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DOI: 10.1055/s-0040-1714745
PAK Membrane Translocation and Phosphorylation Regulate Platelet Aggregation Downstream of Gi and G12/13 Pathways
Funding This work was partially supported by the National Natural Science Foundation of China (grant numbers: 81673429, 81872862, and 81402920) to Z.D. and Y.Z. and Startup Fund from The First Affiliated Hospital of Zhengzhou University to Z.D.![](https://www.thieme-connect.de/media/10.1055-s-00035024/202011/lookinside/thumbnails/10-1055-s-0040-1714745_190747-1.jpg)
Abstract
Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G12/13 depends on agonist concentrations; coactivation of Gi and G12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G12/13, and PAK may represent a potential antiplatelet and antithrombotic target.
Authors' Contributions
Conceptualization: Z.D.; methodology: J.Z., Y.Z., and L.H.; investigation: J.Z., Y.Z., Sh.Z., L.C., G.P., L.H., S.Z., and Z.D.; resources: Z.D. and J.D.; mouse models: J.L.; writing—original draft: Y.Z., J.Z., and Z.D.; writing—review and editing: Z.D. and S.K.; supervision: Z.D.; funding acquisition: Z.D. and Y.Z.
* These authors contributed equally to this study.
Publication History
Received: 24 December 2019
Accepted: 25 June 2020
Article published online:
27 August 2020
© 2020. Thieme. All rights reserved.
Georg Thieme Verlag KG
Stuttgart · New York
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References
- 1 Offermanns S. Activation of platelet function through G protein-coupled receptors. Circ Res 2006; 99 (12) 1293-1304
- 2 Li Z, Delaney MK, O'Brien KA, Du X. Signaling during platelet adhesion and activation. Arterioscler Thromb Vasc Biol 2010; 30 (12) 2341-2349
- 3 Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci U S A 1998; 95 (14) 8070-8074
- 4 Cattaneo M. P2Y12 receptors: structure and function. J Thromb Haemost 2015; 13 (Suppl. 01) S10-S16
- 5 Paul BZ, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors. J Biol Chem 1999; 274 (41) 29108-29114
- 6 Sebastiano M, Momi S, Falcinelli E, Bury L, Hoylaerts MF, Gresele P. A novel mechanism regulating human platelet activation by MMP-2-mediated PAR1 biased signaling. Blood 2017; 129 (07) 883-895
- 7 Kim S, Foster C, Lecchi A. et al. Protease-activated receptors 1 and 4 do not stimulate G(i) signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of G(i) signaling. Blood 2002; 99 (10) 3629-3636
- 8 Moers A, Nieswandt B, Massberg S. et al. G13 is an essential mediator of platelet activation in hemostasis and thrombosis. Nat Med 2003; 9 (11) 1418-1422
- 9 Gong H, Shen B, Flevaris P. et al. G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin “outside-in” signaling. Science 2010; 327 (5963): 340-343
- 10 Jin J, Daniel JL, Kunapuli SP. Molecular basis for ADP-induced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem 1998; 273 (04) 2030-2034
- 11 Dorsam RT, Kunapuli SP. Central role of the P2Y12 receptor in platelet activation. J Clin Invest 2004; 113 (03) 340-345
- 12 Klages B, Brandt U, Simon MI, Schultz G, Offermanns S. Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets. J Cell Biol 1999; 144 (04) 745-754
- 13 Paul BZ, Daniel JL, Kunapuli SP. Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways. Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change. J Biol Chem 1999; 274 (40) 28293-28300
- 14 Kim S, Jin J, Kunapuli SP. Relative contribution of G-protein-coupled pathways to protease-activated receptor-mediated Akt phosphorylation in platelets. Blood 2006; 107 (03) 947-954
- 15 Jin J, Mao Y, Thomas D, Kim S, Daniel JL, Kunapuli SP. RhoA downstream of G(q) and G(12/13) pathways regulates protease-activated receptor-mediated dense granule release in platelets. Biochem Pharmacol 2009; 77 (05) 835-844
- 16 Dangelmaier C, Manne BK, Liverani E, Jin J, Bray P, Kunapuli SP. PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses. Thromb Haemost 2014; 111 (03) 508-517
- 17 Kroner C, Eybrechts K, Akkerman JW. Dual regulation of platelet protein kinase B. J Biol Chem 2000; 275 (36) 27790-27798
- 18 Dittrich M, Birschmann I, Mietner S, Sickmann A, Walter U, Dandekar T. Platelet protein interactions: map, signaling components, and phosphorylation groundstate. Arterioscler Thromb Vasc Biol 2008; 28 (07) 1326-1331
- 19 O'Brien KA, Stojanovic-Terpo A, Hay N, Du X. An important role for Akt3 in platelet activation and thrombosis. Blood 2011; 118 (15) 4215-4223
- 20 Stephens L, Anderson K, Stokoe D. et al. Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science 1998; 279 (5351): 710-714
- 21 Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev 1999; 13 (22) 2905-2927
- 22 Zhang W, Bhetwal BP, Gunst SJ. Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle. J Physiol 2018; 596 (16) 3617-3635
- 23 Aslan JE, Baker SM, Loren CP. et al. The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation. Am J Physiol Cell Physiol 2013; 305 (05) C519-C528
- 24 Higuchi M, Onishi K, Kikuchi C, Gotoh Y. Scaffolding function of PAK in the PDK1-Akt pathway. Nat Cell Biol 2008; 10 (11) 1356-1364
- 25 Badolia R, Manne BK, Dangelmaier C, Chernoff J, Kunapuli SP. Gq-mediated Akt translocation to the membrane: a novel PIP3-independent mechanism in platelets. Blood 2015; 125 (01) 175-184
- 26 Wettschureck N, Offermanns S. Mammalian G proteins and their cell type specific functions. Physiol Rev 2005; 85 (04) 1159-1204
- 27 Dorsam RT, Kim S, Jin J, Kunapuli SP. Coordinated signaling through both G12/13 and G(i) pathways is sufficient to activate GPIIb/IIIa in human platelets. J Biol Chem 2002; 277 (49) 47588-47595
- 28 Nieswandt B, Schulte V, Zywietz A, Gratacap MP, Offermanns S. Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets. J Biol Chem 2002; 277 (42) 39493-39498
- 29 Hu L, Chang L, Zhang Y. et al. Platelets express activated P2Y12 receptor in patients with diabetes mellitus. Circulation 2017; 136 (09) 817-833
- 30 Zhang S, Zhang S, Hu L. et al. Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis. Circulation 2015; 131 (13) 1160-1170
- 31 Zhang Y, Ye J, Hu L. et al. Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12. J Thromb Haemost 2012; 10 (10) 2149-2157
- 32 Zhang SH, Zhang Y, Shen J. et al. Tumor vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid inhibits platelet activation and thrombosis via inhibition of thromboxane A2 signaling and phosphodiesterase. J Thromb Haemost 2013; 11 (10) 1855-1866
- 33 Hu L, Fan Z, Du H. et al. BF061, a novel antiplatelet and antithrombotic agent targeting P2Y12 receptor and phosphodiesterase. Thromb Haemost 2011; 106 (06) 1203-1214
- 34 Takasaki J, Saito T, Taniguchi M. et al. A novel Galphaq/11-selective inhibitor. J Biol Chem 2004; 279 (46) 47438-47445
- 35 Nagy Jr B, Bhavaraju K, Getz T, Bynagari YS, Kim S, Kunapuli SP. Impaired activation of platelets lacking protein kinase C-theta isoform. Blood 2009; 113 (11) 2557-2567
- 36 Kim S, Dangelmaier C, Bhavanasi D. et al. RhoG protein regulates glycoprotein VI-Fc receptor γ-chain complex-mediated platelet activation and thrombus formation. J Biol Chem 2013; 288 (47) 34230-34238
- 37 Yin H, Stojanovic A, Hay N, Du X. The role of Akt in the signaling pathway of the glycoprotein Ib-IX induced platelet activation. Blood 2008; 111 (02) 658-665
- 38 Borst O, Münzer P, Gatidis S. et al. The inflammatory chemokine CXC motif ligand 16 triggers platelet activation and adhesion via CXC motif receptor 6-dependent phosphatidylinositide 3-kinase/Akt signaling. Circ Res 2012; 111 (10) 1297-1307
- 39 Stojanovic A, Marjanovic JA, Brovkovych VM. et al. A phosphoinositide 3-kinase-AKT-nitric oxide-cGMP signaling pathway in stimulating platelet secretion and aggregation. J Biol Chem 2006; 281 (24) 16333-16339
- 40 Kim S, Jin J, Kunapuli SP. Akt activation in platelets depends on Gi signaling pathways. J Biol Chem 2004; 279 (06) 4186-4195
- 41 Woulfe D, Jiang H, Morgans A, Monks R, Birnbaum M, Brass LF. Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2. J Clin Invest 2004; 113 (03) 441-450
- 42 Chen J, De S, Damron DS, Chen WS, Hay N, Byzova TV. Impaired platelet responses to thrombin and collagen in AKT-1-deficient mice. Blood 2004; 104 (06) 1703-1710
- 43 Vidal C, Geny B, Melle J, Jandrot-Perrus M, Fontenay-Roupie M. Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin. Blood 2002; 100 (13) 4462-4469
- 44 Pandey D, Goyal P, Dwivedi S, Siess W. Unraveling a novel Rac1-mediated signaling pathway that regulates cofilin dephosphorylation and secretion in thrombin-stimulated platelets. Blood 2009; 114 (02) 415-424
- 45 Aslan JE, Itakura A, Haley KM. et al. p21 activated kinase signaling coordinates glycoprotein receptor VI-mediated platelet aggregation, lamellipodia formation, and aggregate stability under shear. Arterioscler Thromb Vasc Biol 2013; 33 (07) 1544-1551
- 46 Nurden P, Savi P, Heilmann E. et al. An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function. J Clin Invest 1995; 95 (04) 1612-1622
- 47 Savi P, Beauverger P, Labouret C. et al. Role of P2Y1 purinoceptor in ADP-induced platelet activation. FEBS Lett 1998; 422 (03) 291-295
- 48 Léon C, Hechler B, Freund M. et al. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice. J Clin Invest 1999; 104 (12) 1731-1737
- 49 Aungraheeta R, Conibear A, Butler M. et al. Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor. Blood 2016; 128 (23) 2717-2728
- 50 Manning BD, Toker A. AKT/PKB signaling: navigating the network. Cell 2017; 169 (03) 381-405
- 51 Chang J-K, Ni Y, Han L. et al. Protein kinase D1 (PKD1) phosphorylation on Ser203 by type I p21-activated kinase (PAK) regulates PKD1 localization. J Biol Chem 2017; 292 (23) 9523-9539