HMGB1 binds to activated platelets via the receptor for advanced glycation end products and is present in platelet rich human coronary artery thrombi

Summary

High mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p< 0.05), and TLR4^-/- mice (MFI 2.11 vs 25.65, p< 0.05) but failed to show binding to platelets from RAGE^-/- mice (p > 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p< 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.

Keywords

HMGB1 - platelets - RAGE - ACS

^* Equally contributing authors

• References

• 1 Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002; 418: 191-195.
• 2 Andersson U, Tracey KJ. HMGB1 is a therapeutic target for sterile inflammation and infection. Annu Rev Immunol 2011; 29: 139-162.
• 3 de Souza AW, Westra J, Limburg PC. et al. HMGB1 in vascular diseases: Its role in vascular inflammation and atherosclerosis. Autoimmun Rev 2012; 11: 909-917.
• 4 Wang H, Bloom O, Zhang M. et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science 1999; 285: 248-251.
• 5 Bianchi ME, Manfredi AA. High-mobility group box 1 (HMGB1) protein at the crossroads between innate and adaptive immunity. Immunol Rev 2007; 220: 35-46.
• 6 Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature 2011; 473: 317-325.
• 7 Inoue K, Kawahara K, Biswas KK. et al. HMGB1 expression by activated vascular smooth muscle cells in advanced human atherosclerosis plaques. Cardiovasc Pathol 2007; 16: 136-143.
• 8 Kalinina N, Agrotis A, Antropova Y. et al. Increased expression of the DNA-binding cytokine HMGB1 in human atherosclerotic lesions: role of activated macrophages and cytokines. Arterioscler Thromb Vasc Biol 2004; 24: 2320-2325.
• 9 Kanellakis P, Agrotis A, Kyaw TS. et al. High-mobility group box protein 1 neutralisation reduces development of diet-induced atherosclerosis in apolipoprotein e-deficient mice. Arterioscler Thromb Vasc Biol 2011; 31: 313-319.
• 10 Huo Y, Schober A, Forlow SB. et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat Med 2003; 9: 61-67.
• 11 Langer HF, Gawaz M. Platelet-vessel wall interactions in atherosclerotic disease. Thromb Haemost 2008; 99: 480-486.
• 12 Eisenhardt SU, Habersberger J, Murphy A. et al. Dissociation of pentameric to monomeric C-reactive protein on activated platelets localizes inflammation to atherosclerotic plaques. Circ Res 2009; 105: 128-137.
• 13 Schulz C, Massberg S. Platelets in atherosclerosis and thrombosis. Handb Exp Pharmacol 2012; 111-133.
• 14 Duerschmied D, Suidan GL, Demers M. et al. Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice. Blood 2013; 121: 1008-1015.
• 15 Lievens D, Zernecke A, Seijkens T. et al. Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis. Blood 2010; 116: 4317-4327.
• 16 Peter K, Bobik A. HMGB1 signals danger in acute coronary syndrome: emergence of a new risk marker for cardiovascular death?. Atherosclerosis 2012; 221: 317-318.
• 17 Liu K, Mori S, Takahashi HK. et al. Anti-high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats. FASEB J 2007; 21: 3904-3916.
• 18 Birschmann I, Mietner S, Dittrich M. et al. Use of functional highly purified human platelets for the identification of new proteins of the IPP signaling pathway. Thromb Res 2008; 122: 59-68.
• 19 Libby P. Inflammation in atherosclerosis. Arterioscler Thromb Vasc Biol 2012; 32: 2045-2051.
• 20 Langer HF, Bigalke B, Seizer P. et al. Interaction of platelets and inflammatory endothelium in the development and progression of coronary artery disease. Semin Thromb Hemost 2010; 36: 131-138.
• 21 Semple JW, Italiano Jr. JE, Freedman J. Platelets and the immune continuum. Nat Rev Immunol 2011; 11: 264-274.
• 22 Duerschmied D, Bode C, Ahrens I. Immune functions of platelets. Thromb Haemost 2014; 112: 678-691.
• 23 Maugeri N, Franchini S, Campana L. et al. Circulating platelets as a source of the damage-associated molecular pattern HMGB1 in patients with systemic sclerosis. Autoimmunity 2012; 45: 584-587.
• 24 Rouhiainen A, Imai S, Rauvala H. et al. Occurrence of amphoterin (HMG1) as an endogenous protein of human platelets that is exported to the cell surface upon platelet activation. Thromb Haemost 2000; 84: 1087-1094.
• 25 Maugeri N, Campana L, Gavina M. et al. Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps. J Thromb Haemost 2014; 12: 2074-2088.
• 26 Yan SF, Yan SD, Ramasamy R. et al. Tempering the wrath of RAGE: an emerging therapeutic strategy against diabetic complications, neurodegeneration, and inflammation. Ann Med 2009; 41: 408-422.
• 27 Park JS, Gamboni-Robertson F, He Q. et al. High mobility group box 1 protein interacts with multiple Toll-like receptors. Am J Physiol Cell Physiol 2006; 290: C917-924.
• 28 Harja E, Bu DX, Hudson BI. et al. Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE-/- mice. J Clin Invest 2008; 118: 183-194.
• 29 Soro-Paavonen A, Watson AM, Li J. et al. Receptor for advanced glycation end products (RAGE) deficiency attenuates the development of atherosclerosis in diabetes. Diabetes 2008; 57: 2461-2469.
• 30 Varo N, Libby P, Nuzzo R. et al. Elevated release of sCD40L from platelets of diabetic patients by thrombin, glucose and advanced glycation end products. Diab Vasc Dis Res 2005; 2: 81-87.
• 31 Gawlowski T, Stratmann B, Ruetter R. et al. Advanced glycation end products strongly activate platelets. Eur J Nutr 2009; 48: 475-481.
• 32 Denis MM, Tolley ND, Bunting M. et al. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell 2005; 122: 379-391.
• 33 Zimmerman GA, Weyrich AS. Signal-dependent protein synthesis by activated platelets: new pathways to altered phenotype and function. Arterioscler Thromb Vasc Biol 2008; 28: s17-24.
• 34 Vazzana N, Guagnano MT, Cuccurullo C. et al. Endogenous secretory RAGE in obese women: association with platelet activation and oxidative stress. J Clin Endocrinol Metab 2012; 97: E1726-1730.
• 35 Fukami A, Adachi H, Yamagishi S. et al. Factors associated with serum high mobility group box 1 (HMGB1) levels in a general population. Metabolism 2009; 58: 1688-1693.
• 36 Schulz C, Penz S, Hoffmann C. et al. Platelet GPVI binds to collagenous structures in the core region of human atheromatous plaque and is critical for atheroprogression in vivo. Basic Res Cardiol 2008; 103: 356-367.
• 37 Seizer P, Gawaz M, May AE. Platelet-monocyte interactions-a dangerous liaison linking thrombosis, inflammation and atherosclerosis. Curr Med Chem 2008; 15: 1976-1980.
• 38 Shah PK, Falk E, Badimon JJ. et al. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques. Potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation 1995; 92: 1565-1569.
• 39 Martinet W, Schrijvers DM, De Meyer GR. Molecular and cellular mechanisms of macrophage survival in atherosclerosis. Basic Res Cardiol 2012; 107: 297.
• 40 Schiraldi M, Raucci A, Munoz LM. et al. HMGB1 promotes recruitment of inflammatory cells to damaged tissues by forming a complex with CXCL12 and signaling via CXCR4. J Exp Med 2012; 209: 551-563.
• 41 Zernecke A, Schober A, Bot I. et al. SDF-1alpha/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells. Circulation Res 2005; 96: 784-791.
• 42 Stellos K, Langer H, Daub K. et al. Platelet-derived stromal cell-derived factor-1 regulates adhesion and promotes differentiation of human CD34+ cells to endothelial progenitor cells. Circulation 2008; 117: 206-215.
• 43 Stellos K, Bigalke B, Langer H. et al. Expression of stromal-cell-derived factor-1 on circulating platelets is increased in patients with acute coronary syndrome and correlates with the number of CD34+ progenitor cells. Eur Heart J 2009; 30: 584-593.
• 44 Stellos K, Bigalke B, Borst O. et al. Circulating platelet-progenitor cell coaggregate formation is increased in patients with acute coronary syndromes and augments recruitment of CD34+ cells in the ischaemic microcirculation. Eur Heart J 2013; 34: 2548-2556.
• 45 Schwarz M, Meade G, Stoll P. et al. Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets. Circ Res 2006; 99: 25-33.
• 46 Stoll P, Bassler N, Hagemeyer CE. et al. Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation. Arterioscler Thromb Vasc Biol 2007; 27: 1206-1212.
• 47 Wang X, Hagemeyer CE, Hohmann JD. et al. Novel single-chain antibody-targeted microbubbles for molecular ultrasound imaging of thrombosis: validation of a unique noninvasive method for rapid and sensitive detection of thrombi and monitoring of success or failure of thrombolysis in mice. Circulation 2012; 125: 3117-3126.