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.