Pflügers Archiv - European Journal of Physiology

, Volume 457, Issue 5, pp 1173–1185 | Cite as

Rac1 is essential for phospholipase C-γ2 activation in platelets

  • Irina Pleines
  • Margitta Elvers
  • Amrei Strehl
  • Miroslava Pozgajova
  • David Varga-Szabo
  • Frauke May
  • Anna Chrostek-Grashoff
  • Cord Brakebusch
  • Bernhard Nieswandt
Signaling and Cell Physiology

Abstract

Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cβ or PLCγ2. Active PLCs trigger Ca2+ mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCβ isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCγ2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP3) and intracellular calcium mobilization suggesting inappropriate activation of PLCγ2 despite normal tyrosine phosphorylation of the enzyme. Rac1−/− platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA2 analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1−/− mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCγ2 activation in platelets and that this is critical for thrombus formation in vivo.

Keywords

Ca2+ mobilization G-protein IP3 Phospholipase C Platelets Rac1 

Abbreviations

BSA

bovine serum albumin

CLEC-2

C-type lectin-like receptor

CRP

collagen related peptide

CVX

convulxin

FACS

fluoresence activated cell sorting

FcR

Fc receptor

FITC

fluoresceine isothiocyanate

GP

glycoprotein

HRP

horseradish peroxidase

Ig

immunoglobulin

PAGE

polyacrylamide gel electrophoresis

PLCγ2

phospholipase Cγ2

PMA

phorbol 12-myristate 13-acetate

prp

platelet rich plasma

PVDF

polyvinylidene difluoride

RC

rhodocytin

SDS

sodium dodecyl sulfate

TxA2

thromboxane A2

vWF

von Willebrand factor

References

  1. 1.
    Ruggeri ZM (2002) Platelets in atherothrombosis. Nat Med 8:1227–1234PubMedCrossRefGoogle Scholar
  2. 2.
    Varga-Szabo D, Pleines I, Nieswandt B (2008) Cell adhesion mechanisms in platelets. Arterioscler Thromb Vasc Biol 28:403–412PubMedCrossRefGoogle Scholar
  3. 3.
    Offermanns S, Toombs CF, Hu YH, Simon MI (1997) Defective platelet activation in G alpha(q)-deficient mice. Nature 389:183–186PubMedCrossRefGoogle Scholar
  4. 4.
    Watson SP, Asazuma N, Atkinson B, Berlanga O, Best D, Bobe R, Jarvis G, Marshall S, Snell D, Stafford M, Tulasne D, Wilde J, Wonerow P, Frampton J (2001) The role of ITAM- and ITIM-coupled receptors in platelet activation by collagen. Thromb Haemost 86:276–288PubMedGoogle Scholar
  5. 5.
    Nieswandt B, Watson SP (2003) Platelet-collagen interaction: is GPVI the central receptor? Blood 102:449–461PubMedCrossRefGoogle Scholar
  6. 6.
    Suzuki-Inoue K, Fuller GL, Garcia A, Eble JA, Pohlmann S, Inoue O, Gartner TK, Hughan SC, Pearce AC, Laing GD, Theakston RD, Schweighoffer E, Zitzmann N, Morita T, Tybulewicz VL, Ozaki Y, Watson SP (2006) A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2. Blood 107:542–549PubMedCrossRefGoogle Scholar
  7. 7.
    Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4:517–529PubMedCrossRefGoogle Scholar
  8. 8.
    Varga-Szabo D, Braun A, Kleinschnitz C, Bender M, Pleines I, Pham M, Renne T, Stoll G, Nieswandt B (2008) The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction. J Exp Med 205:1583–1591PubMedCrossRefGoogle Scholar
  9. 9.
    Bird GS, Aziz O, Lievremont JP, Wedel BJ, Trebak M, Vazquez G, Putney JW Jr (2004) Mechanisms of phospholipase C-regulated calcium entry. Curr Mol Med 4:291–301PubMedCrossRefGoogle Scholar
  10. 10.
    Illenberger D, Schwald F, Pimmer D, Binder W, Maier G, Dietrich A, Gierschik P (1998) Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1. EMBO J 17:6241–6249PubMedCrossRefGoogle Scholar
  11. 11.
    Illenberger D, Walliser C, Strobel J, Gutman O, Niv H, Gaidzik V, Kloog Y, Gierschik P, Henis YI (2003) Rac2 regulation of phospholipase C-beta 2 activity and mode of membrane interactions in intact cells. J Biol Chem 278:8645–8652PubMedCrossRefGoogle Scholar
  12. 12.
    Illenberger D, Walliser C, Nurnberg B, Diaz LM, Gierschik P (2003) Specificity and structural requirements of phospholipase C-beta stimulation by Rho GTPases versus G protein beta gamma dimers. J Biol Chem 278:3006–3014PubMedCrossRefGoogle Scholar
  13. 13.
    Piechulek T, Rehlen T, Walliser C, Vatter P, Moepps B, Gierschik P (2005) Isozyme-specific stimulation of phospholipase C-gamma2 by Rac GTPases. J Biol Chem 280:38923–38931PubMedCrossRefGoogle Scholar
  14. 14.
    Offermanns S (2006) Activation of platelet function through G protein-coupled receptors. Circ Res 99:1293–1304PubMedCrossRefGoogle Scholar
  15. 15.
    Klages B, Brandt U, Simon MI, Schultz G, Offermanns S (1999) Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets. J Cell Biol 144:745–754PubMedCrossRefGoogle Scholar
  16. 16.
    Miranti CK, Leng L, Maschberger P, Brugge JS, Shattil SJ (1998) Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1. Curr Biol 8:1289–1299PubMedCrossRefGoogle Scholar
  17. 17.
    McCarty OJ, Larson MK, Auger JM, Kalia N, Atkinson BT, Pearce AC, Ruf S, Henderson RB, Tybulewicz VL, Machesky LM, Watson SP (2005) Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow. J Biol Chem 280:39474–39484PubMedCrossRefGoogle Scholar
  18. 18.
    Azim AC, Barkalow K, Chou J, Hartwig JH (2000) Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor. Blood 95:959–964PubMedGoogle Scholar
  19. 19.
    Gratacap MP, Payrastre B, Nieswandt B, Offermanns S (2001) Differential regulation of Rho and Rac through heterotrimeric G-proteins and cyclic nucleotides. J Biol Chem 276:47906–47913PubMedGoogle Scholar
  20. 20.
    Hartwig JH, Bokoch GM, Carpenter CL, Janmey PA, Taylor LA, Toker A, Stossel TP (1995) Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell 82:643–653PubMedCrossRefGoogle Scholar
  21. 21.
    Soulet C, Gendreau S, Missy K, Benard V, Plantavid M, Payrastre B (2001) Characterisation of Rac activation in thrombin- and collagen-stimulated human blood platelets. FEBS Lett 507:253–258PubMedCrossRefGoogle Scholar
  22. 22.
    Akbar H, Kim J, Funk K, Cancelas JA, Shang X, Chen L, Johnson JF, Williams DA, Zheng Y (2007) Genetic and pharmacologic evidence that Rac1 GTPase is involved in regulation of platelet secretion and aggregation. J Thromb Haemost 5:1747–1755PubMedCrossRefGoogle Scholar
  23. 23.
    Suzuki-Inoue K, Yatomi Y, Asazuma N, Kainoh M, Tanaka T, Satoh K, Ozaki Y (2001) Rac, a small guanosine triphosphate-binding protein, and p21-activated kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin alpha(2)beta(1). Blood 98:3708–3716PubMedCrossRefGoogle Scholar
  24. 24.
    Chrostek A, Wu X, Quondamatteo F, Hu R, Sanecka A, Niemann C, Langbein L, Haase I, Brakebusch C (2006) Rac1 is crucial for hair follicle integrity but is not essential for maintenance of the epidermis. Mol Cell Biol 26:6957–6970PubMedCrossRefGoogle Scholar
  25. 25.
    Kuhn R, Schwenk F, Aguet M, Rajewsky K (1995) Inducible gene targeting in mice. Science 269:1427–1429PubMedCrossRefGoogle Scholar
  26. 26.
    Knight CG, Morton LF, Onley DJ, Peachey AR, Ichinohe T, Okuma M, Farndale RW, Barnes MJ (1999) Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen. Cardiovasc Res 41:450–457PubMedCrossRefGoogle Scholar
  27. 27.
    Nieswandt B, Bergmeier W, Rackebrandt K, Gessner JE, Zirngibl H (2000) Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice. Blood 96:2520–2527PubMedGoogle Scholar
  28. 28.
    Nieswandt B, Brakebusch C, Bergmeier W, Schulte V, Bouvard D, Mokhtari-Nejad R, Lindhout T, Heemskerk JW, Zirngibl H, Fassler R (2001) Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen. EMBO J 20:2120–2130PubMedCrossRefGoogle Scholar
  29. 29.
    Heemskerk JW, Feijge MA, Rietman E, Hornstra G (1991) Rat platelets are deficient in internal Ca2+ release and require influx of extracellular Ca2+ for activation. FEBS Lett 284:223–226PubMedCrossRefGoogle Scholar
  30. 30.
    Sugihara K, Nakatsuji N, Nakamura K, Nakao K, Hashimoto R, Otani H, Sakagami H, Kondo H, Nozawa S, Aiba A, Katsuki M (1998) Rac1 is required for the formation of three germ layers during gastrulation. Oncogene 17:3427–3433PubMedCrossRefGoogle Scholar
  31. 31.
    Nieswandt B, Moser M, Pleines I, Varga-Szabo D, Monkley S, Critchley D, Fassler R (2007) Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo. J Exp Med 204:3113–3118PubMedCrossRefGoogle Scholar
  32. 32.
    Fuller GL, Williams JA, Tomlinson MG, Eble JA, Hanna SL, Pohlmann S, Suzuki-Inoue K, Ozaki Y, Watson SP, Pearce AC (2007) The C-type lectin receptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signaling cascade. J Biol Chem 282:12397–12409PubMedCrossRefGoogle Scholar
  33. 33.
    Nieswandt B, Schulte V, Zywietz A, Gratacap MP, Offermanns S (2002) Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets. J Biol Chem 277:39493–39498PubMedCrossRefGoogle Scholar
  34. 34.
    Moers A, Wettschureck N, Gruner S, Nieswandt B, Offermanns S (2004) Unresponsiveness of platelets lacking both Galpha(q) and Galpha(13). Implications for collagen-induced platelet activation. J Biol Chem 279:45354–45359PubMedCrossRefGoogle Scholar
  35. 35.
    Bergmeier W, Schulte V, Brockhoff G, Bier U, Zirngibl H, Nieswandt B (2002) Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody. Cytometry 48:80–86PubMedCrossRefGoogle Scholar
  36. 36.
    Heemskerk JW, Vuist WM, Feijge MA, Reutelingsperger CP, Lindhout T (1997) Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses. Blood 90:2615–2625PubMedGoogle Scholar
  37. 37.
    Gruner S, Prostredna M, Aktas B, Moers A, Schulte V, Krieg T, Offermanns S, Eckes B, Nieswandt B (2004) Anti-glycoprotein VI treatment severely compromises hemostasis in mice with reduced alpha2beta1 levels or concomitant aspirin therapy. Circulation 110:2946–2951PubMedCrossRefGoogle Scholar
  38. 38.
    Massberg S, Gawaz M, Gruner S, Schulte V, Konrad I, Zohlnhofer D, Heinzmann U, Nieswandt B (2003) A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J Exp Med 197:41–49PubMedCrossRefGoogle Scholar
  39. 39.
    Nieswandt B, Schulte V, Bergmeier W, Mokhtari-Nejad R, Rackebrandt K, Cazenave JP, Ohlmann P, Gachet C, Zirngibl H (2001) Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice. J Exp Med 193:459–470PubMedCrossRefGoogle Scholar
  40. 40.
    Gruner S, Prostredna M, Koch M, Miura Y, Schulte V, Jung SM, Moroi M, Nieswandt B (2005) Relative antithrombotic effect of soluble GPVI dimer compared with anti-GPVI antibodies in mice. Blood 105:1492–1499PubMedCrossRefGoogle Scholar
  41. 41.
    Pozgajova M, Sachs UJ, Hein L, Nieswandt B (2006) Reduced thrombus stability in mice lacking the alpha2A-adrenergic receptor. Blood 108:510–514PubMedCrossRefGoogle Scholar
  42. 42.
    Thodeti CK, Massoumi R, Bindslev L, Sjolander A (2002) Leukotriene D4 induces association of active RhoA with phospholipase C-gamma1 in intestinal epithelial cells. Biochem J 365:157–163PubMedCrossRefGoogle Scholar
  43. 43.
    Chang JS, Seok H, Kwon TK, Min DS, Ahn BH, Lee YH, Suh JW, Kim JW, Iwashita S, Omori A, Ichinose S, Numata O, Seo JK, Oh YS, Suh PG (2002) Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity. J Biol Chem 277:19697–19702PubMedCrossRefGoogle Scholar
  44. 44.
    Kauffenstein G, Bergmeier W, Eckly A, Ohlmann P, Leon C, Cazenave JP, Nieswandt B, Gachet C (2001) The P2Y(12) receptor induces platelet aggregation through weak activation of the alpha(IIb)beta(3) integrin—a phosphoinositide 3-kinase-dependent mechanism. FEBS Lett 505:281–290PubMedCrossRefGoogle Scholar
  45. 45.
    Nonne C, Lenain N, Hechler B, Mangin P, Cazenave JP, Gachet C, Lanza F (2005) Importance of platelet phospholipase Cgamma2 signaling in arterial thrombosis as a function of lesion severity. Arterioscler Thromb Vasc Biol 25:1293–1298PubMedCrossRefGoogle Scholar
  46. 46.
    Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima K, Yatomi Y, Narimatsu H, Ozaki Y (2007) Involvement of the snake toxin receptor CLEC-2 in podoplanin-mediated platelet activation by cancer cells. J Biol Chem 282(36):25993–6001PubMedCrossRefGoogle Scholar
  47. 47.
    Nassar N, Cancelas J, Zheng J, Williams DA, Zheng Y (2006) Structure- function based design of small molecule inhibitors targeting Rho family GTPases. Curr Top Med Chem 6:1109–1116PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Irina Pleines
    • 1
  • Margitta Elvers
    • 1
  • Amrei Strehl
    • 1
  • Miroslava Pozgajova
    • 1
  • David Varga-Szabo
    • 1
  • Frauke May
    • 1
  • Anna Chrostek-Grashoff
    • 2
  • Cord Brakebusch
    • 3
  • Bernhard Nieswandt
    • 1
    • 4
  1. 1.Rudolf Virchow Center for Experimental BiomedicineUniversity of WürzburgWürzburgGermany
  2. 2.Cardiovascular Research CenterUniversity of Virginia Health SystemCharlottesvilleUSA
  3. 3.BRIC, Biomedical InstituteUniversity of CopenhagenCopenhagenDenmark
  4. 4.Institute of Clinical Biochemistry and PathobiochemistryUniversity of WürzburgWürzburgGermany

Personalised recommendations