Differential regulation of the platelet GPIb-IX complex by anti-GPIb beta antibodies

• Type of Material: Article
• Authors: Edward M Quach, Emory University; Wenchun Chen, Emory University; Yingchun Wang, Emory University; Hans Deckmyn, Katholieke Univ Leuven; Francois Lanza, Université de Strasbourg, INSERM, BPPS UMR-S1255; Bernhard Nieswandt, Julius Maximilian University of Wurzburg; Renhao Li, Emory University
• Language: English
• Date: 2021-05-20
• Publisher: WILEY
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2021 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc.
• Final Published Version (URL): http://dx.doi.org/10.1111/jth.15359
• Title of Journal or Parent Work: JOURNAL OF THROMBOSIS AND HAEMOSTASIS
• Volume: 19
• Issue: 8
• Start Page: 2044
• End Page: 2055
• Grant/Funding Information: This work is partly supported by NIH grants HL082808, HL146299 (RL) and HL134241 (MEQ).
• Keywords: MONOCLONAL-ANTIBODY; RECEPTOR; Peripheral Vascular Disease; CYTOSKELETAL REORGANIZATION; Life Sciences & Biomedicine; glycoprotein Ib‐ GLYCOPROTEIN IB-ALPHA; BINDING; IDENTIFICATION; Hematology; Cardiovascular System & Cardiology; filopodia; ADHESION; IX complex; VON-WILLEBRAND-FACTOR; platelet activation; CALCIUM SIGNALS; biomechanics; TRANSMEMBRANE DOMAIN; Science & Technology; microscopy

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.