CLEC-2 Supports Platelet Aggregation in Mouse but not Human Blood at Arterial Shear

Joshua H. Bourne; Christopher W. Smith; Natalie J. Jooss; Ying Di; Helena C. Brown; Samantha J. Montague; Mark R. Thomas; Natalie S. Poulter; Julie Rayes; Steve P. Watson

doi:10.1055/a-1896-6992

Thrombosis and Haemostasis, Inhaltsverzeichnis

CC BY-NC-ND 4.0 · Thromb Haemost 2022; 122(12): 1988-2000
DOI: 10.1055/a-1896-6992

Cellular Haemostasis and Platelets

CLEC-2 Supports Platelet Aggregation in Mouse but not Human Blood at Arterial Shear

Autoren

Joshua H. Bourne

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
Christopher W. Smith

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
Natalie J. Jooss

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

²Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
Ying Di

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
Helena C. Brown

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

³Institute of Experimental Biomedicine I, University Hospital and Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany.
Samantha J. Montague

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
Mark R. Thomas

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

⁴UHB and SWBH NHS Trusts, Birmingham, United Kingdom
Natalie S. Poulter

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

⁵Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, The Midlands, United Kingdom
Julie Rayes

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

⁵Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, The Midlands, United Kingdom
Steve P. Watson

¹Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

⁵Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, The Midlands, United Kingdom

Abstract

C-type lectin-like receptor 2 (CLEC-2) is highly expressed on platelets and a subpopulation of myeloid cells, and is critical in lymphatic development. CLEC-2 has been shown to support thrombus formation at sites of inflammation, but to have a minor/negligible role in hemostasis. This identifies CLEC-2 as a promising therapeutic target in thromboinflammatory disorders, without hemostatic detriment. We utilized a GPIbα-Cre recombinase mouse for more restricted deletion of platelet-CLEC-2 than the previously used PF4-Cre mouse. clec1b^fl/flGPIbα-Cre⁺ mice are born at a Mendelian ratio, with a mild reduction in platelet count, and present with reduced thrombus size post-FeCl₃-induced thrombosis, compared to littermates. Antibody-mediated depletion of platelet count in C57BL/6 mice, to match clec1b^fl/flGPIbα-Cre⁺ mice, revealed that the reduced thrombus size post-FeCl₃-injury was due to the loss of CLEC-2, and not mild thrombocytopenia. Similarly, clec1b^fl/flGPIbα-Cre⁺ mouse blood replenished with CLEC-2-deficient platelets ex vivo to match littermates had reduced aggregate formation when perfused over collagen at arterial flow rates. In contrast, platelet-rich thrombi formed following perfusion of human blood under flow conditions over collagen types I or III, atherosclerotic plaque, or inflammatory endothelial cells were unaltered in the presence of CLEC-2-blocking antibody, AYP1, or recombinant CLEC-2-Fc. The reduction in platelet aggregation observed in clec1b^fl/flGPIbα-Cre⁺ mice during arterial thrombosis is mediated by the loss of CLEC-2 on mouse platelets. In contrast, CLEC-2 does not support thrombus generation on collagen, atherosclerotic plaque, or inflamed endothelial cells in human at arterial shear.

Keywords

CLEC-2 - thrombosis - platelets - AYP1 - thromboinflammation

Volltext

Referenzen

References
1 Rayes J, Watson SP, Nieswandt B. Functional significance of the platelet immune receptors GPVI and CLEC-2. J Clin Invest 2019; 129 (01) 12-23
2 Rayes J, Bourne JH, Brill A, Watson SP. The dual role of platelet-innate immune cell interactions in thrombo-inflammation. Res Pract Thromb Haemost 2019; 4 (01) 23-35
3 Rayes J, Jadoui S, Lax S. et al. The contribution of platelet glycoprotein receptors to inflammatory bleeding prevention is stimulus and organ dependent. Haematologica 2018; 103 (06) e256-e258
4 Lee RH, Bergmeier W. Platelet immunoreceptor tyrosine-based activation motif (ITAM) and hemITAM signaling and vascular integrity in inflammation and development. J Thromb Haemost 2016; 14 (04) 645-654
5 Martin EM, Zuidscherwoude M, Morán LA, Di Y, García A, Watson SP. The structure of CLEC-2: mechanisms of dimerization and higher-order clustering. Platelets 2021; 32 (06) 733-743
6 Lowe KL, Navarro-Núñez L, Bénézech C. et al. The expression of mouse CLEC-2 on leucocyte subsets varies according to their anatomical location and inflammatory state. Eur J Immunol 2015; 45 (09) 2484-2493
7 Wichaiyo S, Lax S, Montague SJ. et al. Platelet glycoprotein VI and C-type lectin-like receptor 2 deficiency accelerates wound healing by impairing vascular integrity in mice. Haematologica 2019; 104 (08) 1648-1660
8 Rayes J, Lax S, Wichaiyo S. et al. The podoplanin-CLEC-2 axis inhibits inflammation in sepsis. Nat Commun 2017; 8 (01) 2239
9 Hitchcock JR, Cook CN, Bobat S. et al. Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets. J Clin Invest 2015; 125 (12) 4429-4446
10 Payne H, Ponomaryov T, Watson SP, Brill A. Mice with a deficiency in CLEC-2 are protected against deep vein thrombosis. Blood 2017; 129 (14) 2013-2020
11 Bourne JH, Beristain-Covarrubias N, Zuidscherwoude M. et al. CLEC-2 prevents accumulation and retention of inflammatory macrophages during murine peritonitis. Front Immunol 2021; 12: 693974
12 May F, Hagedorn I, Pleines I. et al. CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis. Blood 2009; 114 (16) 3464-3472
13 Suzuki-Inoue K. Essential in vivo roles of the platelet activation receptor CLEC-2 in tumour metastasis, lymphangiogenesis and thrombus formation. J Biochem 2011; 150 (02) 127-132
14 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 (05) 926-934
15 Haining EJ, Cherpokova D, Wolf K. et al. CLEC-2 contributes to hemostasis independently of classical hemITAM signaling in mice. Blood 2017; 130 (20) 2224-2228
16 Bourne JH, Colicchia M, Di Y. et al. Heme induces human and mouse platelet activation through C-type-lectin-like receptor-2. Haematologica 2021; 106 (02) 626-629
17 Quintanilla M, Montero-Montero L, Renart J, Martín-Villar E. Podoplanin in inflammation and cancer. Int J Mol Sci 2019; 20 (03) E707
18 Hatakeyama K, Kaneko MK, Kato Y. et al. Podoplanin expression in advanced atherosclerotic lesions of human aortas. Thromb Res 2012; 129 (04) e70-e76
19 Oishi S, Tsukiji N, Otake S. et al. Heme activates platelets and exacerbates rhabdomyolysis-induced acute kidney injury via CLEC-2 and GPVI/FcRγ. Blood Adv 2021; 5 (07) 2017-2026
20 Tiedt R, Schomber T, Hao-Shen H, Skoda RC. Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo. Blood 2007; 109 (04) 1503-1506
21 Pertuy F, Aguilar A, Strassel C. et al. Broader expression of the mouse platelet factor 4-cre transgene beyond the megakaryocyte lineage. J Thromb Haemost 2015; 13 (01) 115-125
22 Calaminus SD, Guitart AV, Sinclair A. et al. Lineage tracing of Pf4-Cre marks hematopoietic stem cells and their progeny. PLoS One 2012; 7 (12) e51361
23 Abram CL, Roberge GL, Hu Y, Lowell CA. Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice. J Immunol Methods 2014; 408: 89-100
24 Ponomaryov T, Payne H, Fabritz L, Wagner DD, Brill A. Mast cells granular contents are crucial for deep vein thrombosis in mice. Circ Res 2017; 121 (08) 941-950
25 Nagy Z, Vögtle T, Geer MJ. et al. The Gp1ba-Cre transgenic mouse: a new model to delineate platelet and leukocyte functions. Blood 2019; 133 (04) 331-343
26 Haining EJ, Lowe KL, Wichaiyo S. et al. Lymphatic blood filling in CLEC-2-deficient mouse models. Platelets 2021; 32 (03) 352-367
27 Gitz E, Pollitt AY, Gitz-Francois JJ. et al. CLEC-2 expression is maintained on activated platelets and on platelet microparticles. Blood 2014; 124 (14) 2262-2270
28 Reininger AJ, Bernlochner I, Penz SM. et al. A 2-step mechanism of arterial thrombus formation induced by human atherosclerotic plaques. J Am Coll Cardiol 2010; 55 (11) 1147-1158
29 Smith CW, Raslan Z, Parfitt L. et al. TREM-like transcript 1: a more sensitive marker of platelet activation than P-selectin in humans and mice. Blood Adv 2018; 2 (16) 2072-2078
30 de Witt SM, Swieringa F, Cavill R. et al. Identification of platelet function defects by multi-parameter assessment of thrombus formation. Nat Commun 2014; 5: 4257
31 van Geffen JP, Brouns SLN, Batista J. et al. High-throughput elucidation of thrombus formation reveals sources of platelet function variability. Haematologica 2019; 104 (06) 1256-1267
32 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 (47) 39474-39484
33 Vardon Bounes F, Mémier V, Marcaud M. et al. Platelet activation and prothrombotic properties in a mouse model of peritoneal sepsis. Sci Rep 2018; 8 (01) 13536
34 Fujita H, Hashimoto Y, Russell S, Zieger B, Ware J. In vivo expression of murine platelet glycoprotein Ibalpha. Blood 1998; 92 (02) 488-495
35 Kroll MH, Harris TS, Moake JL, Handin RI, Schafer AI. von Willebrand factor binding to platelet GpIb initiates signals for platelet activation. J Clin Invest 1991; 88 (05) 1568-1573
36 Hughes CE, Navarro-Núñez L, Finney BA, Mourão-Sá D, Pollitt AY, Watson SP. CLEC-2 is not required for platelet aggregation at arteriolar shear. J Thromb Haemost 2010; 8 (10) 2328-2332
37 Stalker TJ. Mouse laser injury models: variations on a theme. Platelets 2020; 31 (04) 423-431
38 Grover SP, Mackman N. How useful are ferric chloride models of arterial thrombosis?. Platelets 2020; 31 (04) 432-438
39 Woollard KJ, Sturgeon S, Chin-Dusting JP, Salem HH, Jackson SP. Erythrocyte hemolysis and hemoglobin oxidation promote ferric chloride-induced vascular injury. J Biol Chem 2009; 284 (19) 13110-13118
40 Tseng MT, Dozier A, Haribabu B, Graham UM. Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery. Thromb Res 2006; 118 (02) 275-280
41 Hahl P, Davis T, Washburn C, Rogers JT, Smith A. Mechanisms of neuroprotection by hemopexin: modeling the control of heme and iron homeostasis in brain neurons in inflammatory states. J Neurochem 2013; 125 (01) 89-101
42 Poillerat V, Gentinetta T, Leon J. et al. Hemopexin as an inhibitor of hemolysis-induced complement activation. Front Immunol 2020; 11: 1684
43 Hovig T, McKenzie FN, Arfors KE. Measruement of the platelet response to laserinduced microvascular injury. Ultrastructural studies. Thromb Diath Haemorrh 1974; 32 (2-3): 695-703
44 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 (15) e73-e82
45 Dunster JL, Unsworth AJ, Bye AP. et al. Interspecies differences in protein expression do not impact the spatiotemporal regulation of glycoprotein VI mediated activation. J Thromb Haemost 2020; 18 (02) 485-496
46 Zeiler M, Moser M, Mann M. Copy number analysis of the murine platelet proteome spanning the complete abundance range. Mol Cell Proteomics 2014; 13 (12) 3435-3445
47 Jamasbi J, Megens RT, Bianchini M. et al. Cross-linking GPVI-Fc by anti-Fc antibodies potentiates its inhibition of atherosclerotic plaque- and collagen-induced platelet activation. JACC Basic Transl Sci 2016; 1 (03) 131-142
48 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 (04) 356-367
49 Shao B, Hoover CM, Shi H. et al. Deletion of platelet CLEC-2 decreases GPIbα-mediated integrin aIIbb3 activation and decreases thrombosis in TTP. Blood 2022; 139 (16) 2523-2533
50 Brown HC, Beck S, Navarro S. et al. Antibody-mediated depletion of human CLEC-2 in a novel humanised mouse model. bioRxiv. 2021 . Doi: 2021.10.03.462933
51 Chauhan A, Sheriff L, Hussain MT. et al. The platelet receptor CLEC-2 blocks neutrophil mediated hepatic recovery in acetaminophen induced acute liver failure. Nat Commun 2020; 11 (01) 1939
52 Nicolson PLR, Nock SH, Hinds J. et al. Low-dose Btk inhibitors selectively block platelet activation by CLEC-2. Haematologica 2021; 106 (01) 208-219

Zusatzmaterial

Supplementary Material (PDF) (opens in new window)