Genetic deletion of platelet glycoprotein Ib alpha but not its extracellular domain protects from atherosclerosis

Summary

The pathogenesis of atherosclerosis involves the interplay of haematopoietic, stromal and endothelial cells. Platelet interactions with endothelium and leukocytes are pivotal for atherosclerosis promotion. Glycoprotein (GP) Ibα is the ligand-binding subunit of the platelet GPIb-IX-V receptor complex; its deficiency causes the Bernard-Soulier syndrome (BSS), characterised by absent platelet GPIb-IX-V, macrothrombocytopenia and bleeding. We designed this study to determine the role of platelet GPIbα in the pathogenesis of atherosclerosis using two unique knockout models. Ldlr^-/- mice were reconstituted with wild-type (wt), GPIbα^-/- (lacks GPIbα) or chimeric IL-4R/GPIbα-Tg (lacks GPIbα extracellular domain) bone marrow and assayed for atherosclerosis development after feeding with pro-atherogenic “western diet”. Here, we report that Ldlr^-/- mice reconstituted with GPIbα^-/- bone marrow developed less atherosclerosis compared to wt controls; accompanied by augmented accumulation of pro-inflammatory CD11b+ and CD11c+ myeloid cells, reduced oxLDL uptake and decreased TNF and IL 12p35 gene expression in the aortas. Flow cytometry and live cell imaging in whole blood-perfused microfluidic chambers revealed reduced platelet-monocyte aggregates in GPIbα^-/- mice, which resulted in decreased monocyte activation. Interestingly, Ldlr ^-/- mice reconstituted with IL-4R/GPIbα-Tg bone marrow, producing less abnormal platelets, showed atherosclerotic lesions similar to wt mice. Platelet interaction with blood monocytes and accumulation of myeloid cells in the aortas were also essentially unaltered. Moreover, only complete GPIbα ablation altered platelet microparticles and CCL5 chemokine production. Thus, atherosclerosis reduction in mice lacking GPIbα may not result from the defective GPIbα-ligand binding, but more likely is a consequence of functional defects of GPIbα^-/- platelets and reduced blood platelet counts.

Keywords

Aorta - atherosclerosis - inflammation - myeloid cells - platelets

• References

• 1 Koenen RR, von Hundelshausen P, Nesmelova IV. et al. Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice. Nat Med 2009; 15: 97-103.
• 2 Huo Y, Schober A, Forlow SB. et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nature Med 2003; 09: 61-67.
• 3 Gawaz M, Langer H, May AE. Platelets in inflammation and atherogenesis. J Clin Invest 2005; 115: 3378-3384.
• 4 Theilmeier G, Michiels C, Spaepen E. et al. Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia. Blood 2002; 99: 4486-4493.
• 5 Massberg S, Brand K, Gruner S. et al. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med 2002; 196: 887-896.
• 6 Gawaz M. Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium. Cardiovasc Res 2004; 61: 498-511.
• 7 Sarma J, Laan CA, Alam S. et al. Increased platelet binding to circulating monocytes in acute coronary syndromes. Circulation 2002; 105: 2166-2171.
• 8 Henn V, Slupsky JR, Grafe M. et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 1998; 391: 591-594.
• 9 von Hundelshausen P, Weber KS, Huo Y. et al. RANTES deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium. Circulation 2001; 103: 1772-1777.
• 10 Gawaz M, Page S, Massberg S. et al. Transient platelet interaction induces MCP-1 production by endothelial cells via I kappa B kinase complex activation. Thromb Haemost 2002; 88: 307-314.
• 11 Gawaz M, Neumann FJ, Dickfeld T. et al. Activated platelets induce monocyte chemotactic protein-1 secretion and surface expression of intercellular adhesion molecule-1 on endothelial cells. Circulation 1998; 98: 1164-1171.
• 12 Bombeli T, Schwartz BR, Harlan JM. Adhesion of activated platelets to endothelial cells: evidence for a GPIIbIIIa-dependent bridging mechanism and novel roles for endothelial intercellular adhesion molecule 1 (ICAM-1), alphavbeta3 integrin, and GPIbalpha. J Exp Med 1998; 187: 329-339.
• 13 Gawaz M, Brand K, Dickfeld T. et al. Platelets induce alterations of chemotactic and adhesive properties of endothelial cells mediated through an interleukin-1-dependent mechanism. Implications for atherogenesis. Atherosclerosis 2000; 148: 75-85.
• 14 Frenette PS, Johnson RC, Hynes RO. et al. Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin. Proc Natl Acad Sci USA 1995; 92: 7450-7454.
• 15 Frenette PS, Denis CV, Weiss L. et al. P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo. J Exp Med 2000; 191: 1413-1422.
• 16 Braun OO, Slotta JE, Menger MD. et al. Primary and secondary capture of platelets onto inflamed femoral artery endothelium is dependent on P-selectin and PSGL-1. Eur J Pharmacol 2008; 592: 128-132.
• 17 Burger PC, Wagner DD. Platelet P-selectin facilitates atherosclerotic lesion development. Blood 2003; 101: 2661-2666.
• 18 Manka D, Forlow SB, Sanders JM. et al. Critical role of platelet P-selectin in the response to arterial injury in apolipoprotein-E-deficient mice. Arterioscl Thromb Vasc Biol 2004; 24: 1124-1129.
• 19 Phillips JW, Barringhaus KG, Sanders JM. et al. Single injection of P-selectin or P-selectin glycoprotein ligand-1 monoclonal antibody blocks neointima formation after arterial injury in apolipoprotein E-deficient mice. Circulation 2003; 107: 2244-2249.
• 20 Du X. Signaling and regulation of the platelet glycoprotein Ib-IX-V complex. Curr Opin Hematol 2007; 14: 262-269.
• 21 Andrews RK, Gardiner EE, Shen Y. et al. Glycoprotein Ib-IX-V. Intern J Biochem Cell Biol 2003; 35: 1170-1174.
• 22 Romo GM, Dong JF, Schade AJ. et al. The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin. J Exp Med 1999; 190: 803-814.
• 23 Simon DI, Chen Z, Xu H. et al. Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18). J Exp Med 2000; 192: 193-204.
• 24 Wang Y, Sakuma M, Chen Z. et al. Leukocyte engagement of platelet glycoprotein Ibalpha via the integrin Mac-1 is critical for the biological response to vascular injury. Circulation 2005; 112: 2993-3000.
• 25 Ghasemzadeh M, Kaplan ZS, Alwis I. et al. The CXCR1/2 ligand NAP-2 promotes directed intravascular leukocyte migration through platelet thrombi. Blood 2013; 121: 4555-4566.
• 26 Andrews RK, Berndt MC. Bernard-soulier syndrome: an update. Semin Thromb Hemost 2013; 39: 656-662.
• 27 Ware J, Russell S, Ruggeri ZM. Generation and rescue of a murine model of platelet dysfunction: the Bernard-Soulier syndrome. Proc Natl Acad Sci USA 2000; 97: 2803-2808.
• 28 Strassel C, Nonne C, Eckly A. et al. Decreased thrombotic tendency in mouse models of the Bernard-Soulier syndrome. Arterioscl Thromb Vasc Biol 2007; 27: 241-247.
• 29 Kahn ML, Diacovo TG, Bainton DF. et al. Glycoprotein V-deficient platelets have undiminished thrombin responsiveness and Do not exhibit a Bernard-Soulier phenotype. Blood 1999; 94: 4112-4121.
• 30 Abdulla A, Awla D, Hartman H. et al. Platelets regulate P-selectin expression and leukocyte rolling in inflamed venules of the pancreas. Eur J Pharmacol 2012; 682: 153-160.
• 31 Iannacone M, Sitia G, Isogawa M. et al. Platelets mediate cytotoxic T lymphocyte-induced liver damage. Nature Med 2005; 11: 1167-1169.
• 32 Hime NJ, Black AS, Bulgrien JJ. et al. Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr-/- mice expressing CETP. J Lipid Res 2008; 49: 2113-2123.
• 33 Koltsova EK, Kim G, Lloyd KM. et al. IL-27 Receptor Limits Atherosclerosis in Ldlr-/- Mice. Circulation Res 2012; 111: 1274-1285.
• 34 Kanaji T, Russell S, Ware J. Amelioration of the macrothrombocytopenia associated with the murine Bernard-Soulier syndrome. Blood 2002; 100: 2102-2107.
• 35 Koltsova EK, Garcia Z, Chodaczek G. et al. Dynamic T cell-APC interactions sustain chronic inflammation in atherosclerosis. J Clin Invest 2012; 122: 3114-3126.
• 36 Sundd P, Gutierrez E, Pospieszalska MK. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nature Meth 2010; 07: 821-824.
• 37 Sundd P, Gutierrez E, Koltsova EK. et al. ’Slings’ enable neutrophil rolling at high shear. Nature 2012; 488: 399-403.
• 38 Sundd P, Gutierrez E, Petrich BG. et al. Live cell imaging of paxillin in rolling neutrophils by dual-color quantitative dynamic footprinting. Microcirculation 2011; 18: 361-372.
• 39 Chiang EY, Hidalgo A, Chang J. et al. Imaging receptor microdomains on leukocyte subsets in live mice. Nature Meth 2007; 04: 219-222.
• 40 Sumagin R, Prizant H, Lomakina E. et al. LFA-1 and Mac-1 define characteristically different intralumenal crawling and emigration patterns for monocytes and neutrophils in situ. J Immunol 2010; 185: 7057-7066.
• 41 An G, Wang H, Tang R. et al. P-selectin glycoprotein ligand-1 is highly expressed on Ly-6Chi monocytes and a major determinant for Ly-6Chi monocyte recruitment to sites of atherosclerosis in mice. Circulation 2008; 117: 3227-3237.
• 42 Tasaka S, Richer SE, Mizgerd JP. et al. Very late antigen-4 in CD18-independent neutrophil emigration during acute bacterial pneumonia in mice. Am J Resp Crit Care Med 2002; 166: 53-60.
• 43 Mantovani A, Garlanda C, Locati M. Macrophage diversity and polarization in atherosclerosis: a question of balance. Arterioscler Thromb Vasc Biol 2009; 29: 1419-1423.
• 44 Rautou PE, Vion AC, Amabile N. et al. Microparticles, vascular function, and atherothrombosis. Circ Res 2011; 109: 593-606.
• 45 Woollard KJ, Geissmann F. Monocytes in atherosclerosis: subsets and functions. Nat Rev Cardiol 2010; 07: 77-86.
• 46 Simon DI, Zidar D. Neutrophils in atherosclerosis: alarmin evidence of a hit and run?. Circulation Res 2012; 110: 1036-1038.
• 47 Freedman JE, Loscalzo J. Platelet-monocyte aggregates: bridging thrombosis and inflammation. Circulation 2002; 105: 2130-2132.
• 48 Vintersten K, Monetti C, Gertsenstein M. et al. Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals. Genesis 2004; 40: 241-246.
• 49 Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 2003; 19: 71-82.
• 50 Knight CJ. Antiplatelet treatment in stable coronary artery disease. Heart 2003; 89: 1273-1278.
• 51 Massberg S, Schurzinger K, Lorenz M. et al. Platelet adhesion via glycoprotein IIb integrin is critical for atheroprogression and focal cerebral ischemia: an in vivo study in mice lacking glycoprotein IIb. Circulation 2005; 112: 1180-1188.
• 52 Fujimura Y, Titani K, Holland LZ. et al. von Willebrand factor. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib. J Biol Chem 1986; 261: 381-385.
• 53 Ruggeri ZM, Zarpellon A, Roberts JR. et al. Unravelling the mechanism and significance of thrombin binding to platelet glycoprotein Ib. Thromb Haemost 2010; 104: 894-902.
• 54 Methia N, Andre P, Denis CV. et al. Localized reduction of atherosclerosis in von Willebrand factor-deficient mice. Blood 2001; 98: 1424-1428.
• 55 Zarpellon A, Celikel R, Roberts JR. et al. Binding of alpha-thrombin to surface-anchored platelet glycoprotein Ib (alpha) sulfotyrosines through a two-site mechanism involving exosite I. Proc Natl Acad Sci USA 2011; 108: 8628-8633.
• 56 Borissoff JI, Spronk HM, ten Cate H. The hemostatic system as a modulator of atherosclerosis. N Engl J Med 2011; 364: 1746-1760.
• 57 Ruggeri ZM, De Marco L, Gatti L. et al. Platelets have more than one binding site for von Willebrand factor. J Clin Invest 1983; 72: 1-12.
• 58 Dejana E, Lampugnani MG, Giorgi M. et al. Von Willebrand factor promotes endothelial cell adhesion via an Arg-Gly-Asp-dependent mechanism. J Cell Biol 1989; 109: 367-375.
• 59 Wolf D, Hohmann JD, Wiedemann A. et al. Binding of CD40L to Mac-1’s I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis--but does not affect immunity and thrombosis in mice. Circulation Res 2011; 109: 1269-1279.
• 60 Andre P, Prasad KS, Denis CV. et al. CD40L stabilizes arterial thrombi by a beta3 integrin--dependent mechanism. Nature Med 2002; 08: 247-252.
• 61 Kahn ML, Zheng YW, Huang W. et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-694.
• 62 Cranmer SL, Ashworth KJ, Yao Y. et al. High shear-dependent loss of membrane integrity and defective platelet adhesion following disruption of the GPIbalphafilamin interaction. Blood 2011; 117: 2718-2727.
• 63 Ley K, Miller YI, Hedrick CC. Monocyte and macrophage dynamics during atherogenesis. Arterioscl Thromb Vasc Biol 2011; 31: 1506-1516.
• 64 Sinauridze EI, Kireev DA, Popenko NY. et al. Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets. Thromb Haemost 2007; 97: 425-434.
• 65 Shantsila E, Kamphuisen PW, Lip GY. Circulating microparticles in cardiovascular disease: implications for atherogenesis and atherothrombosis. J Thromb Haemost 2010; 08: 2358-2368.
• 66 Strassel C, Hechler B, Bull A. et al. Studies of mice lacking the GPIb-V-IX complex question the role of this receptor in atherosclerosis. J Thromb Haemost 2009; 07: 1935-1938.