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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.    2016, Vol. 10 Issue (1) : 76-84    https://doi.org/10.1007/s11684-015-0426-7
RESEARCH ARTICLE
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
Wenjing Zhang,Fei Gao,Donghe Lu,Na Sun,Xiaoxue Yin,Meili Jin,Yanhong Liu()
Department of Laboratory Diagnosis, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China
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Abstract

Anti-β2 glycoprotein I (anti-β2GP I ) antibodies are important contributors to thrombosis, especially in patients with antiphospholipid syndrome (APS). However, the mechanism by which anti-β2GP I antibodies are involved in the pathogenesis of thrombosis is not fully understood. In this report, we investigated the role of anti-β2GP I antibodies in complexes with β2GP I as mediators of platelet activation, which can serve as a potential source contributing to thrombosis. We examined the involvement of the apolipoprotein E receptor 2' (apoER2') and glycoprotein I ba (GP I bα) in platelet activation induced by the anti-β2GP I /β2GP I complex. The interaction between the anti-β2GP I /β2GP I complex and platelets was examined using in vitro methods, in which the Fc portion of the antibody was immobilized using protein A coated onto a microtiter plate. Platelet activation was assessed by measuring GP II b/ III a activation and P-selectin expression and thromboxane B2 production as well as p38 mitogen-activated protein kinase phosphorylation. Our results revealed that the anti-β2GP I /β2GP I complex was able to activate platelets, and this activation was inhibited by either the anti-GP I bα antibody or the apoER2' inhibitor. Results showed that the anti-β2GP I /β2GP I complex induced platelet activation via GP I bα and apoER2', which may then contribute to the prothrombotic tendency in APS patients.

Keywords anti-β2GP I /β2GP I complex      platelet      GP I bα      apoER2'      thrombosis     
Corresponding Author(s): Yanhong Liu   
Just Accepted Date: 09 November 2015   Online First Date: 01 December 2015    Issue Date: 31 March 2016
 Cite this article:   
Wenjing Zhang,Fei Gao,Donghe Lu, et al. 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.
 URL:  
https://academic.hep.com.cn/fmd/EN/10.1007/s11684-015-0426-7
https://academic.hep.com.cn/fmd/EN/Y2016/V10/I1/76
Fig.1  Specific of b2GP I and anti-b2GP I antibodies, and the anti-b2GP I antibodies and b2GP I binding assay. (A) b2GP I (Sino Biological Inc.) or purified b2GP I were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and incubated in the primary antibodies against b2GP I. (B) b2GP I (Sino Biological Inc.) was subjected to SDS-PAGE and incubated in the primary antibodies against b2GP I (Sino Biological Inc.) or purified anti-b2GP I antibodies. b2GP I was then incubated in HRP-conjugated goat anti-mouse. (C) Different concentrations of dilution anti-b2GP I antibodies (2, 5, 10, 20, 50, and 100 mg/ml) interacted with b2GP I (100 mg/ml), which was immobilized to plates. The data are expressed as mean±SD. n = 10; *P<0.05 versus the other groups.
Fig.2  The anti-b2GP I/b2GP I complex induced CD62P expression and GP II b/III a activation as well as TXB2 production of platelet. Human platelets were treated with PBS, the IgG/b2GP I complex (10/100 mg/ml), the anti-b2GP I/BSA complex (10/100 mg/ml), or the anti-b2GP I/b2GP I complex (10/100 mg/ml). (A) The cells were imaged via flow cytometry. (B) TXB2 production in human platelets was monitored using ELISA text. The data are expressed as mean±SD. In A and B, n = 10; *P<0.05 versus the other treatment.
Fig.3  CD62P expression and GP II b/III a activation, and TXB2 production of platelet inhibited by AK2 and RAP. Human platelets were treated with PBS, thrombin (20 mmol/L), anti-b2GP I /b2GP I complex, anti-b2GP I/b2GP I complex and RAP (0.45 mmol/L), anti-b2GP I/b2GP I complex and AK2 (0.2 mmol/L), or anti-b2GP I/b2GP I complex and RAP and AK2. (A) The cells were imaged via flow cytometry. (B) TXB2 production in human platelets was monitored using ELISA text. The data are expressed as mean±SD. In A and B, n = 10; *P<0.05 versus the IgG treatment; **P<0.05 versus the anti-b2GP I/b2GP I complex treatment.
Fig.4  The anti-b2GP I/b2GP I complex induced p38MAPK phosphorylation and TXB2 production of platelets. (A) Ratio of P-p38MAPK to total p38MAPK in platelets after treatment with the anti-b2GP I/b2GP Icomplex (10/100 mg/ml). A slight increase in P-p38MAPK expression from 5 min to 40 min, which was significant at 30 min. (B) Ratio of P-p38MAPK to total p38MAPK in washed human platelets after stimulation with PBS, IgG/b2GP I complex, anti-b2GP I/BSA complex, or anti-b2GP I/b2GP Icomplex for 30 min. (C) Ratio of P-p38MAPK to total p38MAPK in human platelets after treatment with PBS, thrombin (20 mmol/L), anti-b2GP I/b2GP I complex (10/100 mg/ml), or anti-b2GP I/b2GP I complex and SB203580 (20 mmol/L) for 30 min. (D) TXB2 expression in human platelets after injection with PBS, IgG/b2GP I complex, anti-b2GP I/BSA complex, or anti-b2GP I/b2GP I complex. The data are expressed as mean±SD. In A and B, n = 6; *P<0.05 versus the other treatment. In C and D, n = 6; *P<0.05 versus the PBS treatment; **P<0.05 versus the anti-b2GP I/b2GP I complex treatment.
Fig.5  AK2 and RAP reduced the p38MAPK phosphorylation in platelets. Ratio of P-p38MAPK to total p38MAPK in human platelets after stimulation with PBS, thrombin (20 mmol/L), anti-b2GP I/b2GP I complex, anti-b2GP I/b2GP I complex and RAP, anti-b2GP I/b2GP I complex and AK2, or anti-b2GP I/b2GP I complex and RAP and AK2. The data are expressed as mean±SD. n = 6; *P<0.05 versus IgG treatment; **P<0.05 versus anti-b2GP I/b2GP I complex treatment.
Fig.6  The anti-b2GP I/b2GP I complex induces platelet activation via apoER2' and GP Ibα. When one anti-b2GP I antibody interacts with two molecules of b2GP I, the resulting anti-b2GP I/b2GP I complex increases the affinity for phospholipids on the outer surface of the platelet membrane, which then allows for a coalescence with apoER2' and GP Ibα due to the effects of mass action. The interaction between the anti-b2GP I/b2GP I complex binding to apoER2' and GP Ibα then activates the p38MAPK signaling pathway. The initiation of this p38MAPK pathway promotes GP II b/III a activation and P-selectin expression as well as TXB2 production, which subsequently induces platelet activation either through direct or indirect mechanisms.
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