title: Epitope mapping of human VWF A3 recognized by monoclonal antibody SZ-123 and SZ-125 using MALDI mass spectrometry.
abstract: von Willebrand factor (VWF) serves as a molecular bridge between the constituents of the subendothelium, such as collagen, and receptors of the platelet membrane, primarily GPIb. We have previously reported two monoclonal antibodies (mAbs), SZ-123 and SZ-125, which specifically bind the VWF A3 domain and block the interaction of VWF with collagen type III and ristocetin- or botrocetin-induced platelet aggregation. Here, we identified the epitopes recognized by SZ-123 and SZ-125 using matrix-assisted laser desorption ionization mass spectrometry in combination with proteolysis protection assays. Our results demonstrated that SZ-123 recognizes a discontinuous epitope, involving the residues (989)AHLLSLVDVMQR(1000) and (1017)YLTSEMHGARPGASK(1031) of VWF. SZ-125 recognizes a linear epitope, encompassing the sequence (1001)EGGPSQIGDALGFAVR(1016). Immunoassays further indicated that the synthetic peptide, NH2-EGGPSQIGDALGFAVR-COOH, is sufficient for the binding to SZ-125. These results provide insight into the mechanistic basis for the inhibition of VWF binding to collagen by SZ-123/SZ-125.
authors.affiliation: Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou, 215006, China.
authors.forename: Miao
authors.initials: M
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou, 215006, China. zhaoyimingbox@163.com.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou, 215006, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Shen
authors.affiliation: Nanjing Medical University, Nanjing, China.
authors.forename: Fuqiang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou, 215006, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: He
authors.affiliation: Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou, 215006, China.
authors.forename: Changgeng
authors.initials: C
authors.lastname: Ruan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014841:von Willebrand Factor / Q000737:chemistry* / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D014841:von Willebrand Factor