biostudies-literatureQuach MENHLBI NIH HHSNational Heart, Lung, and Blood Institute2044-2055https://www.ebi.ac.uk/biostudies/studies/S-EPMC8324530biostudies-literatureUnknown19(8)<h4>Background</h4>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.<h4>Objectives</h4>To characterize a novel rat monoclonal antibody 3G6 that binds GPIbβ.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.Journal of thrombosis and haemostasis : JTHDifferential regulation of the platelet GPIb-IX complex by anti-GPIbβ antibodies.PMC8324530HL146299R21 HL146299HL134241HL082808F31 HL134241R01 HL082808Lanza FNieswandt BQuach MEChen WLi RWang YDeckmyn HfalseDifferential regulation of the platelet GPIb-IX complex by anti-GPIbβ antibodies.<h4>Background</h4>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.<h4>Objectives</h4>To characterize a novel rat monoclonal antibody 3G6 that binds GPIbβ.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.2021-01-01T00:00:00Z2021 Aug2025-04-27T00:06:02.679Z2025-04-06T17:47:57.991ZS-EPMC83245303391503110.1111/jth.15359