vWA proteins of Leptospira interrogans induce hemorrhage in leptospirosis by competitive inhibition of vWF/GPIb-mediated platelet aggregation.
vWA proteins of Leptospira interrogans induce hemorrhage in leptospirosis by competitive inhibition of vWF/GPIb-mediated platelet aggregation.
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问号钩端螺旋体的 vWA 蛋白通过竞争性抑制 vWF/GPIb 介导的血小板聚集而诱导钩端螺旋体病出血。
DOI：
10.1016/j.ebiom.2018.10.033
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发表时间：
2018-11
期刊：
影响因子：
11.1
通讯作者：
Yan J
中科院分区：
文献类型：
--
作者：
Fang JQ;Imran M;Hu WL;Ojcius DM;Li Y;Ge YM;Li KX;Lin X;Yan J
钩端螺旋体病是一种世界性的人畜共患病，其病原体主要是问号钩端螺旋体。出血是钩端螺旋体病的典型病理特征。血管性血友病因子（vWF）与血小板糖蛋白Ib α（GPIbα）的结合是血小板聚集启动的关键步骤。L.问号线虫vwa-I和vwa-II基因含有vWF-A结构域，但它们诱导出血的能力尚未确定。L.表达的重组蛋白与人血小板和人GPIb α的结合能力。应用流式细胞仪、表面等离子体共振（SPR）和等温滴定量热法（ITC）检测问号线虫Lai株vwa-I和vwa-II基因（rLep-vWA-I和rLep-vWA-II）。采用光聚集法、Western分析、分光光度法和共聚焦显微镜检测人血小板聚集及其信号激酶和活性成分。通过SPR/ITC测量鉴定了rLep-vWA-I和rLep-vWA-II中的Hu-GPibα结合位点。rLep-vWA-I和rLep-vWA-II均能抑制rHu-vWF/瑞斯托霉素诱导的Hu-血小板聚集，但Hu-GPIb α-IgG、rLep-vWA-I-IgG和rLep-vWA-II-IgG阻断了这种结合或抑制作用。SPR和ITC显示Hu-GPIbα与rLep-vWA-I/rLep-vWA-II之间存在紧密的相互作用，KD值为3.87 × 10−7-8.65 × 10−8 M。rL-vWA-I/rL-vWA-II的Hu-GPIbα结合既不激活血小板中的PI 3 K/AKT-ERK和PLC/PKC激酶，也不影响NO、cGMP、ADP、Ca ~（2+）和TXA_2水平。Lep-vWA-I中的G13/R36/G47和Lep-vWA-II中的G76/Q126被确认为Hu-GPIbα结合位点。小鼠注射rLep-vWA-I或rLep-vWA-II可导致弥漫性肺和局灶性肾出血，但这种出血可被rLep-vWA-I-IgG或rLep-vWA-II-IgG阻断。L.问号线虫vwa-I和vwa-II基因通过竞争性抑制vWF介导的Hu-血小板聚集诱导出血。
Leptospira interrogans is the major causative agent of leptospirosis, a worldwide zoonotic disease. Hemorrhage is a typical pathological feature of leptospirosis. Binding of von Willebrand factor (vWF) to platelet glycoprotein-Ibα (GPIbα) is a crucial step in initiation of platelet aggregation. The products of L. interrogans vwa-I and vwa-II genes contain vWF-A domains, but their ability to induce hemorrhage has not been determined. Human (Hu)-platelet- and Hu-GPIbα-binding abilities of the recombinant proteins expressed by L. interrogans strain Lai vwa-I and vwa-II genes (rLep-vWA-I and rLep-vWA-II) were detected by flowcytometry, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Hu-platelet aggregation and its signaling kinases and active components were detected by lumiaggregometry, Western analysis, spectrophotometry and confocal microscopy. Hu-GPIbα-binding sites in rLep-vWA-I and rLep-vWA-II were identified by SPR/ITC measurements. Both rLep-vWA-I and rLep-vWA-II were able to bind to Hu-platelets and inhibit rHu-vWF/ristocetin-induced Hu-platelet aggregation, but Hu-GPIbα-IgG, rLep-vWA-I-IgG and rLep-vWA-II-IgG blocked this binding or inhibition. SPR and ITC revealed a tight interaction between Hu-GPIbα and rLep-vWA-I/rLep-vWA-II with KD values of 3.87 × 10−7-8.65 × 10−8 M. Hu-GPIbα-binding of rL-vWA-I/rL-vWA-II neither activated the PI3K/AKT-ERK and PLC/PKC kinases nor affected the NO, cGMP, ADP, Ca2+ and TXA2 levels in Hu-platelets. G13/R36/G47 in Lep-vWA-I and G76/Q126 in Lep-vWA-II were confirmed as the Hu-GPIbα-binding sites. Injection of rLep-vWA-I or rLep-vWA-II in mice resulted in diffuse pulmonary and focal renal hemorrhage but this hemorrhage was blocked by rLep-vWA-I-IgG or rLep-vWA-II-IgG. The products of L. interrogans vwa-I and vwa-II genes induce hemorrhage by competitive inhibition of vWF-mediated Hu-platelet aggregation.
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