Identification of a nucleotide-binding site on glycoprotein IIb. Relationship to ADP-induced platelet activation.
Formalin-fixed platelets have been used to study the binding of adenine nucleotides in order to avoid the complications of nucleotide metabolism and to achieve steady-state binding. Sp-adenosine-5'-(1-thiotriphosphate) (Sp-ATP-alpha-S) binds to platelets at two sites (Kd1 3 nM; 31,000 sites/platelet; Kd2 200 nM; 300,000 sites/platelet) as compared with values for ADP under these conditions (Kd1 30 nM; 25,000 sites/platelet and Kd2 3 microM; 400,000 sites/platelet) (bound/total approximately 0.1). Competition binding experiments showed that both of the ATP-alpha-S sites were accessible to ADP and vice versa. [35S]ATP-alpha-S was photoaffinity cross-linked to unfixed platelets by direct irradiation with ultraviolet light. A single radiolabeled component (120 kDa) was identified and shown to be identical with the alpha subunit of GPIIb based on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting with anti-GPIIb monoclonal antibodies, by isoelectric focusing (pI 4.5-5.5), by immunoaffinity adsorption using monoclonal anti-GPIIb/IIIa antibodies coupled to Sepharose, and by crossed immunoelectrophoresis. Amino-terminal sequencing of a tryptic fragment labeled with [35S]ATP-alpha-S identified an 18-kDa domain beginning at Tyr-198 in the primary sequence of GPIIb alpha. These studies demonstrate the presence of an adenine nucleotide-binding site on GPIIb alpha.
Greco
N J
NJ
Cell Biology Laboratory, American Red Cross, Rockville, Maryland 20855.
Yamamoto
N
N
Jackson
B W
BW
Tandon
N N
NN
Moos
M
M
Jamieson
G A
GA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D001665:Binding Sites; D001667:Binding, Competitive; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008970:Molecular Weight; D010777:Photochemistry; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism
D000345:Affinity Labels; D010980:Platelet Membrane Glycoproteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate