Molecular Mechanism Involved In Zeolite Accelerated Coagulation Process

Under the circumstances of traffic accidents,natural disasters and battlefields,the uncontrollable hemorrhage is the leading cause of death.The lack of efficient hemostatic agents suitable for massive blood loss like artery bleeding in current pre-hospital first-aid,results in vast life lost before reaching hospitals.Therefore,the research and developments of emergency hemostatic agents is critical to reduce hemorrhage-related mortality.As inorganic mineral materials,zeolite molecular sieve was found to be procoagulant in 1980s,by Francis X.Hursey.In 2002,the first zeolite based hemostatic agent,QuikClot was approved by FDA,and distributed to U.S.military in various wars.For more than a decade,zeolite based hemostatic agent has saved thousands of lives in battlefields and daily emergencies,while its procoagulant mechanism is still far from understood.Recently,the knowledge of biomolecular layer absorbed on materials surface—protein corona,provided a new perspective to understand the linkage between material's synthetic physiochemical properties and its biological responses.This work was focused on interactions between zeolite and blood plasma proteins,including the dynamic changes of components and biological functions of protein corona on zeolite,thereby providing a new molecular-based mechanism for zeolite's procoagulant actionFirst,the major influence factors of zeolite's procoagulant action,calcium cations and zeolite surface were explored briefly.It was found that calcium exchanged zeolite can release Ca2+ into plasma by ion exchange process,which consequently determines the clotting time by participating thrombin activation.In addition,thrombin activation was found to be rely on bare zeolite surface.Then,we made the first comprehensive resolution of zeolite's protein corona during coagulation by proteomics analysis.Zeolite exhibited a high selectivity towards adsorbed plasma proteins,in which coagulation related proteins were enriched to 3.03%from 0.66%.Especially,a full intrinsic coagulation pathway including factors FXII,FXI,FIX,FX,FV,FII and FXIII were identified,which confirmed zeolite's induced coagulation pathway.During the early stage of coagulation,factor FV and FX were found to assemble prothrombinase complex on zeolite surface,which was responsible for activation of prothrombin to thrombin.Also,the on-site surface activated thrombin showed unusual stability.We thereby proposed a mechanism of zeolite surface-initiated coagulation,to illustrate the roles of zeolite inorganic surface in multiple protein activation process.Furthermore,we investigated the specific interactions between zeolite and thrombin molecules.The zeolite adsorbed thrombin induced 7%decrease in ?-helix and 13%increase in ?-sheet,resulting a decreased activity towards chromogenic substrate.This activity loss was found to be resolved by precoating of BSA on zeolite surface,which also enhanced its resistance towards antithrombin ?.By employing dimethyl labeling analysis,we identified three lysine residues,K345,K350 and K440 might be the potential binding sites between thrombin and zeolite.In the end,we employed zeolite and its composite with protein corona into challenging applications,like hemophilia A and gastric ulcer bleeding.For the systematic coagulopathy of hemophilia A,the full intrinsic pathway and active thrombin in protein corona can induce fibrin formation directly by bypassing the traditional pathway,which decreased clotting time from over 60mins to 5mins in a FVlll deficient hemophilia A mice docking model.For the gastric ulcer bleeding,zeolite immobilized thrombin exhibited superior stability after gastric juice treatment.It decreased the ulcer area from 35.1±4.4%to 17.8±2.0%under dosage of 2.5 g/kg,and increased gastric pH from 2.0±0.5 to 3.5±0.5 in a HCl-ethanol induced gastric ulcer model in mice.In summary,this work creatively put forward a molecular level mechanism for zeolite's procoagulant action based on dynamic analysis of protein corona components and functions.Further investigation of specific bio-nano interactions revealed that the formation of protein corona not only transformed zeolite surface,but also built a promising bio-inorganic hemostat,which was applicable to hemophilia A and gastric ulcer bleeding.