Attempts to photoaffinity label platelet ADP receptors with 2-azidoADP have not been successful possibly due to the absence of a spacer arm between the nucleotide and the photolabile group. We have synthesized a probe having a long spacer arm by coupling 2-(3-aminopropylthio)-ADP to succinimidyl 4-3H-azidobenzoate. Labeling competable by ADP could not demonstrated with intact platelets. With isolated platelet membranes, three bands (Mr 140,000, 110,000 and 46,000) were labeled that were not competed by ADP while three other bands (Mr 188,000, 92,000 and 51,000) were competable by 100 uM ADP.Another problem in characterizing ADP receptors has been complications due to ADP metabolism and secretion from the dense granules. To avoid this problem we have measured the binding of ADP and analogues to formalin-fixed platelets. ADP bound to two sites (Kl, 0.35 ± 0.04 uM; R1, 160,000 ± 20,000 sites/platelet; K2 7.9 ± 2.0 uM; R2, 400,000 ± 40,000 sites/platelet) with low non-specific binding: these values are in agreement with ADP concentrations required for activation. Affinity at the high affinity site was in the sequence ADP(0.35 uM)=ATP(0.4 uM)›2-MeS.ADP(6.8 uM)› GDP(49 uM) › AMP(360 uM); adenosine did not compete. Binding at the high affinity site was blocked by pMBS (EC50 250 uM) and 5-fluoro-sulfonylbenzoyladenosine (EC50 1 mM). This is the first report of photoaffinity labeling of putative ADP receptors. Our experiments with fixed platelets suggest that they may be useful in testing agonists, antagonists and inhibitors in the absence of complications due to secretion and metabolism.
Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states
