scholarly journals Immunolocalization of beta 1 integrins in platelets and platelet- derived microvesicles

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1197-1203 ◽  
Author(s):  
JD Wencel-Drake ◽  
MG Dieter ◽  
SC Lam
Keyword(s):  
Cellular Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Fluorescent Labeling ◽  
Platelet Glycoprotein ◽  
Resting Cells ◽  
Intact Cells ◽  
Platelet Surface ◽  
Permeabilized Cells

Abstract Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.

scholarly journals Immunolocalization of beta 1 integrins in platelets and platelet- derived microvesicles

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1197-1203 ◽  
Author(s):  
JD Wencel-Drake ◽  
MG Dieter ◽  
SC Lam
Keyword(s):  
Cellular Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Fluorescent Labeling ◽  
Platelet Glycoprotein ◽  
Resting Cells ◽  
Intact Cells ◽  
Platelet Surface ◽  
Permeabilized Cells

Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.

scholarly journals Plasmin-induced redistribution of platelet glycoprotein Ib

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2005-2010 ◽  
Author(s):  
AD Michelson ◽  
MR Barnard
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glycoprotein Ib ◽  
Autologous Plasma ◽  
Proteolytic Fragment ◽  
Platelet Surface ◽  
Microfilament System

Platelet membrane glycoprotein Ib (GPIb), a receptor for von Willebrand factor and thrombin, is present on the platelet surface membrane, in intraplatelet stores, and in plasma (as the proteolytic fragment glycocalicin). We examined the hypothesis that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool. Incubation of washed platelets with plasmin (1 hour, 22 degrees C) resulted in loss of platelet surface GPIb, but further incubation (3 hours, 37 degrees C) in autologous plasma resulted in restoration of platelet surface GPIb, as determined by ristocetin- induced platelet agglutination and a flow cytometric assay of platelet binding of three GPIb-specific monoclonal antibodies. Despite the restoration of platelet surface GPIb after the 3-hour incubation of plasmin-treated platelets in autologous plasma, the whole platelet GPIb content (measured by enzyme-linked immunosorbent assay [ELISA], sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and flow cytometry) remained reduced, quantitatively corresponding to an increase in plasma glycocalicin concentration (measured by ELISA). The loss and restoration of platelet surface GPIb occurred on all platelets and, as evidenced by lack of inhibition by prostaglandin E1, EDTA, and cytochalasins, was not mediated by cyclic AMP, extracellular Ca2+, or the platelet microfilament system. In summary, this study shows that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool by recruitment of GPIb molecules from the intraplatelet pool (or from a sequestered surface site).

scholarly journals Plasmin-induced redistribution of platelet glycoprotein Ib

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2005-2010 ◽  
Author(s):  
AD Michelson ◽  
MR Barnard
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glycoprotein Ib ◽  
Autologous Plasma ◽  
Proteolytic Fragment ◽  
Platelet Surface ◽  
Microfilament System

Abstract Platelet membrane glycoprotein Ib (GPIb), a receptor for von Willebrand factor and thrombin, is present on the platelet surface membrane, in intraplatelet stores, and in plasma (as the proteolytic fragment glycocalicin). We examined the hypothesis that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool. Incubation of washed platelets with plasmin (1 hour, 22 degrees C) resulted in loss of platelet surface GPIb, but further incubation (3 hours, 37 degrees C) in autologous plasma resulted in restoration of platelet surface GPIb, as determined by ristocetin- induced platelet agglutination and a flow cytometric assay of platelet binding of three GPIb-specific monoclonal antibodies. Despite the restoration of platelet surface GPIb after the 3-hour incubation of plasmin-treated platelets in autologous plasma, the whole platelet GPIb content (measured by enzyme-linked immunosorbent assay [ELISA], sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and flow cytometry) remained reduced, quantitatively corresponding to an increase in plasma glycocalicin concentration (measured by ELISA). The loss and restoration of platelet surface GPIb occurred on all platelets and, as evidenced by lack of inhibition by prostaglandin E1, EDTA, and cytochalasins, was not mediated by cyclic AMP, extracellular Ca2+, or the platelet microfilament system. In summary, this study shows that after plasmin-mediated cleavage of platelet surface GPIb, platelets can replenish their surface GPIb pool by recruitment of GPIb molecules from the intraplatelet pool (or from a sequestered surface site).

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Snatashree Mohanty ◽  
M. Makesh ◽  
K. V. Rajendran ◽  
P. P. Suresh Babu ◽  
Deepika Anand ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Catla Catla ◽  
Cirrhinus Mrigala ◽  
Indian Major Carps ◽  
Sds Page ◽  
Igg1 Isotype ◽  
Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

scholarly journals Generation of a Monoclonal Antibody against D-Dimer Using HTS-Based LiCA

2019 ◽  
Vol 25 (3) ◽  
pp. 310-319
Author(s):  
Yuan Dong ◽  
Hanjin Hou ◽  
An Chen ◽  
Wei Ma ◽  
Moli Yin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Enzyme Hydrolysis ◽  
Clinical Samples ◽  
Sds Page ◽  
Thrombotic Diseases ◽  
D Dimer

D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.

Additional Glycoprotein Defects In Glanzmann’s Thrombasthenia Platelets

1981 ◽  
Author(s):  
J L McGregor ◽  
K J Clemetson ◽  
E James ◽  
A Capitanio ◽  
M Dechavanne ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Gel Electrophoresis ◽  
Lens Culinaris ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Platelet Surface ◽  
Glanzmann’S Thrombasthenia ◽  
Healthy Donors ◽  
Glanzmann's Thrombasthenia ◽  
Dimensional Electrophoresis

Glanzmann’s thrombasthenia (G.T.) platelets are deficient in 2 major membrane GP (IIb and IIIa). In order to investigate if these are the only defects in this disorder, platelets from G.T. patients and from healthy donors were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetylgalactosamine residues). Labelled or unlabelled platelets were solubilized in sodium dodecyl sulphate (SDS) and separated by 2-dimensional polyacrylamide gel electrophoresis, first according to isoelectric point and then according to molecular weight. Glycoproteins from unlabelled platelets separated by 2-dimensional electrophoresis were identified by binding of 125I-labelled Lens culinaris lectin (mannose, glucose specific) GPIIbA1 and IIIaA1 were absent in one G.T. patient while in others lower amounts of 2 GP were found in positions similar to these GP. Major membrane GP (IbA1, IbA2, IbB1 and IIIbA1) had more intensely labelled terminal sialic acid moieties in G.T. platelets than in normals. A major membrane GP designated Ic had an altered pi and its penultimate galactose/N-acetyl galactosamine residues labelled more intensely in G.T. platelets than in controls. One high M.Wt. GP and a number of lower M.Wt. GP (IVa, IVb and VII) normally found in platelets of healthy donors were absent in G.T. platelets. These results indicate strongly that there is a major perturbation of the platelet surface in G.T.

scholarly journals Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1072-1080 ◽  
Author(s):  
B Rucinski ◽  
A Poggi ◽  
P James ◽  
JC Holt ◽  
S Niewiarowski
Keyword(s):  
Amino Acid ◽  
Basic Protein ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Heterologous Antigens

Abstract Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6–0.9 M NaCl and at 1–1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000–7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno- precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

scholarly journals Characterization of Proteins in the Outer Membrane Preparation of a Murine Pathogen, Helicobacter bilis

2001 ◽  
Vol 69 (5) ◽  
pp. 3502-3506 ◽  
Author(s):  
Zhongming Ge ◽  
Peter Doig ◽  
James G. Fox
Keyword(s):  
Outer Membrane ◽  
Wide Spectrum ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Membrane Preparation ◽  
Laboratory Animals ◽  
Helicobacter Bilis ◽  
H Pylori

ABSTRACT Helicobacter bilis is a bacterial pathogen associated with multifocal hepatitis and inflammatory bowel disease in certain strains of mice. This bacterium colonizes the liver, bile, and lower intestine in mice and has also been isolated from a wide spectrum of laboratory animals. In this study, proteins present in the outer membrane preparation (OMP) of four H. bilis strains isolated from a mouse, a dog, a rat, and a gerbil were characterized and compared with that of Helicobacter pylori, a human gastric pathogen. All four H. bilis strains had similar OMP protein profiles that were distinct from those of H. pylori. Immunoblotting demonstrated that OMP proteins fromH. bilis and H. pylori have little cross-reactivity, except for their flagellins. Nine major immunogenic polypeptides were present in the H. bilis OMPs. By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five heat-modifiable proteins with molecular masses of 82, 66, 52, 47 and 37 kDa were identified. The N-terminal sequences of the 46- and 47-kDa OMP proteins had no homology with protein sequences available in public databases. These results indicate that H. bilis has a conserved, unique OMP protein profile that is distinct from those of H. pylori.

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