Adenine nucleotide binding and photoincorporation in Glanzmann's thrombasthenia platelets

Abstract

Adenosine 5′-(1-thiotriphosphate) (ATPαS) binds to about 25 000 high affinity sites in platelets ( K d ∼ 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 α chain of glycoprotein IIb (GPIIba) 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, 20000 sites/platelet) but, in this case, photoincorporation occurred equally in GPIIb and GPIIIa. To determine the possible function of GPIIba in ADP-induced activation, platelets were isolated from two Glanzmann's thrombasthenia patients whose platelets contain ∼6% of normal levels of GPIIb. ADP and ATPaS bound to intact, formaldehyde-fixed Glanzmann's platelets at high affinity sites with dissociation constants of ∼30 nM and ∼2 nM, respectively. Both nucleotides also bound to low affinity sites with dissociation constants of ∼2 μM: these values are similar to those obtained with control platelets. ATPαS antagonized the shape ADP-induced shape change response of Glanzmann's platelets (EC 50 5 μM) indicating that it bound to the P 2T (ADP) receptor. However, photoincorporation was low (∼7% of control) similar to their content of GPIIba. 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 GPIIbα.