Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibalpha 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 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.

• Authors: Yang Shen, Susan L Cranmer, Andrea Aprico, James C Whisstock, Shaun P Jackson, Michael C Berndt, Robert K Andrews
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 281 Issue 36 Pg. 26419-23 (Sep 08 2006) ISSN: 0021-9258 [Print] United States
• PMID: 16774914 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Crotalid Venoms
• Hemagglutinins
• Leucine-Rich Repeat Proteins
• Membrane Glycoproteins
• Membrane Proteins
• Platelet Glycoprotein GPIb-IX Complex
• Proteins
• Recombinant Fusion Proteins
• adhesion receptor
• von Willebrand Factor
• Glutamic Acid
• botrocetin
• Leucine

Topics

• Animals
• CHO Cells
• Cell Adhesion (physiology)
• Cricetinae
• Crotalid Venoms (metabolism)
• Dogs
• Glutamic Acid (metabolism)
• Hemagglutinins (metabolism)
• Humans
• Leucine (metabolism)
• Leucine-Rich Repeat Proteins
• Membrane Glycoproteins
• Membrane Proteins (chemistry, genetics, metabolism)
• Models, Molecular
• Platelet Glycoprotein GPIb-IX Complex
• Protein Conformation
• Proteins (chemistry, genetics, metabolism)
• Recombinant Fusion Proteins (genetics, metabolism)
• Repetitive Sequences, Amino Acid
• Shear Strength
• von Willebrand Factor (metabolism)