LETTERS

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

Alexandra Moers1,Bernhard Nieswandt2,Steffen Massberg3,Nina Wettschureck1, Sabine Grüner2, Ildiko Konrad3,Valerie Schulte2,Barsom Aktas2,Marie-Pierre Gratacap1,5,Melvin I Simon4, Meinrad Gawaz3 & Stefan Offermanns1

Platelet activation at sites of vascular injury is essential for nation of both Gna13flox alleles results in a phenotype characteris- primary hemostasis, but also underlies arterial thrombosis tic of the Gna13– null allele10 (data not shown). 1,2 α α leading to myocardial infarction or stroke . Platelet activators To generate mice lacking G 12 and G 13 in platelets, we used a such as adenosine diphosphate, thrombin or thromboxane mouse line expressing the Cre under the control of the inter- 13 A2 (TXA2) activate receptors that are coupled to heterotrimeric feron-inducible Mx promoter (Mx-Cre) .Deletion of the Gna13 http://www.nature.com/naturemedicine G proteins1,3. Activation of platelets through these receptors gene in mice carrying Mx-Cre was induced by intraperitoneal involves signaling through Gq, Gi and Gz (refs. 4–6). However, injections of polyinosinic-polycytidylic acid (PIPC). Treatment the role and relative importance of G12 and G13, which are with PIPC resulted in almost complete recombination in liver, bone activated by various platelet stimuli7–9, are unclear. Here we marrow and spleen, as assessed by Southern blotting (data not α α α show that lack of G 13, but not G 12, severely reduced the shown). Four weeks after the last PIPC injection, there was no G 13 potency of thrombin, TXA2 and collagen to induce platelet detectable in platelets, liver or spleen, whereas levels of shape changes and aggregation in vitro. These defects were other G-protein α-subunits were unchanged (Fig. 1d and data not α accompanied by reduced activation of RhoA and inability to shown). Platelet counts were not affected by G 13 deficiency, and form stable platelet thrombi under high shear stress ex vivo. surface expression of prominent membrane receptors, including α β β G 13 deficiency in platelets resulted in a severe defect in glycoprotein Ib-Ix (GPIb-Ix), GPV, GPVI, 1 and 3 integrins and primary hemostasis and complete protection against arterial CD9, was normal (data not shown). thrombosis in vivo. We conclude that G13-mediated signaling Because previous studies suggested a role for G12 and G13 in the processes are required for normal hemostasis and thrombosis receptor-mediated platelet shape change response7,we exposed

© Group 2003 Nature Publishing α α α α and may serve as a new target for antiplatelet drugs. platelets deficient in both G 12 and G 13 (G 12/G 13-deficient) to increasing concentrations of various platelet activators. In wild- G12 and G13 constitute a subfamily of heterotrimeric G type platelets, thrombin, collagen and the TXA2 mimetic U46619 that are activated through various receptors, including those led to platelet shape change at concentrations one to two orders of α α involved in many platelet stimuli. Mice lacking G 13, the -subunit magnitude lower than those required to induce aggregation. In α α of G13, die in utero because of a defect in angiogenesis, whereas contrast, there was no shape change in G 12/G 13-deficient α 10,11 G 12-deficient mice are phenotypically normal .To circumvent platelets in response to low agonist concentrations (Fig. 2a,b and α embryonic lethality of mice deficient in G 13 and to study the role Supplementary Fig. 1 online). However, at high agonist concentra- α α α α of G 12- and G 13-mediated signaling in platelet activation, we tions, G 12/G 13-deficient platelets underwent shape change and used Cre/loxP-mediated recombination to conditionally inactivate all stimuli induced aggregation, although with considerably lower α α Gna13, the gene encoding G 13, alone or in a G 12-deficient potency than in wild-type platelets. Surprisingly, platelets lacking –/– ta α (Gna12 ) background. We generated a Gna13 allele, Gna13 ,con- only G 13 showed defects indistinguishable from those observed in α α α taining three loxP sites and a cassette containing the neomycin G 12/G 13-deficient platelets, whereas platelets lacking only G 12 r α α resistance (neo ) and the thymidine kinase gene (tk), by gene target- behaved like wild-type. Thus, G 13,but not G 12, is required for ing of embryonic stem cells (Fig. 1a). The Gna13ta allele was con- the induction of shape change and aggregation in response to low verted into a conditional allele, in which exon 2 is flanked by loxP and intermediate concentrations of TXA2, thrombin and collagen. flox − α sites (Gna13 ), or into a deleted allele (Gna13 ) by crossing these To test whether the aggregation defect observed in G 13 and α α animals with the Cre-deleter mouse strain EIIa-Cre (ref. 12; G 12/G 13-deficient platelets was caused by defective inside-out flox/flox α β Fig. 1a–c). Gna13 mice were normal, whereas full recombi- activation of IIb 3 integrin, we directly assessed this process by

1Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. 2Vascular Biology, Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, 97078 Würzburg, Germany. 3Deutsches Herzzentrum und 1. Medizinische Klinik, Technische Universität München, D-80636 München, Germany. 4Division of Biology 147-75, California Institute of Technology, Pasadena, California 91125, USA. 5Present address: INSERM, Unité 326, Hôpital Purpan, 31059 Toulouse Cedex, France. Correspondence should be addressed to S.O. ([email protected]).

Published online 5 October 2003; doi:10.1038/nm943

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Figure 1 Generation of Gna13flox allele abc and Cre-mediated recombination in mice. (a) Targeting strategy (see Supplementary Methods online for details). , loxP sites; neortk, neomycin resistance/thymidine kinase cassette; Gna13+, wild-type allele; Gna13ta, targeted allele; Gna13–, deleted allele; Gna13flox, floxed allele. S, SpeI; Sc, SacI; K, KpnI; H, HindIII; N, NheI; A, probe A; B, probe B. (b) HindIII- d digested genomic DNA of indicated embryonic stem cell clones hybridized with probe B. (c) Identification of Gna13− and Gna13flox alleles after Cre-mediated recombination in SpeI-digested tail-tip DNA hybridized with probe A. (d) Western blots of platelet lysates from wild-type, Gna12–/–, PIPC-induced Mx-Cre Gna13flox/flox or Mx-Cre Gna12–/–Gna13flox/flox mice probed with antibodies to G-protein α-subunits (anti-Gα). fl, floxed allele. Arrows indicate 42-kDa marker protein.

flow cytometry. Because this analysis was conducted in diluted (Fig. 2c and data not shown), thrombin-induced secretion was not α α platelet suspensions, which largely excludes the accumulation of affected by G 12 and/or G 13 deficiency (data not shown). α β released mediators, U46619 does not lead to significant IIb 3 acti- Thrombin exerts its effects on mouse platelets primarily through http://www.nature.com/naturemedicine vation and degranulation unless Gi-type G proteins are activated by the protease-activated receptor (PAR)-4 (ref. 15). P-selectin expo- coadministration of adrenaline or adenosine diphosphate14. sure and serotonin secretion in response to the PAR-4-activating α β α Thrombin-induced IIb 3 activation was indistinguishable peptide AYPGKF was clearly reduced in platelets lacking G 13 α α between wild-type and G 12/G 13-deficient platelets, and there (Fig. 2d). This suggests that thrombin induces additional, PAR-4- α α α was only a very small decrease in G 13 and G 12/G 13-deficient independent signaling pathways that compensate for the lack of α α α platelets, as compared with wild-type and G 12-deficient platelets, G 12 and/or G 13 in the degranulation response, but not in aggre- when exposed to U46619 in the presence of adrenaline (data not gation and shape change. GPIb, which has a role in thrombin- α 16 shown). Thus, the defective aggregation response of G 13- and induced activation processes ,does not seem to be involved, as α α G 12/G 13-deficient platelets is not caused by impaired inside-out proteolytic removal of the 45-kDa N-terminal ligand-binding α β α activation of IIb 3. domain of GPIb- did not affect the ability of thrombin to induce α α α We next examined whether the absence of G 12,G 13 or both secretion in G 13-deficient platelets (data not shown). had any effect on platelet degranulation. Whereas U46619- or Because the Rho- and Rho kinase–mediated signaling pathway adrenaline-induced P-selectin exposure and serotonin secretion leading to light chain (MLC) phosphorylation is activated

© Group 2003 Nature Publishing α α α was markedly reduced in G 13 and G 12/G 13-deficient platelets through G12 and G13 together (ref. 17), and because Rho and Rho kinase are involved in the induction of platelet shape change7,18,19,we tested whether activa- abc tion of RhoA and phosphorylation of MLC α α were affected in G 12/G 13-deficient platelets. In wild-type platelets, U46619 induced RhoA activation and MLC phosphorylation, with a α maximal effect at 10 nM. Again, G 12- deficient platelets were indistinguishable from wild-type platelets in this regard. In α α α G 12/G 13- or G 13-deficient platelets, how- ever, no RhoA activation or MLC phosphory- lation was observed in response to 10 nM d U46619 (Fig. 3a), indicating that RhoA activa-

Figure 2 Ex vivo analysis of wild-type and α α G 12/G 13-deficient platelets. (a,b) Platelets were stimulated with increasing concentrations of thrombin (a) or U46619 (b). Data are shown as aggregometric traces; addition of stimuli is indicated by arrows. (c,d) Secretion induced by U46619 and adrenaline (c) or PAR-4 peptide (d) was analyzed by flow cytometry using FITC-labeled antibodies against P-selectin. Stimuli were given at the indicated concentrations. WT, wild-type; 12–/–, Gna12–/–; 13–/–, Gna13–/–; 12/13–/–, Gna12–/–Gna13–/–.

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α α Figure 3 Ex vivo analysis of wild-type and G 12/G 13-deficient platelets. (a) Detection of activated Rho in platelets stimulated with U46619, as a described in Methods. Arrows indicate 20-kDa marker protein. WT, wild type; IB, immunoblot; anti-RhoA, anti-P-MLC, anti-MLC, antibodies to RhoA, phosphorylated MLC and MLC, respectively. (b) Phase-contrast α –/– microscopy of whole blood from wild-type, G 12-deficient (Gna12 ), PIPC- induced Mx-Cre Gna12–/–Gna13flox/flox (Gna12/13–/–) or PIPC-induced Mx- Cre Gna12+/+Gna13flox/flox (Gna13–/–) mice, perfused over a collagen-coated surface. (c,d) Analysis of flow chamber experiments showing mean ± s.d. of percentage of surface area covered by thrombi (c) and numbers of single attached platelets per microscopic field (d; n = 5–8). b

tion and subsequent MLC phosphorylation is primarily induced through G13 in platelets. We next studied the potential role of the G13-mediated signaling pathway in thrombus formation during perfusion of whole blood over collagen under high shear conditions ex vivo (Fig. 3b). Wild- α type and G 12-deficient platelets adhered to collagen fibers, and adherent platelets initiated the formation of platelet aggregates α α α within 2 min. Initial adhesion of G 13- or G 12/G 13-deficient platelets was indistinguishable from that observed with wild-type platelets. However, platelet thrombus formation was severely impaired, and virtually no thrombi could be observed after rinsing http://www.nature.com/naturemedicine the chamber (Fig. 3b–d). These findings suggest that the formation and stabilization of platelet thrombi under high shear flow condi- tions requires intact signaling through the G13-mediated pathway. The defective activation of RhoA in the absence of G13 may con- cd tribute to the observed defect in the formation of stable platelet aggregates, as RhoA may be required for platelet aggregation under high shear conditions and for irreversible aggregation of platelets in suspension20,21. α α α To test whether defects observed in G 13- and G 12/G 13-defi- cient platelets in vitro would have any effect under in vivo condi- tions, we determined tail-bleeding times as a measure for primary α α α hemostasis (Fig. 4a). Mice with G 13- or G 12/G 13-deficient interferon-responsive tissues had massively increased bleeding

© Group 2003 Nature Publishing α times compared with wild-type or G 12-deficient mice (Fig. 4a). To bone marrow cells derived from PIPC-induced Mx-Cre α –/– flox/flox flox/flox rule out the possibility that G 13 deficiency in endothelial cells, Gna12 Gna13 or Mx-CreGna13 mice into irradiated hepatocytes or other interferon-responsive organs contributed to wild-type recipient animals. Four weeks after the transfer, all trans- the increased bleeding time, we restricted the Mx-Cre-induced planted animals had normal platelet counts, but bleeding times of α α α deletion of Gna13 to the hematopoietic system by transferring mice transplanted with G 13- or G 12/G 13-deficient bone mar-

a bcd e Time (min)

f

Figure 4 In vivo analysis of hemostasis and thrombosis. (a) Bleeding times of PIPC-induced mice with the indicated genotypes, as described in Methods. Each point represents one individual mouse. fl, floxed allele. (b–d) Bleeding time experiments (b), platelet adhesion (c) and thrombus formation (d) in wild-type mice (Recip.) transplanted with bone marrow from the following animals (Donor): noninduced Mx-Cre Gna12+/+Gna13flox/flox (WT), PIPC-induced Mx-Cre Gna12–/–Gna13flox/flox (12/13–/–) or PIPC-induced Mx-Cre Gna12+/+Gna13flox/flox (13–/–). n =6 for c and d. (e,f) Representative cross-sections of carotid arteries of wild-type mice (e) and mice α with G 13-deficient platelets (f).

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row were massively prolonged (>20 min) compared with mice collected from the retro-orbital plexus in acid citrate dextrose (150 µl per transplanted with bone marrow from noninduced Mx-Cre ml blood) from anesthetized mice. Blood was diluted with half the volume +/+ flox/flox of HEPES–Tyrode buffer [137 mM NaCl, 2 mM KCl, 12 mM NaHCO , 0.3 Gna12 Gna13 mice (Fig. 4b). This indicates that G13-medi- 3 ated signaling is required for normal hemostasis. mM NaH2PO4,2 mM CaCl2,1 mM MgCl2, 5.5 mM glucose and 5 mM HEPES (pH 7.3)] containing 0.35% human serum albumin. Platelet-rich We next assessed the role of Gα in arterial thrombus formation 13 plasma was washed once, and optical aggregation experiments were con- by in vivo fluorescence microscopy of the injured carotid artery. ducted in a two-channel aggregometer (Chrono-Log). The experiment was conducted using wild-type animals trans- α 6 planted with wild-type or G 13-deficient bone marrow. After Flow cytometry. Washed platelets (2 × 10 ) were incubated with the indi- induction of injury by ligation of the carotid artery, wild-type cated stimuli for 2 min at 37 °C, stained with fluorophore-conjugated mon- platelets adhered to the subendothelial surface and massive oclonal antibodies (emfret Analytics) at saturating concentrations for thrombi subsequently developed in the carotid artery (Fig. 4c–e). 15 min at room temperature and directly analyzed on a FACSCalibur α (Becton Dickinson). Platelets were gated by forward and side scatter14. Platelets lacking G 13 also adhered rapidly to the injured vessel wall (Fig. 4c). However, the total number of platelets that firmly 14 × 8 ∼ Secretion of 5-hydroxy[ C]tryptamine. Platelets (5.8 10 /ml) were adhered was reduced by 50% compared with wild-type platelets. loaded with 5-hydroxy[14C]tryptamine (200 nCi/ml) and stimulated with α In contrast to wild-type animals, mice with G 13-deficient platelets U46619 for 45 s. After fixation and centrifugation, 5-hydroxy[14C]trypta- did not show any thrombi after vascular injury, indicating that mine release was determined by liquid-scintillation counting. α G 13-deficient platelets were not able to form stable thrombi in vivo (Fig. 4d,f). This is consistent with the data obtained ex vivo and Determination of activated cellular Rho and MLC phosphorylation. The shows that the initial adhesion of platelets to the injured vessel wall amount of activated cellular Rho was determined by precipitation with a was not substantially altered, whereas the subsequent formation fusion protein consisting of glutathione-S-transferase (GST) and the Rho- binding domain of rhotekin (GST-RBD)26. Platelets were stimulated with and stabilization of platelet thrombi was altered by the absence of 10 nM of U46619 for 10 s, lysed and subjected to GST-rhotekin pull-down. G13-mediated signaling. Precipitated GTP-bound RhoA and lysates were immunoblotted with anti- Platelet activation during hemostasis and thrombosis is a com- bodies against RhoA, MLC or the phosphorylated form of MLC (all from http://www.nature.com/naturemedicine plex process involving many stimuli that act on platelets at sites of Santa Cruz Biotechnology). vascular injury. Most of these stimuli directly or indirectly use G- protein-mediated signaling pathways to induce platelet activation. Flow chamber experiments. Experiments were conducted as described27. Defining the relative importance of signaling through different Briefly, transparent flow chambers with a slit depth of 50 µm, equipped with heterotrimeric G proteins is useful in understanding the molecular Horm-type collagen-coated cover slips, were connected to a syringe filled mechanisms of platelet activation. Our data clearly indicate that with anticoagulated blood. Perfusion was done using a pulse-free pump under high shear stress equivalent to a wall shear rate of 1000 s–1 (4 min). The G -mediated signaling is necessary for platelet activation in hemo- 13 chambers were then rinsed with a 4 min perfusion of HEPES buffer contain- stasis and thrombosis. Whereas the phenotypic changes in platelets ing 2 mM Ca2+, at the same shear stress, and phase-contrast images were α α 6,22,23 from G i2- or G z-deficient mice seem to be less severe , the recorded from at least five different microscope fields (×40 objectives). α defects observed in mice with G 13-deficient platelets are compa- Shown are experiments representative of five to eight experiments per group. α rable with the in vivo defects in mice lacking G q (ref. 4). Thus, platelet activation seems to be an integrated process that requires Determination of bleeding times. Adult mice were anesthetized with intraperitoneal pentobarbital (50 mg per kg body weight) and intramuscu- different G-protein signaling pathways involving Gq,Gi,Gz and

© Group 2003 Nature Publishing lar xylazin (3 mg/kg), and the tail was cut 5–6 mm from the tip and G13.Although signaling through two of these pathways can be suf- ficient to induce some platelet activation5,14,24,all three pathways immersed in saline at 37 °C. Bleeding time was defined as the time at which all visible signs of bleeding from the incision had stopped. The experiment seem to be required for efficient platelet activation under physio- was stopped after 20 min. logical and pathological conditions. The severe defects observed in α mice with G 13-deficient platelets may be caused by the decreased Carotid artery ligation and assessment of platelet adhesion and aggrega- α 28 responsiveness of G 13-deficient platelets to various stimuli. tion. Experiments were conducted as described . Platelets were isolated and Reduced potency of platelet activators may become limiting, espe- labeled with 5-carboxyfluorescein diacetate succinimidyl ester as described. cially under high flow conditions that lead to rapid clearance of sol- Labeled platelets (200 × 106 platelets per 250 µl) were infused intravenously uble stimuli. We also observed impaired stabilization of platelet into anesthetized mice of the same genotype as those used to prepare the thrombi under high shear stress, a process that may require acute platelets. The right common carotid artery was dissected free and ligated vig- and continuous activation of RhoA-mediated signaling pathways. orously near the carotid bifurcation for 5 min to induce vascular injury. Adhesion of autogeneous platelets was assessed before and after carotid The fact that Gα deficiency protected animals against thrombosis 13 injury by in vivo video microscopy28.Videotaped images were evaluated suggests that inhibition of the G13-mediated signaling pathway in using a computer-assisted image analysis program28 (Cap Image 7.4, H. platelets is a promising strategy to prevent or treat platelet activa- Zeintl). The number of adherent platelets was assessed by counting the cells tion in thrombosis. that did not move or detach from the endothelial surface within 10 s. The area covered by thrombi was also quantified and is presented in µm2. METHODS Conditional inactivation of the Gna13 gene. See Supplementary Methods Histology. Carotid arteries were perfusion-fixed in situ with 4% online. paraformaldehyde (pH 7.0) 15 min after induction of injury, excised, fixed in 0.1 M cacodylate-buffered Karnovsky solution (2.5% glutaraldehyde and 1% Western blot analysis. Platelet lysates were subjected to SDS-PAGE. After paraformaldehyde) overnight at room temperature and postfixed in 1% α blotting, nitrocellulose membranes were probed with antibodies to G 12 osmium tetroxide for 2 h at pH 7.3. The samples were dehydrated in graded α α α (ref. 25) and to G 13,G i and G q/11 (Santa Cruz Biotechnology). ethanols and embedded in EmBed-812 epoxy resin (all reagents from Science Services). After 48 h of heat polymerization at 60 °C, semithin (0.8 µm) sec- Platelet preparation and aggregation. All animal experiments and care were tions were cut with a diamond knife (Diatome) and double stained with approved by the local Animal Care & Use Committee. Whole blood was aqueous solutions of 1% toluidine blue and basic fuchsin (60 °C, 1 min).

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α Bone marrow transplantation. Bone marrow cells from mice (10–12 weeks impaired cell chemokinesis as a result of G 13 deficiency. Science 275, 533–536 × 6 (1997). old) were removed aseptically from femurs and tibias. Cells (5 10 ) were α 11. Gu, J.L., Müller, S., Mancino, V., Offermanns, S. & Simon, M.I. Interaction of G 12 with resuspended in DMEM and transplanted by tail-vein infusion into lethally α α G 13 and G q signaling pathways. Proc. Natl. Acad. Sci. USA 99, 9352–9357 (2002). irradiated (10 Gy) recipients (C57BL/6; Charles River) 1 d after irradiation. 12. Lakso, M. et al. Efficient in vivo manipulation of mouse genomic sequences at the α zygote stage. Proc. Natl. Acad. Sci. USA 93, 5860–5865 (1996). G 13 deficiency was verified by western blotting. 13. Kühn, R., Schwenk, F., Aguet, M. & Rajewsky, K. Inducible gene targeting in mice. Note: Supplementary information is available on the Nature Medicine website. Science 269, 1427–1429 (1995). 14. Nieswandt, B., Schulte, V., Zywietz, A., Gratacap, M.-P. & Offermanns, S. Costimulation of G - and G /G -mediated signaling pathways induces integrin alpha IIbbeta 3 acti- ACKNOWLEDGEMENTS i 12 13 vation in platelets. J. Biol. Chem. 277, 39493–39498 (2002). We thank A. Rogatzki and A. Rippberger for expert technical help and B. Arnold 15. Sambrano, G.R., Weiss, E.J., Zheng, Y.W., Huang, W. & Coughlin, S.R. Role of thrombin for kind help with bone marrow transplantation experiments. This work was signaling in platelets in hemostasis and thrombosis. Nature 413, 74–82 (2001). supported by the Deutsche Forschungsgemeinschaft and the Volkswagen 16. Savage, B., Cattaneo, M. & Ruggeri, Z.M. Mechanisms of platelet aggregation. Curr. Foundation. Opin. Hematol. 8, 270–276 (2001). 17. Sah, V.P., Seasholtz, T.M., Sagi, S.A. & Brown, J.H. The role of Rho in -coupled COMPETING INTERESTS STATEMENT receptor . Annu. Rev. Pharmacol. Toxicol. 40, 459–489 (2000). 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