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

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 GPIb alpha, one GPIX, and two GPIb beta subunits. Once platelets are immobilized to the subendothelial matrix, shear generated by blood flow unfolds a membrane-proximal mechanosensory domain (MSD) in GPIb alpha, exposing a conserved trigger sequence and activating the receptor. Currently, GPIb alpha 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 GPIb beta and GPIX in signal transduction remain murky. Objectives To characterize a novel rat monoclonal antibody 3G6 that binds GPIb beta. 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-GPIb beta antibody, RAM.1. Results Both RAM.1 and 3G6 bind to purified GPIb beta 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 GPIb alpha. 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 GPIb beta, strongly suggest that changes in the conformation of GPIb beta underlie outside-in activation via GPIb-IX.

Automated summary

The study identified a novel rat monoclonal antibody 3G6 that acts as a positive modulator of GPIb beta, enhancing GPIb-IX binding and activation. In contrast, another antibody RAM.1 inhibits GPIb-IX signaling, suggesting that changes in GPIb beta conformation may be crucial for outside-in activation via GPIb-IX.