J. Biol. Chem., Vol. 266, Issue 21, 13627-13633, 07, 1991

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

NJ Greco, N Yamamoto, BW Jackson, NN Tandon, M Moos Jr and GA Jamieson
Cell Biology Laboratory, American Red Cross, Rockville, Maryland 20855.

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.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				N. J. Greco, G. Tonon, W. Chen, X. Luo, R. Dalal, and G. A. Jamieson Novel structurally altered P2X1 receptor is preferentially activated by adenosine diphosphate in platelets and megakaryocytic cells Blood, July 1, 2001; 98(1): 100 - 107. [Abstract] [Full Text] [PDF]

				E. L. Saenko, D. Scandella, A. V. Yakhyaev, and N. J. Greco Activation of Factor VIII by Thrombin Increases Its Affinity for Binding to Synthetic Phospholipid Membranes and Activated Platelets J. Biol. Chem., October 23, 1998; 273(43): 27918 - 27926. [Abstract] [Full Text] [PDF]

				N. J. Greco Functional Expression of a P2T ADP Receptor in Xenopus Oocytes Injected With Megakaryocyte (CMK 11-5) RNA Arterioscler. Thromb. Vasc. Biol., April 1, 1997; 17(4): 769 - 777. [Abstract] [Full Text]

				R. N. Puri, A. Kumar, H. Chen, R. F. Colman, and R. W. Colman Inhibition of ADP-induced Platelet Responses by Covalent Modification of Aggregin, a Putative ADP Receptor, by 8-(4-Bromo-2,3-dioxobutylthio)ADP J. Biol. Chem., October 13, 1995; 270(41): 24482 - 24488. [Abstract] [Full Text] [PDF]