Neuroprotective Roles And Mechanisms Of Action Of VWF-A2 In Experimental Traumatic Brain Injury

Background and Objective: Traumatic brain injury(TBI)is a devastating health problem with high mortality and morbidity,and the effective clinical pharmacotherapy remains insufficient.The pathogenesis of TBI is highly complex,secondary injury including TBI associated coagulopathy(TBI-AC)and blood-brain barrier(BBB)disruption have been implicated to exacerbate neurological impairment and associated positively with poor clinical outcomes.Compelling data indicated that plasma levels and adhesive activity of adhesive ligand von Willibrand factor(VWF)were significantly increased following TBI.Furthermore,VWF became microvesicle(MV)-bound and mediated MV-induced endothelial injury and TBI-AC.Previous studies indicated that the recombinant A2 domain polypeptide specifically inhibited platelet interaction with fibrin and VWF.The current study aims to purify recombinant VWF-A2 domain,evaluate the effects of VWF-A2 on BBB disruption and TBI-AC,and to explore the possible mechanisms,providing a therapeutic approach for patients with TBI.Methods:(1)plasmid encoding 6x-His-tagged VWF-A2 domain was transformed,induced,and purified,and the purity was identified by SDS-PAGE and Western blot.Biological function was assessed by ADMATS-13 cleavage assay.(2)C57BL/6J mice were subjected to TBI using a fluid percussion injury device.VWF-A2(4 mg/kg)was administered by intraperitoneal injection at 30 min post-TBI,and the concerntration of circulating VWF-A2 was measured.Neurological deficits were evaluated using the modified neurological severity score(m NSS)and 3-day survival was monitored.(3)The levels of circulating MVs were measured by flow cytometery.Evan's blue dye extravasation assay was used to measure post-TBI BBB disruption.Hematoxylin eosin staining was used to determine the lung exudation.Transwell culture system and cell immunofluorescence were used to evaluate the effect of VWF-A2 on MVs,purified from TBI mouse blood or BDMVs from mouse brains subjected to freezing and thaw injury,induced endothelial disruption.(4)Enzyme linked immunosorbent assays were used to measured the expression of VWF antigen,D-dimer,fibrinogen,and the ability of VWF binding to collagen.Fibrin deposition in kidney was observed by phosphotungstic acid hematoxylin staining.MV-mediated pro-coagulant effect was determined by thrombin generation and activated factor X clotting time assays.Flow cytometry was used to evaluate platelet activation and platelet count was also measured.In addition,tail bleeding test was used to determin the post-TBI bleeding tendency.(5)Co-immunoprecipitation and surface plasmon resonance assays were used to determin the interaction of VWF-A2 and VWF in vivo and in vitro.In addition,the effect of VWF-A2 on ristocetin cofactor activity and shear-induced platelet aggregation were also measured.Results: The recombinant VWF-A2 protein was purified suceessfully,and the purity was 90-95%.VWF-A2 was also identified to have the ability of being cleaved by ADAMTS13 in vitro.VWF-A2 reduced the release of brain-,platelet-,and endothelial-derived MVs,and inhibited VWF mediated MV-induced endothelial injury and activation.VWF-A2 significantly improved survival and reduced neurological deficits following TBI.More important,VWF-A2 reduced VWF antigen and adhesive reactivity,alleviated VWF-induced platelet activation and post-TBI thrombocytopenia.In addition,VWF-A2 could reduce post-TBI hypercoaugulable and hyperfibrinolytic state,including reduced post-TBI plasma fibrinogen depletion,elevation of plasma D-dimer,shortened clotting time,thrombin generation.Moreover,VWF-A2 reduced bleeding time following TBI,and did not induce bleeding tendency in normal mice.Finally,in vivo and in vitro experiments indicated that VWF-A2 bound to VWF-A1,and this A1-A2 interaction blocked ristocetin cofactor activity and in shear-induced platelet aggregation.Conclusions: Our results demonstrated that purified VWF-A2 could reduce BBB disruption and TBI-AC,improve survival and neurological deficits,partially through binding to the active conformation of the A1 domain in VWF without impairing basal hemostasis.This study offered a new therapeutic strategy for TBI.In addition,A2 may also be used to specifically detect hyper-adhesive VWF in Thrombotic Thrombocytopenic Purpura and severe infection.