Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibα regulate shear-dependent cell adhesion to von Willebrand factor

Abstract

Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbα (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbα expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4(Leu60-Glu 128)were crucial for GPIbα-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbα N-terminal domain and GPIbα-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbα with human GPIbα containing a homology domain swap of canine for human sequence within Leu 60-Glu128 and a reverse swap (canine GPIbα with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbα antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbα, cells expressing GPIbα, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s-1. Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbα, is essential for GPIbα·VWF-dependent adhesion as hydrodynamic shear increases. Show more