Studies on The Function of Platelet Surface Antigens

1979 ◽  
Author(s):  
J Connellan ◽  
I. Smith ◽  
B. Barlow ◽  
P.A. Castaldi
Keyword(s):  
Gel Electrophoresis ◽  
Antiplatelet Antibody ◽  
Platelet Surface ◽  
Platelet Membranes ◽  
Coomassie Blue ◽  
Rabbit Igg ◽  
Centrifuge Tube ◽  
Agar Gel Electrophoresis ◽  
Membrane Components ◽  
Sds Electrophoresis

Heterologous antibodies to platelets and isolated membranes have influences on the response of intact platelets to aggregating stimuli. This study is concerned with identification of the active antigens in an effort to assign a functional role to specified membrane components. Antisera were produced in rabbits to membranes obtained from washed platelets. Platelets were labelled with 125I by using lactoperoxidase and DD125ISA. 4 major labelled proteins were obtained on SDS gel electrophoresis. Several of these labelled proteins reacted with the antiplatelet membrane antibody. These proteins were isolated by treating a mixture of platelet membranes with antiplatelet antibody and subsequently incubated with goat anti-rabbit IgG. Two proteins were detected in this fraction on SDS electrophoresis. IgG was purified from antisera by QAE sephadex chromatography and insolubilized on CNBr-Sepharose. Disrupted platelets or platelet membranes were exposed to the immobilized IgG in a centrifuge tube. The Sepharoae-IgG-antigen complex was sedimented, washed with Tris ph 7.5, 3M NaCl and finally Guanidine HCl. Three proteins were detected on SDS electrophoresis of the Guanidine-HCl wash. Crossover Agar gel electrophoresis was performed on platelet membranes and whole platelets solubilized in triton. Two protein reactants, with a line of partial identity, were evident when the gels were stained with Coomassie Blue. since F (ab)2 fragments isolated from the antiserum have inhibitory effects in the response to ristocetin and collagen, it is possible that the proteins under study may be involved in some platelet surface reactions.

scholarly journals Platelet antithrombin defect in malignancy: platelet protein alterations

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 479-485 ◽  
Author(s):  
EM Sloand ◽  
DM Kenney ◽  
FC Chao ◽  
J Lawler ◽  
JL Tullis
Keyword(s):  
Sodium Dodecyl ◽  
Test System ◽  
Clotting Time ◽  
Platelet Surface ◽  
Platelet Protein ◽  
Coomassie Blue ◽  
Patient Groups ◽  
Protein Alterations ◽  
Sds Electrophoresis ◽  
Tritium Labeling

Abstract Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)- electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti- human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.

scholarly journals Platelet antithrombin defect in malignancy: platelet protein alterations

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 479-485
Author(s):  
EM Sloand ◽  
DM Kenney ◽  
FC Chao ◽  
J Lawler ◽  
JL Tullis
Keyword(s):  
Sodium Dodecyl ◽  
Test System ◽  
Clotting Time ◽  
Platelet Surface ◽  
Platelet Protein ◽  
Coomassie Blue ◽  
Patient Groups ◽  
Protein Alterations ◽  
Sds Electrophoresis ◽  
Tritium Labeling

Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)- electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti- human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.

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 46DIFFERENTIAL PATTERNS OF PROTEIN SYNTHESIS BETWEEN NORMAL AND CLONED PLACENTAE

2004 ◽  
Vol 16 (2) ◽  
pp. 145
Author(s):  
H.R. Kim ◽  
J.K. Kang ◽  
J.T. Yoon ◽  
H.H. Seong ◽  
C.S. Park ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gel Electrophoresis ◽  
Optical Resolution ◽  
Protein Patterns ◽  
Molecular Weight Range ◽  
Animal Cloning ◽  
Coomassie Blue ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Native Cattle

Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by extremely low success rate. Most clones die before birth and survivors frequently display abnormalities. It is speculated that epigenetic reprogramming is somehow defective in reconstituted embryos (Reik W et al., 2003 Theriogenology 59 21–32; Han YM et al., 2003 Theriogenology 59, 33–44). It is likely that placental anomalies are directly or indirectly responsible for the death of cloned fetus and neonates. To address this question, we analyzed protein patterns of two placentae obtained after postnatal death of fetuses from SCNT of Korean Native Cattle and two normal placentae obtained after birth of AI fetuses. Global proteomics approach was employed by using 2-D gel electrophoresis and mass spectrometry to separate the different placenta proteins. Proteins within an isoelectric point range of 4.0 to 7.0 and a molecular weight range of 20–100kDa were analyzed by means of 2-D gel electrophoresis with three replications of each sample. The stained gels were scanned and calibrated at an optical resolution of 63.5μm/pixel using a GS-710 (Bio-Rad Laboratories, Hercules, CA, USA). Approximately 480 spots were detected in placental 2-D gel stained with coomassie-blue. Then, image analysis by Malanie III (Swiss Institute for Bioinformatics, Geneva, Switzerland) was performed to detect variations in protein spots between normal and SCNT placentae. In the comparison of normal and SCNT samples, at least 15 protein spots were identified as regulated differentially. Using MALDI-TOF-MS (PerSeptive Biosystems, Framinham, MA, USA), 10 spots were identified as up-regulated proteins in SCNT placentae including BPLP-I, Rho GDI 2, osteoclast stimulating factors, SM22, 60S Acidic Ribosomal and Protein P2, whereas five spots were down-regulated proteins such as Peroxiredoxin 2. Mass spectrometry with sequencing was used to further analyze the uncharacterized proteins. Most identified proteins in this analysis appeared to be related to cell proliferation and differentiation, fetal growth and development or metabolism. Further, specific functions of proteins in placenta have been investigated at the molecular levels during pregnancy.

POLYETHYLENE GLYCOL ON AGAR-GEL ELECTROPHORESIS

The Lancet ◽  
1974 ◽  
Vol 304 (7892) ◽  
pp. 1321-1322
Author(s):  
W.H. Taylor ◽  
D.J. Etherington
Keyword(s):  
Polyethylene Glycol ◽  
Gel Electrophoresis ◽  
Agar Gel ◽  
Agar Gel Electrophoresis
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