Characterization of a monoclonal antibody ITI-Pl 1 directed against human platelet membrane glycoprotein IIb using extracts of whole platelets and platelet surface and intracellular membranes.
A monoclonal antibody (mAb) termed ITI-Pl 1 has been prepared by the hybridoma procedure. Using immuno-absorption and crossed immunoelectrophoresis of Triton X-100 extracts of untreated and EDTA-treated human platelets it was shown to be directed against the surface membrane glycoprotein IIb (GP IIb). This mAb binds to whole platelets independently of ADP-stimulation and the presence of Ca2+-ions. It saturates at around 870 ng/10(8) cells corresponding to approximately 35,800 molecules/platelet. ITI-Pl 1 did not significantly inhibit GP IIb-IIIa dependent functions such as platelet aggregation or fibrinogen binding. Immunofluorescence could be demonstrated using ITI-Pl 1 and intact normal platelets, but not with platelets from a Glanzmann's thrombasthenia patient. Crossed immuno-electrophoresis with platelet extracts from four different thrombasthenic patients gave a line precipitate in the intermediate gel with 125I-labelled ITI-Pl 1 and autoradiography indicating trace amounts of free GP IIb or the GP IIb-IIIa complex. The epitope on GP IIb detected by ITI-Pl 1 is not destroyed by neuraminidase treatment. Thus the mAb also interacts with neuraminidase-treated GP IIb-IIIa complex in highly purified platelet surface membrane fractions as well as with GP IIb-IIIa from untreated internal membranes isolated by continuous flow electrophoresis.
Thorsen
L I
LI
Research Institute for Internal Medicine, Rikshospitalet, Oslo, Norway.
Gaudernack
G
G
Hack
N
N
Wilkinson
J M
JM
Brosstad
F
F
Solum
N O
NO
Crawford
N
N
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001792:Blood Platelets / Q000276:immunology*; D002462:Cell Membrane / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007163:Immunosorbent Techniques; D007425:Intracellular Membranes / Q000276:immunology; D051379:Mice; D010980:Platelet Membrane Glycoproteins / Q000276:immunology*; D013915:Thrombasthenia / Q000276:immunology
D000911:Antibodies, Monoclonal; D010980:Platelet Membrane Glycoproteins