Ribophorin II is involved in the tissue factor expression mediated by phosphatidylserine-dependent antiprothrombin antibody on monocytes
Ribophorin II is involved in the tissue factor expression mediated by phosphatidylserine-dependent antiprothrombin antibody on monocytes
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DOI：
10.1093/rheumatology/kew005
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发表时间：
2016-06-01
期刊：
影响因子：
5.5
通讯作者：
Atsumi, Tatsuya
中科院分区：
文献类型：
--
作者：
Fujieda, Yuichiro;Amengual, Olga;Atsumi, Tatsuya
目的。磷脂酰丝氨酸依赖性的，也称为aPS - PT，可识别与抗磷脂综合征（APS）相关的磷脂酰丝氨酸 - 凝血酶原复合物。我们先前已报道aPS - PT通过p38丝裂原活化蛋白激酶途径诱导单核细胞上组织因子（TF）的表达。然而，凝血酶原与aPS - PT之间涉及细胞信号通路激活的细胞表面相互作用仍不清楚。本研究的目的是确定参与凝血酶原和aPS - PT与单核细胞表面结合以及诱导TF表达的膜蛋白。
方法。将带有FLAG标签的凝血酶原的RAW264.7细胞进行孵育，并使用抗FLAG抗体偶联的琼脂糖珠通过亲和色谱法进行分离。然后通过在线纳流液相色谱 - 串联质谱法分析免疫纯化的蛋白质。通过酶联免疫吸附测定（ELISA）和表面等离子体共振分析凝血酶原与所鉴定的蛋白质核糖核蛋白II（RPN2）之间的结合。为了阐明RPN2在TF表达中的作用，通过实时定量聚合酶链反应（qPCR）测定用RPN2小干扰RNA处理的RAW264.7细胞中的TF mRNA水平。
结果。RPN2被鉴定为参与凝血酶原与细胞表面结合的候选分子。通过ELISA和表面等离子体共振证实了凝血酶原与RPN2之间的结合。用RPN2小干扰RNA处理的RAW264.7细胞显示出由凝血酶原和一种小鼠单克隆aPS - PT介导的TF表达显著降低。
结论。我们确定RPN2是单核细胞表面上的凝血酶原结合蛋白之一，这表明RPN2参与APS患者血栓形成的病理生理学过程。
Objective. Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphati dylserine-prothrombin complex, which is associated with APS. We have previously reported that aPS-PT induces tissue factor (TF) expression on monocytes through the p38 mitogen-activated protein kinase pathway. However, the cell surface interaction between prothrombin and aPS-PT, which is involved in the activation of cell-signalling pathways, has remained unknown. The objective of this study was to identify membrane proteins involved in the binding of prothrombin and aPS-PT to monocyte surfaces as well as the induction of TF expression.Methods. RAW264.7 cells with FLAG-tagged prothrombin were incubated and separated using affinity chromatography with anti-FLAG antibody-conjugated Sepharose beads. Immunopurified proteins were then analysed by an online nano-liquid chromatography-tandem mass spectrometry. The binding between prothrombin and the identified protein, ribophorin II (RPN2), was analysed by ELISA and surface plasmon resonance. To elucidate the role of RPN2 in TF expression, the TF mRNA level in RAW264.7 cells treated with RPN2 small interfering RNA was determined by quantitative real-time PCR (qPCR).Results. RPN2 was identified as a candidate molecule involved in the binding of prothrombin to the cell surface. The binding between prothrombin and RPN2 was confirmed by ELISA and surface plasmon resonance. RAW264.7 cells treated with RPN2 small interfering RNA showed significant reduction of the TF expression mediated by prothrombin and a mouse monoclonal aPS-PT.Conclusion. We identified that RPN2 is one of the prothrombin-binding proteins on monocyte surfaces, suggesting that RPN2 is involved in the pathophysiology of thrombosis in patients with APS.