Unresponsiveness of Platelets Lacking Both G q and G 13 IMPLICATIONS FOR COLLAGEN-INDUCED PLATELET ACTIVATION* Received for publication, August 5, 2004, and in revised form, August 18, 2004 Published, JBC Papers in Press, August 23, 2004, DOI 10.1074/jbc.M408962200 Alexandra Moers‡, Nina Wettschureck‡, Sabine Gru ¨ ner§, Bernhard Nieswandt§, and Stefan Offermanns‡¶ From the ‡Pharmakologisches Institut, Universita ¨ t Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany, and §Rudolf-Virchow-Zentrum fu ¨ r Experimentelle Biomedizin, Versbacher St. 9, 97078 Wu ¨ rzburg, Germany The diffusible platelet stimuli ADP and thromboxane A 2 activate multiple G protein-mediated signaling path- ways and function as important secondary mediators of platelet activation as they are released from activated platelets. Because they can also increase their own for- mation and release, their effects are amplified; eventu- ally, all major G protein-mediated signaling pathways are activated. The multiple positive feedback mecha- nisms operating during platelet activation have ob- scured the exact analysis of the roles individual G pro- tein-mediated signaling pathways play during the platelet activation process. In this report, we show that platelets lacking G q and G 13 are completely unrespon- sive to diffusible stimuli such as ADP, thromboxane A 2 , or thrombin, even when applied at very high concentra- tions in combination, whereas all stimuli are able to induce platelet aggregation, shape change, and RhoA activation in platelets lacking only one G subunit. This shows that G q or G 13 is required to induce some platelet activation, whereas the activation of G i -mediated sig- naling alone is not sufficient to induce activation of mouse platelets. In addition, platelets lacking G q and G 13 adhered normally to collagen under high shear but did not aggregate any more in response to collagen, indicating that collagen-induced platelet activation but not platelet adhesion requires intact G protein-medi- ated signaling pathways. Platelet adhesion and activation at sites of vascular injury and subsequent formation of a platelet plug is initiated by a multistep process involving the interaction of platelets with adhesive macromolecules, such as collagen and von Willebrand factor, at the subendothelial surface (1, 2). The initial interac- tion of platelets with the extracellular matrix under the condi- tion of high shear rates is mediated by GPIb-V-IX, which in- teracts with von Willebrand factor bound to subendothelial collagen (3) and induces rolling of platelets to the subendothe- lium. During the rolling process in particular, the collagen receptor glycoprotein VI (GPVI) 1 interacts with the extracellu- lar matrix and via the FcR chain induces signaling pathways involving phospholipase C2 and phosphoinositide-3 kinase (4). These signaling processes promote some inside-out activa- tion of such integrins as IIb3 (GPIIb-IIIa) or 21 (GPIa- IIa), which results in the firm adhesion of platelets to the injured vessel wall (5). Although collagen is able to induce some platelet activation, the recruitment of additional platelets to the growing platelet plug involves the local accumulation of diffusable mediators, which are produced or released once platelet adhesion has been initiated and some level of activa- tion through platelet adhesion receptors has occurred (6). These mediators include ADP/ATP and thromboxane A 2 (TxA 2 ) which are secreted or released from activated platelets as well as thrombin, which is produced on the surface of activated platelets. These platelet stimuli have in common their action through G protein-coupled receptors. Whereas ADP induces the activation of G q and G i via P2Y 1 and P2Y 12 receptors (7, 8), the activated TxA 2 receptor couples to G q and G 12 /G 13 (9, 10). G protein-coupled protease-activated receptors, which are acti- vated by thrombin, are functionally coupled to G q ,G 12 /G 13 , and, in some cases, G i (11). Progress has recently been made using genetic mouse models in understanding the role of indi- vidual G protein-mediated signaling pathways during platelet activation. On the basis of platelets from mice lacking individ- ual G protein -subunits, three G proteins have been identified as major mediators of platelet activation via ADP and TxA 2 receptors: G q ,G i2 , and G 13 (12–15). Other G proteins, such as G z ,G i3 , or G 12 play only minor roles or are not coupled to the main platelet-activating receptors (12, 14, 16, 17). However, even in the absence of G q ,G i2 , or G 13 , some platelet activation can still be induced. For instance, platelets lacking G q still undergo the platelet shape change reaction in response to var- ious stimuli (10, 13), and costimulation of G i - and G 13 -mediated signaling pathways results in integrin IIb3 activation in the absence of G q (18, 19). In the absence of G i2 , ADP and thrombin can still induce platelet activation; however, the ef- fects are clearly impaired and require higher ligand concentra- tions than in wild-type platelets (12, 15). Finally, lack of G 13 in platelets severely reduces the potency of thrombin, TxA 2 , and collagen to induce platelet shape change and aggregation but does not render them completely unresponsive (14). In the case of G q and G 13 deficiency, in vivo functions are severely affected (13, 14). Thus, platelet activation through G protein- coupled receptors is an integrated process that requires differ- ent G protein signaling pathways involving at least G q ,G i , and G 13 . Although all three pathways seem to be required for effi- cient platelet activation, signaling through two of these path- ways is still sufficient to induce platelet activation. To further define the minimal requirement of G protein-mediated platelet activation, we have generated platelets lacking both G q , and G 13 . Herein, we show that in the absence of both G q and G 13 , ADP, TxA 2 , or thrombin can induce no platelet activation. This * This study was funded by the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence should be addressed. Tel.: 49-6221- 548246; Fax: 49-6221-548549; E-mail: stefan.offermanns@urz.uni- heidelberg.de. 1 The abbreviations used are: GPVI, glycoprotein VI; TxA 2 , throm- boxane A 2 ; MLC, myosin light chain. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 45354 –45359, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 45354 by guest on April 24, 2020 http://www.jbc.org/ Downloaded from