Key result
Cells expressing GPIbα with a canine sequence swap in Leu60-Glu128 became increasingly ineffective at supporting adhesion to VWF as shear increased from 50 to 2000 s-1 compared to wild-type.
The LRR2-4 region (Leu60-Glu128) of human GPIbalpha is essential for GPIbalpha-VWF-dependent adhesion under increasing hydrodynamic shear stress.
Pinpoints LRR2-4 for shear-dependent GPIbα-VWF adhesion; leaves open therapeutic targeting in human platelet disorders.
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 GPIbalpha (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 GPIbalpha expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbalpha-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbalpha N-terminal domain and GPIbalpha-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 GPIbalpha with human GPIbalpha containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbalpha with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbalpha 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 GPIbalpha, cells expressing GPIbalpha, 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 GPIbalpha, is essential for GPIbalpha.VWF-dependent adhesion as hydrodynamic shear increases.
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Shen et al. (2006) studied Platelet adhesion and thrombosis. Human GPIbα with canine sequence swap within Leu60-Glu128 vs. Wild-type human GPIbα was evaluated on Adhesion to von Willebrand factor under flow (shear rates from 50 to 2000 s-1). Cells expressing GPIbα with a canine sequence swap in Leu60-Glu128 became increasingly ineffective at supporting adhesion to VWF as shear increased from 50 to 2000 s-1 compared to wild-type.
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