Biphasic myosin II light chain activation during clot retraction

Marion Egot; Alexandre Kauskot; Dominique Lasne; Pascale Gaussem; Christilla Bachelot-Loza

doi:10.1160/TH13-04-0335

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2013; 110(06): 1215-1222
DOI: 10.1160/TH13-04-0335

Platelets and Blood Cells

Schattauer GmbH

Biphasic myosin II light chain activation during clot retraction

Marion Egot

¹Inserm UMR_S U765, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, France

,

Alexandre Kauskot

¹Inserm UMR_S U765, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, France

,

Dominique Lasne

¹Inserm UMR_S U765, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, France

³AP-HP, Laboratoire d’hématologie, Hôpital Necker, Paris, France

,

Pascale Gaussem

¹Inserm UMR_S U765, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, France

⁴AP-HP, Service d’hématologie biologique, Hôpital Européen Georges Pompidou, Paris, France

,

Christilla Bachelot-Loza

¹Inserm UMR_S U765, Paris, France

²Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, France

› Institutsangaben

Abstract

Summary

Clot retraction is an essential step during primary haemostasis, thereby promoting thrombus stability and wound healing. Integrin αIIbβ3 plays a critical role in clot retraction, by inducing acto-myosin interactions that allow platelet cytoskeleton reorganisation. However, the signalling pathways that lead to clot retraction are still misunderstood. In this study, we report the first data on the kinetics of myosin II light chain (MLC) phosphorylation during clot retraction. We found an early phosphorylation peak followed by a second peak. By using specific inhibitors of kinases and small G proteins, we showed that MLC kinase (MLCK), RhoA/ROCK, and Rac-1 were involved in clot retraction and in the early MLC phosphorylation peak. Only Rac-1 and actin polymerisation, controlled by outside-in signalling, were crucial to the second MLC phosphorylation peak.

Keywords

Clot retraction - cytoskeleton - myosin light chain - kinases - signal transduction

Volltext

Referenzen

References
1 Li Z, Delaney MK, O’Brien KA. et al. Signaling during platelet adhesion and activation. Arterioscler Thromb Vasc Biol 2010; 30: 2341-2349.
2 Flevaris P, Li Z, Zhang G. et al. Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signaling pathway. Blood 2009; 113: 893-901.
3 Payrastre B, Missy K, Trumel C. et al. The integrin alpha IIb/beta 3 in human platelet signal transduction. Biochem Pharmacol 2000; 60: 1069-1074.
4 Cohen I, Gerrard JM, White JG. Ultrastructure of clots during isometric contraction. J Cell Biol 1982; 93: 775-787.
5 Schoenwaelder SM, Yuan Y, Cooray P. et al. Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin alphaIIbbeta3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots. J Biol Chem 1997; 272: 1694-1702.
6 Chen YP, O’Toole TE, Leong L. et al. Beta 3 integrin-mediated fibrin clot retraction by nucleated cells: differing behavior of alpha IIb beta 3 and alpha v beta 3. Blood 1995; 86: 2606-2615.
7 Kunitada S, FitzGerald GA, Fitzgerald DJ. Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction. Blood 1992; 79: 1420-1427.
8 Sellers JR, Spudich JA, Sheetz MP. Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments. J Cell Biol 1985; 101: 1897-1902.
9 Ridley AJ. Rho: theme and variations. Curr Biol 1996; 6: 1256-1264.
10 Suzuki-Inoue K, Hughes CE, Inoue O. et al. Involvement of Src kinases and PLCgamma2 in clot retraction. Thromb Res 2007; 120: 251-258.
11 Leon C, Eckly A, Hechler B. et al. Megakaryocyte-restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion. Blood 2007; 110: 3183-3191.
12 Leng L, Kashiwagi H, Ren XD. et al. RhoA and the function of platelet integrin alphaIIbbeta3. Blood 1998; 91: 4206-4215.
13 Schoenwaelder SM, Hughan SC, Boniface K. et al. RhoA sustains integrin alpha IIbbeta 3 adhesion contacts under high shear. J Biol Chem 2002; 277: 14738-14746.
14 Burridge K, Wennerberg K. Rho and Rac take center stage. Cell 2004; 116: 167-179.
15 Hartwig JH, Bokoch GM, Carpenter CL. et al. Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell 1995; 82: 643-653.
16 Azim AC, Barkalow K, Chou J. et al. Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor. Blood 2000; 95: 959-964.
17 Johnson GJ, Leis LA, Krumwiede MD. et al. The critical role of myosin IIA in platelet internal contraction. J Thromb Haemost 2007; 5: 1516-1529.
18 Somlyo AP, Somlyo AV. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 2000; 522: 177-185.
19 Amano M, Ito M, Kimura K. et al. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 1996; 271: 20246-20249.
20 Severin S, Gratacap MP, Lenain N. et al. Deficiency of Src homology 2 domain-containing inositol 5-phosphatase 1 affects platelet responses and thrombus growth. J Clin Invest 2007; 117: 944-952.
21 Randriamboavonjy V, Isaak J, Fromel T. et al. AMPK alpha2 subunit is involved in platelet signaling, clot retraction, and thrombus stability. Blood 2010; 116: 2134-2140.
22 Osdoit S, Rosa JP. Fibrin clot retraction by human platelets correlates with alpha(IIb)beta(3) integrin-dependent protein tyrosine dephosphorylation. J Biol Chem 2001; 276: 6703-6710.
23 Mazharian A, Roger S, Berrou E. et al. Protease-activating receptor-4 induces full platelet spreading on a fibrinogen matrix: involvement of ERK2 and p38 and Ca2+ mobilization. J Biol Chem 2007; 282: 5478-5487.
24 McCarty OJ, Larson MK, Auger JM. et al. Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow. J Biol Chem 2005; 280: 39474-39484.
25 Pandey D, Goyal P, Dwivedi S. et al. Unraveling a novel Rac1-mediated signaling pathway that regulates cofilin dephosphorylation and secretion in thrombin-stimulated platelets. Blood 2009; 114: 415-424.
26 Zhang LJ, Tao BB, Wang MJ. et al. PI3K p110alpha isoform-dependent Rho GTPase Rac1 activation mediates H2S-promoted endothelial cell migration via actin cytoskeleton reorganization. PLoS One 2012; 7: e44590.
27 Carr Jr. ME, Carr SL, Hantgan RR. et al. Glycoprotein IIb/IIIa blockade inhibits platelet-mediated force development and reduces gel elastic modulus. Thromb Haemost 1995; 73: 499-505.
28 Nurden AT. Inherited abnormalities of platelets. Thromb Haemost 1999; 82: 468-480.
29 Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelet paradigm. Blood 1998; 91: 2645-2657.
30 Parise LV. Integrin alpha(IIb)beta(3) signaling in platelet adhesion and aggregation. Curr Opin Cell Biol 1999; 11: 597-601.
31 Law DA, DeGuzman FR, Heiser P. et al. Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function. Nature 1999; 401: 808-811.
32 Toth-Zsamboki E, Oury C, Cornelissen H. et al. P2X1-mediated ERK2 activation amplifies the collagen-induced platelet secretion by enhancing myosin light chain kinase activation. J Biol Chem 2003; 278: 46661-46667.
33 Flevaris P, Stojanovic A, Gong H. et al. A molecular switch that controls cell spreading and retraction. J Cell Biol 2007; 179: 553-565.
34 Torti M, Bertoni A, Sinigaglia F. et al. The platelet cytoskeleton regulates the aggregation-dependent synthesis of phosphatidylinositol 3,4-bisphosphate induced by thrombin. FEBS Lett 2000; 466: 355-358.
35 Bauer M, Retzer M, Wilde JI. et al. Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets. Blood 1999; 94: 1665-1672.
36 Kohama K, Okagaki T, Hayakawa K. et al. A novel regulatory effect of myosin light chain kinase from smooth muscle on the ATP-dependent interaction between actin and myosin. Biochem Biophys Res Commun 1992; 184: 1204-1211.
37 Ye LH, Hayakawa K, Lin Y. et al. The regulatory role of myosin light chain kinase as an actin-binding protein. J Biochem 1994; 116: 1377-1382.
38 Sato M, Ye LH, Kohama K. Myosin light chain kinase from vascular smooth muscle inhibits the ATP-dependent interaction between actin and myosin by binding to actin. J Biochem 1995; 118: 1-3.
39 Chou CL, Christensen BM, Frische S. et al. Non-muscle myosin II and myosin light chain kinase are downstream targets for vasopressin signaling in the renal collecting duct. J Biol Chem 2004; 279: 49026-49035.
40 Kunieda Y, Nakagawa K, Nishimura H. et al. HMG CoA reductase inhibitor suppresses the expression of tissue factor and plasminogen activator inhibitor-1 induced by angiotensin II in cultured rat aortic endothelial cells. Thromb Res 2003; 110: 227-234.
41 Rolfe BE, Worth NF, World CJ. et al. Rho and vascular disease. Atherosclerosis 2005; 183: 1-16.
42 Calaminus SD, Auger JM, McCarty OJ. et al. MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability. J Thromb Haemost 2007; 5: 2136-2145.
43 Machesky LM, Hall A. Rho: a connection between membrane receptor signalling and the cytoskeleton. Trends Cell Biol 1996; 6: 304-310.
44 Missy K, Plantavid M, Pacaud P. et al. Rho-kinase is involved in the sustained phosphorylation of myosin and the irreversible platelet aggregation induced by PAR1 activating peptide. Thromb Haemost 2001; 85: 514-520.
45 Pleines I, Hagedorn I, Gupta S. et al. Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis. Blood 2012; 119: 1054-1063.
46 Paul BZ, Kim S, Dangelmaier C. et al. Dynamic regulation of microtubule coils in ADP-induced platelet shape change by p160ROCK (Rho-kinase). Platelets 2003; 14: 159-169.
47 Godin CM, Ferguson SS. The angiotensin II type 1 receptor induces membrane blebbing by coupling to Rho A, Rho kinase, and myosin light chain kinase. Mol Pharmacol 2010; 77: 903-911.
48 Stefanini L, Boulaftali Y, Ouellette TD. et al. Rap1-Rac1 Circuits Potentiate Platelet Activation. Arterioscler Thromb Vasc Biol 2012; 32: 434-441.
49 Hall A. Rho GTPases and the control of cell behaviour. Biochem Soc Trans 2005; 33: 891-895.
50 Dwivedi S, Pandey D, Khandoga AL. et al. Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in human blood stimulated by atherosclerotic plaque. J Transl Med 2010; 8: 128.

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