Von Willebrand Factor Enhanced Microvesicle-induced Vascular Leakage And Coagulopathy In Mice With Traumatic Brain Injury

Objective: This study was designed to test the hypothesis that high basal ADAMTS-13 activity prevents TBI-induced disruption of BBB integrity and systemic coagulopathy.This hypothesis was tested by preconditioning mice with recombinant ADAMTS-13 before exposing them to TBI.However,a subset of mice was also given rADAMTS-13 after TBI to test its therapeutic potential.Methods:(1)In this experiment,a severe TBI model of mice was constructed by a hydraulic percussion instrument.The mortality and modified neurological severity score tests(m NSS)of mice between the groups between were tested to assess whether rADAMTS-13 can improve neurological function and survival rate.(2)Evens blue(EB)dye were injected through tail vein to observe whether the injection of rADAMTS-13 into tail vein of TBI mice could reduce the leakage of blood-brain barrier.(3)BBB models in vitro were built using transwell to verify whether VWF has the function of destroying BBB.(4)ADAMTS-13 deficient mice on CASA/Rk background(ADAMTS-13-/-)and their littermates were similarly tested to study the impact of uncleaved and hyperactive VWF on FPI-induced cerebral and systemic changes.(5)Cellulose deposition in lung tissue of mice was observed by phosphotungstic acid hematoxylin(PTAH)staining.(6)Hematoxylin eosin(HE)staining was used to observe the exudation of lung tissue in mice.(7)Enzyme linked immunosorbent assay(enzyme linked immunosorbent assay(ELISA))was used to detect the changes of D-dimers,fibrinogen and VWF antigen levels in plasma.(8)The adhesion activity of VWF was detected by VWF combined with Collagen binding assay(CBA).(9)The coagulation factor Xa coagulation time experiment and the mouse tail vein bleeding time test(mouse tail vein bleeding time)were used to detect whether rADAMTS-13 could reduce the occurrence of coagulation dysfunction after TBI.(10)The change of blood cell content after TBI was studied by complete blood count(CBC).(11)The content of microvesicles(MVs)in plasma was detected by flow cytometry after TBI.(12)The rADAMTS-13 protein levels in plasma of mice before and after TBI was detected by ELISA.(13)Western-blot technology is used to verify that VWF can be bound by microvesicles(MVs).(14)The rADAMTS-13 protein injected through tail vein before and after injury was used to study the feasibility of rADAMTS-13 protein therapy.(15)Lactadherin was injected through mice tail vein before injury to study the binding of VWF to MVs.Results:(1)significant vascular leakage occurred at the impact site and in the surrounding tissue and extensive pulmonary microvascular bleeding were caused following severe FPI.(2)Mice after FPI developed a consumptive coagulopathy defined by significantly reduced plasma fibrinogen,increased plasma D-dimer,and an MV-induced shortening of clotting time.(3)Preconditioning mice with rADAMTS-13 prevented injury-induced vascular leakage and systemic coagulopathy,resulting in improved neurological function and survival without change in tail bleeding.(4)Plasma levels and adhesive activity of circulating VWF were significantly but transiently increased,reaching peak levels at 3 hrs post FPI followed by steady decline.(5)VWF antigen level was moderately decreased in mice preconditioned with rADAMTS-13,even further reduced in the VWF activity.(6)Abnormal activation of platelets can be reduced in mice preconditioned with rADAMTS-13,(7)Plasma collected from mice 3 hrs after FPI,but not that from sham mice induced significant leakage of cultured HUVECs in the transwell system.(8)MVs but MV-free plasma can induce significant leakage of cultured HUVECs in the transwell system and this can be blocked by a VWF antibody.(9)Purified VWF failed to induce the vascular leakage.(10)VWF was detected in both MV and MV-free fractions of plasma from FPI mice,but only in the MV-free fraction of sham mice.(11)Flow cytometry further identified VWF-bound microvesicles from the brain,platelets,and endothelial cells reached the highest level 3 hours post PFI.(12)Lactadherin reduced plasma VWF:Ag and VWF:CB level.(13)The level of plasma VWF antigen and VWF activity in ADAMTS-13-/-mice increased significantly compared with that in C57 BL/6J mice subjected to the same FPI,and also reached the highest level at 3 hours after injury.(14)ADAMTS-13-/-mice preconditioned with rADAMTS-13 developed less severe vascular leakage,had reduced plasma D-dimer,and milder fibrinogen depletion,leading to improved neurological function and survival.(15)The injection of rADAMTS-13 30 minutes post FPI,significantly reduced VWF:CB without reducing plasma VWF:Ag.(16)The injection of rADAMTS-13 30 minutes post FPI,corrected FPI-induced fibrinogen depletion,D-dimer production,platelet activation,severe cerebral vascular leakage,improved their neurological function and their survival.Conclusion:(1)ADAMTS-13 prevented FPI-induced vascular leakage and coagulopathy.(2)ADAMTS-13 reduced FPI-induced VWF adhesive hyperactivity.(3)MV-bound VWF disrupted the integrity of endothelial barrier.(4)ADAMTS-13 deficiency worsened the impact of FPI.(5)ADAMTS-13 was therapeutic.