Electron Microscope Analysis of the Surface Charge of Human Platelets; Stored and Aggregated by Polymeric Bases

1975 ◽  
Author(s):  
Y. Taketomi ◽  
A. Ihara ◽  
A. Kuramoto ◽  
H. Uchino
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Plasma Membranes ◽  
Particle Density ◽  
Average Distance ◽  
Human Platelets ◽  
Degree Of Polymerization ◽  
Surface Coat ◽  
Cationic Polymers ◽  
Neuraminidase Treatment

The stainability of the surface coat of human platelets with positively charged colloidal Thorotrast was studied on the stored and aggregated platelets by some aggregating agents. During storage, the total sialic acid decreased and fell to 65% of initial value on 5th day. The sialic acid released from fresh platelets by neuraminidase treatment was 80% of the total. The density of Thorotrast particles on surface coat did not change during storage up to 7 days. These particles were abolished to some extent from the surface of neuraminidase treated platelets. These particle density did not change significantly in ADP and collagen induced aggregates but decreased in aggregates induced by cationic polymers such as protamine sulfate and polylysine. At the same concentration of polylysines of different degree of polymerization, maximal aggregation was greater with the higher molecular weight. Average distance between the plasma membranes of two adjacent cells in the aggregate was rather wider when the higher molecular weight polylysine was used.

scholarly journals Ultrastructural Studies on the Surface Coat of Human Platelets Aggregated by Polylysine and Dextran

1977 ◽  
Author(s):  
Y. Taketomi ◽  
A. Kuramoto
Keyword(s):  
Platelet Aggregation ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Average Distance ◽  
Human Platelets ◽  
Surface Coat ◽  
High Concentration ◽  
Platelet Suspension ◽  
Ultrastructural Studies ◽  
Induced Aggregation

The surface coat of human platelets was stained with positive charged colloidal Thorotrast particles and ruthenium red to elucidate the mechanism of platelet aggregation by macromolecules.Positive charged macromolecule, polylysine (Mw.l5,000; 23,000; 180,000) could induce the aggregation in low concentration but high concentration was needed in the case of neutral macromolecule, dextran (Mw.40,000; 250,000; 2000,000). The larger molecules of polylysine and dextran were more effective in inducing platelet aggregation. In the dextran induced aggregation, Thorotrast particles on the cell surface did not decrease significantly. On the other hand, the surface membranes of platelets treated with neuraminidase and aggregated by polylysine were essentially devoid of bound particles. These findings suggested that polylysine induced aggregation more effectively than dextran by reducing the negative surface charge and giving stronger adsorption force on cell surface.In washed platelet suspension, polylysine induced aggregation without release reaction of 14C-serotonin, but average distance between plasma membranes of aggregated platelets did not vary with the degrees of polymerization. In this respect, it seems probable that after forming macromolecular bridging of cell surface, another interaction may follow between cell surfaces.

scholarly journals Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain

1976 ◽  
Vol 156 (1) ◽  
pp. 143-150 ◽  
Author(s):  
R H Quarles
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Weight Class ◽  
Developmental Difference ◽  
Adult Rats ◽  
Neuraminidase Treatment

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.

Inhibition of the Platelet Reaction by a High Molecular Weight Phosphoglycoprotein Isolated from Human Platelet Plasma Membranes

1981 ◽  
Vol 45 (02) ◽  
pp. 130-135 ◽  
Author(s):  
R Apitz-Castro ◽  
G Fonseca ◽  
V Michelena ◽  
M R Cruz
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Secondary Wave ◽  
Platelet Reaction ◽  
Induced Aggregation ◽  
External Calcium

SummaryThe effect of a phospho-glycoprotein (HMW-GP), obtained from human platelet plasma membranes, on the aggregation and secretion of human platelets was studied. Incubation of PRP with 4 to 16 μg/ml of HMW-GP results in inhibition of ADP-, Epinephrine-, Collagen-, and Thrombin-induced platelet aggregation. The effect is mainly reflected on the secondary wave of aggregation. The inhibitory effect is partially overcome by higher concentration of the inducers, however, even under these conditions, a clear tendency towards disaggregation is observed. 5HT release (Col-induced) is strongly decreased from 50% to 4.5. The inhibitory effect on Thrombin-induced aggregation is markedly dependent on external calcium, being maximal at 5 mM calcium. The HMW-GP does not bind ADP or Thrombin. Membrane conformation is markedly affected, as evidenced by the effect of HMW-GP on the iodination of surface polypeptides of intact platelets. It is suggested that interaction of HMW-GP with the platelet membrane blocks the signal(s) transmission that links stimulus to activation. The inhibition observed might just represent an experimental amplification of the endogenous modulatory function that has been proposed for this high molecular weight phosphoglycoprotein.

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

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.

scholarly journals Functional Analysis of the Streptococcus gordonii DL1 Sialic Acid-Binding Adhesin and Its Essential Role in Bacterial Binding to Platelets

2004 ◽  
Vol 72 (7) ◽  
pp. 3876-3882 ◽  
Author(s):  
Yukihiro Takahashi ◽  
Ayako Yajima ◽  
John O. Cisar ◽  
Kiyoshi Konishi
Keyword(s):  
Sialic Acid ◽  
Essential Role ◽  
Signal Sequence ◽  
Complete Removal ◽  
Human Platelets ◽  
Microbial Colonization ◽  
Streptococcus Gordonii ◽  
Rich Region ◽  
Neuraminidase Treatment ◽  
Sialic Acid Binding

ABSTRACT Bacterial recognition of host sialic acid-containing receptors plays an important role in microbial colonization of the human oral cavity. The sialic acid-binding adhesin of Streptococcus gordonii DL1 was previously associated with the hsa gene encoding a 203-kDa protein. The predicted protein sequence consists of an N-terminal nonrepetitive region (NR1), including a signal sequence, a relatively short serine-rich region (SR1), a second nonrepetitive region (NR2), a long serine-rich region (SR2) containing 113 dodecapeptide repeats, and a C-terminal cell wall anchoring domain. In the present study, the contributions of SR1, NR2, and SR2 to Hsa-mediated adhesion were assessed by genetic complementation. Adhesion of an hsa chromosomal deletion mutant to sialic acid-containing receptors was restored by plasmids containing hsa constructs encoding Hsa that lacked either the N- or C-terminal portion of SR2. In contrast, hsa constructs that lacked the coding sequences for SR1, NR2, or the entire SR2 region failed to restore adhesion. Surface expression of recombinant Hsa was not affected by removal of SR1, NR2, or a portion of SR2 but was greatly reduced by complete removal of SR2. Wheat germ agglutinin, a probe for Hsa-specific glycosylation, reacted with recombinant Hsa lacking SR1, NR2, or SR2 but not with recombinant Hsa lacking both SR1 and SR2. Significantly, the aggregation of human platelets by S. gordonii DL1, an interaction implicated in the pathogenesis of infective endocarditis, required the expression of hsa. Moreover, neuraminidase treatment of the platelets eliminated this interaction, further supporting the hypothesis that Hsa plays an essential role in the bacterium-platelet interaction.

Immunochemical Detection of the Thomsen-Friedenreich Antigen (T-Antigen) on Platelet Plasma Membranes

1978 ◽  
Vol 39 (01) ◽  
pp. 186-192 ◽  
Author(s):  
Wolfgang M Glöckner ◽  
Hermann D Kaulen ◽  
Gerhard Uhlenbruck
Keyword(s):  
Gas Chromatography ◽  
Sialic Acid ◽  
Haemolytic Uraemic Syndrome ◽  
Agaricus Bisporus ◽  
Plasma Membranes ◽  
Helix Pomatia ◽  
T Antigen ◽  
Con A ◽  
Neuraminidase Treatment ◽  
Immunochemical Detection

SummaryPlatelet plasma membranes were found to possess the disaccharide β--galactosyl(1−3)-N-acetyl-D-galactosamine which was measured by gas chromatography after release by alkaline borohydride treatment and desialylation. Immunological evidence using the specific lectins from Arachis hypogoea and Agaricus bisporus and an anti-T serum confirmed the presence of this disaccharide, the immunodominant group of the Thomsen-Friedenreich antigen (T-antigen). This receptor was only found after prior neuraminidase treatment indicating that it is normally a cryptic antigen, i.e. masked by sialic acid in the native membrane. Evidence for a second receptor with terminal N-acetylgalactosamine was obtained using the lectin from Helix pomatia. The binding of myxovirus and the lectins from Phaseolus vulgaris (PHA) and Canavalia ensiformis (Con A) to platelet membrane was also demonstrated.The implication of the T-antigen in elimination of the platelets and its role in the haemolytic-uraemic syndrome is discussed.

scholarly journals Chemical characterization of the proteins and glycoproteins of mouse liver plasma membranes solubilized by sequential extraction with aqueous and organic solvents

1972 ◽  
Vol 126 (3) ◽  
pp. 459-466 ◽  
Author(s):  
J. W. Gurd ◽  
W. H. Evans ◽  
H. R. Perkins
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Membrane Protein ◽  
Mouse Liver ◽  
Plasma Membranes ◽  
Sodium Dodecyl ◽  
Insoluble Residue ◽  
Sialic Acid Content ◽  
Bicarbonate Buffer ◽  
Liver Plasma Membranes

1. A procedure for the stepwise fractionation of the proteins of mouse liver plasma membranes is described. 2. Of the membrane protein 20–25% was soluble in 50mm-sodium carbonate–bicarbonate buffer (pH9.7). This fraction contained a large number of proteins but only 1 major glycoprotein. It was low in sialic acid, amino sugars and phospholipid. 3. Extraction of the alkali-insoluble residue with aq. 33% pyridine solubilized an additional 30–35% of the membrane protein. The pyridine-soluble membrane components were enriched in sialic acid and glucosamine and it was shown that this procedure resulted in the selective extraction of glycoproteins. 4. Gel filtration in sodium dodecyl sulphate resolved the pyridine-soluble proteins into five fractions of decreasing molecular weight and an inverse relationship between molecular weight and sialic acid content was indicated.

scholarly journals Identification and characterization of podocalyxin--the major sialoprotein of the renal glomerular epithelial cell.

1984 ◽  
Vol 98 (4) ◽  
pp. 1591-1596 ◽  
Author(s):  
D Kerjaschki ◽  
D J Sharkey ◽  
M G Farquhar
Keyword(s):  
Sialic Acid ◽  
Alcian Blue ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Protein Composition ◽  
Cationic Dyes ◽  
Surface Coat ◽  
Colloidal Iron ◽  
Neuraminidase Treatment ◽  
Glomerular Epithelial Cell

The glomerular epithelial polyanion is a specialized cell surface component found on renal glomerular epithelial cells (podocytes) that is rich in sialoprotein(s), as detected by staining with cationic dyes (colloidal iron, alcian blue) and wheat germ agglutinin (WGA). We have isolated rat glomeruli and analyzed their protein composition by SDS PAGE in 5-10% gradient gels. When the gels were stained with alcian blue or "Stains All," a single band with an apparent Mr of 140,000 was detected that also stained very prominently with silver, but not with Coomassie Blue. This band predominated in fluorograms of gels of isolated glomeruli that had been labeled in their sialic acid residues by periodate-[3H]borohydride. In lectin overlays, the 140-kilodalton (kd) band was virtually the only one that bound [125I]wheat germ agglutinin, and this binding could be prevented by predigestion with neuraminidase. [125I]Peanut lectin bound exclusively to the 140-kd band after neuraminidase treatment. An antibody was prepared that specifically recognizes only the 140-kd band by immunoprecipitation and immuneoverlay. By immunoperoxidase and immunogold techniques, it was localized to the surface coat of the glomerular epithelium and, less extensively, to that of endothelial cells. When analyzed (after electroelution from preparative SDS gels), the 140-kd band was found to contain approximately 20% hexose and approximately 4.5% sialic acid. These findings indicate that the 140-kd protein is the major sialoprotein of the glomerulus, and it is the only component of glomerular lysates with an affinity for cationic dyes and lectins identical to that defined histochemically for the epithelial polyanion in situ. Since this molecule is a major component of the cell coat or glycocalyx of the podocytes, we have called it "podocalyxin."

scholarly journals Interaction of platelet plasma membranes with thrombin-activated platelets

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 305-312 ◽  
Author(s):  
HR Prasanna ◽  
HH Edwards ◽  
DR Phillips
Keyword(s):  
Human Erythrocyte ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Erythrocyte Membranes ◽  
Functional Sites ◽  
Activated Platelets ◽  
Platelet Membranes ◽  
Human Erythrocyte Membranes

Abstract This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin- activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

scholarly journals Metabolic Profiling of Xylooligosaccharides by Lactobacilli

Polymers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2387
Author(s):  
Ilia Iliev ◽  
Tonka Vasileva ◽  
Veselin Bivolarski ◽  
Albena Momchilova ◽  
Iskra Ivanova
Keyword(s):  
Plasma Membranes ◽  
Meat Products ◽  
Lactobacillus Brevis ◽  
Degree Of Polymerization ◽  
Acid Fermentation ◽  
Carbohydrate Utilization ◽  
Beneficial Effects ◽  
Mixed Acid ◽  
Rat Liver Plasma Membranes ◽  
Different Types

Three lactic acid bacteria (LAB) strains identified as Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus sakei isolated from meat products were tested for their ability to utilize and grow on xylooligosaccharides (XOSs). The extent of carbohydrate utilization by the studied strains was analyzed by HPLC. All three strains showed preferences for the degree of polymerization (DP). The added oligosaccharides induced the LAB to form end-products of typical mixed-acid fermentation. The utilization of XOSs by the microorganisms requires the action of three important enzymes: β-xylosidase (EC 3.2.1.37) exo-oligoxylanase (EC 3.2.1.156) and α-L-arabinofuranosidase (EC 3.2.1.55). The presence of intracellular β-D-xylosidase in Lb. brevis, Lb. plantarum, and Lb. sakei suggest that XOSs might be the first imported into the cell by oligosaccharide transporters, followed by their degradation to xylose. The studies on the influence of XOS intake on the lipids of rat liver plasma membranes showed that oligosaccharides display various beneficial effects for the host organism, which are probably specific for each type of prebiotic used. The utilization of different types of oligosaccharides may help to explain the ability of Lactobacillus strains to compete with other bacteria in the ecosystem of the human gastrointestinal tract.

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