Download PDF
Thromb Haemost 2011; 105(05): 873-882
DOI: 10.1160/TH10-10-0630
Cardiovascular Biology and Cell Signalling
Schattauer GmbH

Expression of the vitamin K-dependent proteins GAS6 and protein S and the TAM receptor tyrosine kinases in human atherosclerotic carotid plaques

Begoña Hurtado
1   Translational Research Laboratory, Catalan Institute of Oncology-IDIBELL, L’Hospitalet de Llobregat, Spain
2   Department of Cell Death and Proliferation, IIBB-CSIC-IDIBAPS, Barcelona, Spain
,
Xavier Muñoz
1   Translational Research Laboratory, Catalan Institute of Oncology-IDIBELL, L’Hospitalet de Llobregat, Spain
,
Pedro Recarte-Pelz
2   Department of Cell Death and Proliferation, IIBB-CSIC-IDIBAPS, Barcelona, Spain
,
Nadia Garcia
1   Translational Research Laboratory, Catalan Institute of Oncology-IDIBELL, L’Hospitalet de Llobregat, Spain
,
Anna Luque
3   Cardiovascular Research Center, ICCC, Barcelona, Spain
,
Jerzy Krupinski
3   Cardiovascular Research Center, ICCC, Barcelona, Spain
4   Department of Neurology, Stroke Unit, Hospital Universitari Mutua de Terrassa, Terrassa, Spain
5   Fundacio IDIBELL, Barcelona, Spain
6   School of Biology, Chemistry and Health Science, and Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK
,
Núria Sala
1   Translational Research Laboratory, Catalan Institute of Oncology-IDIBELL, L’Hospitalet de Llobregat, Spain
,
Pablo García de Frutos
2   Department of Cell Death and Proliferation, IIBB-CSIC-IDIBAPS, Barcelona, Spain
› Author Affiliations Financial support: We acknowledge grants SAF2006–07681 and BFU2007–61699/BFI from the Spanish Ministry of Science and Innovation; Marató de TV3 2008–121 from Fundació la Marató de TV3, PI081420 from the ISCIII, Spanish Ministry of Health. XM, NG and NS are members of the ISCIII network RTICCC (ISCIII DR06/0020). XM was supported by ISCIII CA06/0200 and BH has been recipient (2004–2008) of a grant from the Institut d’Investigació Biomèdica de Bellvitge (IDIBELL06/IDB-001).
Further Information

Publication History

Received: 01 October 2010

Accepted after major revision: 25 January 2011

Publication Date:
28 November 2017 (online)

    Permissions and Reprints

Summary

The GAS6/ProS-TAM system is composed of two vitamin K-dependent ligands (GAS6 and protein S) and their three protein tyrosine kinase receptors TYRO3, AXL and MERTK, known as the TAM receptors. The system plays a prominent role in conditions of injury, inflammation and repair. In murine models of atherosclerotic plaque formation, mutations in its components affect atherosclerosis severity. Here we used Taqman low-density arrays and immunoblotting to study mRNA and protein expression of GAS6, ProS and the TAM receptors in human carotid arteries with different degrees of atherosclerosis. The results show a clear down-regulation of the expression of AXL in atheroma plaques with respect to normal carotids that is matched by decreased abundance of AXL in protein extracts detected by immunoblotting. A similar decrease was observed in PROS1 mRNA expression in atherosclerotic carotids compared to the normal ones, but in this case protein S (ProS) was clearly increased in protein extracts of carotid arteries with increasing grade of atherosclerosis, suggesting that ProS is carried into the plaque. MERTK was also increased in atherosclerotic carotid arteries with respect to the normal ones, suggesting that the ProS-MERTK axis is functional in advanced human atherosclerotic plaques. MERTK was expressed in macrophages, frequently in association with ProS, while ProS was abundant also in the necrotic core. Our data suggest that the ProS-MERTK ligand-receptor pair was active in advanced stages of atherosclerosis, while AXL signalling is probably down-regulated.

Keywords

PROS1 - GAS6 - TYRO3 - AXL - MERTK, - arterioesclerosis - atheroma - carotid plaque - vitamin K-dependent proteins - receptor tyrosine kinases

 

  • References

  • 1 Hansson K, Stenflo J. Post-translational modifications in proteins involved in blood coagulation. J Thromb Haemost 2005; 3: 2633-2648.
    CrossrefPubMedGoogle Scholar
  • 2 Linger RM, Keating AK, Earp HS. et al. TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer. Adv Cancer Res 2008; 100: 35-83.
    PubMedGoogle Scholar
  • 3 Lemke G, Rothlin CV. Immunobiology of the TAM receptors. Nat Rev Immunol 2008; 8: 327-336.
    CrossrefPubMedGoogle Scholar
  • 4 Castoldi E, Hackeng TM. Regulation of coagulation by protein S. Curr Opin Hematol 2008; 15: 529-536.
    CrossrefPubMedGoogle Scholar
  • 5 Garcia de Frutos P, Fuentes-Prior P, Hurtado B. et al. Molecular basis of protein S deficiency. Thromb Haemost 2007; 98: 543-556.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 6 Varnum BC, Young C, Elliott G. et al. Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6. Nature 1995; 373: 623-626.
    CrossrefPubMedGoogle Scholar
  • 7 Stitt TN, Conn G, Gore M. et al. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 1995; 80: 661-670.
    CrossrefPubMedGoogle Scholar
  • 8 Godowski PJ, Mark MR, Chen J. et al. Reevaluation of the roles of protein S and Gas6 as ligands for the receptor tyrosine kinase Rse/Tyro 3. Cell 1995; 82: 355-358.
    CrossrefPubMedGoogle Scholar
  • 9 Lu Q, Lemke G. Homeostatic regulation of the immune system by receptor tyro-sine kinases of the Tyro 3 family. Science 2001; 293: 306-311.
    CrossrefPubMedGoogle Scholar
  • 10 Scott RS, McMahon EJ, Pop SM. et al. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 2001; 411: 207-211.
    CrossrefPubMedGoogle Scholar
  • 11 Burstyn-Cohen T, Heeb MJ, Lemke G. Lack of protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis. J Clin Invest 2009; 119: 2942-2953.
    CrossrefPubMedGoogle Scholar
  • 12 Saller F, Brisset AC, Tchaikovski SN. et al. Generation and phenotypic analysis of protein S-deficient mice. Blood 2009; 114: 2307-2314.
    CrossrefPubMedGoogle Scholar
  • 13 Evenas P, Garcia de Frutos P, Nicolaes GA. et al. The second laminin G-type domain of protein S is indispensable for expression of full cofactor activity in activated protein C-catalysed inactivation of factor Va and factor VIIIa. Thromb Haemost 2000; 84: 271-277.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Balogh I, Hafizi S, Stenhoff J. et al. Analysis of Gas6 in human platelets and plasma. Arterioscler Thromb Vasc Biol 2005; 25: 1280-1286.
    CrossrefPubMedGoogle Scholar
  • 15 Ming Cao W, Murao K, Imachi H. et al. Phosphatidylinositol 3-OH kinase-Akt/ protein kinase B pathway mediates Gas6 induction of scavenger receptor a in immortalized human vascular smooth muscle cell line. Arterioscler Thromb Vasc Biol 2001; 21: 1592-1597.
    CrossrefPubMedGoogle Scholar
  • 16 Cavet ME, Smolock EM, Ozturk OH. et al. Gas6-axl receptor signaling is regulated by glucose in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2008; 28: 886-891.
    CrossrefPubMedGoogle Scholar
  • 17 Ganopolsky JG, Abid MR, Aird WC. et al. GAS6-induced signaling in human endothelial cells is mediated by FOXO1a. J Thromb Haemost 2008; 6: 1804-1811.
    CrossrefPubMedGoogle Scholar
  • 18 Tjwa M, Bellido-Martin L, Lin Y. et al. Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets, and leukocytes. Blood 2008; 111: 4096-4105.
    CrossrefPubMedGoogle Scholar
  • 19 Melaragno MG, Wuthrich DA, Poppa V. et al. Increased expression of Axl tyrosine kinase after vascular injury and regulation by G protein-coupled receptor agonists in rats. Circ Res 1998; 83: 697-704.
    CrossrefPubMedGoogle Scholar
  • 20 Korshunov VA, Mohan AM, Georger MA. et al. Axl, a receptor tyrosine kinase, mediates flow-induced vascular remodeling. Circ Res 2006; 98: 1446-1452.
    CrossrefPubMedGoogle Scholar
  • 21 Konishi A, Aizawa T, Mohan A. et al. Hydrogen peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells. J Biol Chem 2004; 279: 28766-28770.
    CrossrefPubMedGoogle Scholar
  • 22 Cosemans JM, van Kruchten R, Olieslagers S. et al. Potentiating role of Gas6 and TAM receptors in human and murine platelet activation and thrombus stabilization. J Thromb Haemost 2010; 8: 1797-1808.
    CrossrefPubMedGoogle Scholar
  • 23 Fernandez-Fernandez L, Bellido-Martin L, Garcia de Frutos P. Growth arrest-specific gene 6 (GAS6). An outline of its role in haemostasis and inflammation. Thromb Haemost 2008; 100: 604-610.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 24 Angelillo-Scherrer A, de Frutos P, Aparicio C. et al. Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis. Nat Med 2001; 7: 215-221.
    CrossrefPubMedGoogle Scholar
  • 25 Galkina E, Ley K. Immune and inflammatory mechanisms of atherosclerosis. Annu Rev Immunol 2009; 27: 165-197.
    CrossrefPubMedGoogle Scholar
  • 26 Tjwa M, Moons L, Lutgens E. Pleiotropic role of growth arrest-specific gene 6 in atherosclerosis. Curr Opin Lipidol 2009; 20: 386-392.
    CrossrefPubMedGoogle Scholar
  • 27 Lutgens E, Tjwa M, Garcia de Frutos P. et al. Genetic loss of Gas6 induces plaque stability in experimental atherosclerosis. J Pathol 2008; 216: 55-63.
    CrossrefPubMedGoogle Scholar
  • 28 Thorp E, Cui D, Schrijvers DM. et al. Mertk receptor mutation reduces efferocytosis efficiency and promotes apoptotic cell accumulation and plaque necrosis in atherosclerotic lesions of apoe-/- mice. Arterioscler Thromb Vasc Biol 2008; 28: 1421-1428.
    CrossrefPubMedGoogle Scholar
  • 29 Ait-Oufella H, Pouresmail V, Simon T. et al. Defective mer receptor tyrosine kinase signaling in bone marrow cells promotes apoptotic cell accumulation and accelerates atherosclerosis. Arterioscler Thromb Vasc Biol 2008; 28: 1429-1431.
    CrossrefPubMedGoogle Scholar
  • 30 Hurtado B, Abasolo N, Munoz X. et al. Association study between polymorphims in GAS6-TAM genes and carotid atherosclerosis. Thromb Haemost 2010; 104: 592-598.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 31 Muñoz X, Sumoy L, Ramirez-Lorca R. et al. Human vitamin K-dependent GAS6: gene structure, allelic variation, and association with stroke. Hum Mutat 2004; 23: 506-512.
    CrossrefPubMedGoogle Scholar
  • 32 Muñoz X, Obach V, Hurtado B. et al. Association of specific haplotypes of GAS6 gene with stroke. Thromb Haemost 2007; 98: 406-412.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 Jiang L, Liu CY, Yang QF. et al. Plasma level of growth arrest-specific 6 (GAS6) protein and genetic variations in the GAS6 gene in patients with acute coronary syndrome. Am J Clin Pathol 2009; 131: 738-743.
    CrossrefPubMedGoogle Scholar
  • 34 Bladin CF, Alexandrov AV, Murphy J. et al. Carotid Stenosis Index. A new method of measuring internal carotid artery stenosis. Stroke 1995; 26: 230-234.
    CrossrefPubMedGoogle Scholar
  • 35 Stary HC, Chandler AB, Dinsmore RE. et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation 1995; 92: 1355-1374.
    CrossrefPubMedGoogle Scholar
  • 36 Bamford J, Sandercock P, Dennis M. et al. Classification and natural history of clinically identifiable subtypes of cerebral infarction. Lancet 1991; 337: 1521-1526.
    CrossrefPubMedGoogle Scholar
  • 37 Hellemans J, Mortier G, De Paepe A. et al. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 2007; 8: R19.
    CrossrefPubMedGoogle Scholar
  • 38 Oksjoki R, Kovanen PT, Mayranpaa MI. et al. Complement regulation in human atherosclerotic coronary lesions. Immunohistochemical evidence that C4b-binding protein negatively regulates the classical complement pathway, and that C5b-9 is formed via the alternative complement pathway. Atherosclerosis 2007; 192: 40-48.
    CrossrefPubMedGoogle Scholar
  • 39 Luque A, Turu M, Juan-Babot O. et al. Overexpression of hypoxia/inflammatory markers in atherosclerotic carotid plaques. Front Biosci 2008; 13: 6483-6490.
    PubMedGoogle Scholar
  • 40 Nelken NA, Coughlin SR, Gordon D. et al. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest 1991; 88: 1121-1127.
    CrossrefPubMedGoogle Scholar
  • 41 Krupinski J, Turu MM, Martinez-Gonzalez J. et al. Endogenous expression of C-reactive protein is increased in active (ulcerated noncomplicated) human carotid artery plaques. Stroke 2006; 37: 1200-1204.
    CrossrefPubMedGoogle Scholar
  • 42 Sluimer JC, Gasc JM, van Wanroij JL. et al. Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis. J Am Coll Cardiol 2008; 51: 1258-1265.
    CrossrefPubMedGoogle Scholar
  • 43 Budagian V, Bulanova E, Orinska Z. et al. Soluble Axl is generated by ADAM10-dependent cleavage and associates with Gas6 in mouse serum. Mol Cell Biol 2005; 25: 9324-9339.
    CrossrefPubMedGoogle Scholar
  • 44 Oksala N, Levula M, Airla N. et al. ADAM-9, ADAM-15, and ADAM-17 are up-regulated in macrophages in advanced human atherosclerotic plaques in aorta and carotid and femoral arteries--Tampere vascular study. Ann Med 2009; 41: 279-290.
    CrossrefPubMedGoogle Scholar
  • 45 Sather S, Kenyon KD, Lefkowitz JB. et al. A soluble form of the Mer receptor tyro-sine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation. Blood 2007; 109: 1026-1033.
    PubMedGoogle Scholar
  • 46 Hutter R, Valdiviezo C, Sauter BV. et al. Caspase-3 and tissue factor expression in lipid-rich plaque macrophages: evidence for apoptosis as link between inflammation and atherothrombosis. Circulation 2004; 109: 2001-2008.
    CrossrefPubMedGoogle Scholar
  • 47 Melaragno MG, Fridell YW, Berk BC. The Gas6/Axl system: a novel regulator of vascular cell function. Trends Cardiovasc Med 1999; 9: 250-253.
    CrossrefPubMedGoogle Scholar
  • 48 Fridell YW, Villa Jr. J, Attar EC. et al. GAS6 induces Axl-mediated chemotaxis of vascular smooth muscle cells. J Biol Chem 1998; 273: 7123-7126.
    CrossrefPubMedGoogle Scholar
  • 49 Murao K, Imachi H, Sayo Y. et al. A product of growth arrest-specific gene 6 modulates scavenger receptor expression in human vascular smooth muscle cells. FEBS Lett 1999; 459: 363-366.
    CrossrefPubMedGoogle Scholar
  • 50 Prasad D, Rothlin CV, Burrola P. et al. TAM receptor function in the retinal pigment epithelium. Mol Cell Neurosci 2006; 33: 96-108.
    CrossrefPubMedGoogle Scholar
  • 51 Uehara H, Shacter E. Auto-oxidation and oligomerization of protein S on the apoptotic cell surface is required for Mer tyrosine kinase-mediated phagocytosis of apoptotic cells. J Immunol 2008; 180: 2522-2530.
    CrossrefPubMedGoogle Scholar
  • 52 Liao D, Wang X, Li M. et al. Human protein S inhibits the uptake of AcLDL and expression of SR-A through Mer receptor tyrosine kinase in human macrophages. Blood 2009; 113: 165-174.
    CrossrefPubMedGoogle Scholar