vWA proteins of Leptospira interrogans induce hemorrhage in leptospirosis by competitive inhibition of vWF/GPIb-mediated platelet aggregation

EBioMedicine. 2018 Nov;37:428-441. doi: 10.1016/j.ebiom.2018.10.033. Epub 2018 Oct 15.

Abstract

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 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.

Interpretation: The products of L. interrogans vwa-I and vwa-II genes induce hemorrhage by competitive inhibition of vWF-mediated Hu-platelet aggregation.

Keywords: Competitive inhibition; Hemorrhage; Leptospira interrogans; Leptospirosis; Platelet aggregation; von Willebrand factor; vwa-I and vwa-II genes.

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Female
  • Hemorrhage / genetics
  • Hemorrhage / metabolism*
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Leptospira interrogans / genetics
  • Leptospira interrogans / metabolism*
  • Leptospirosis / genetics
  • Leptospirosis / metabolism*
  • Mice
  • Platelet Glycoprotein GPIb-IX Complex / genetics
  • Platelet Glycoprotein GPIb-IX Complex / metabolism*
  • von Willebrand Factor / genetics
  • von Willebrand Factor / metabolism*

Substances

  • Bacterial Proteins
  • Platelet Glycoprotein GPIb-IX Complex
  • adhesion receptor
  • von Willebrand Factor