Abstract
BackgroudLeptospira 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.MethodsHuman (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.FindingsBoth 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.InterpretationThe 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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