| Integrins are members of a large family of functionally conserved cell adhesion receptors,mainly composed of non-covalent binding of ? subunit and ? subunit.Among them the ?3 subunit can combine with the ?IIb subunit or the ?v subunit to form ?IIb?3 or ?v?3 integrins.Integrin ?v?3 is widely expressed,while integrin ?IIb?3 is specifically expressed on the surface of platelets and megakaryocytes.As the most abundant receptor on the platelet membrane,?IIb?3 is regarded as the final signaling of platelet activation and plays a crucial role in the process of bidirectional signal transduction of platelets.Integrins are mainly composed of large extracellular domains,transmembrane helix domains and short cytoplasmic domains.The ?3 subunit cytoplasmic domain is very important in integrin signal transduction.The ? subunit cytoplasmic domain is short and non-enzymatic,but contains highly conserved domains,including membrane proximal ? helix,membrane proximal NPx Y motif and membrane distal Nxx Y motif.Our previous studies have shown that Chinese hamster ovary(CHO)cells with ?3E726Q mutant had a defect in spreading on immobilized fibrinogen and complicated with membrane blebbing.Moreover,this blebbing could be inhibited by the myosin light chain ATPase inhibitor blebbistatin.CHO cell expressing ?IIb?3 is the most common alternative model of platelets.However,due to the lack of platelet-specific receptors and skeleton proteins,CHO cells cannot perfectly substitute platelets.Therefore,we employed CRISPR/Cas9 technique and homologous recombination repair methods to introduce ?3E726Q-point mutations,establishing transgenic mouse models.Platelet functions of integrin ?3E726Q transgenic mice were assessed to determine whether integrin ?3E726Q affects the signal transduction and skeleton protein recombination within the platelets.Objective: By breeding and identifying integrin ?3E726Q transgenic mice,to assess platelet functions and to observe the role of the ?3E726Q mutation in signal transduction and skeleton protein recombination.Methods: 1.Construct,breed and identify by PCR to obtain a certain amount of transgenic mice with ?3E726Q.2.To detect the platelet function of transgenic mice with ?3E726Q: obtain blood from mice by cardiac puncture,detect the bidirectional signal transduction of the platelet,including the inside-out signal transduction event,soluble fibrinogen binding,and the outside-in signal transduction event,including platelet spreading on immobilized fibrinogen,platelet aggregation,clot retraction and closure time,etc.Results: 1.Integrin ?3E726Q transgenic mice were successfully identified by PCR and sequencing.The results of sequencing showed that the amplified products of heterozygous mice had "double peaks" at their mutation sites,and both homozygotes and wild types had "single peaks".According to the sequence information of the mutation sites,if "GAA" is mutated into "CAA",it would be mutant type,and if it was still "GAA",it would be wild type.2.The fibrinogen binding of platelets from integrin ?3E726Q transgenic mice was unchanged compared with that from wild type mice,suggesting that the inside-out signal transduction is normal.3.The wild-type mice platelets spread well on immobilized fibrinogen,while transgenic mice with ?3E726Q showed poor platelet spreading ability on immobilized fibrinogen.4.The retraction rate of fibrin clot in ?3E726Q transgenic mice was similar to that in wild type mice.5.We used PFA-200 to detect the closure time on a membrane coated with Col/Epi and Col/ADP.The results showed that the blood closure time(CT)of wild-type mice was normal,while the whole blood of ?3E726Q transgenic mice wasn't closed until whole blood exhausted.Conclusion: The platelets of transgenic mice with ?3E726Q manifested normal fibrinogen binding,normal function of fibrin clot reaction,poor spreading ability on immobilized fibrinogen,and a decline in thrombogenesis. |
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