Adenine nucleotide binding and photoincorporation in Glanzmann's thrombasthenia platelets.
Adenosine 5'-(1-thiotriphosphate) (ATP alpha S) binds to about 25,000 high affinity sites in platelets (Kd approximately 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 alpha 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 (Kd 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 approximately 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 approximately 30 nM and approximately 2 nM, respectively. Both nucleotides also bound to low affinity sites with dissociation constants of approximately 2 microM: 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 (EC50 5 microM) indicating that it bound to the P2T (ADP) receptor. However, photoincorporation was low (approximately 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.
Greco
N J
NJ
American Red Cross, Cell Biology Laboratory, Rockville, MD 20855, USA.
Tandon
N N
NN
Jackson
B
B
Jamieson
G A
GA
D000227:Adenine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D015539:Platelet Activation / Q000187:drug effects; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology*; D013915:Thrombasthenia / Q000378:metabolism*
D000227:Adenine Nucleotides; D000345:Affinity Labels; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate