钩端螺旋体 interrogans 的 vWA 蛋白通过竞争性抑制 vWF/GPIb 介导的血小板聚集诱导钩端螺旋体病中的出血。
vWA proteins of Leptospira interrogans induce hemorrhage in leptospirosis by competitive inhibition of vWF/GPIb-mediated platelet aggregation.
机构信息
Division of Basic Medical Microbiology, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, PR China; Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, PR China.
Division of Basic Medical Microbiology, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, PR China; Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, PR China.
出版信息
EBioMedicine. 2018 Nov;37:428-441. doi: 10.1016/j.ebiom.2018.10.033. Epub 2018 Oct 15.
BACKGROUD
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.
METHODS
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.
FINDINGS
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 K values of 3.87 × 10-8.65 × 10 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, Ca and TXA 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.
INTERPRETATION
The products of L. interrogans vwa-I and vwa-II genes induce hemorrhage by competitive inhibition of vWF-mediated Hu-platelet aggregation.
背景
钩端螺旋体是引起钩端螺旋体病的主要病原体，这是一种全球性的人畜共患病。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 Ibα（GPIbα）的结合是血小板聚集启动的关键步骤。钩端螺旋体 vwa-I 和 vwa-II 基因的产物含有 vWF-A 结构域，但它们诱导出血的能力尚未确定。
方法
通过流式细胞术、表面等离子体共振（SPR）和等温热力学滴定（ITC）检测由钩端螺旋体菌株 Lai vwa-I 和 vwa-II 基因表达的重组蛋白（rLep-vWA-I 和 rLep-vWA-II）对人（Hu）血小板和 Hu-GPIbα 的结合能力。通过发光聚集测定法、Western 分析、分光光度法和共聚焦显微镜检测 Hu 血小板聚集及其信号转导激酶和活性成分。通过 SPR/ITC 测量鉴定 rLep-vWA-I 和 rLep-vWA-II 中的 Hu-GPIbα 结合位点。
结果
rLep-vWA-I 和 rLep-vWA-II 均能与人血小板结合并抑制 rHu-vWF/瑞斯托菌素诱导的人血小板聚集，但 Hu-GPIbα-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，K 值为 3.87×10-8 至 6.55×10-8M。rL-vWA-I/rL-vWA-II 与人 GPIbα 的结合既不激活 PI3K/AKT-ERK 和 PLC/PKC 激酶，也不影响 Hu 血小板中的 NO、cGMP、ADP、Ca 和 TXA 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。在小鼠中注射 rLep-vWA-I 或 rLep-vWA-II 会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。
解释
钩端螺旋体 vwa-I 和 vwa-II 基因的产物通过竞争性抑制 vWF 介导的 Hu 血小板聚集来诱导出血。
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