Platelet Integrin Activation Mediated By VWF-A1/GP?b Under Flow Field And Intrinsic Mechanism Of A1 Mutation Leading To Dysfunction Of A Domain

The combination of von Willebrand factor(VWF)and platelet glycoprotein ?b?(GP?b?)mediates platelet adhesion and activation.The 2B and 2M mutations that occur on the VWFA1 domain can cause severe hemorrhagic diseases.Here we used a parallel plate flow chamber device that can simulate the physiological environment to analyze the platelet movement behavior mediated by wild-type WT-A1,2B mutant R1308 L,and 2M mutant G1324 S.Due to the electrostatic adsorption of the bottom plate,the conformation of fibrinogen(Fg)changes,which in turn causes its biological function to change.We labeled VWF-A1 and Fg with biotin.The experimental results show that,compared with the single-plated wild-type WT-A1,the single-plated 2B mutant R1308 L significantly enhances the platelet adhesion ratio,prolongs the platelet's instantaneous binding time and reduces the platelet rolling speed;while the singleplated 2M type The mutant G1324 S decreased the adhesion rate of platelets and the transient binding time,and the rolling speed increased.When Shuangpu WT-A1+Fg and R1308L+Fg,the platelet adhesion ratio was increased from 0.92% and 1.18% of single shop VWF-A1 and R1308 L to 1.31% and 1.96% respectively;The fitting analysis shows that WT-A1+Fg and R1308L+Fg of Shuangpu can activate integrin at 0.18 s and 0.14 s,respectively,and reduce their rolling speed;however,the activation of integrin does not occur in G1324 S,compared with G1324 S in single shop.There was no significant difference in platelet adhesion ratio,instant tethering time and rolling speed on G1324S+Fg and Shuangpu.This indicates that two different mutations cause similar bleeding characteristics with different mechanisms.In order to further explore the effect of mutations on the platelet function of VWF adhesion,we used molecular dynamics simulation to reveal the internal molecular mechanism of A1 mutation leading to dysfunction of A domain.First,a complex model of A1/A2 was constructed by HADDOCK using flexible docking method,and a specific site mutation was performed by computer to obtain 2M mutant G1324S-A1/A2,2B mutant R1308L-A1/A2 complex model.Through conformational analysis,we found that the A2 domain is located on the side of the binding surface of A1 and GP?b? and occupies most of the binding site of A1 and GP?b?.The specific tensile dissociation process and tensile dissociation force of the WT-A1/A2 composite and its mutants were obtained through constant speed tensile simulation.Compared with WTA1/A2 complex,R1308L-A1/A2 complex has lower mechanical stability and faster dissociation time,while G1324S-A1/A2 mutant is significantly enhanced and has longer dissociation time.Three times of 100 ns free molecular dynamics simulations confirmed the main hydrogen bond and salt bridge network in the A1/A2 complex;the R1308L-A1/A2 mutant reduced the ARG571,LYS572,GLU576 amino acid residues and A2 structure The interaction between the ?3 helix and ?4?5-loop loop on the domain reduces the stability of the molecule.The G1324S-A1/A2 mutant enhances LYS572-ASP1587(survival rate>84.12%)and ARG571-GLU1598(survival rate>22.67 %)The stability of the formed salt bridge and a new interaction between ARG571-GLU1598 salt bridge(survival rate>41%).The above research results prove the order of the stability of the complex G1324S-A1/A2> WT-A1/A2> R1308L-A1/A2,and the key residues are obtained.This provides a possible explanation for the mechanism of affinity regulation caused by changes in local kinetic properties caused by single amino acid mutations,and also provides some guidance for the design and development of related thrombotic drugs.