P-selectin-mediated Integrin LFA-1 Activation And Adhesive Rolling Of Neutrophils On The Immobilized Platelets Under Flows

A variety of adhesion molecules are involved in inflammatory reaction when inflammation occurs in the body.Selectins expressed on vascular endothelial cells mediate initial leukocytes adhesion by recognizing PSGL-1 on the leukocyte surface which triggers downstream cell signal transduction and activates integrin LFA-1 of leukocytes.The activation of integrin is a key event in the vascular inflammatory chain reaction and is regulated by the hemodynamic environment.However,the mechanism and signaling pathway of selectin-mediated LFA-1activation have not been elucidated.In the first part of the paper,we mainly explored P-selectin-mediated integrin LFA-1 activation and signal pathway under flows.We use a parallel-plate flow chamber(PPFC)system that simulates the human blood flow environment.We perfused the treated(piceatannol,staurosporine,cytochalasin B and MNS inhibited syk,moesin,actin and talin,respectively)or no treated neutrophils into flow chamber on the substrates of P-selectin or P-selectin+ICAM-1 under the condition of wall shear stress(0.1~0.45 dyn/cm~2).High speed camera(100 fps)was used to observe cells adhesion events and record tether lifetime(molecular bond lifetime)on the substrates.Integrin activation was detected by the tether lifetime of P-selectin+ICAM-1 substrates.The results showed that P-selectin coupling force prolongs the tether lifetime of cells.The activation of LFA-1 mediated by P-selectin was a rapid event with a trigger time of less than 0.7 second.With the increase of wall shear stress,the lifetime of neutrophils mediated by P-selectin and ICAM-1 was extended and then shortened.The results show that the P-selectin-mediated integrin LFA-1 rapid activation involves mechanosensitive signal transduction pathway PSGL-1?moesin?actin?talin?LFA-1.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 neutrophils.The A1 domain of von Willebrand factor(vWF)secreted by the damaged site binds to the platelet glycoprotein receptor GPIb?which triggers downstream signaling and leads to platelet activation under the condition of high shear force.Activated platelets secrete adhesion molecules,among which P-selectin can bind to PSGL-1 to recruit neutrophils.Neutrophils undergo a multi-step cascade on platelets mediated by adhesion molecules while the rolling adhesion behavior of neutrophils on platelets and related mechanical and biological mechanisms under flow environment have not been clarified.In the second part of this paper,we mainly explored adhesive rolling of neutrophils on the immobilized platelets under flows.We used the PPFC technique and adhered platelets to the flow chamber substrates coated with vWF-A1 molecules.Platelets were activated for different time(0 min,2.5 min and 7.5min)by loading 10 dyn/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 under 0.5 dyn/cm~2 wall shear stress.We extracted parameters such as the number of adhesion events,tether lifetime of cells,and rolling velocity.The results showed that neutrophils can specifically bind to activated platelets.The tether lifetime of neutrophils on platelets is independent on mechanical stimulation,whereas mechanical stimulation reduces the velocity of neutrophils rolling on activated platelets.We investigated P-selectin-mediated activation of neutrophils integrin LFA-1 and its signaling pathway under flows and analyzed the relevant kinetic parameters,which will deepen the understanding of leukocyte immune and inflammatory response process.At the same time,we explored adhesive rolling of neutrophils on the immobilized platelets under flows,which helps to understand the mechanism of platelet-neutrophils interaction and the process of atherosclerosis.The researches in this paper can provide reference for the discovery of inflammation-related drug targets and the design of anti-thrombotic drugs for cardiovascular and cerebrovascular diseases.