Platelet adhesion to podoplanin under flow is mediated by the receptor CLEC-2 and stabilised by Src/Syk-dependent platelet signalling

Summary

Platelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in bloodfilled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following α∥bβ3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγR∥A receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice.

Keywords

Platelet physiology - rheology - lymphatics - adhesion molecules - signal transduction

Video 1 Suppl. Video 1: Src and Syk inhibitors reduce stability of platelet adhesion to LECs. To analyse real-time adhesion of platelets to LECs, heparinized whole blood was incubated with FITC-conjugated anti-human CD41 and CD42b antibodies at 1/100 dilution and inhibitors or vehicle control as appropriate for 10 minutes and then perfused over LEC monolayers at 50 s^-1. Digital images were captured at 500 ms interval with 500 ms exposure time and 2×2 binning settings to create 1 minute videos. Upper left: vehicle; upper right: 9 µM integrilin; lower left: 10 µM dasatinib; lower right: 30 µM PRT060318.

Video 2 Suppl. Video 2: Src and Syk inhibitors reduce stability of platelet adhesion to hFcPDPN. To analyse real-time adhesion of platelets to hFcPDPN, heparinized whole blood was incubated with 4 µM DiOC6, 10µg/ml IV.3 antibody and inhibitors or vehicle control as appropriate for 10 minutes and then perfused over hFcPDPN-coated channels at 50 s^-1. Digital images were captured at 250 ms interval with 200 ms exposure time and 2×2 binning settings to create 1 minute videos. Upper left: vehicle; upper right: 9 µM integrilin; lower left: 10 µM dasatinib; lower right: 30 µM PRT060318.

Conflicts of interest

None declared.

• References

• 1 Hägerling R, Pollmann C, Andreas M. et al. A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy. EMBO J 2013; 32: 629-644.
• 2 Yang Y, Garcia-Verdugo JM, Soriano-Navarro M. et al. Lymphatic endothelial progenitors bud from the cardinal vein and intersomitic vessels in mammalian embryos. Blood 2012; 120: 2340-2348.
• 3 Tober J, Koniski A, McGrath KE. et al. The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis. Blood 2007; 109: 1433-1441.
• 4 Bertozzi CC, Schmaier AA, Mericko P. et al. Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling. Blood 2010; 116: 661-670.
• 5 Bohmer R, Neuhaus B, Buhren S. et al. Regulation of developmental lymphangiogenesis by Syk(+) leukocytes. Dev Cell 2010; 18: 437-449.
• 6 Finney BA, Schweighoffer E, Navarro-Núñez L. et al. CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development. Blood 2012; 119: 1747-1756.
• 7 Ichise H, Ichise T, Ohtani O. et al. Phospholipase Cgamma2 is necessary for separation of blood and lymphatic vasculature in mice. Development 2009; 136: 191-195.
• 8 Schacht V, Ramirez MI, Hong YK. et al. T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J 2003; 22: 3546-3556.
• 9 Uhrin P, Zaujec J, Breuss JM. et al. Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation. Blood 2010; 115: 3997-4005.
• 10 Fu J, Gerhardt H, McDaniel JM. et al. Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice. J Clin Invest 2008; 118: 3725-3737.
• 11 Carramolino L, Fuentes J, Garcia-Andres C. et al. Platelets play an essential role in separating the blood and lymphatic vasculatures during embryonic angiogenesis. Circ Res 2010; 106: 1197-1201.
• 12 Kiefer F, Brumell J, Al-Alawi N. et al. The Syk protein tyrosine kinase is essential for Fcgamma receptor signaling in macrophages and neutrophils. Mol Cell Biol 1998; 18: 4209-4220.
• 13 El Zawahry MD, Sayed NM, El-Awady HM. et al. A study of the gross, microscopic and functional anatomy of the thoracic duct and the lympho-venous junction. Int Surg 1983; 68: 135-138.
• 14 Hess PR, Rawnsley DR, Jakus Z. et al. Platelets mediate lymphovenous hemostasis to maintain blood-lymphatic separation throughout life. J Clin Invest 2014; 124: 273-284.
• 15 Bender M, May F, Lorenz V. et al. Combined in vivo depletion of glycoprotein VI and C-type lectin-like receptor 2 severely compromises hemostasis and abrogates arterial thrombosis in mice. Arterioscler Thromb Vasc Biol 2013; 33: 926-934.
• 16 May F, Hagedorn I, Pleines I. et al. CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis. Blood 2009; 114: 3464-3472.
• 17 Andre P, Morooka T, Sim D. et al. Critical role for Syk in responses to vascular injury. Blood 2011; 118: 5000-5010.
• 18 Law DA, Nannizzi-Alaimo L, Ministri K. et al. Genetic and pharmacological analyses of Syk function in SOHIIbβ3 signaling in platelets. Blood 1999; 93: 2645-2652.
• 19 Pan Y, Yago T, Fu J. et al. Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets. Blood 2014; 124: 3656-3665.
• 20 Hughes CE, Navarro-Núñez L, Finney BA. et al. CLEC-2 is not required for platelet aggregation at arteriolar shear. J Thromb Haemost 2010; 08: 2328-2332.
• 21 Ventura A, Kirsch DG, McLaughlin ME. et al. Restoration of p53 function leads to tumour regression in vivo. Nature 2007; 445: 661-665.
• 22 Séverin S, Pollitt AY, Navarro-Núñez L. et al. Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signaling. J Biol Chem 2011; 286: 4107-4116.
• 23 Turner M, Mee PJ, Costello PS. et al. Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 1995; 378: 298-302.
• 24 Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. J Am Med Assoc 1999; 282: 2035-2042.
• 25 Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012; 09: 671-675.
• 26 Bazzoni G, Dejana E. Endothelial cell-to-cell junctions: molecular organisation and role in vascular homeostasis. Physiol Rev 2004; 84: 869-901.
• 27 Huang J, Roth R, Heuser JE. et al. Integrin alpha(v)beta(3) on human endothelial cells binds von Willebrand factor strings under fluid shear stress. Blood 2009; 113: 1589-1597.
• 28 Mangin P, Yuan Y, Goncalves I. et al. Signaling role for phospholipase C gamma 2 in platelet glycoprotein Ib alpha calcium flux and cytoskeletal reorganisation. Involvement of a pathway distinct from FcR gamma chain and Fc gamma RIIA. J Biol Chem 2003; 278: 32880-32891.
• 29 Borgognone A, Navarro-Núñez L, Correia JN. et al. CLEC-2-dependent activation of mouse platelets is weakly inhibited by cAMP but not by cGMP. J Thromb Haemost 2014; 12: 550-559.
• 30 Pollitt AY, Grygielska B, Leblond B. et al. Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerisation, secondary mediators, and Rac. Blood 2010; 115: 2938-2946.
• 31 Reilly MP, Sinha U, Andre P. et al. PRT-060318, a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model. Blood 2011; 117: 2241-2246.
• 32 Ruggeri ZM, Mendolicchio GL. Adhesion mechanisms in platelet function. Circ Res 2007; 100: 1673-1685.
• 33 Navarro-Núñez L, Langan SA, Nash GB. et al. The physiological and pathophysiological roles of platelet CLEC-2. Thromb Haemost 2013; 109: 991-998.
• 34 Auger JM, Kuijpers MJ, Senis YA. et al. Adhesion of human and mouse platelets to collagen under shear: a unifying model. FASEB J 2005; 19: 825-827.
• 35 Melis E, Bonnefoy A, Daenens K. et al. αIIbβ3 antagonism vs. antiadhesive treatment to prevent platelet interactions with vascular subendothelium. J Thromb Haemost 2004; 02: 993-1002.
• 36 Burkhart JM, Vaudel M, Gambaryan S. et al. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012; 120: e73-e82.
• 37 Gitz E, Pollitt AY, Gitz-Francois JJ. et al. CLEC-2 expression is maintained on activated platelets and on platelet microparticles. Blood 2014; 124: 2262-2270.
• 38 Zawieja DC. Contractile physiology of lymphatics. Lymphat Res Biol 2009; 07: 87-96.
• 39 Hughes CE, Pollitt AY, Mori J. et al. CLEC-2 activates Syk through dimerisation. Blood 2010; 115: 2947-2955.
• 40 Pollitt AY, Poulter NS, Gitz E. et al. Syk and Src family kinases regulate CLEC-2 mediated clustering of podoplanin and platelet adhesion to lymphatic endothelial cells. J Biol Chem 2014; 289: 35695-35710.
• 41 Suzuki-Inoue K, Fuller GL, Garcia A. et al. A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2. Blood 2006; 107: 542-549.
• 42 Kaur M, Singh M, Silakari O. Inhibitors of switch kinase ‘spleen tyrosine kinase’ in inflammation and immune-mediated disorders: A review. Eur J Med Chem 2013; 67: 434-446.
• 43 Goldblatt M, Huggins JT, Doelken P. et al. Dasatinib-induced pleural effusions: a lymphatic network disorder?. Am J Med Sci 2009; 338: 414-417.
• 44 Hochhaus A, Kantarjian H. The development of dasatinib as a treatment for chronic myeloid leukemia (CML): from initial studies to application in newly diagnosed patients. J Cancer Res Clin Oncol 2013; 139: 1971-1984.
• 45 Falanga A, Russo L, Verzeroli C. Mechanisms of thrombosis in cancer. Thromb Res 2013; 131 (Suppl. 01) S59-S62.
• 46 Konstantopoulos K, Thomas SN. Cancer cells in transit: the vascular interactions of tumor cells. Annu Rev Biomed Eng 2009; 11: 177-202.
• 47 Cueni LN, Hegyi I, Shin JW. et al. Tumor lymphangiogenesis and metastasis to lymph nodes induced by cancer cell expression of podoplanin. Am J Pathol 2010; 177: 1004-1016.
• 48 Kunita A, Kashima TG, Morishita Y. et al. The platelet aggregation-inducing factor aggrus/podoplanin promotes pulmonary metastasis. Am J Pathol 2007; 170: 1337-1347.
• 49 Kerrigan AM, Navarro-Núñez L, Pyz E. et al. Podoplanin-expressing inflammatory macrophages activate murine platelets via CLEC-2. J Thromb Haemost 2012; 10: 484-486.
• 50 Hatakeyama K, Kaneko MK, Kato Y. et al. Podoplanin expression in advanced atherosclerotic lesions of human aortas. Thromb Res 2012; 129: e70-e76.