Study On The Interaction And The Mechanism Of Mechanical Regulation Between The Transmembrane Proteins(PSGL-1,CD40l) With Receptors That Mediate The Immune Response Of Circulating Leukocytes

During the process of platelet hemostasis after vascular damage,it involves the key events in hemodynamic environment such as platelet adhesion,activation,aggregation and interaction with leukocytes.In the hemodynamic environment,circulating platelets and white blood cells will interact with each other under mediating of some adhesive molecules and chemokine factors,be involved in the early formation and development of coronary plaque and thrombosis after plaque rupture.CD40L and P-selectin secreted by platelets are key molecules that mediate the interaction between platelets and leukocytes,but the intracellular mechano-chemical signal transduction process is not yet clear.First,we observed and analyzed the adhesion and rolling of leukocytes on activated platelets under different shear stress(0.1?0.5 dyn/cm~2)using the parallel-plate flow chamber(PPFC)system.In the experiment,platelets adhered to flow chamber substrates coated with v WF-A1 molecules.Platelets were activated for different time(0 min,2.5 min and 7.5 min)by loading 10dyn/cm~2 wall shear stress.Subsequently,the neutrophils suspension was perfusion into the flow chamber experimental area.High-speed camera was used to observe and record the rolling adhesion of neutrophils on activated platelets.We extracted parameters such as the number of adhesion events,tether lifetime of cells,and rolling velocity.The results showed that the leukocytes rolling velocity decreased first and then increased with the increase of shear force.However,the tether lifetime of leucocytes on platelets was lengthened and then shortened.The rolling adhesion properties of this biphasical force-dependent leukocytes to activated platelets is not only related to the secretion of key platelet molecules(P-selectin and CD40L),but also related to“catch-slip”bond mechanism of complexes(PSGL-1/P-selectin,CD40L/CD40).At the same time,increased activation time of platelets stimulated by blood shear stress will shorten the rolling velocity of leukocytes on platelets,suggesting that the intensity or time of mechanical stimulation will regulate the secretion of key molecules(P-selectin and CD40L)on platelets.Then,atomic force microscope(AFM)technique was used to measure the adhesion frequency of CD40L to receptor CD40(monomer and dimer)and?M?2 at single-molecule level,and to extract and analyze the single-bond lifetime and dissociation constants of the adhesion complexes(CD40L/CD40,CD40L/?M?2)at different tension levels.Biphasic force-dependent CD40L-CD40,CD40L-?M?2 molecular bond dissociation constant prompted that,under the force threshold,the binding affinity between CD40L/CD40 and CD40L/?M?2 receptor-ligands increased with the increase of force,and when the force exceeded the force threshold,the binding affinity between CD40L/CD40 and CD40L/?M?2receptor-ligands decreased with increasing force.Dimerization of CD40 had significantly enhanced the bond lifetime of CD40L-CD40 complex and increased the maximum rupture force it can withstand.Both CD40 dimerization-and“Catch bond”-induced enhancement of affinity of CD40 with CD40L may be necessary for stable cross talks between two cells.This finding may be useful for understanding CD40L/CD40(or?M?2)-induced events importantly in physiological or pathological processes at molecular and cellular level.The mechanical regulation mechanism of CD40L interacting with its receptors(CD40 and?M?2)is revealed to better understand the physiological and pathological processes of cardiovascular and cerebrovascular diseases.Finally,we used molecular dynamics(MD)simulations along with an intermolecular hydrogen bond analysis method based on statistics to study the interaction of PSGL-1juxtamembrane peptide with the radixin FERM domain in mechanical environment.MD simulations have provided atomic-level details of intermolecularmotions as a function of time to figure out the dynamic behavior of PSGL-1/ERM complex.The average number of hydrogen bonds and the relative dissociation probability indicate that the"Catch-slip"bond and force jointly regulate the binding affinity of PSGL-1 and ERM protein interaction.Both conformations analysis and solventaccessible surface(SASA)variant showed that mechanical signal transduction through the PSGL-1 cytoplasmic region,can mediate the exposure of phosphorylation site Y205 on ITAM-like motif,expect for the phosphorylation site Y191.These results of this study demonstrated a mechanical signal pathway that activates Syk via the PSGL-1 intracellular domain,and expounds the interaction mechanism of PSGL-1/ERM/Syk at the atomic level.In this paper,we focus on the frontier scientific issues of interaction between leukocytes and platelets.We carried out systematic and in-depth research on the events with the strategy which intends to integrate the theoretical principles,experimental techniques and analytical methods of biochemistry and molecular biology,cell molecular biomechanics,combined with atomic force microscope,parallel-plate flow chamber and molecular dynamics simulations.To demonstrate how the force regulates the rolling adhesion of leukocytes on activated platelets,reveals the mechanical regulation mechanism of the interaction between platelet transmembrane protein CD40L and its receptors(CD40,?M?2),and explores the force-chemical coupling regulation mechanism of the interaction between PSGL-1 and ERM proteins on cells.It aims to better understand the physiological and pathological processes of cardiovascular and cerebrovascular diseases,provide solutions for the clinical treatment and rehabilitation of patients with cardiovascular and cerebrovascular diseases,the discovery of related molecular drug targets and the new design of antibody drugs.