M Edward Quach1, Wenchun Chen1, Yingchun Wang1, Hans Deckmyn2, François Lanza3, Bernhard Nieswandt4, Renhao Li1. 1. Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA, USA. 2. Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium. 3. Université de Strasbourg, INSERM, BPPS UMR-S1255, Strasbourg, France. 4. Rudolf Virchow Center, Julius Maximilian University of Wurzburg, Würzburg, Germany.
Abstract
BACKGROUND: Platelets' initial recognition of endothelial damage proceeds through the interaction between collagen, plasma von Willebrand factor (VWF), and the platelet glycoprotein (GP)Ib-IX complex (CD42). The GPIb-IX complex consists of one GPIbα, one GPIX, and two GPIbβ subunits. Once platelets are immobilized to the subendothelial matrix, shear generated by blood flow unfolds a membrane-proximal mechanosensory domain (MSD) in GPIbα, exposing a conserved trigger sequence and activating the receptor. Currently, GPIbα appears to solely facilitate ligand-induced activation because it contains both the MSD and the binding sites for all known ligands to GPIb-IX. Despite being positioned directly adjacent to the MSD, the roles of GPIbβ and GPIX in signal transduction remain murky. OBJECTIVES: To characterize a novel rat monoclonal antibody 3G6 that binds GPIbβ. METHODS: Effects of 3G6 on activation of GPIb-IX are characterized in platelets and Chinese hamster ovary cells expressing GPIb-IX (CHO-Ib-IX) and compared with those of an inhibitory anti-GPIbβ antibody, RAM.1. RESULTS: Both RAM.1 and 3G6 bind to purified GPIbβ and GPIb-IX with high affinity. 3G6 potentiates GPIb-IX-associated filopodia formation in platelets or CHO-Ib-IX when they adhere VWF or antibodies against the ligand-binding domain (LBD) of GPIbα. Pretreatment with 3G6 also increased anti-LBD antibody-induced GPIb-IX activation. Conversely, RAM.1 inhibits nearly all GPIb-IX-related signaling in platelets and CHO-Ib-IX cells. CONCLUSIONS: These data represent the first report of a positive modulator of GPIb-IX activation. The divergent modulatory effects of 3G6 and RAM.1, both targeting GPIbβ, strongly suggest that changes in the conformation of GPIbβ underlie outside-in activation via GPIb-IX.
BACKGROUND: Platelets' initial recognition of endothelial damage proceeds through the interaction between collagen, plasma von Willebrand factor (VWF), and the platelet glycoprotein (GP)Ib-IX complex (CD42). The GPIb-IX complex consists of one GPIbα, one GPIX, and two GPIbβ subunits. Once platelets are immobilized to the subendothelial matrix, shear generated by blood flow unfolds a membrane-proximal mechanosensory domain (MSD) in GPIbα, exposing a conserved trigger sequence and activating the receptor. Currently, GPIbα appears to solely facilitate ligand-induced activation because it contains both the MSD and the binding sites for all known ligands to GPIb-IX. Despite being positioned directly adjacent to the MSD, the roles of GPIbβ and GPIX in signal transduction remain murky. OBJECTIVES: To characterize a novel rat monoclonal antibody 3G6 that binds GPIbβ. METHODS: Effects of 3G6 on activation of GPIb-IX are characterized in platelets and Chinese hamster ovary cells expressing GPIb-IX (CHO-Ib-IX) and compared with those of an inhibitory anti-GPIbβ antibody, RAM.1. RESULTS: Both RAM.1 and 3G6 bind to purified GPIbβ and GPIb-IX with high affinity. 3G6 potentiates GPIb-IX-associated filopodia formation in platelets or CHO-Ib-IX when they adhere VWF or antibodies against the ligand-binding domain (LBD) of GPIbα. Pretreatment with 3G6 also increased anti-LBD antibody-induced GPIb-IX activation. Conversely, RAM.1 inhibits nearly all GPIb-IX-related signaling in platelets and CHO-Ib-IX cells. CONCLUSIONS: These data represent the first report of a positive modulator of GPIb-IX activation. The divergent modulatory effects of 3G6 and RAM.1, both targeting GPIbβ, strongly suggest that changes in the conformation of GPIbβ underlie outside-in activation via GPIb-IX.
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