Appearance of additional ouabain binding sites on platelet surfaces following activation.
Na,K-ATPase is an integral membrane protein complex involved in the regulation of intracellular Na+ and K+ ion levels. The number of accessible Na,K-ATPase complexes on the surface of human platelets before and after activation by adenosine 5'-diphosphate (ADP) was quantitated using ouabain, a highly specific inhibitor of this enzyme. Studies with [3H]ouabain revealed an increase in the number of accessible ouabain binding sites from approximately 55 per resting platelet to approximately 625 per ADP-activated platelet. Ultrastructural localization of this ion transport complex was also studied in resting human platelets or following activation by ADP. Localization was performed using a novel colloidal gold affinity probe which employed ouabain (ouabain-GAP) as a ligand specific for the Na,K-ATPase. Qualitatively, the number of surface-associated ouabain-GAP labels per platelet was observed to increase following activation by ADP. Fracture-labeling studies of frozen preparations of resting and freshly activated platelets suggested ouabain binding sites were associated with intracellular platelet structures during the early stages of activation. Together these results suggest an increase in the amount of ouabain binding sites at the surface of human platelets as an event of activation and that at least some of these additional sites may originate from intracellular sites.
Department of Biology, University of Oregon, Eugene 97403.
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Mrsny
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D010042:Ouabain / Q000378:metabolism*; D015539:Platelet Activation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D015854:Up-Regulation
D010042:Ouabain; D000244:Adenosine Diphosphate; D000254:Sodium-Potassium-Exchanging ATPase