Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex

1986 ◽  
Vol 6 (3) ◽  
pp. 323-333 ◽  
Author(s):  
Christopher Bird ◽  
Marion Callus ◽  
Lynne Trickett ◽  
Robin Thorpe
Keyword(s):  
Monoclonal Antibody ◽  
Fibroblast Cell ◽  
Cytoskeletal Proteins ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Membrane Glycoprotein ◽  
Polymorphonuclear Leucocytes ◽  
Microfilament System ◽  
Fibroblast Cell Lines

We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin.Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system.

Ristocetin and Botrocetin Involve Two Distinct Domains of von Willebrand Factor for Binding to Platelet Membrane Glycoprotein lb

1990 ◽  
Vol 64 (02) ◽  
pp. 326-332 ◽  
Author(s):  
J P Girma ◽  
Y Takahashi ◽  
A Yoshioka ◽  
J Diaz ◽  
D Meyer
Keyword(s):  
Von Willebrand Factor ◽  
Synthetic Peptides ◽  
Competitive Inhibition ◽  
Final Concentration ◽  
Binding Domain ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Membrane Glycoprotein ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryWe have evidence that ristocetin and botrocetin mediate binding of von Willebrand Factor (vWF) to platelet glycoprotein lb (GPIb) through two distinct domains on the vWF molecule. This was established by using monoclonal antibodies (MAbs) to vWF and synthetic peptides derived from the sequence of vWF. MAb 322 and MAb NMC/vW 4 both recognize native vWF as well as fragments containing the GPIb-binding domain of vWF, obtained with the following enzymes: trypsin (116 kDa), V-8 pro tease (Spill, 320 kDa) and V-8 protease plus subtilisin (33-28 kDa). Nevertheless, the lack of reciprocal displacement between the two MAbs in experiments of competitive inhibition for binding to vWF demonstrate that their respective epitopes are separate. Both MAbs inhibit 125I-vWF binding to platelet membrane GPIb and vWF-dependent platelet agglutination induced by ristocetin. However, only MAb NMC/vW4 inhibits these functions in the presence of botrocetin and when ristocetin-induced platelet agglutination is inhibited by MAb 322, botrocetin is still able to restore the agglutination. The involvement of two distinct domains of vWF for binding to GPIb in the presence of ristocetin or botrocetin was confirmed in experiments of binding of 125I-vWF to platelets using as competitor synthetic peptides corresponding to the GPIb binding domain of vWF (Cys 474 to Pro 488 and Ser 692 to Pro 708). At a final concentration of 2.5 mM both peptides inhibit more than 90% of the binding of vWF to ristocetin-treated platelets but are unable to modify this binding in the presence of botrocetin. In conclusion our data suggest that botrocetin and ristocetin involve distinct sites on vWF for binding to GPIb.

Generation and Characterization of a Murine Monoclonal Antibody Specific for the Human T1-Cd5 Molecule

1987 ◽  
Vol 2 (3) ◽  
pp. 143-150 ◽  
Author(s):  
Federico Genzano ◽  
Ada Funaro ◽  
Massimo Alessio ◽  
Lucia B. De Monte ◽  
Graziella Bellone ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
T Lymphocytes ◽  
Membrane Glycoprotein ◽  
Functional Features ◽  
Specific Membrane ◽  
Murine Monoclonal Antibodies ◽  
Selection Of

Murine monoclonal antibodies (MoAbs) have found widespread applications in the characterization of the molecular and functional features of lymphocyte differentiation antigens. The present paper summarizes the results of our work dealing with the production and selection of a murine MoAb recognizing a molecule expressed during the whole differentiative life of T lymphocytes. The MoAb CB01 resulted to be specific for an apparently unique epitope of the T-cell specific membrane glycoprotein T1-CD5.

Engineering and Characterization of a Chimeric Anti-platelet Glycoprotein Ibα Monoclonal Antibody and Preparation of Its Fab Fragment

Hybridoma ◽  
2010 ◽  
Vol 29 (2) ◽  
pp. 125-132 ◽  
Author(s):  
Jianfeng Yang ◽  
Shundong Ji ◽  
Ningzheng Dong ◽  
Yiming Zhao ◽  
Changgeng Ruan
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Glycoprotein ◽  
Fab Fragment

Immunological characterization of cytoskeletal proteins associated with the basal body, axoneme and flagellum attachment zone of Trypanosoma brucei

Parasitology ◽  
1995 ◽  
Vol 111 (1) ◽  
pp. 77-85 ◽  
Author(s):  
R. Woodward ◽  
M. J. Carden ◽  
K. Gull
Keyword(s):  
Monoclonal Antibody ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Complex Mixture ◽  
Cytoskeletal Proteins ◽  
Bovine Sperm ◽  
Phosphatase Treatment ◽  
Body Complex ◽  
Attachment Zone

SUMMARYThe monoclonal antibody BS7, raised to bovine sperm flagellum cytoskeletal antigens in a previous study, is here reported to detect flagellum-associated structures in Trypanosoma brucei and Crithidia fasciculata. Immunoblotting showed that BS7 cross-reacts with several cytoskeletal T. brucei proteins but phosphatase treatment did not diminish this complex immunoblot reactivity. To characterize further the cross-reactive proteins recognized in T. brucei-cytoskeletons by BS7 each was excised from preparative gels and used as an immunogen for antiserum production. Two proteins, with apparent sizes around 43 and 47 kDa, produced antisera shown to be monospecific by immunoblotting total T. brucei flagellum preparations. Each of these detected the basal body-associated immunofluorescence in T. brucei. Identification of the smaller, 43 kDa, component as a basal body-associated product was supported by the behaviour of a second monoclonal antibody, BBA4, which was also shown to detect the T. brucei basal body complex by immunofluorescence and immunoblots the 43 kDa polypeptide. These observations reveal new components of the trypanosome cytoskeleton. Also, they provide a further example of an immunological approach for identification of interesting, rare components of the T. brucei cytoskeleton starting from a complex mixture of proteins.

scholarly journals Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: Model systems for equine sarcoids

Virology ◽  
2008 ◽  
Vol 373 (2) ◽  
pp. 352-361 ◽  
Author(s):  
Z.Q. Yuan ◽  
E.A. Gault ◽  
P. Gobeil ◽  
C. Nixon ◽  
M.S. Campo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fibroblast Cell ◽  
Model Systems ◽  
Fibroblast Cell Lines

scholarly journals Unravelling the mechanism and significance of thrombin binding to platelet glycoprotein Ib

2010 ◽  
Vol 104 (11) ◽  
pp. 894-902 ◽  
Author(s):  
Alessandro Zarpellon ◽  
James Roberts ◽  
Richard Mc Clintock ◽  
Hua Jing ◽  
G. Loredana Mendolicchio ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Vascular Injury ◽  
Platelet Membrane ◽  
Active Enzyme ◽  
Platelet Glycoprotein ◽  
Membrane Glycoprotein ◽  
Main Question ◽  
Glycoprotein Ib ◽  
Platelet Membrane Glycoprotein

SummaryThe main question concerning the mechanism of α-thrombin binding to platelet membrane glycoprotein (GP)Ib is whether it involves both thrombin exosite I and exosite II. The solution of two independent crystal structures suggests alternative explanations that may actually reflect different modes of binding with distinct pathophysiological significance. With respect to function, it is still unclear whether thrombin binding to GPIb promotes procoagulant and prothrombotic pathways of re-sponse to vascular injury or limits such responses by sequestering, at least temporarily, the active enzyme. We review here published information on these topics and touch upon ongoing studies aimed at finding definitive answers to outstanding questions relevant for a better understanding of thrombosis and haemostasis.

Characterization of Three Alboaggregins Purified from Trimeresurus albolabris Venom

1992 ◽  
Vol 67 (06) ◽  
pp. 702-707 ◽  
Author(s):  
Manling Peng ◽  
Weiqi Lu ◽  
Edward P Kirby
Keyword(s):  
Monoclonal Antibody ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Glycoprotein ◽  
Molecular Weights ◽  
New Family ◽  
Competitive Inhibitors ◽  
Ion Exchange Hplc ◽  
Formalin Fixed

SummaryAlboaggregins (AL-A, AL-B, AL-C) isolated from Trimeresurus albolabris snake venom represent a new family of proteins which bind to platelet glycoprotein lb (GPIb). These alboaggregins were purified to homogeneity with ion exchange HPLC (Mono-Q column) and hydrophobic HPLC (TSK Phenyl-5PW column). On SDS-polyacrylamide gel electrophoresis, apparent molecular weights of AL-A, AL-B and AL-C were 52 kDa, 26 kDa, and 121 kDa respectively, under nonreducing conditions. Upon reduction, each alboaggregin showed two types of chains with apparent molecular weights in the range of 15-20 kDa. All three alboaggregins agglutinated formalin-fixed platelets. Agglutination activities and binding of labeled alboaggregins to GPIb were specifically inhibited by the monoclonal antibody AK2 which is directed against the 45 kDa N-terminal region on GPIb, but not by monoclonal antibodies against other epitopes on GPIb. 125I-alboaggregin binding to platelets was not altered by the presence of thrombin. Alboaggregins did not bind to GPIIb/IIIa. Alboaggregins were competitive inhibitors for 125I-bovine vWF binding to platelets. Mutual competition studies between AL-A, AL-B and AL-C for the binding of labeled bovine vWF and AL-B to platelets demonstrated that AL-B and AL-C had a significantly higher affinity than AL-A.

scholarly journals Necessity of extracellular domain of W (c-kit) receptors for attachment of murine cultured mast cells to fibroblasts

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 650-656 ◽  
Author(s):  
S Adachi ◽  
Y Ebi ◽  
S Nishikawa ◽  
S Hayashi ◽  
M Yamazaki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Mast Cells ◽  
Point Mutations ◽  
Fibroblast Cell ◽  
Extracellular Domain ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Murine Embryos ◽  
Intracellular Tyrosine Kinase Domain ◽  
Fibroblast Cell Lines

Abstract The receptor encoded by the W (c-kit) locus (W receptor) is expressed on the surface of cultured mast cells (CMC) derived from normal (+/+) mice, whereas its ligand encoded by the Sl locus (Sl ligand) is expressed on the surface of fibroblast cell lines derived from murine embryos. Involvement of W receptors and Sl ligands in attachment of CMC to fibroblasts was investigated. CMC were cocultured with fibroblasts; nonattaching CMC were removed and the remaining CMC were counted. CMC derived from mice of the W/W genotype did not express the extracellular domain of W receptors, and attachment of W/W CMC to +/+ fibroblasts was significantly impaired. Fibroblasts derived from embryos of the Sl/Sl genotype did not express Sl ligands, and the attachment of +/+ CMC to Sl/Sl fibroblasts was also impaired. The Wv and W42 alleles are point mutations at the intracellular tyrosine kinase domain. Attachment of either Wv/Wv, W/Wv, or W/W42 CMC to +/+ fibroblasts was comparable with that of +/+ CMC. Moreover, the addition of monoclonal antibody against the extracellular domain of W receptors inhibited the attachment of +/+ CMC to +/+ fibroblasts. Thus, the extracellular domain of W receptors appeared to be necessary for attachment of CMC to fibroblasts.

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