The Role of Calcium ION in Platelet Aggregation

1979 ◽  
Author(s):  
N. Deguchi ◽  
Y. Ikeda ◽  
K. Toyama ◽  
T. Hirano ◽  
K. Watanabe ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Membrane Vesicles ◽  
Platelet Membrane ◽  
Ultrastructural Changes ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Attachment Sites ◽  
Precise Role

Calcium is required for platelet aggregation but the precise role of Ca++ is not yet known. We have investigated the role of Ca++ in platelet aggregation, performing an ultra-structural and biochemical analysis of Ca++ treated platelet membrane vesicles. Human platelet membrane vesicles isolated by glycerol lysis technique according to the method of Barber and Jamieson, were suspended in 10 mM tris buffered saline, pH 7.4. Aggregation of platelet membrane vesicles was clearly observed under the phase-contrast microscopy in the presence of 5 mM CaCl2. Aggregated membranes were solubilized in 1Ï SDS and 1% 2-mer-captoethanol, and electfophorcsed on 5% SDS-polyacrylamide gels. Comparison of polypeptides of aggregated and native membranes revealed that the polypeptide of molecular weight 200,000 Daltons disappeared, instead, coomassie blue staining became visualized at the top of the gel in aggregated membranes. Ultrastructural observations were performed after fixation of membrane vesicles with 2% glutaraldehyde for both thin-section and freeze-fracturing. Freeze-fractured image of native membranes exhibit a smooth surface with a random distribution of intramembranous particles (IMP). Membrane vesicles treated with 5 mM CaCl2 possess round depressions suggestive of areas of mutual contact. These circular attachment sites are free of IMP, which accumulated at the periphery. It is suggested that these ultrastructural changes induced by Ca++ may be regarded as crucial events in platelet aggregation.

Aggregation of Platelet Membrane Vesicles by ADP

1979 ◽  
Author(s):  
K. Watanabe ◽  
M. Yamamoto ◽  
Y. Ando ◽  
Y. Ikeda ◽  
K. Toyama ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Staining Intensity ◽  
Protein Band ◽  
Disc Electrophoresis ◽  
Platelet Membrane ◽  
Blue Staining ◽  
P Value ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Biophysical Changes

The study was performed to examine biochemical and biophysical changes in platelet membrane vesicles(PMV) when aggregated by ADP. PMV was isolated from normal donors by using glycerol lysis technique, and was suspended in 10mM Tris, 0.15M NaCl buffer, pH 7,4. Aggregation of PMV was readily observed under phasecontrastmlcroscopy within 3 min after addition of ADP(5mM), while AMP, UDF or GDP was without effect. Light scattering Intensity of PMV suspension determined by Lasernepherometer increased with addition of ADPCJmM), reaching plateau within 2 to 3 min, also indicating that vesicles were rapidly aggregating in suspending medium. In electronmicroscopic observation, amorphous floccuent materials were seen between aggregated PMV by ADP. This PMV aggregation by ADP was inhibited by the treatment of PMV with either neuraminidase(0.1u/ml) or trypsin(20μg/ml) and was hardly observed in PMV from a patient with thrombasthenia.The addition of ADF(10mM) to PMV suspension resulted in the reduction in electronhoretic mobility of PW(0.301±0.057μ·cm/sec·V, control ;1.194±0.037) and increase in membrane microviscositv (p value; 0.277, control; 0.255).Changes in protein profiles in aggregated PMV were examined by SDS Polyacrylamide gel disc electrophoresis. One protein band(M.W. 200,000) disappeared and instead, there observed increased coomassie blue staining intensity at the top of the gel.The results suggest that these biochemical and biophysical changes in PMV may in part explain the mechanisms of platelet aggregation By ADP.

PRODUCTION AND CHARACTERISATION OF A MONOCLONAL ANTIBODY AGAINST HUMAN ANTI-THROMBIN III

1987 ◽  
Author(s):  
Deborah A Rathjen ◽  
Carolyn L Geczy
Keyword(s):  
Cultured Cells ◽  
Factor Xa ◽  
Lymphocyte Activation ◽  
Blue Staining ◽  
Aortic Endothelial Cells ◽  
Tissue Sections ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
At Iii

To study the role of anticoagulants, particularly antithrombin III (AT III) and heparin, on the activation of coagulation by monocytes/macrophages which have been stimulated with a soluble lymphocyte activation product, macrophage procoagulant inducing factor, we have prepared monoclonal antibodies (MAbs) to human AT III.In fusion experiments, in contrast to wells containing peritoneal feeder cells, positive hybrids were only found in wells containing medium conditioned by the macrophage cell line J774 (Rathjen and Geczy, 1986). Of 5 hybrids which initially produced antibody, only one hybrid, showed stable Ab production. The MAb, designated 22/23, was not cross-reactive with either 1 antitrypsin or ovalbumin and did not inhibit the biological activity of AT III in chromogenic assays which measured inhibition of thrombin and Factor Xa by AT III. An immunoadsorbent prepared using MAb 22/23 depleted AT III activity from a purified AT III preparation. Reduction and alkylat ion of the disulphide bonds of the protein portion of AT III completely abbrogated MAb binding indicating that the native configuration of AT III was important. Isoelectric focussing of AT III, followed by transfer of the focussed protein to nitrocellulose by diffusion and probing with MAb 22/23, revealed at least 8 bands in the region of pH 5.2 to 5.85. Coomassie blue staining of a gel run in parallel showed 9 bands in this region. The MAb provides a useful tool for the detection of AT III on both cultured cells (bovine aortic endothelial cells are positive by immunofluorescence) and tissue sections.Rathjen, D.A. and Geczy, C.L. Hybridomo. 5s 255-261 (1986)

Analysis of Bufo arenarum oviductal secretion during the sexual cycle

Zygote ◽  
2009 ◽  
Vol 17 (4) ◽  
pp. 329-340 ◽  
Author(s):  
Claudia A. Crespo ◽  
Inés Ramos ◽  
Marcela F. Medina ◽  
Silvia N. Fernández
Keyword(s):  
Electrophoretic Pattern ◽  
Sexual Cycle ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Sds Page ◽  
Bufo Arenarum ◽  
Ovulatory Period

SummaryBufo arenarum oocytes are oviposited surrounded by jelly coats, one component of the extracellular matrix required for fertilization. The secretion, released to the oviductal lumen, was analysed by SDS-PAGE. The coomassie blue staining evidenced an electrophoretic pattern with molecules ranging between 300 and 19 kDa that showed variations in their secretion profiles during the sexual cycle. In the preovulatory period the densitometric analysis showed the presence of nine peaks with marked predominance of the 74 kDa molecule. Once ovulation has occurred, the jelly coats become arranged around the oocytes during their transit throughout the oviductal pars convoluta (PC), revealing the addition of three proteins only observed during this period, which suggests a differential secretion. Some of these proteins could not diffuse under any extraction treatment, indicating for them a structural or in situ function. Proteins of low molecular mass diffused totally while others showed a partial diffusing capacity. After ovulation a marked decrease in the relative amount of all the proteins released to the lumen, especially the 74 kDa protein, could be detected. During this period, unlike the other stages of the sexual cycle, a differential secretion pattern was observed along the PC. The histochemical analysis performed during the ovulatory period showed the presence of glycoconjugates including both acidic and neutral groups. The present results are in agreement with previous ultrastructural and histochemical studies that describe the role of Bufo arenarum jelly coats in fertilization.

Coomassie blue staining for high sensitivity gel-based proteomics

2013 ◽  
Vol 90 ◽  
pp. 96-106 ◽  
Author(s):  
Victoria J. Gauci ◽  
Matthew P. Padula ◽  
Jens R. Coorssen
Keyword(s):  
High Sensitivity ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

scholarly journals Heating Greatly Speeds Coomassie Blue Staining and Destaining

BioTechniques ◽  
2000 ◽  
Vol 28 (3) ◽  
pp. 426-432 ◽  
Author(s):  
Connie Wong ◽  
Srinavas Sridhara ◽  
James C.A. Bardwell ◽  
Ursula Jakob
Keyword(s):  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

The reactivity to N-ethyl maleimide of the subunits of cytochrome oxidase

1977 ◽  
Vol 55 (9) ◽  
pp. 988-994 ◽  
Author(s):  
A. McGeer ◽  
B. Lavers ◽  
G. R. Williams
Keyword(s):  
Electron Transport ◽  
Cytochrome Oxidase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Blue Staining ◽  
Beef Heart ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Beef heart cytochrome oxidase (EC 1.9.3.1) prepared in this laboratory consistently presents 10 Coomassie blue staining zones on SDS–polyacrylamide gel electrophoresis. At pH 7.0 only two of these polypeptides (III and VIa) are labelled by radioactive N-ethyl maleimide (NEM). The labelling of VIa is variable and correlates with the activity of particular oxidase preparations. When cytochrome oxidase is isolated from alkylated membranes, either mitochondria or electron transport particles, polypeptide VIa is found not to be labelled; polypeptide III is more strongly labelled than when isolated oxidase is alkylated, and label now appears in polypeptide I which is not alkylated upon treatment of isolated oxidase with NEM.

scholarly journals Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1212-1219 ◽  
Author(s):  
N Kieffer ◽  
B Boizard ◽  
D Didry ◽  
JL Wautier ◽  
AT Nurden
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Blue Staining ◽  
Igg Antibody ◽  
Immune Disorder ◽  
Immunochemical Characterization ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Abstract We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle

1992 ◽  
Vol 4 (2) ◽  
pp. 249 ◽  
Author(s):  
A Paliwal ◽  
B Malaviya ◽  
VP Kamboj
Keyword(s):  
Menstrual Cycle ◽  
Protein Profile ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Protein Patterns ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Oviductal Fluid

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

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