Characterization of human platelet glycoprotein antigens giving rise to individual immunoprecipitates in crossed-immunoelectrophoresis

Abstract Washed human platelets were labeled with 125I by the lactoperoxidase- catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.

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Characterization of human platelet glycoprotein antigens giving rise to individual immunoprecipitates in crossed-immunoelectrophoresis

Blood ◽

10.1182/blood.v58.6.1190.bloodjournal5861190 ◽

1981 ◽

Vol 58 (6) ◽

pp. 1190-1197 ◽

Cited By ~ 1

Author(s):

TJ Kunicki ◽

AT Nurden ◽

D Pidard ◽

NR Russell ◽

JP Caen

Keyword(s):

Sodium Dodecyl ◽

Electrophoretic Analysis ◽

Human Platelets ◽

Soluble Proteins ◽

Platelet Glycoprotein ◽

Membrane Glycoprotein ◽

Membrane Glycoproteins ◽

Reference Pattern ◽

Crossed Immunoelectrophoresis ◽

Normal Individuals

Washed human platelets were labeled with 125I by the lactoperoxidase- catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.

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Immunoelectrophoretic Analysis of Membrane Glycoproteins in Cultured Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645214 ◽

1990 ◽

Vol 63 (02) ◽

pp. 303-311

Author(s):

Tone Børsum

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Rabbit Antiserum ◽

Platelet Glycoprotein ◽

Membrane Glycoproteins ◽

Human Endothelial Cells ◽

Immunoelectrophoretic Analysis ◽

Glycoprotein Complex ◽

Crossed Immunoelectrophoresis ◽

Triton X

SummaryHuman endothelial cells isolated from umbilical cordswere solubilized in Triton X-100 and examined by crossedimmunoelec-trophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelialcell proteins with 14Cmannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques.Two monoclonal antibodies against the platelet glycoprotein complex Ilb-IIIa and glycoprotein IlIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex Ilb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.

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Human Blood Platelet Membrane Glycoproteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657050 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1490-1502 ◽

Cited By ~ 3

Author(s):

C S P Jenkins ◽

E F Ali-Briggs ◽

G T E Zonneveld ◽

A Sturk ◽

J Clemetson

Keyword(s):

Specific Problem ◽

Sodium Dodecyl ◽

Normal Subjects ◽

Blood Platelet ◽

Platelet Membrane ◽

Buffer System ◽

Membrane Glycoprotein ◽

Membrane Glycoproteins ◽

Human Blood Platelet ◽

Electrophoretic Techniques

SummaryThe separation of the major platelet membrane glycoproteins of normal subjects and subjects with well defined platelet membrane glycoprotein abnormalities have been examined using four different polyacrylamide gel electrophoretic techniques (continuous and discontinuous). The mobilities of the resolved glycoprotein bands have been correlated with the glycoprotein nomenclature proposed for the conventional sodium dodecyl sulphate- phosphate buffer system. Since the glycoprotein distribution varies depending on the system used, the merits of each method should be considered before application to a specific problem.

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Further Characterization Of Platelet Membrane Glycoproteins

10.1055/s-0038-1652282 ◽

1981 ◽

Author(s):

P Clezardin ◽

J L McGregor ◽

K J Clemetson ◽

M Dechavanne ◽

E F Lüscher

Keyword(s):

Ricinus Communis ◽

Lectin Binding ◽

Sodium Dodecyl ◽

Human Platelets ◽

Galactose Oxidase ◽

Membrane Glycoproteins ◽

Neuraminidase Treatment ◽

Platelet Surface ◽

Reducing Conditions ◽

Surface Labelling

The binding of 125I-labelled lectins to major and minor platelet glycoproteins (GP) and their subunits has been investigated. Human platelets were isolated, washed, solubilized in sodium dodecyl sulphate (SDS) under non-reducing conditions and separated on 5, 7.5 and 10 % non-reduced/reduced 2-D polyacrylamide gels. The gels were incubated with 125I-labelled lectins; Lens culinaris lectin (LCL), concanavalin A (ConA) wheat germ agglutinin (WGA) or Ricinus communis agglutinin (RCA-120), then washed extensively dried and exposed to X-ray film by indirect autoradiography. Surface-labelled platelets were similarly separated. WGA and RCA bound predominantly to GPIbα but also to two minor bands above and below it which were affected by neuraminidase treatment. One of them bound two 125I-lectins (LCL and ConA) while GPIbα did not. Additional GP bands were detected by lectin binding and by surface-labelling beneath GPIIIβ (IV). With platelets labelled by the neuraminidase/galactose oxidase/NaB3H4 method a GP was detected between Ila and Ilia which was not found with periodate/ NaB3H4 labelling (not affected by reduction). Two spots on the diagonal bound LCL and ConA. GP Ibβ bound LCL more strongly than IIbp. GPIbp also bound WGA and RCA. GPIcβ apparently bound only ConA. GPIbβ and IIbβ were labelled equally strongly by surface labelling techniques, Icβ was apparently not labelled. Further GP subunits were detected one below Ibβ and IIbβ and another which originated in the GPVII region. These techniques demonstrate that the platelet surface is even more complex than previously thought.

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Isolation and structural characterization of the polypeptide subunits of membrane glycoprotein IIb-IIIa from human platelets

Blood ◽

10.1182/blood.v59.1.80.80 ◽

1982 ◽

Vol 59 (1) ◽

pp. 80-85 ◽

Cited By ~ 44

Author(s):

RP McEver ◽

JU Baenziger ◽

PW Majerus

Keyword(s):

High Performance ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Structural Features ◽

Human Platelets ◽

Membrane Glycoprotein ◽

Radiolabeled Peptides ◽

The Relationship ◽

Peptide Maps

Abstract We have previously demonstrated the isolation of platelet membrane glycoprotein IIb-IIIa by affinity chromatography with a specific monoclonal antibody. We have now separated the polypeptide subunits IIb and IIIa of the isolated glycoprotein by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and have compared their structural features. Both IIb and IIIa contain approximately 15% carbohydrate, but IIIa contains a larger percentage of mannose residues, suggesting the presence of high mannose as well as complex N- linked oligosaccharide chains. The amino acid compositions are sufficiently similar to imply areas of sequence homology between the two subunits. To examine further the relationship between the subunits, we digested a mixture of 125I-IIb and 131I-IIIa with trypsin and then separated the radiolabeled peptides by high performance liquid chromatography. The resultant peptide maps of IIb and IIIa are completely different. This indicates that neither subunit is derived from the other and suggests that polypeptides IIb and IIIa are products of separate genes.

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Proteins from different classes of liver nuclei in normal and thioacetamide-treated rats

Biochemical Journal ◽

10.1042/bj1330441 ◽

1973 ◽

Vol 133 (3) ◽

pp. 441-455 ◽

Cited By ~ 16

Author(s):

F. Gonzalez-Mujica ◽

A. P. Mathias

Keyword(s):

Nuclear Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Specific Radioactivity ◽

Electrophoretic Analysis ◽

Soluble Proteins ◽

Adolescent And Young Adult ◽

Adult Rats ◽

Chromosomal Proteins ◽

Liver Nuclei

1. In normal rats the amounts of each of the main types of nuclear protein, i.e. soluble proteins, histones, non-histone chromosomal proteins and residual proteins, vary within the different classes of rat liver nuclei fractionated by zonal centrifugation. 2. Heterogeneity is observed in the non-histone chromosomal proteins prepared from different classes of liver nuclei. These differences were observed by analysis of the proteins both by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and electrofocusing electrophoresis. They are most evident between the non-histone chromosomal proteins obtained from stromal and parenchymal nuclei. However, some differences are also found for the parenchymal nuclei, between the diploid parenchymal and the tetraploid parenchymal, and between them and the nuclei involved in the synthesis of DNA respectively. 3. Drastic alterations in the nuclear proteins are found after the administration of thioacetamide. The changes observed are complex and not uniform. They vary with the age of the animal and the type of nucleus. In general an increase in the soluble proteins and non-histone chromosomal proteins and a decrease in the residual proteins is observed. There is a decrease in the specific radioactivity of soluble and residual proteins. 4. Electrophoretic analysis of the non-histone chromosomal proteins showed that specific changes occurred after administration of thioacetamide, which are different in adolescent and young adult rats.

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Isolation and structural characterization of the polypeptide subunits of membrane glycoprotein IIb-IIIa from human platelets

Blood ◽

10.1182/blood.v59.1.80.bloodjournal59180 ◽

1982 ◽

Vol 59 (1) ◽

pp. 80-85 ◽

Cited By ~ 1

Author(s):

RP McEver ◽

JU Baenziger ◽

PW Majerus

Keyword(s):

High Performance ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Structural Features ◽

Human Platelets ◽

Membrane Glycoprotein ◽

Radiolabeled Peptides ◽

The Relationship ◽

Peptide Maps

We have previously demonstrated the isolation of platelet membrane glycoprotein IIb-IIIa by affinity chromatography with a specific monoclonal antibody. We have now separated the polypeptide subunits IIb and IIIa of the isolated glycoprotein by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and have compared their structural features. Both IIb and IIIa contain approximately 15% carbohydrate, but IIIa contains a larger percentage of mannose residues, suggesting the presence of high mannose as well as complex N- linked oligosaccharide chains. The amino acid compositions are sufficiently similar to imply areas of sequence homology between the two subunits. To examine further the relationship between the subunits, we digested a mixture of 125I-IIb and 131I-IIIa with trypsin and then separated the radiolabeled peptides by high performance liquid chromatography. The resultant peptide maps of IIb and IIIa are completely different. This indicates that neither subunit is derived from the other and suggests that polypeptides IIb and IIIa are products of separate genes.

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The formation of Ca++-dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis

Blood ◽

10.1182/blood.v58.2.268.bloodjournal582268 ◽

1981 ◽

Vol 58 (2) ◽

pp. 268-278 ◽

Cited By ~ 1

Author(s):

TJ Kunicki ◽

D Pidard ◽

JP Rosa ◽

AT Nurden

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Human Platelets ◽

Tetraacetic Acid ◽

Rabbit Antibody ◽

Membrane Glycoproteins ◽

Normal Platelet ◽

Crossed Immunoelectrophoresis ◽

Glycoprotein Antigen ◽

Glycoprotein Iiia ◽

Triton X

Triton X-100 soluble proteins from 125I-labeled human platelets were studied by crossed immunoelectrophoresis employing a multispecific rabbit antibody raised against whole normal platelets. Emphasis was placed upon an analysis of immunoprecipitates containing 125I-labeled major membrane glycoproteins, and in particular, a prominent immunoprecipitate containing a glycoprotein antigen (s) previously designated as protein 16. SDS-polyacrylamide gel electrophoresis of protein 16 precipitated by a monospecific alloantibody. IgG L . . . , confirmed the presence of both glycoproteins IIb and IIIa. 125I-IgG L . . . , at concentration below that capable of precipitating protein 16 by itself, bound specifically to the precipitate containing protein 16 produced by the multispecific rabbit antibody. No other precipitates formed by the rabbit antibody contained either glycoprotein IIb or IIIa. When platelet proteins, incubated with optimum concentrations of ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol bis (B- aminoethylether) NN1-tetraacetic acid (EGTA), were electrophoresed against the rabbit antibody, previously unobserved immunoprecipitates that contained either free glycoprotein IIb or free glycoprotein IIIa were detected. Upon readdition of excess Ca++, but not Mg++, to the same protein samples, a single immunoprecipitate containing both glycoproteins was once again observed. It is thus demonstrated that glycoproteins IIb and IIIa can form Ca++-dependent complexes (protein 16) in Triton X-100 extracts of normal platelets. The potential significance of the reversible association of these glycoproteins to normal platelet function is discussed.

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A platelet membrane glycoprotein (GP) deficiency in healthy blood donors: Naka- platelets lack detectable GPIV (CD36)

Blood ◽

10.1182/blood.v76.9.1698.1698 ◽

1990 ◽

Vol 76 (9) ◽

pp. 1698-1703 ◽

Cited By ~ 106

Author(s):

N Yamamoto ◽

H Ikeda ◽

NN Tandon ◽

J Herman ◽

Y Tomiyama ◽

...

Keyword(s):

Binding Sites ◽

Gel Electrophoresis ◽

Blood Donors ◽

Platelet Membrane ◽

Platelet Glycoprotein ◽

Membrane Glycoprotein ◽

Two Dimensional ◽

Platelet Concentrates ◽

Two Dimensional Gel Electrophoresis ◽

Crossed Immunoelectrophoresis

Abstract It has recently been shown that the Naka antigen, which is absent in 3% to 11% of Japanese blood donors, is expressed on platelet glycoprotein IV (GPIV; CD36) (Tomiyama et al, BLOOD, 75:684, 1990). In the present studies, flow cytometry was used to distinguish differences in the reactivity of Naka+ and Naka- platelets with both OKM5, a monoclonal antibody that recognizes an epitope on GPIV, and with polyclonal anti- GPIV antibody. OKM5 was also used to screen 871 platelet concentrates prepared from healthy US blood donors. Three of these showed markedly deficient binding of 125I-OKM5 or an incidence of 0.34%. Two of these donors were re-accessed and showed less than 1% binding of 125I-OKM5 as compared with 10,300 +/- 1,500 binding sites per platelet in controls (n = 4). Platelets from these two US donors were radiolabeled (125I, 3H) and compared with control platelets and with platelets from Japanese Naka+ and Naka- donors by crossed immunoelectrophoresis, protein blots, immunoprecipitation, and two-dimensional gel electrophoresis. GPIV could not be detected by any of these techniques in the Naka- platelets nor in the donors whose platelets showed deficient binding of OKM5. These results suggest that GPIV functions as an isoantigen rather than an alloantigen in immunizing Naka- platelet recipients. This is the first report of the absence of a major platelet membrane GP in healthy blood donors.

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Comparison Of The Major Membrane Glycoproteins Of Human, Rabbit And Rat Platelets

10.1055/s-0038-1652279 ◽

1981 ◽

Author(s):

B Toor ◽

J L McGregor ◽

K J Clemetson ◽

L McGregor ◽

M Dechavanne ◽

...

Keyword(s):

Sialic Acid ◽

Surface Composition ◽

Sodium Dodecyl ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Neuraminidase Treatment ◽

Platelet Surface ◽

Rabbit Platelets ◽

Rat Platelets

Rabbit and rat platelets have been extensively investigated under in vitro or in vivo conditions to try to understand the pathology of thrombosis in man. Here, surface-labelling techniques have been used to find out if the platelet surface has a similar composition in these two animals and in man or not. Human, rabbit and rat platelets were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetyl galactosamine residues). Labelled platelets were solubilized in sodium dodecyl sulphate and separated under reducing conditions on 7.5 % Laemmli polyacrylamide gels. Dried gels were exposed to film by fluorography or indirect autoradiography. Terminal Gal/Gal NAc residues (no neuraminidase treatment) were strongly labelled with rat and rabbit platelets compared to human platelets which labelled very poorly. Terminal sialic acid labelling with rat and rabbit platelets showed a weak labelling of a glycoprotein (GP) with the same M.Wt. as GPIb which is the most intensely labelled GP in man. However two GP (with rabbits) and one GP (in rats) were intensely labelled at a M.Wt. similar to that of GPIa in man. These GP had a different M.Wt. with terminal Gal/Gal NAc labelling. Bands with a similar M.Wt. to GPIIb and IIIa in man were strongly iodinated with rabbit platelets but with rat platelets only a single band at the position of GPIIb was strongly iodinated. These results strongly indicate that there are considerable differences in surface composition between rabbit, rat and human platelets.

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