The Activation Mechanism Of VWF-A And The Platelet P-selectin Surface Expression Induced By VWF-A1 In The Flow

When blood vessels are injured,endothelial cells secrete the von Willebrand factor(vWF),which binds to exposed subendothelial collagen.vWF stretches from a loose spherical conformation to an activated linear conformation under the flow and exposes the GPIb?binding site on A1.Studies have shown that self-inhibitory interactions within vWF can inhibit vWF from binding to platelets and participate in the activation of vWF-A domains.Besides,mutations on vWF-A1 also affect the affinity of vWF to platelets.The gain of function mutation(GOF)R1308L promotes the vWF/GPIb? interaction,and the loss of function mutation(LOF)G1324S does the opposite.Through the interaction of vWF-A1 and GPIb?,circulating platelets can be recruited to the injured vascular site and activated under the flow shear stress.The surface expression of P-selectin is an important sign of platelet activation,which promotes the adhesion of leukocytes by binding to PSGL-1 on leukocytes.In addition to agonists,shear stress is also one of the regulatory factors of platelet P-selectin surface expression,and it depends on the interaction of vWF and GPIb?.However,the vWF-A domain activation in flow and the machinal-chemical regulation mechanism of vWF-A1 induced platelet P-selectin surface expression remains unclear.In response to the above scientific problems,this thesis first uses the parallel plate flow chamber and atomic force microscope scanning technology to detect the functional and conformational transitions of vWF-A1A2A3 after different flow shear stress pretreatments.We found that the ability of vWF-A1A2A3 binds to GPIb? first weakened and then strengthened with the increase of shear stress.At the same time,the conformation of vWF-A1A2A3 contracted first and then stretched when the shear stress exceeded the threshold force.The GOF mutant R1308L has a significantly stronger ability of binding to GPIb? than the wild-type vWF-A1A2A3,and a looser conformation;the LOF mutant G1324S has a more compact conformation and binds to GPIb? weakly.Single molecule force spectroscopy experiments confirmed that the self-inhibition of vWF-A1A2A3 mainly exists between A1/A2 and A1/A3.R1308L down-regulated the binding affinity of A1 and A2,leading to an activated stretched conformation of the vWF-A domain.In contrast,G1324S enhanced the stability of the closed conformation of the vWF-A domain by up-regulating the binding affinity between A1 and A2,making vWF more difficult to activate.The biphasic force-dependent A1/A2 molecular bond dissociation constant suggests that below the force threshold,the force inhibits the activation of vWF by enhancing the stability of the vWF-A closure structure,and when the force exceeds the mechanical threshold,the force decrease the stability of the vWF-A structure and promotes the transformation of the vWF-A domain into the stretched activated conformation,revealing the mechanical regulation and molecular mechanism of vWF-A1A2A3 activation.The A1/A2 interaction within vWF can not only regulate the activation of vWF-A1A2A3 but also improve the bleeding disease by inhibiting the binding of vWF-A1 to GPIb?,becoming a drug target.Therefore,understanding the key residues of the A1/A2 interaction is of great significance.To explore the binding surface information of A1/A2 and A1/A3 in vWF-A1A2A3,we have obtained the possibility conformation of vWF-A1A2A3 through flexible docking and screening.The interaction between A1/A2 and A1/A3 was analyzed by molecular dynamics simulation,and the residues on the binding surface were sorted according to the residue interaction index.We found that GLU1598,VAL1546,ASP1587,SER1596,LYS1617,GLY1595,and ARG1583 on A2 domain,and SER2,LYS185,ASP160,GLN3,PRO157,SER161,and HSD184 onA3 domain participated in the combination of vWF-A1,which could provide a theoretical basis for target drug design.To explore the regulatory effect of vWF-A1 on platelet P-selectin surface expression,we performed flow cytometry,parallel plate flow chamber,and immunofluorescence staining experiments.Our experimental results proved that flow shear stress initiated vWF-A1-induced platelet P-selectin surface expression through the PI3K/Akt signaling pathway,and the level of platelet P-selectin surface expression promoted with the increase of shear stress and mechanical stimulus time.GOF mutant R1308Lcould not only promote platelet adhesion but also increase platelet P-selectin expression,while GOF mutant G1324S did the opposite,indicating that vWF-A1 mediates platelet P-selectin surface expression in the flow was kinetic-dependent.Besides,we found that when the shear stress accumulation(the product of shear stress and mechanical stimulus time,SSA)value was equal,the level of platelet P-selectin surface expression was very similar,implying that SSA regulated the platelet P-selectin surface expression mediated by vWF-A1.This thesis studied the mechanical regulation of vWF-A1A2A3 activation in the bloodstream environment and analyzed the binding surface information of vWF-A1A2A3 internal interactions.We also investigated the machinal-chemical regulation mechanism of vWF-A1-induced platelet P-selectin surface expression in flow.This study will deepen the understanding of platelet hemostasis and thrombosis and provide a theoretical basis for the development and design of cardiovascular disease drugs.