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Expression of platelet glycoprotein Ib alpha in HEL cells.
We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.
Fibrin monomer induces binding of endogenous platelet von Willebrand factor to the glycocalicin portion of platelet glycoprotein IB.
This study demonstrates that when platelets are stimulated by thrombin in the presence of low concentrations of purified human fibrinogen (10 to 20 micrograms/mL, final concentration) binding of released platelet von Willebrand factor (plt-vWF) to the platelet membrane is enhanced. This effect appears to be mediated by fibrin monomer produced by the action of thrombin on the fibrinogen in the incubation suspension. When fibrin polymerization is inhibited, the binding of released plt-vWF to the platelets is markedly increased. This enhanced binding is dependent on platelet glycoprotein Ib (GPIb) as shown by a decreased response with Bernard-Soulier platelets and inhibition by both monoclonal and polyclonal antibodies against glycocalicin. The binding of fibrin to thrombin-activated platelets preincubated with monoclonal antibody against GPIIb/IIIa is increased when the predominant form of fibrin is fibrin monomer. The fibrin binding is also decreased in the presence of antibody against glycocalicin. Our data demonstrate that fibrin monomer facilitates plt-vWF binding to the glycocalicin portion of platelet GPIb on thrombin-stimulated platelets and that binding of fibrin monomer to glycocalicin is necessary for this response to occur.
[Blood platelet membrane glycoproteins].
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[Some current topics on platelet membrane glycoproteins].
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The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets.
A soluble radioreceptor assay has been developed to characterize thrombin receptor activities of the human platelet membrane. 125I-Thrombin was added to platelet membranes solubilized in 1% Triton X-100, and thrombin bound to platelet receptors was separated from free thrombin by precipitation with wheat germ agglutinin (WGA) in the presence of alpha 1-acid glycoprotein as carrier. Both high affinity binding (Ki, 0.09 nM; R1, 0.30 pmol/mg protein) and moderate affinity binding (K2, 38 nM; R2, 72 pmol/mg protein) were detected in the detergent-solubilized membrane preparations and these binding parameters were in excellent agreement with values previously determined using intact platelets (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). Using the soluble radioreceptor assay, both high and moderate affinity binding was detected in highly purified preparations of glycoprotein Ib (GPIb) and glycocalicin, and the binding isotherms were identical with those of the crude detergent-solubilized membrane preparation. Treatment of detergent-solubilized membranes with increasing concentrations of a monospecific polyclonal antibody to glycocalicin resulted in the stepwise depletion of GPIb and concomitant reductions of thrombin binding activity. These results demonstrate that both high and moderate affinity binding of thrombin to platelets is completely expressed in the glycocalicin portion of GPIb.
Involvement of membrane sugar receptors and membrane glycoconjugates in the adhesion of 3LL cell subpopulations to cultured pulmonary cells.
Lewis lung carcinoma cells are able to bind sugar residues, mainly alpha-D-glucosyl and alpha-D-mannosyl derivatives as assessed by fluorescent neoglycoproteins binding assay. We have investigated the binding efficiency and shown that: 3LL tumor cells are heterogeneous with regards to their capability to recognize neoglycoproteins, as shown by fluorescence microscopy and flow cytofluorometry analyses; basically two distinct subpopulations could be evidenced which were called glucose-receptor-rich (or glucose-specific lectin-rich, GLR 3LL) and glucose-receptor-poor (or glucose-specific lectin-poor, GLP 3LL) cells; those two subpopulations could be separated on the basis of their binding properties to neoglycoprotein-substituted microcarriers onto which GLR 3LL cells were able to rapidly adhere (2 h) while GLP 3LL cells were not. Some aspects of the biological behavior of these two selected populations were investigated in order to determine the possible involvement of 3LL cell membrane sugar receptors in cell-cell recognition and adhesion to other cells: namely C57 B1/6 mouse pulmonary cells maintained in primary culture. The two 3LL sublines bind to pulmonary cells but their adhesion kinetics were markedly different. Adhesion inhibition studies showed the adhesion process to be dependent upon the specificity of membrane lectins present on both the tumor cell surface (alpha-D-glucose-specific) and on the pulmonary cells (alpha-L-fucose-specific). Surface sugar-specific receptors on mouse pulmonary cells were shown to bind beta-D-galactose-, alpha-L-fucose and alpha-L-rhamnose substituted serum albumin. A neoglycoprotein bearing alpha-L-rhamnose residues was an efficient binder under the conditions of cell adhesion experiments and a potent cell adhesion inhibitor. A fucose-containing neoglycoprotein was shown to have a high inhibitory activity when used concomitantly to alpha-D-glucose-containing neoglycoproteins. Adhesion inhibition experiments, performed with cells the sugar specific receptors of which have been selectively inactivated, showed that the alpha-L-fucose specific receptors on pulmonary cell surface are partly responsible for the specificity of this cell-cell recognition process.
Identification of receptors in the interaction between P. falciparum merozoites and erythrocytes.
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The role of glycosylation in protein recognition. Warner-Lambert Parke-Davis Award lecture.
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[Role of the von Willebrand factor in the physiopathology of primary hemostasis].
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[Platelet membrane receptors].
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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.
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.
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.
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.
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.
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.