Identification of a nucleotide-binding site on glycoprotein IIb. Relationship to ADP-induced platelet activation

• Published in: The Journal of biological chemistry 1991-07, Vol.266 (21), p.13627-13633
• Main Authors: N J Greco, N Yamamoto, B W Jackson, N N Tandon, M Moos, Jr, G A Jamieson
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - metabolism; Adenosine Triphosphate - metabolism; Affinity Labels; Analytical, structural and metabolic biochemistry; Binding Sites; Binding, Competitive; Biological and medical sciences; Electrophoresis, Gel, Two-Dimensional; Fundamental and applied biological sciences. Psychology; Glycoproteins; Humans; Immunoelectrophoresis, Two-Dimensional; In Vitro Techniques; Isoelectric Point; Molecular Weight; Photochemistry; Platelet Activation; Platelet Membrane Glycoproteins - chemistry; Platelet Membrane Glycoproteins - metabolism; Proteins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)92746-8

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.