Leptospira Interrogans Vwa Gene Products Induce Hemorrhage By Competitive Inhibition Of VWF/GPIb?-mediated Platelet Aggregation

Background:Leptospirosis caused by pathogenic Leptospira is a worldwide zoonotic infectious disease.Hemorrhage and jaundice are the two typical pathological features of the disease,but until now the pathogenesis of hemorrhage in leptospirosis remains unknown.von Willebtand factor is an important blood coagulation factor to initate platelet aggregation after biding to a-subunit of memebrane glucoprotein-Ib(GPIba)on platelets.The products of vwa-? and vwa-? genes in Serogroup Icterohaemorrhagiae serovar Lai strain Lai of L.interrogans,a pathogenic leptospiral genospecies,are annotated as vWF-A-domain-containing proteins,but the role and mechanism of the two proteins in the hemorrhagic process during leptospirosis have not been reported.Methods:The prokaryotic expression of vwa-? and vwa-? genes from L.interrogans strain Lai were generated using pET42a as the expression vector and Escherichia coli BL21DE3 as the expression host bacteria.Ni-NTA affinity chromatography was applied to extract the recombinant proteins rLep-vWA-? and rLep-vWA-? expressed by the vwa-? and vwa-? genes.The possible contaminated E.coli lipopolysaccharide(LPS)in the rLep-vWA-? and rLep-vWA-? extracts was detected and then removed using limulus test and a Detoxi-gel LPS-affinity chromatography.Flow cytometry,surface plasmon resonance(SPR)examination and isothermal titration calorimetry(ITC)were used to detect the abilities of rLep-vWA-?and rLep-vWA-? binding to human platelets and GPIba.The target proteins of rHu-GPIb? from total proteins of L.interrogans strain Lai were captured and identified by coprecipitation test plus LC-MS/MS.The role of rLep-vWA-? and rLep-vWA-? in induction of human platelet aggregation was detected using a lumiaggregometer.Western Blot assay,spectrophotometry and laser confocal microscopy were applied to dtect the phosphorylation of kinases in platelet aggregation-associated PI3K/AKT-ERK and PLC-PKC signaling pathways and active mediators in human platelets.The expression level changes of vwa-? and vwa-? genes and extretion of the two gene products were detected using real-time luorescent quantitation RT-PCR and Western Blot assay.The point-muation plus SPR or ITC were used to deterime the sites in rLep-vWA-? and rLep-vWA-? binding to recombinant human GPIba(rHu-GPIba).The hemorrhages in lung,liver and kidney tissue samples as well as the activated partial thromboplastin time(APTT),prothrombin time(PT)and thrombin time(TT)of peripheral blood samples of rLep-vWA-?-or rLep-vWA-?-injected C3H/HeJ mice were detected using a microscope after HE-staining and an auto-blood clotting analyzer.Results:The prokaryotic expression systems of L.interrogans strain Lai vwa-? and vwa-? genes could efficiently express the target recombinant proteins rLep-vWA-? and rLep-vWA-?.After treatment with Detoxi-gel affinity column,rLep-vWA-? and rLep-vWA-? extracts were undetectable for LPS.rLep-vWA-? and rLep-vWA-? could bind to 81.7%-94.7%of human platelets,but this binding was blocked by rLep-vWA-?-IgG and rLep-vWA-?-IgG.The SPR and ITC detection confirmed the high Hu-GPIba-binding ability of rLep-vWA-? and rLep-vWA-? with the KD values of 3.87×10-8.65×10-8 M.The products of vwa-? and vwa-? genes were captured by rHu-GPIba from the total proteins of L.interrogans strain Lai.rLep-vWA-? and rLep-vWA-? did not induce human platelet aggregation but blocked the Hu-rvWF and its co-operator ristocetin mediated platelet aggregation in vitro.After treatment with rLep-vWA-? and rLep-vWA-?,the phosphorylation levels of AKT,ERK,PLC and PKC,the kinases in human platelet aggregation-associated signaling pathways as well as the aggregation-associated NO,cGMP,Ca2+,ADP and TXA2 levels had no significant changes.The mRNA level as well as the protein expression and excretion of vwa-? and vwa-? genes were significantly increased during infection of HUVEC with L.interrogans strain Lai.When the G13,G47 or R36 in Lep-vWA-? and G76 or Q126 in Lep-vWA-? was mutated,the abilities of rLep-vWA-? and rLep-vWA-? binding to rHu-GPIb? and inhibiting Hu-rvWF/ristocetin-mediated platelet aggregation were significantly attenuated even absent.After intravenous injection with rLep-vWA-? or rLep-vWA-?,severe diffuse hemorrhage in the lung tissues and focal hemorrhage in the kidney tissues of mice were presented and APTT,PT and TT of the peripheral blood samples had 2.36-5.63-fold extension.Conclusion:The products of L.interrogans vwa-? and vwa-? genes can block vWF-mediated human platelet aggregation by competitive binding to platelet GPIb?,which causes hemorrhage in human leptospirosis.