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 specif ic photoactivatable substituent, is directly photoincorporated into a specific 18 kDa domain beginning at Tyr-198 in the Lu chain of glycopr otein IIb (GPIIb alpha) following ultraviolet irradiation of fresh unf ixed 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 oc curred equally in GPIIb and GPIIIa. To determine the possible function of GPIIb alpha in ADP-induced activation, platelets were isolated fro m two Glanzmann's thrombasthenia patients whose platelets contain simi lar to 6% of normal levels of GPIIb. ADP and ATP alpha S bound to inta ct, formaldehyde-fixed Glanzmann s platelets at high affinity sites wi th dissociation constants of similar to 30 nM and similar to 2 nM, res pectively. Both nucleotides also bound to low affinity sites with diss ociation constants of similar to 2 mu M: these values are similar to t hose obtained with control platelets. ATP alpha S antagonized the shap e 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 photo incorporation are occurring at different sites on the platelet surface but suggest that the ADP binding site may be located in proximity to GPIIb alpha.