Attempts to Define a Platelet ADP Receptor with 203Hg-p-Mercuribenzene Sulphonate (MBS)

ADP inhibits stimulation of platelet adenylate cyclase through a receptor with specificity and affinity indistinguishable from those of the ADP receptor mediating aggregation. The former, but not the latter effect is blocked by MBS (0.3–0.6 μM in PRP) indicating involvement of an externally oriented thiol. To remove effects of plasma thiols, aspirin treated platelets, labelled with 14C-adenine, were washed successively by centrifugation into albumin and passage through sepharose 2B into Ca-free Tyrode’s solution; this preserved their responses to PGE1 and ADP. Platelets incubated with 203Hg-MBS were pelleted through a layer of radiographic contrast medium. MBS binding showed a saturable high affinity component (ca. 250,000 sites/cell: 1/2 saturated at 8–10 μM). Excess cysteine reduced but did not eliminate non-saturable binding. Treatment of the platelets with Ellmans reagent (DTNB) reduced the apparent number of high affinity sites to ca. 100,000 and increased the apparent affinity of binding to ca. 2.5 μM. Effects of MBS on adenyl cyclase, monitored in the same experiment, correlated closely with high affinity binding. SDS electrophoresis of unreduced platelets on gradient Polyacrylamide gel slabs showed preferential labelling of several proteins by MBS, but the results suggested some redistribution of the label during extraction and separation. The number of high affinity sites for MBS found indicates the maximum number of thiols involved in ADP effects on adenylate cyclase. The true number is probably much smaller as MBS will label all externally available thiols.