title: Functional Effect of the Mutations Similar to the Cleavage during Platelet Activation at Integrin β3 Cytoplasmic Tail when Expressed in Mouse Platelets.
abstract: Previous studies in Chinese hamster ovary cells showed that truncational mutations of β3 at sites of F754 and Y759 mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F754 to C-terminus and the conservative N756ITY759 motif in platelet function have yet to be elaborated. Mice expressing β3 with F754 and Y759 truncations, or NITY deletion (β3-ΔTNITYRGT, β3-ΔRGT, or β3-ΔNITY) were established through transplanting the homozygous β3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different β3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the β3-ΔTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the β3-ΔNITY platelets. Behaviors of the β3-ΔRGT platelets were consistent with the previous observations in the β3-ΔRGT knock-in platelets. The adhesion impairment of platelets with the β3 mutants under flow was in different orders of magnitude shown as: β3-ΔTNITYRGT>β3-ΔRGT>β3-ΔNITY to fibrinogen-coated surface, and β3-ΔTNITYRGT>β3-ΔNITY>β3-ΔRGT to collagen-coated surface. To evaluate the interaction of the β3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that β3-ΔRGT interacted with kindlin but not c-Src, β3-ΔNITY interacted with c-Src but not kindlin, while β3-ΔTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin β3, i.e. T755NITYRGT762, R760GT762, and N756ITY759 participate in bidirectional, outside-in, and inside-out signaling, respectively and the association of c-Src or kindlin with β3 integrin may regulate these processes.
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.|Department of Hematology, Affiliated Hospital of Jiangsu University, Zhenjiang, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Jichun
authors.initials: J
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Xiongying
authors.initials: X
authors.lastname: Cui
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.|Department of Hematology, Institute of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
authors.forename: Jiansong
authors.initials: J
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Zhangbiao
authors.initials: Z
authors.lastname: Long
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Yulan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Lanlan
authors.initials: L
authors.lastname: Tao
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Ruan
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.|Department of Hematology, Affiliated Hospital of Jiangsu University, Zhenjiang, China.
authors.forename: Dongya
authors.initials: D
authors.lastname: Li
authors.affiliation: Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Suzhou, 215006, China.
authors.forename: Kesheng
authors.initials: K
authors.lastname: Dai
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Mao
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Collaborative Innovation Center of Hematology, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Xi
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D005340:Fibrinogen / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018056:Hemorheology; D055767:Immobilized Proteins / Q000378:metabolism; D039661:Integrin beta3 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D015539:Platelet Activation / Q000235:genetics*; D010973:Platelet Adhesiveness; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011485:Protein Binding; D012190:Retroviridae / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D014162:Transfection
chemical_list: D055767:Immobilized Proteins; D039661:Integrin beta3; D050505:Mutant Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D049452:Green Fluorescent Proteins; D005340:Fibrinogen