vWA Proteins of Leptospira Interrogans Induce Hemorrhage in Leptospirosis by Competitive Inhibition of vWF/GPIB-Mediated Platelet Aggregation

Backgroud: Leptospira interrogans is the causative agent of leptospirosis, a worldwide zoonotic disease. Hemorrhage is a typical pathological feature of leptospirosis. Binding of von Willebrand factor (vWF) to human platelet glycoprotein-Ibα (Hu-GPIbα) is a crucial step in initiation of Hu-platelet aggregation. The products of L. interrogans vwa-I and vwa-II genes have been shown to contain vWF-A domains, but their ability to induce hemorrhage had not been examined. Methods: The Hu-platelet- and Hu-GPIbα-binding abilities of the recombinant proteins encoded by vwa-I and vwa-II genes (rLep-vWA-I and rLep-vWA-II) of L. interrogans were detected by flow cytometry, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Hu-platelet aggregation-associated signaling kinases and active components were detected by Western Blot assay, spectrophotometry and confocal microscopy. Hu-GPIbα-binding sites in rLep-vWA-I and rLep-vWA-II were identified by point-mutation and SPR/ITC measurements. Findings: rLep-vWA-I and rLep-vWA-II could bind to Hu-platelets and inhibit rHu-vWF/ristocetin-mediated platelet aggregation, but Hu-GPIbα-IgG, rLep-vWA-I-IgG or rLep-vWA-II-IgG blocked this binding or reversed inhibition. SPR and ITC confirmed the high Hu-GPIbI±-binding ability of rLep-vWA-I and rLep-vWA-II with KD values of 3.87x10-7-8.65x10-8 M. Binding of rL-vWA-I or 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, acted as the Hu-GPIbα-binding sites. Moreover, rLep-vWA-I and rLep-vWA-II caused diffuse pulmonary and focal renal hemorrhage in mice. Interpretation: The products of L. interrogans vwa-I and vwa-II genes induce hemorrhage by competitive inhibition of vWF-mediated Hu-platelet aggregation. Funding: National Natural Science Foundation of China. Declaration of Interest: No reported conflicts. Ethical Approval: All subjects gave written informed consent, and the study was approved by the Human Ethics Committee of Zhejiang University School of Medicine, and complied with the Declaration of Helsinki. All the animal experimental protocols were approved by the Ethics Committee for Animal Experiment of Zhejiang University School of Medicine.