G-PROTEINS OF THE G(12) FAMILY ARE ACTIVATED VIA THROMBOXANE A(2) AND THROMBIN RECEPTORS IN HUMAN PLATELETS

Using subtype-specific antisera, we were able to identify the recently described alpha subunits of G(12) and G(13) in platelet membranes as 43-kDa proteins. Activation of the thromboxane A(2) and the thrombin receptors in platelet membranes led to increased incorporation of the photoreactive GTP analogue [alpha-P-32]GTP azidoanilide into immunoprecipitated alpha(12) and alpha(13), indicating that both receptors couple to G(12) and G(13). In addition, both activated receptors were demonstrated to couple to one or more members of the G(q) family. In the absence of receptor agonists, incorporation of [alpha-P-32]GTP azidoanilide into alpha(12) and alpha(13) was low over a long time period (up to 45 min) due to an obviously low basal nucleotide exchange rate, whereas an agonist-stimulated photolabeling of alpha(12) and alpha(13) could be observed after 4-8 min and reached a maximum after 30-45 min. Effective activation of G(12) and G(13) via the thromboxane A(2) and the thrombin receptors was not dependent on the presence of GDP. Our results provide evidence that G(12) and G(13) play a functional role in transmembrane signal transduction and suggest that both proteins are involved in pathways leading to platelet activation.