ADENINE-NUCLEOTIDE BINDING AND PHOTOINCORPORATION IN GLANZMANNS-THROMBASTHENIA PLATELETS

Adenosine 5'-(1-thiotriphosphate) (ATP alpha S) binds to about 25 000 high affinity sites in platelets (K-d similar to 3 nM), competes fully in inhibiting the binding of ADP and, despite the absence of a specific photoactivatable substituent, is directly photoincorporated into a specific 18 kDa domain beginning at Tyr-198 in the Lu chain of glycoprotein IIb (GPIIb alpha) following ultraviolet irradiation of fresh unfixed platelets (Greco et al. (1991) J. Biol. Chem. 266, 13627-13633). 8-azido ATP has now been shown to have similar binding parameters (K-d 8 nM, 20 000 sites/platelet) but, in this case, photoincorporation occurred equally in GPIIb and GPIIIa. To determine the possible function of GPIIb alpha in ADP-induced activation, platelets were isolated from two Glanzmann's thrombasthenia patients whose platelets contain similar to 6% of normal levels of GPIIb. ADP and ATP alpha S bound to intact, formaldehyde-fixed Glanzmann s platelets at high affinity sites with dissociation constants of similar to 30 nM and similar to 2 nM, respectively. Both nucleotides also bound to low affinity sites with dissociation constants of similar to 2 mu M: these values are similar to those obtained with control platelets. ATP alpha S antagonized the shape ADP-induced shape change response of Glanzmann's platelets (EC(50) 5 mu M) indicating that it bound to the P-2T (ADP) receptor. How ever, photoincorporation was low (similar to 7% of control) similar to their content of GPIIb alpha, These results show that ADP binding and photoincorporation are occurring at different sites on the platelet surface but suggest that the ADP binding site may be located in proximity to GPIIb alpha.