The Mechanical Stability Of VWF-A2 Domain Studied By Molecular Dynamics Simulations

Cardiovascular disease has become one of the diseases that threaten human lives and health.Physiological hemostasis and pathological thrombosis are the essential parts involved in the cardiovascular disease.von Willebrand factor（VWF）plays a very important role in the above processes.In the case of vascular injury,VWF could mediate platelet adhesion,aggregation,and cross-linking to maintain hemostasis.The hemostatic potential of VWF multimers depends on its size,the newly secreted VWF usually exists as ultra-large VWF multimers,which are of the strongest hemostatic potential.ADAMTS13（A disintegrin and metalloproteinase with a thrombospondin type 1 motif,member 13）could cleavage the buried peptide bond（TYR1605-MET1606）,thus regulating the hemostatic potential of VWF multimers.The unfolding of VWF-A2 domain exposures the proteolysis sites,which plays as the preconditions of ADAMTS13 proteolysis.The reported researches suggest that shear force could regulate the above proteolysis processes.In this study,we selected four types of VWF-A2 molecules: the crystal structures of VWF-A2 domain not containing calcium（A2,PDB ID: 3GXB）and with calcium bound(A2/Ca²⁺,PDB ID: 3ZQK)were downloaded from the Protein data bank.The two type 2A VWD mutants(R1597W-A2 and R1597W-A2/Ca²⁺)were constructed through computational site-directed mutation,based on the crystal structures of the above two,respectively.We used steered dynamics simulations under constant forces to compare the conformational changes during the equilibrations,the unfolding pathway differences,and the exposure degree variance of the proteolysis sites,aiming to investigate how calcium and mutant R1597 W affect the mechanical stability of VWF-A2 domain.The crystal data and data of the dynamic equilibrations showed that calcium binding and mutant R1597 W did not largely change the global conformation of VWF-A2 domain,instead lowed and enhanced the localized flexibility of the α3β4-loop,respectively.The data of the steered dynamics simulations under constant forces showed that stretch force could induce the exposure of the proteolysis sites.Calcium enhanced the mechanical stability of VWF-A2 domain through maintaining its hydrophobic core,hindering the exposure of proteolysis sites.Type 2A VWD mutant R1597 W lowed the mechanical stability of VWF-A2 domain to a certain extent,accelerating the unfolding process and the exposure of proteolysis sites during the prelimitary stages.However,during the middle and later stages of unfolding,the α3β4-loop with high flexibility increased the movements of its surrounding region,slowing down the unfolging processes,thus affecting the complete exposure of proteolysis sites.Our results indicate that stretch force could induce the unfolding of the β5-strand in VWF-A2 domain and the exposure of proteolysis sites.Calcium binding could enhance the mechanical stability of VWF-A2 domain and hindering the exposure of proteolysis sites,through strengthening the center hydrophobic core,leading to reduced proteolysis efficiency of ADAMTS13.On the contrary,mutant R1597 W lowed the mechanical stability of VWF-A2 to a certain extent,accelerating the exposuring process of proteolysis sites,resulting in enhaced proteolysis efficiency.The α3β4-loop was the key element in the regulation of mechanical stability of VWF-A2 domain.These results may help to understand how ADAMTS13 cleavages the VWF-A2 domain and regulates the hemostatic potential of VWF,and help to develop the relevant anti-thrombus drugs at the same time.