Molecular Dynamics Simulation Study On The Adsorption And Activation Mechanism Of Key Coagulation Factors On The Zeolite Surface

Hemorrhage from severe trauma can cause significant incidence and mortality.Hemostasis is the body’s natural response to injury,but once massive bleeding occurs,the body needs the help of hemostatic materials to effectively stop the bleeding.Inorganic hemostatic materials such as zeolite show increasing promise for use in scenes such as battlefield and pre-hospital emergencies due to their excellent properties.Recent studies have shown that zeolite acts as a platelet to enable the assembly of the prothrombinase complex.The prothrombinase complex is formed by factor Xa,its cofactor Va,and calcium ions on the membrane(of the platelet).The formed prothrombinase complex can activate prothrombin into thrombin and contribute to blood clotting.It also has become a consensus that in situ generated thrombin in the protein corona of zeolites plays a key role in the zeolite-initiated hemostasis.Zeolite acts as a type of reinforced activated inorganic platelet,but the mechanism of zeoliteinitiated blood coagulation at the molecular level remains obscure.In recent years,molecular dynamics(MD)simulations have become an important tool to study protein adsorption and activation on material surfaces.Here,MD simulations were performed to investigate the adsorption and activation mechanism of key coagulation factors on the zeolite surface.Our works may provide new insights into the design of zeolite-based hemostasis agents.(1)Previous studies have shown that factor Va(FVa)C1-C2 domains play a key role in the assembly and activity of the prothrombinase complex in the natural plateletbased blood coagulation cascade.Here,MD simulations were performed to investigate the binding details of the FVa light chain(A3-C1-C2 domains)on the phosphatidylserine(PS)membranes(of platelets)and Ca2+ LTA-type(CaA)zeolite surface.We have described the FVa C1-C2 domains and PS membrane interaction that are closely related to its cofactor activity.Different from the natural PS membrane surface,FVa light chain repeatedly exhibits a strong C2 domain anchoring interaction in its binding mode on the CaA surface.It is found that the porous surface structure of CaA zeolite and local highly dense solvation water clusters on the CaA zeolite surface restrict the movement of some lysine resides on the bottom of C2 domain.Our results show that the anchoring interaction can be slightly suppressed,so that FVa light chain can change from single-foot(strong C2 domain anchoring)to double-foot(both C1-C2 domain)adsorption states on the zeolite surface.This double-foot adsorption state is similar to natural PS membrane systems,which may make FVa have higher cofactor activity.(2)Prothrombin can be activated at two cleavage sites,R271 and R320,which generates two intermediates:prethrombin-2 via the initial cleavage at R271,and meizothrombin via the first cleavage at R320.Inconsistent activation pathways will affect the rate of thrombin generation and further affect the coagulation process.The interaction of the prothrombin cleavage site sequence with factor Xa(FXa)may play an important role in the selection of this activation pathway.Here,we used metadynamics with an advanced sampling technique to construct the free energy surface(FES)map of the interaction between the prothrombin cleavage site sequences(Pep271 or Pep320)and the catalytic site of FXa.The binding modes for the binding of Pep271 or Pep320 to the FXa catalytic triad were studied.Further molecular dynamics simulations revealed that corresponding to different binding modes,the binding stability of key residues R320 and R271 to the FXa catalytic triad would be different.This will affect the choice of prothrombin activation pathway on the zeolite and cell membrane surface.(3)It has been known that the zeolite-initiated hemostasis process involves a complex series of zymogen activation on the zeolite surface,and the special conformation of thrombin may contribute to its unusual activity.However,the molecular interaction details between thrombin and zeolite,which would be crucial in explaining the procoagulant activity of the zeolites,are far away from distinct.Here,we proposed a combined computational and experimental approach to determine the adsorbed sites and orientations of thrombin binding to Ca2+-exchanged LTA-type(CaA)zeolite.We have implemented fourteen ensembles of simulated annealing molecular dynamics(SAMD)simulations and experimental surface residue microenvironment analysis to reduce the starting orientations needed for further MD simulations.The combined MD simulations and procoagulant activity characterization also reveal the consequent corresponding deactivation of thrombin on CaA zeolite.It is mainly caused by two aspects:Firstly,the secondary structure of thrombin can change after its adsorption on the CaA zeolite.Secondly,the positively charged area of thrombin mediates the preferential interaction between thrombin and CaA zeolite.Some thrombin substrate sites are thus blocked by zeolite after its adsorption.The combined use of computational and experimental approaches,on the one hand,provides a promising method for characterizing the interaction between protein and nanoparticles.On the other hand,it reveals the deactivation mechanism of thrombin on the CaA zeolite surface,which can provide new ideas for the design and application of zeolite with high procoagulant activity.(4)Based on the deactivation mechanism of thrombin on the CaA zeolite surface,we extended our study to the regulation of thrombin adsorption orientation by MD simulations.It was found that the distribution of-NH3+groups in the z-direction and xy plane of the zeolite can be regulated by the concentration of the lysine peptide.The interaction between thrombin’s lysine residues and the zeolite can be inhibited accordingly.SAMD simulations were used to study the interaction of lysine peptidemodified zeolite and thrombin,it can be found that the adsorption orientation of thrombin changed with the increase of the concentration of the peptide.Long-time MD simulations also found that lysine peptide can inhibit the interaction between positively charged residues of thrombin(e.g.K459 and K618)and zeolite,regulating the thrombin adsorption orientation.As revealed by the above work,we can turn the activity of thrombin by regulating the thrombin adsorption orientation.