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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

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
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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 Authors: 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:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-018-0673-5
http://academic.hep.com.cn/fmd/EN/Y2019/V13/I6/680
Variable Control group Anti-b2GPI antibodies ( + ) patients Anti-b2GPI antibody (−) thrombotic patients
No thrombus Thrombotic
N 15 10 11 9
Mean age-years 40±5 35±2 37±5 45±3
Female sex-no.(%) 5 (33.3) 7 (70.0) 4 (36.4) 3 (33.3)
Platelets (254±22) × 109/L (225±35) × 109/L (304±52) × 109/L (284±40) × 109/L
Venous thromboembolic-no. 3 5
Pulmonary embolism-no.(%) 1 (33.3) 3 (60.0)
Deep vein thrombosis-no.(%) 2 (66.7) 2 (40.0)
Arterial thromboembolic-no. 8 4
Stroke-no.(%) 4 (50.0) 2 (50.0)
Acute myocardial infarction-no.(%) 3 (37.5) 1 (25.0)
Renal artery thrombosis-no.(%) 1 (12.5) 1 (25.0)
No thrombosis
SLE-no.(%) 5 (50.0)
Viral meningitis-no.(%) 2 (20.0)
Cerebral hemorrhage-no.(%) 3 (30.0)
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.
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