prepared, and immunoprecipitation and membrane immunofluorescence assay were performed on various cell lines. SGP140 was detected on P12/Ichikawa, Raji, P3HR-1, Daudi, Namalva, BALL-1, MOLT-4B, TALL-1, NALL-1, and K562 cells, but was not detected on HL-60 cells. When HL-60 cells were treated with dimethyl sulfoxide, retinoic acid, or 12-O-tetradecanoylphorbol-13-acetate, SGP140 was detected on cell surfaces. We discuss the possibility that SGP140 may be a differentiation antigen.

Animals↗
PubMed1984

Effects of iron loading and bacillus Calmette-Guerin on a glycoprotein recognition system on rat hepatic sinusoidal cells.

Experiments were performed to determine the effects of agents that modify Kupffer cells on the mannose-N-acetylglucosamine-glycoprotein receptor on hepatic sinusoidal cells. Cells were prepared by collagenase liver perfusion, centrifugation on Percoll gradients, and centrifugal elutriation. The uptake of 125I-labeled agalacto-orosomucoid (125I-AGOR), an N-acetylglucosamine-terminated glycoprotein, was greatest (53% of total uptake) by elutriator fractions containing equal proportions of endothelial and Kupffer cells ("mixed cell" fraction). Uptake was specific and time and concentration dependent. The apparent Km (0.4 mumol/L) and the patterns of inhibition by monosaccharides were similar in all the elutriator fractions, suggesting that only one class of receptor was present. The highest apparent maximal velocity (18 pmol/hr/5 X 10(6) cells) was found in the mixed cell fraction, indicating this fraction contained the highest proportion of receptor-bearing cells. Latex (0.8 micron) and bacillus Calmette-Guerin pretreatments did not influence the hepatic uptake of the glycoprotein in vivo. Iron sorbitol significantly reduced hepatic glycoprotein uptake and caused a twofold increase in the proportion of the ligand remaining in the circulation. Uptake of 125I-agalacto-orosomucoid by cells from latex-treated rats was similar to controls, but uptake by bacillus Calmette-Guerin-treated rat cells was only 25% of control uptake. This was related to a marked increase in sinusoidal cell number caused by bacillus Calmette-Guerin. In contrast, iron sorbitol caused a selective suppression of 125I-agalacto-orosomucoid uptake (10% of control uptake) by cells in the mixed cell fraction. This study showed that maximal uptake of 125I-agalacto-orosomucoid was by elutriator fractions containing equal proportions of endothelial and Kupffer cells and that iron sorbitol suppressed ligand uptake by these cells, possibly by influencing the mannose-N-acetylglucosamine receptor on Kupffer cells.

Animals↗
PubMed2026

Inherited Platelet GPIV Deficiency: First Description of a Series of Unrelated Patients with Bleeding Diathesis.

GPIV (CD36) is a multifunctional membrane protein expressed on various cells, including platelets, where it plays a role in adhesion and activation through the interaction with its ligands, including collagen types I and III and thrombospondin 1. Inherited GPIV deficiency, historically recognized in anti-Naka alloimmunized East Asian donors, is considered asymptomatic and associated with normal platelet aggregation, although impaired adhesion under high-flow conditions has been reported. Here, we reconsider the molecular basis, epidemiology and functional consequences of GPIV deficiency and report four unrelated patients in whom heterozygous CD36 variants are associated with markedly reduced platelet GPIV expression and a clinically relevant mucocutaneous bleeding diathesis. Patients suffered lifelong bleeding symptoms despite normal light-transmission aggregometry and platelet granule content and release and displayed decreased GPIV expression. Three of them showed slightly decreased VWF. Platelet adhesion to Type I collagen was reduced at high shear. These cases suggest for the first time an association between CD36 gene variants and bleeding and underscore the importance of including GPIV in the diagnostic workup of inherited platelet disorders, particularly when conventional assays do not reveal abnormalities.

Humans↗
PubMed1982

Further studies on the interaction between thrombin and GP Ib using crossed immunoelectrophoresis. Effect of thrombin inhibitors.

The platelet surface protein GP Ib (glycocalicin-related protein) has been shown to be retarded by thrombin-Sepharose 4B in a crossed immunoelectrophoresis system. The interaction between GP Ib and thrombin was abolished when thrombin was blocked either at the active serine site with tosyl-lysine-chloromethyl-ketone (TLCK) or phenylmethylsulfonylfluoride (PMSF) or at the fibrinogen binding site (macromolecular binding site) with N-bromosuccinimide (NBS) or heparin, indicating that both sites have to be freely accessible for the retention of the glycocalicin-related protein by thrombin.

Animals↗
PubMed1984

Structural studies on the O-linked carbohydrate chains of human platelet glycocalicin.

Glycocalicin (140 kDa), constituting the main part of glycoprotein Ib (160 kDa), was released from the human platelet membrane by the action of a Ca2+-dependent protease, present in the platelet cytoplasm and liberated during sonication of the platelet suspension. After activation of the protease by Ca2+, the sonicated platelet suspension was subjected to differential centrifugation. The supernatant was applied to a column of wheat germ agglutinin linked to Sepharose 4B; glycocalicin was eluted from the column with 2.5% (w/v) N-acetylglucosamine. Glycocalicin was found to contain 40% carbohydrate by weight, representing N- as well as O-glycosidically linked carbohydrate chains. The O-glycosidic chains were split off by alkaline cleavage in the presence of 3H-labelled NaBH4. The liberated 3H-labelled oligosaccharide-alditols were fractionated on a DEAE-Sephadex A-25 column. The structures of the oligosaccharide-alditols were investigated by 500-MHz 1H-NMR spectroscopy. The major compound was identified as NeuAc alpha(2----3)Ga1 beta(1----3)[NeuAc alpha(2----3)Ga1 beta(1----4)GlcNAc beta(1----6)]GalNAc-ol. Two minor compounds were found to be NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol and NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol.

Blood Platelets↗
PubMed1983

The carbohydrate moiety of human platelet glycocalicin.

Glycocalicin, a predominant glycoprotein on the human platelet surface, has been purified from a platelet suspension by sonication, ammonium sulfate precipitation and acid treatment followed by chromatography on columns of wheat germ agglutinin-Sepharose and Sephacryl S-300. Ser/Thr-linked (O-linked) oligosaccharides were released by alkaline borohydride treatment, and fractionated by high performance liquid chromatography with an anion-exchange resin. The structure of a major oligosaccharide alditol separated by high performance liquid chromatography was investigated by a combination of compositional analyses, methylation and glycosidase treatments, and proposed to be a hexasaccharide alditol, NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-6(NeuAc alpha 2-3Gal beta 1-3) N-acetylgalactosaminitol. We also found some sugar units which appeared to be intermediates in the biosynthetic pathway of the major hexasaccharide.

Blood Platelets↗
PubMed1992

Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane.

Platelet glycoprotein (GP) V is a Mr 82,000 plasma membrane protein of unknown function that is cleaved by the potent platelet agonist, thrombin, to yield a Mr 69,500 fragment (GPVf1). Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor. Association between GPV and GPIb-IX has been suggested by the finding that both proteins are deficient in the Bernard-Soulier syndrome, a bleeding disorder characterized by giant platelets and defective interaction with von Willebrand factor. Here we report that GPV and GPIb-IX are coprecipitated by monoclonal antibodies (mAbs) against GPV, GPIb, or GPIX when platelets are solubilized in the mild detergent, digitonin. Treatment of digitonin immunopreciptates with the nonionic detergent, Nonidet P-40, released GPV from anti-GPIb and anti-GPIX mAb precipitates and GPIb-IX from the anti-GPV mAb precipitate. Removal of the Mr 45,000 amino-terminal part of GPIb alpha by treatment with elastase did not abrogate association of GPV with GPIb-IX, showing that the leucine-rich repeat sequences in GPIb alpha are not required for complex formation. Binding studies with 125I-labeled mAbs showed the presence of 24,370 GPIb-IX complexes and 11,170 molecules of GPV/platelet (n = 5). These data show that the leucine-rich glycoproteins GPV and GPIb-IX form a noncovalent complex in the platelet membrane. GPV may play a role in the interaction of platelets with von Willebrand factor.

Animals↗
PubMed1991

Characterization of the binding domains on platelet glycoproteins Ib-IX and IIb/IIIa complexes for the quinine/quinidine-dependent antibodies.

Sera of 12 patients with quinine/quinidine-induced thrombocytopenia showed drug-dependent antibody binding to glycoprotein (GP) Ib-IX complex. The reaction with GPIb-IX complex of 11 of these 12 sera was strongly inhibited by the complex-specific monoclonal antibodies (MoAbs) AK1 and SZ1. The exception was a quinine-induced serum designated BU. The reaction of the six quinidine-induced sera was also partially blocked by an anti-GPIX MoAb, FMC25. Only 3 of the 12 patient sera showed drug-dependent antibody binding to GPIIb/IIIa, which was strongly inhibited by the anti-GPIIIa MoAb 22C4, and the anti-GPIIb alpha MoAb SZ22. With detergent-solubilized Serratia metalloprotease-treated platelets, quinine/quinidine-induced sera, except BU, immunoprecipitated a membrane-bound proteolytic fragment of GPIb-IX complex. In contrast, BU immunoprecipitated glycocalicin and a 40-Kd peptide tail fragment of GPIb alpha from the cell supernatant. Using purified GPIb-IX complex or its components as the target antigen, all the quinine-induced sera, except BU, immunoprecipitated GPIb-IX complex but failed to immunoprecipitate GPIb, GPIX, or the complex reformed from GPIb and GPIX. The quinidine-induced sera strongly immunoprecipitated purified GPIb-IX complex, weakly immunoprecipitated purified GPIX and the recombined complex, but did not immunoprecipitate purified GPIb. The combined data suggest that one quinine-dependent antibody (BU) recognizes an epitope in the peptide tail region of GPIb alpha and the other five quinine-dependent antibodies react with a complex-specific epitope on the membrane-associated region of GPIb-IX complex, whereas each of the six quinidine-induced sera contain two drug-dependent antibodies, one reactive with the GPIb-IX complex-specific epitope and the other reactive with GPIX. The binding domain(s) on GPIIb/IIIa for the quinine/quinidine-dependent antibodies appear to be sterically close to the epitopes for 22C4 and SZ22.

Antibodies↗