The Force Regulation Mechanism Of GOF/WT ADAMTS13 Interaction With VWF A2

VWF（von Willebrand Factor,VWF）is a force-sensitive glycoprotein with hemostatic function.It can capture platelets to the damaged blood vessel and initiate primary hemostasis.If the activity of VWF is not regulated,it will capture a large number of platelets and even blood cells,eventually leading to the formation of thrombosis.ADAMTS13 specifically cleaves the Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ peptide bond embedded in the VWF A2 domain,regulates the size and activity of the multimeric VWF prevents thrombus formation.Force-sensitive protein VWF changes from a spherical conformation to an elongated protein in response to high shear stress,which exposes the VWF A2 cleavage site and promotes cleavage.Gain-of-function（GOF ADAMTS13）ADAMTS13 Spacer domain variant（R568K/F592Y/R660K/Y661F/Y665F）was more enzyme cleavage activity than WT ADAMTA13.Biochemical experiments revealed the binding site between the enzyme and the substrate.But biochemical experiments required two molecules to incubate for a long time,which far exceeded the interaction process of instantaneous contact under real physiological conditions.The interaction between ADAMTS13 and VWF on the micro time scale and the influence of the force in it are not clear yet.Therefore,in this study,VWF A2 and ADAMTS13 with different functional types were taken as the research object.These rapid,transient and mechanically regulated molecular interaction process was detected by atomic force microscopy using monomolecular technology.To simulate the interaction between WT/GOF ADAMTS13and VWF A2 in a real hemodynamic environment.To explore the mechanism of mechanical regulation.Our results showed that GOF/WT ADAMTS13 interaction with VWF A2 had a high binding affinity at a very short contact time of 0.8 s.The rupture force of GOF/WT ADAMTS13interaction with VWF A2 was 23.5±0.8 p N and 27.4±0.9 p N,respectively,both two complexes had high mechanical strength.At the same time,the dynamics parameters were obtained by fitting the Bell-Evans model.AFM clamp mode was used to investigate the intermolecular bonding survival time.The results showed that the GOF/WT ADAMTS13interaction with VWF A2 exhibited a biphasic force-dependent binding characteristic,that is,with the increase of external forces,the mechanism of the bonding life from"catch bonds"to"slip bonds".This is consistent with the reported biphasic force-dependent mechanism that regulates the interaction between VWF A1A2A3 and ADAMTS13.Suggesting that the VWF A2 monomer domain plays an important role in the interaction between VWF and ADAMTS13.Finally,the SⅡtag was fused into the C-terminal of VWF A1,A2,and A3 monomer proteins through homologous construction.The recombinant proteins VWF A1-SⅡand VWF A3-SⅡwere successfully expressed by prokaryotic expression system and high purity target protein was obtained.Our study on the mechanical regulation mechanism of the WT/GOF ADAMTS13 binding to VWF A2 will help to further understand the physiological and pathological processes of hemostasis and thrombosis,and provide new vision and ideas for the design and screening of molecular drugs for related diseases.