Anti-β<sub>2</sub>GPI/β<sub>2</sub>GPI complexes induce platelet activation and promote thrombosis via p38MAPK: a pathway to targeted therapies

doi:10.1007/s11684-018-0673-5

Front. Med.

2019, Vol. 13

Issue (6) : 680-689 https://doi.org/10.1007/s11684-018-0673-5

RESEARCH ARTICLE

Anti-β₂GPI/β₂GPI complexes induce platelet activation and promote thrombosis via p38MAPK: a pathway to targeted therapies

Wenjing Zhang¹, Caijun Zha¹, Xiumin Lu¹, Ruichun Jia¹, Fei Gao¹, Qi Sun², Meili Jin¹, Yanhong Liu¹(

)

¹. Department of Laboratory Diagnosis, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China
². Department of Emergency, The First Affiliated Hospital of Heilongjiang University of Chinese Medicine, Harbin 150040, China

Download: PDF(3836 KB) HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

Abstract

Anti-β₂ glycoprotein I (anti-β₂GPI) antibodies are important contributors to the development of thrombosis. Anti-β₂GPI antibody complexes with β₂GPI are well known to activate monocytes and endothelial cells via the intracellular NF-kB pathway with prothrombotic implications. By contrast, the interaction of anti-β₂GPI/β₂GPI complexes with platelets has not been extensively studied. The p38 mitogen-activated protein kinase (MAPK) pathway has been recognized to be an important intracellular signaling pathway in the coagulation cascade and an integral component of arterial and venous thrombosis. The present study reveals that levels of anti-β₂GPI/β₂GPI complexes in sera are positively associated with p38MAPK phosphorylation of platelets in thrombotic patients. Furthermore, SB203580 inhibits anti-β₂GPI/β₂GPI complex-induced platelet activation. Thrombus formation decreased in p38MAPK⁻^/⁻ mice after treatment with anti-β₂GPI/β₂GPI complexes. In conclusion, p38MAPK may be a treatment target for anti-β₂GPI antibody-associated thrombotic events.

Keywords anti-β₂GPI antibody β₂GPI platelet p38MAPK thrombosis complex

Corresponding Author(s): Yanhong Liu

Just Accepted Date: 16 January 2019 Online First Date: 28 February 2019 Issue Date: 16 December 2019

Cite this article:

Wenjing Zhang,Caijun Zha,Xiumin Lu, et al. Anti-β₂GPI/β₂GPI complexes induce platelet activation and promote thrombosis via p38MAPK: a pathway to targeted therapies[J]. Front. Med., 2019, 13(6): 680-689.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-018-0673-5
https://academic.hep.com.cn/fmd/EN/Y2019/V13/I6/680

Tab.1 Characteristics of the patients

Fig.1 p38MAPK activation of platelets in anti-b₂GPI antibody-positive patients. (A) p38MAPK activation of platelets from anti-b₂GPI antibody-positive patients with or without thrombus, anti-b₂GPI antibody-negative thrombotic patients, and healthy controls was determined by Western blot as described in Materials and methods. Data are shown as the mean±SEM and taken from a single experiment representative of three independent experiments. (B) Anti-b₂GPI/b₂GPI complexes were measured in thrombotic patients, anti-b₂GPI antibody-positive patients with or without thrombus, and healthy controls. (C) Correlation between levels of p38MAPK activation and anti-b₂GPI/b₂GPI complexes in anti-b₂GPI antibody-positive patients. *P<0.05, ^#P<0.01, and ns= not significant.

Fig.2 p38MAPK activation is associated with the anti-b₂GPI/b₂GPI complexes. p38MAPK activation was measured in (A) control platelets stimulated with anti-b₂GPI/b₂GPI complexes or anti-b₂GPI antibodies or in (B) control or Fc receptor- blocked platelets treated with the anti-b₂GPI/b₂GPI complexes. Data are shown as the mean±SEM and taken from a single experiment representative of three independent experiments. *P<0.05, ^#P<0.01, and ns= not significant.

Fig.3 Anti-b₂GPI/b₂GPI complexes stimulated platelet activation, secretion, and aggregation via the p38 MAPK pathway. (A) P-selectin and GPIIb/IIIa were identified by anti-CD62P-PE antibody and anti-PAC-1-FITC antibody, respectively. The percentage of CD62P or PAC-1 positive platelets was then determined by flow cytometry. (B) Platelet-derived microparticles were stained with anti-CD62P-PE and anti-CD41-FITC antibodies and imaged via confocal microscopy. Numerous small projections were observed emanating from the platelet surface (marked by the white arrow). Scale bar: 10 mm. The percentage of CD62 and CD41-positive platelet microparticles was calculated by ImageJ. (C) Platelet aggregation was monitored using a turbidimetric aggregometer. Data are shown as the mean±SEM and taken from a single experiment representative of three independent experiments. *P<0.05, **P<0.001.

Fig.4 Anti-b₂GPI/b₂GPI complex-mediated p38MAPK activation and platelet activation can be decreased by inhibition of TLR4. Control platelets were stimulated with the anti-b₂GPI/b₂GPI complexes. Some samples were pretreated with TAK-242, a TLR4 inhibitor. (A) Phosphorylated-p38MAPK of platelet was detected by Western blot. (B) The percentage of CD62P or PAC-1 positive platelets was detected by flow cytometry. (C) Platelet-derived microparticles were imaged via confocal microscopy. Numerous small projections were observed emanating from the platelet surface (marked by the white arrow). Scale bar: 10 mm. The percentage of CD62 and CD41-positive platelet microparticles was calculated by ImageJ. (D) Platelet aggregation was monitored using a turbidimetric aggregometer. Data are shown as the mean±SEM and taken from a single experiment representative of three independent experiments. *P<0.05, ns= not significant.

Fig.5 Anti-b₂GPI/b₂GPI complex-induced platelet activation in vivo is mediated by p38MAPK. WT mice were i.v. injected with human IgG or anti-b₂GPI/b₂GPI complexes. p38MAPK⁻^/− mice were injected with anti-b₂GPI/b₂GPI complexes. (A) p38MAPK phosphorylation was tested in treated platelets by Western blot. (B) P-selectin and GPIIb/IIIa were identified by goat anti-mouse anti-CD62P-PE antibody and anti-PAC-1-FITC antibody. The anti-CD62P antibody and anti-PAC-1 antibody-positive platelets was detected by flow cytometry. (C) Platelet aggregation was monitored using a turbidimetric aggregometer. Data are shown as the mean±SEM and taken from a single experiment representative of three independent experiments. *P<0.05, **P<0.001.

Fig.6 Inhibiting p38MAPK can prevent anti-b₂GPI/b₂GPI complex-induced thrombosis in vivo. The common carotid arteries were exposed, and filter paper saturated with 10% FeCl₃ was placed under the right vessel throughout the procedure. (A) Carotid artery thrombosis was measured at 1, 5, and 10 min via laser Doppler flowmetry. The rate of blood perfusion is shown as decreasing from red to blue. (B) Changes in carotid artery blood flow were measured over a duration of 10 min via laser Doppler flowmetry and identified by PIMsoft software. (C) The arteries were removed after treatment with 10% FeCl₃ for 5 min, and hematoxylin–eosin staining was used to measure the thrombus size. Representative photograph of a carotid artery cross-section showing the maximal thrombus size following anti-b₂GPI/b₂GPI complex treatment, which was reduced by p38MAPK knockout. Thirteen mice per group; data are from a single experiment representative of two independent experiments. ? shows the presence of a thrombus.

1	MM Bala, EM Paszek, D Wloch-Kopec, W Lesniak, A Undas. Antiplatelet and anticoagulant agents for primary prevention of thrombosis in individuals with antiphospholipid antibodies. The Cochrane Library. John Wiley & Sons, Ltd., 2017
2	R Forastiero. Antiphospholipid syndrome: new light comes from in vivo studies. J Thromb Haemost 2007; 5(9): 1825–1827 https://doi.org/10.1111/j.1538-7836.2007.02686.x pmid: 17723120
3	C Zha, W Zhang, F Gao, J Xu, R Jia, J Cai, Y Liu. Anti-b2GPI/b2GPI induces neutrophil extracellular traps formation to promote thrombogenesis via the TLR4/MyD88/MAPKs axis activation. Neuropharmacology 2018; 138: 140–150 https://doi.org/10.1016/j.neuropharm.2018.06.001 pmid: 29883691
4	Y Zhang, W Zhang, C Zha, Y Liu. Platelets activated by the anti-b2GPI/b2GPI complex release microRNAs to inhibit migration and tube formation of human umbilical vein endothelial cells. Cell Mol Biol Lett 2018; 23(1): 24 https://doi.org/10.1186/s11658-018-0091-3 pmid: 29785186
5	E Raschi, CB Chighizola, C Grossi, N Ronda, R Gatti, PL Meroni, MO Borghi. b2-glycoprotein I, lipopolysaccharide and endothelial TLR4: three players in the two hit theory for anti-phospholipid-mediated thrombosis. J Autoimmun 2014; 55: 42–50 https://doi.org/10.1016/j.jaut.2014.03.001 pmid: 24685231
6	J Zhang, KR McCrae. Annexin A2 mediates endothelial cell activation by antiphospholipid/anti-b2 glycoprotein I antibodies. Blood 2005; 105(5): 1964–1969 https://doi.org/10.1182/blood-2004-05-1708 pmid: 15471954
7	T Shi, B Giannakopoulos, X Yan, P Yu, MC Berndt, RK Andrews, J Rivera, GM Iverson, KA Cockerill, MD Linnik, SA Krilis. Anti-b2-glycoprotein I antibodies in complex with b2-glycoprotein I can activate platelets in a dysregulated manner via glycoprotein Ib-IX-V. Arthritis Rheum 2006; 54(8): 2558–2567 https://doi.org/10.1002/art.21968 pmid: 16868978
8	R Seger, EG Krebs. The MAPK signaling cascade. FASEB J 1995; 9(9): 726–735 https://doi.org/10.1096/fasebj.9.9.7601337 pmid: 7601337
9	R Bassi, R Heads, MS Marber, JE Clark. Targeting p38-MAPK in the ischaemic heart: kill or cure? Curr Opin Pharmacol 2008; 8(2): 141–146 https://doi.org/10.1016/j.coph.2008.01.002 pmid: 18289939
10	I Canobbio, S Reineri, F Sinigaglia, C Balduini, M Torti. A role for p38 MAP kinase in platelet activation by von Willebrand factor. Thromb Haemost 2004; 91(1): 102–110 https://doi.org/10.1160/TH03-02-0083 pmid: 14691575
11	H Cakmak, Y Seval-Celik, S Arlier, O Guzeloglu-Kayisli, F Schatz, A Arici, UA Kayisli. p38 mitogen-activated protein kinase is involved in the pathogenesis of endometriosis by modulating inflammation, but not cell survival. Reprod Sci 2018; 25(4): 587–597 https://doi.org/10.1177/1933719117725828 pmid: 28845752
12	W Zhang, F Gao, D Lu, N Sun, X Yin, M Jin, Y Liu. Anti-b2 glycoprotein I antibodies in complex with b2 glycoprotein I induce platelet activation via two receptors: apolipoprotein E receptor 2′ and glycoprotein I ba. Front Med 2016; 10(1): 76–84 https://doi.org/10.1007/s11684-015-0426-7 pmid: 26620053
13	A Biasiolo, P Rampazzo, T Brocco, F Barbero, A Rosato, V Pengo. [Anti-b2 glycoprotein I-b2 glycoprotein I] immune complexes in patients with antiphospholipid syndrome and other autoimmune diseases. Lupus 1999; 8(2): 121–126 https://doi.org/10.1191/096120399678847506 pmid: 10192506
14	G Andonegui, SM Kerfoot, K McNagny, KV Ebbert, KD Patel, P Kubes. Platelets express functional Toll-like receptor-4. Blood 2005; 106(7): 2417–2423 https://doi.org/10.1182/blood-2005-03-0916 pmid: 15961512
15	F Fischetti, P Durigutto, V Pellis, A Debeus, P Macor, R Bulla, F Bossi, F Ziller, D Sblattero, P Meroni, F Tedesco. Thrombus formation induced by antibodies to β2-glycoprotein I is complement dependent and requires a priming factor. Blood 2005; 106(7): 2340–2346 https://doi.org/10.1182/blood-2005-03-1319 pmid: 15956288
16	PR Ames, J Delgado Alves, LR Lopez, F Gentile, A Margarita, L Pizzella, J Batuca, G Scenna, V Brancaccio, E Matsuura. Antibodies against β2-glycoprotein I complexed with an oxidised lipoprotein relate to intima thickening of carotid arteries in primary antiphospholipid syndrome. Clin Dev Immunol 2006; 13(1): 1–9 https://doi.org/10.1080/17402520600554930 pmid: 16603439

[1]	Mengxue Huang, Jingjing Wang, Runshun Zhang, Zhuying Ni, Xiaoying Liu, Wenwen Liu, Weilian Kong, Yao Chen, Tiantian Huang, Guihua Li, Dan Wei, Jianzhong Liu, Xuezhong Zhou. Symptom network topological features predict the effectiveness of herbal treatment for pediatric cough[J]. Front. Med., 2020, 14(3): 357-367.
[2]	Yinlong Zhang, Guangna Liu, Jingyan Wei, Guangjun Nie. Platelet membrane-based and tumor-associated platelet- targeted drug delivery systems for cancer therapy[J]. Front. Med., 2018, 12(6): 667-677.
[3]	Isabel Andia, Michele Abate. Platelet-rich plasma: combinational treatment modalities for musculoskeletal conditions[J]. Front. Med., 2018, 12(2): 139-152.
[4]	Jichun Yang, Kaiyue Jin, Jiajun Xiao, Jing Ma, Duan Ma. Endogenous tissue factor pathway inhibitor in vascular smooth muscle cells inhibits arterial thrombosis[J]. Front. Med., 2017, 11(3): 403-409.
[5]	Yan Chen,Wenyan Li,Wen Li,Renjie Chai,Huawei Li. Spatiotemporal expression of Ezh2 in the developing mouse cochlear sensory epithelium[J]. Front. Med., 2016, 10(3): 330-335.
[6]	Yan Ma,Kehua Zhou,Jing Fan,Shuchen Sun. Traditional Chinese medicine: potential approaches from modern dynamical complexity theories[J]. Front. Med., 2016, 10(1): 28-32.
[7]	Wenjing Zhang,Fei Gao,Donghe Lu,Na Sun,Xiaoxue Yin,Meili Jin,Yanhong Liu. Anti-β₂ glycoprotein I antibodies in complex with β₂ glycoprotein I induce platelet activation via two receptors: apolipoprotein E receptor 2' and glycoprotein I bα[J]. Front. Med., 2016, 10(1): 76-84.
[8]	Yan Ma,Shuchen Sun,Chung-Kang Peng. Applications of dynamical complexity theory in traditional Chinese medicine[J]. Front. Med., 2014, 8(3): 279-284.
[9]	Xuezhong Zhou,Yubing Li,Yonghong Peng,Jingqing Hu,Runshun Zhang,Liyun He,Yinghui Wang,Lijie Jiang,Shiyan Yan,Peng Li,Qi Xie,Baoyan Liu. Clinical phenotype network: the underlying mechanism for personalized diagnosis and treatment of traditional Chinese medicine[J]. Front. Med., 2014, 8(3): 337-346.
[10]	Yumei Wen, Xuanyi Wang, Bin Wang, Zhenhong Yuan. Vaccine therapies for chronic hepatitis B: can we go further?[J]. Front Med, 2014, 8(1): 17-23.
[11]	Guocheng LIU MD, Shouhua YANG MD, Zehua WANG MD, . The relationship between platelet-derived growth factor expression and angiogenesis/lymphangiogenesis in cervical cancer[J]. Front. Med., 2009, 3(4): 447-451.
[12]	Ouyan SHI PhD, Guodong XU BM, Chunxiang WANG BM, . Giant renal angiomyolipoma with tuberous sclerosis complex[J]. Front. Med., 2009, 3(4): 495-498.
[13]	Rui ZHU, Lin SHEN. Expression of protease-activated receptors on platelets in healthy individuals[J]. Front Med Chin, 2009, 3(2): 236-239.
[14]	XIA Dasheng, SONG Yanqiu, LI Chao, ZHANG Feng, WEI Minxin. The change of serum leptin and its relationship with platelet membrane glycoprotein Ib in patients with coronary heart disease[J]. Front. Med., 2007, 1(4): 352-355.
[15]	GONG Xiaowei, WEI Jie, LI Yusheng, CHENG Weiwei, DENG Peng, JIANG Yong. Involvement of p38 mitogen-activated protein kinase in the regulation of platelet-derived growth factor -induced cell migration[J]. Front. Med., 2007, 1(3): 248-252.

Viewed

Full text

Abstract

Cited

Shared

Discussed