Binding of adenosine diphosphate to intact human platelets.

1. Human platelet-rich plasma was incubated at 37 degrees C with [8-14C]ADP and with human serum albumin labelled with 125I. The platelets were rapidly separated by centrifugation through silicone oil. From radioactivity determinations of plasma and platelet pellets the uptake of ADP, without or with break-down products, by the platelets was calculated on the assumption that the 125I radioactivity in the pellet represented trapped plasma. 2. ADP radioactivity was taken up by platelets within 10 sec and increased with time of incubation. The uptake of other nucleotide diphosphates was less initially and increased much more slowly. 3. Radioactivity added as ADP was recovered as ATP to the extent of 60%; as ADP of 30%; and as AMP of 10%. 4. Prostaglandin E1 which inhibited platelet aggregation had no effect on the initial or subsequent uptake or on this distribution of radioactivity. 5. The rate of rise in uptake was much slower when platelets were resuspended in plasma heated to 56 degrees C for 30 min. 6. Unlabelled adenosine inhibited the later, but not the initial, uptake while unlabelled ADP inhibited both. Dipyridamole, which blocks adenosine uptake, prevented the later but not the initial uptake. 7. [alpha-32P]ADP radioactivity was taken up at the earliest sampling time and the extent of uptake did not further increase. 8. Guinea-pig platelets, which do not take up adenosine, took up [8-14C]ADP radioactivity from purine-labelled ADP initially. 9. It was concluded that the initial uptake represented binding of ADP and that the later uptake represented labelled adenosine originating as a break-down product of ADP. 10. A Scatchard plot of ADP uptake indicated more than one type of binding site. There were approximately 88,000 high affinity sites per platelet which had an affinity constant of 5-41 X 10(5) M-1.