WT-A1 And Mutants Mediated Calcium Signaling Of Platelets Under Shear Flow

Von Willebrand factor?VWF?is the largest plasma glycoprotein,and its A1 domain contains the binding site of platelet glycoprotein?b?.During physiological hemostasis,the interaction between GP?b?and VWF A1 is a crucial first step to initiate the coagulation and hemostasis cascade.It also mediates platelet thrombosis on the blood vessel wall and also induces the release of intracellular calcium and ADP,which further activates integrin???b?3and induces extracellular calcium influx and promotes stable platelet adhesion.As the second messenger of intracellular signal transduction,calcium ion plays an indispensable role in the physiological process of cell initial adhesion,rolling cytoskeleton rearrangement,stable adhesion and aggregation.Therefore,given that the VWF A1 molecule could mediate the initial adhesion of platelets and that the mutation of a specific site in VWF A1 leads to the occurrence of bleeding disorders and the role of the calcium ions in cell activation and other functions and actions.In this paper,a parallel plate flow chamber is used to simulate the human blood flow environment to explore the differences in platelet adhesion and intracellular calcium release mediated by WT-A1 and two mutants under shear flow.First,E.coli M15 was induced to express WT-A1,2M mutant A1G1324S and 2B mutant A1R1308L protein molecules by IPTG.A single target protein was obtained by nickel column affinity chromatography using the six His-tags carried by the protein,and the expressed protein was identified by SDS-PAGE and Western Blot,and the protein concentration was detected by BCA.We obtained 0.77 mg of WT-A1,1 mg of A1G1324S,0.57 mg of AR1308L.Then,Platelets was perfused on different substrates?PBS,2%BSA,WT-A1+2%BSA,A1R1308L+2%BSA,and A1G1324S+2%BSA?by using a parallel-plate flow chamber.The results showed that PBS can adsorb a large amount of platelets through electrostatic interactions,but 2%BSA can effectively block no-specific adhesion.The three proteins expressed in the experiment can mediate platelet adhesion and the number of platelet adhesions increased sequentially on A1G1324S,WT-A1,and A1R1308L.This indicated that 2%BSA does not affect the specific adhesion of platelets to proteins.Thus,the method to functionalize flow chamber floor is reasonable,we can use this method to study platelets calcium response.Finally,Flou-4 AM as the fluorescent indicator of intracellular calcium,the intracellular calcium concentrations of adherent platelets on WT-A1 and two mutants were detected by parallel plate flow chamber system combined with fluorescence microscope under 0⁵00 s^-1shear rate.By extracting and analyzing the experimental characteristic parameters,such as activation ratio,delay time,and fluorescence increase ratio of platelets.To explore the effects of shear stress,chemical signal and molecular dynamics on the intensity and the speed of calcium response.The results showed that shear rate and molecular concentrations positively regulation of platelets activation ratio and negatively regulation of startup time of calcium response.Compared with static conditions,the activation ratio was increased by 37 times at500 s^-1.When the concentration of WT-A1 was increased from 60?g·mL^-11 to 240?g·mL^-1,the delay time was shortened by 10 s.In addition,changes in the affinity of VWF A1 to platelets caused by the mutation affect the activation rate and delay time of the calcium response.Compared with WT-A1,the type 2B mutant A1R1308L increased the activation ratio and shortened the delay time,whereas the 2M type mutation A1G1324S had the opposite effect,and the activation ratio was decreased,and startupe time of calcium response was increased.