A monoclonal antibody to a membrane glycoprotein binds only to activated platelets.
Rearrangements of membrane glycoproteins are believed to occur during platelet activation, but these changes have not been well defined. We have developed a monoclonal antibody, named S12, which demonstrates dramatically enhanced binding to platelets after thrombin activation. Unstimulated gel-filtered platelets from 12 normal individuals bound only 800 +/- 470 (S.D.) 125I-S12 molecules/cell, while platelets stimulated with 0.5 unit/ml of thrombin bound 9,600 +/- 2,600 molecules/cell (KD = 1.5 nM). Increasing thrombin concentrations produced similar increases in platelet 125I-S12 binding and [14C]serotonin secretion. S12 binding was not dependent on divalent cations. ADP and epinephrine, which caused no [14C]serotonin secretion, had little or no effect on S12 binding. We isolated the S12 binding protein by affinity chromatography of Lubrol PX-solubilized human platelet membranes on S12-agarose. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the isolated protein stains with both periodic acid-Schiff and Coomassie Blue and has an apparent molecular weight of 138,000 (unreduced) and 148,000 (reduced). After radioiodination of intact platelets the protein was also labeled, with apparently equal intensity in both control and thrombin-stimulated cells. The protein's staining, radiolabeling properties, and mobility on sodium dodecyl sulfate gels relative to glycoprotein IIb-IIIa fit previously defined criteria for membrane glycoprotein IIa. Our studies provide further evidence for alterations in membrane glycoproteins after platelet stimulation and suggest that S12 may serve as a useful probe of in vivo platelet activation.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D001792:Blood Platelets / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004837:Epinephrine / Q000494:pharmacology; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D013917:Thrombin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins; D000244:Adenosine Diphosphate; D013917:Thrombin; D004837:Epinephrine