Conserved residues in the GPIbα mechanosensory domain are important for its expression and signaling

Abstract

Abstract Background: Glycoprotein Ib-IX (GPIb-IX), the primary receptor for von Willebrand factor (VWF) on platelets, mediates initial platelet adhesion and activation at vascular injury sites. Within this complex, the mechanosensory domain (MSD) of GPIbα is essential for force-dependent VWF binding and downstream signal transduction. But the contribution of conserved amino acid residues within it to platelet function is unknown. Methods: Each of the residues in the MSD conserved between human and mice was mutated to alanine (Ala) in murine GPIbα. These were stably expressed along with wild-type GPIbβ and GPIX in CHO cell lines. Surface expression of GPIbα was quantified by flow cytometry and western blot, and GPIb-IX complex signaling was assessed via botrocetin-dependent filopodia formation. Subsequently, these conserved residues were mutated to Ala in the human GPIbα to verify their functional conservation. Results: Among 16 stably transfected CHO lines expressing WT or mutant murine GPIbα (15 alanine substitutions), two mutations significantly decreased the expression of GPIbα, two increased the expression, whereas the others little impacted. In addition, five mutations enhanced filopodia formation; Notably, two mutations concurrently elevated both surface expression and filopodia formation. These seven mutations were introduced in the human GPIba gene, and corresponding stable cell lines were constructed. The most notable aspect is that point mutations in the corresponding human genes reproduced these functional characteristics, confirming the conserved role of MSD between species and the conservation of important sites during evolution. Conclusion: This study demonstrates that specific, evolutionarily conserved amino acid residues in MSD are vital nodes regulating receptor expression and signal transduction. Furthermore, the functional conservation observed between mouse and human homologous sequences underlines the importance of the MSD region of GPIbα across species.