Atomic Force Microscopy Imaging Reveals The Conformation Of Metalloprotease ADAMTS13

Platelet adhesion to the damaged vessel wall is an important process in hemostasis and thrombosis.The interaction between von Willebrand Factor?VWF?and the platelet receptor GPIb?,which mediates platelet adhesion and rolling adhesion,plays a very important role in the initial stage of platelet adhesion.VWF activity and its molecular size,molecular weight,the greater its activity.?A disintegrin and metalloprotease with a thrombospondin type 1motifs 13?ADAMTS13 regulates the size of VWF by digesting the Tyr1605-Met1606 peptide bond in the center of the VWF A2 domain and thereby modulates VWF active.At present,studies have shown that ADAMTS13 has a low-affinity closed conformation and a high-affinity open conformation;five specific sites in the spacer domain of ADAMTS13?R568K/F592Y/R660K/Y661F/Y665F?are called gain of function?GOF?,its enzymatic activity was increased by about 2.5-fold compared with WT.In addition,low pH enhances ADAMTS13 activity,divalent metal ions such as Ca²⁺,Mg²⁺and Zn²⁺could regulate the activity of ADAMTS13 digested VWF-A2.ADAMTS13 under various conditions in the enzyme digestion activity change is due to its own conformation change?Whether ADAMTS13 molecules exist multiple conformations,low pH induces a conformational change in ADAMTS13,and divalent metal ions affect ADAMTS13 conformation?In this study,morphological changes of WT-ADAMTS13 and GOF-ADAMTS13 under different conditions were observed by atomic force microscopy.The data show that under different conditions WT-ADAMTS13 and GOF-ADAMTS13 Volume,Projected Area,Maximum length compared to the difference is not obvious.However,the Aspect Ratio of WT-ADAMTS13 protein particles was significantly less than that of GOF-ADAMTS13.The histogram distribution of the aspect ratio data and Gaussian function fitting,WT-ADAMTS13 aspect ratio was bimodal distribution,GOF-ADAMTS13 aspect ratio was three-peak distribution.We define each peak as a different type?state I,state II,state III?.We propose that state I is a"closed"conformation,state III is an"open"conformation,and state II is an intermediate conformation.The data show that WT-ADAMTS13 adopts two different state states?state II?at pH 6 and p H 7.5 whereas another state?state III?is observed at GOF-ADAMTS13.The percentage of state III of WT-ADAMTS13 and that of GOF-ADAMTS13 increased at pH 6 compared to pH 7.5,state I and state II of WT-ADAMTS13 and GOF-ADAMTS13 decreased,indicating that low pH can induce a more open conformation of ADAMTS13.When chelated with EDTA,the conformational distribution of both state II and state III of ADAMTS13 increased significantly without affecting the conformational distribution of GOF.When removing EDTA and adding different divalent ions,we found that the state I of WT-ADAMTS13 significantly increased and the conformation became more compact after adding Ca²⁺and Mg²⁺;After the addition of Mg²⁺to GOF,the state I also increased significantly;Zn²⁺had no effect on the conformation of the two proteins,indicating that Zn²⁺does not affect the enzyme activity of ADAMTS13 by changing its conformation.In this study,by statistical analysis of the conformational data of ADAMTS13 under different conditions,we conclude that there are three conformational states of ADAMTS13,and clarify the conformations of ADAMTS13 under various conditions.The conformation of ADAMTS13 Bivalent metal ion regulation,these findings further deepen our understanding of ADAMTS13 hydrolysis VWF,and regulation of its activity,At the same time provide guidance for the understanding of ADAMTS13 related thrombotic diseases and the development of antithrombotic drugs..