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.
