Immunoelectrophoretic Analysis of Membrane Glycoproteins in Cultured Human Endothelial Cells

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.

The Interaction of β2 Glycoprotein-I and Heparin and Its Effect on β2 Glycoprotein-I Antiphospholipid Antibody Cofactor Function in Plasma

1994 ◽  
Vol 72 (04) ◽  
pp. 578-581 ◽  
Author(s):  
T McNally ◽  
S E Cotterell ◽  
I J Mackie ◽  
D A Isenberg ◽  
S J Machin
Keyword(s):  
Solid Phase ◽  
Pharmaceutical Preparations ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antiphospholipid Antibody ◽  
Biological Functions ◽  
Dermatan Sulphate ◽  
Glycoprotein I ◽  
Crossed Immunoelectrophoresis ◽  
Calcium Heparin ◽  
Cofactor Activity

Summaryβ2 glycoprotein-I (β2GPI), a cofactor for antiphospholipid antibody (aPA) binding, binds to many anionic macromolecules including heparin. The nature of this interaction with heparin is not well understood and its effect on the purported biological functions of β2GPI is unknown.We have examined the interactions of dermatan sulphate (DS) and different pharmaceutical preparations of heparin with β2GPI by crossed immunoelectrophoresis (CIE) and investigated the effect of these agents on plasma levels of p2GPI antigen (β2GPI: Ag) by a standardised enzyme linked immunosorbent assay (ELISA). P2GPI aPA cofactor activity (β2GPI:Cof) was also measured using a modified solid phase an-ti-phosphatidylserine (aPS) ELISA. CIE results confirmed a heparin-β2GPI interaction with unfractionated (UF) heparin. β2GPI:Ag levels were unaffected by any of the preparations investigated. There were no significant differences in β2GPI:Cof activities of the samples containing LMW heparins or DS but levels of β2GPI:Cof were increased in samples containing UF sodium and calcium heparin preparations (0.5 IU/ml Monoparin, p <0.05, and 10 IU/ml Liquemin and Calcipa-rine, p <0.05).

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

Diversity of Glycoprotein Deficiencies in Glanzmann’s Thrombasthenia

1985 ◽  
Vol 54 (03) ◽  
pp. 626-629 ◽  
Author(s):  
M Meyer ◽  
F H Herrmann
Keyword(s):  
High Resolution ◽  
Gel Electrophoresis ◽  
Type Ii ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Structural Variant ◽  
Glanzmann’S Thrombasthenia ◽  
Crossed Immunoelectrophoresis ◽  
Glanzmann's Thrombasthenia ◽  
Platelet Proteins

SummaryThe platelet proteins of 9 thrombasthenic patients from 7 families were analysed by high resolution two-dimensional gel electrophoresis (HR-2DE) and crossed immunoelectrophoresis (CIE). In 7 patients both glycoproteins (GPs) IIb and Ilia were absent or reduced to roughly the same extent. In two related patients only a trace of GP Ilb-IIIa complex was detected in CIE, but HR-2DE revealed a glycopeptide in the position of GP Ilia in an amount comparable to type II thrombasthenia. This GP Ilia-like component was neither recognized normally by anti-GP Ilb-IIIa antibodies nor labeled by surface iodination. In unreduced-reduced two-dimensional gel electrophoresis two components were observed in the region of GP Ilia. The assumption of a structural variant of GP Ilia in the two related patients is discussed.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

Antithrombin III Alger: A New Homozygous AT III Variant

1986 ◽  
Vol 55 (02) ◽  
pp. 218-221 ◽  
Author(s):  
A M Fischer ◽  
P Cornu ◽  
C Sternberg ◽  
F Mériane ◽  
M D Dautzenberg ◽  
...  
Keyword(s):  
Family History ◽  
Antithrombin Iii ◽  
Antigen Concentration ◽  
Crossed Immunoelectrophoresis ◽  
The Family ◽  
At Iii ◽  
Molecular Abnormality ◽  
Slow Moving ◽  
Cofactor Activity ◽  
Plasma Heparin

SummaryA qualitative abnormality of antithrombin III (AT III) was found in the plasma of a 41-year old patient. The plasmatic AT III antigen concentration was 130% and the progressive anti-F IIa and anti-F Xa activities were normal (105% and 137%). The plasma heparin cofactor activity was less than 10%, when measured by F Ila or F Xa inhibition. Crossed immunoelectrophoresis of AT III in the presence of heparin revealed in the plasma an abnormal slow-moving peak. When tested by affinity chromatography on heparin Sepharose, this abnormal AT III did not bind to heparin. Among the investigated relatives, 5 subjects had normal AT III levels, whatever the test used, the nine others having reduced levels of antithrombin heparin cofactor activity (45-61%) but normal levels of immunoreactive AT III (97-122%). Consanguinity was found in the family history. We therefore considered our patient as homozygous for an AT III molecular abnormality affecting the binding site for heparin.

Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

1986 ◽  
Vol 56 (03) ◽  
pp. 349-352 ◽  
Author(s):  
A Tripodi ◽  
A Krachmalnicoff ◽  
P M Mannucci
Keyword(s):  
Venous Thromboembolism ◽  
Active Site ◽  
Inhibitory Activity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Molecular Defect ◽  
Affinity Adsorption ◽  
Italian Family ◽  
Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

α2-Antiplasmin: Functional Characterization and Metabolism in a Heterozygote Deficient Patient

1986 ◽  
Vol 55 (03) ◽  
pp. 375-378 ◽  
Author(s):  
E A R Knot ◽  
J W ten Cate ◽  
R J Lamping ◽  
Liem Kian Gie
Keyword(s):  
Half Life ◽  
Functional Characterization ◽  
Bleeding Tendency ◽  
Normal Range ◽  
Crossed Immunoelectrophoresis ◽  
Synthetic Rate ◽  
Catabolic Rate ◽  
Deficient Patient ◽  
Antigen Activity ◽  
Bleeding History

SummaryAn 81-year-old male with a mild life-long bleeding history and an α2-antiplasmin (α2-AP) plasma level of 55% biological activity and 41% antigen activity (normal range 80-140%) was studied. The ratio of plasminogen binding (PB): non-plasminogen binding (NPB) α2-AP assayed by modified crossed immunoelectrophoresis (CIE) was 7.3/2.7 (controls 6.3 ± 0.49 SD/3.7 ± 0.49 SD). The patient’s α2-AP showed decreased affinity for fibrin, i. e. 8.3% versus 32.4% of normal control α2-AP associated with fibrin during clotting of plasma. A metabolic study performed with human purified 125I-α2-AP(PB/NPB 7.7/2.3) showed a plasma radioactivity disappearance half-life of 72.9 h (n 60.1 ± 5.3 h) with a normal fractional catabolic rate and a reduced absolute catabolic (synthetic) rate of 0.70 mg/kg/day (n 2.10 ± 0.60 mg/kg/day). The exchange between the central and third compartment was increased. The increased α2-AP PB form and the increased plasma radioactivity disappearance half-life are suggestive of a slower conversion of the PB form into the NPB form and/or slower degradation of the PB form. The bleeding tendency in this patient could be explained by decreased synthesis of α2-AP and decreased binding to fibrin.

Immunochemical Characterization of Setaria cervi Microfilarial Antigens Using Novel Antibodies

2019 ◽  
Vol 19 (14) ◽  
pp. 1263-1274 ◽  
Author(s):  
Anuradha Kalani ◽  
Komal Kalani ◽  
Poonam Chaturvedi ◽  
Pankaj Chaturvedi
Keyword(s):  
Adult Worm ◽  
Diagnostic Tests ◽  
Protective Immunity ◽  
Complex Nature ◽  
Immune Recognition ◽  
Setaria Cervi ◽  
Crossed Immunoelectrophoresis ◽  
Study Results ◽  
Immune Rabbit

Background:Filariasis affects millions of people in tropical and subtropical regions of the world and is caused by nematode roundworm. In order to develop a vaccine and specific diagnostic tests, it is important to characterize different stages of the filarial worms. Microfilariae (Mf) stage of the roundworm is found in host’s blood or lymph vessels and can be important not only for developing better immunodiagnostics but also for understanding immune recognition and its relevance to immunepathogenesis and protective immunity.Objective:The present study aimed to immunocharacterize Mf and adult worm antigens that could be helpful in future diagnostic tests.Method:Four different immune sera against Setaria cervi intact live, intact live with adjuvant, intact glutaraldehyde fixed with adjuvant and total somatic Mf were prepared and used for the immunocharacterization of Mf antigens.Results:Our study results suggest that compared to fixed intact Mf, live intact Mf are more immunogenic, as the immune sera generated against intact live Mf showed high ELISA reactivity with Setaria cervi Mf and adult worm antigens. All the four immune sera IgG fractions had surface specificity as determined through considerable ELISA reactivity with S. cervi intact Mf. When tested under native conditions (immunoelectrophoresis and crossed immunoelectrophoresis), all the four immune rabbit sera were able to detect antigens of S. cervi Mf and adult stages.Conclusion:These results can be useful in detailed understanding of the complex nature of the Mf and adult antigens, which are prerequisites in the development of vaccine and more specific diagnostic tests.

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