title: 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.affiliation: Department of Immunology and the Department of Biochemistry and Molecular Biology, Monash University, Victoria, 3800, Australia.
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.forename: Susan L
authors.initials: SL
authors.lastname: Cranmer
authors.forename: Andrea
authors.initials: A
authors.lastname: Aprico
authors.forename: James C
authors.initials: JC
authors.lastname: Whisstock
authors.forename: Shaun P
authors.initials: SP
authors.lastname: Jackson
authors.forename: Michael C
authors.initials: MC
authors.lastname: Berndt
authors.forename: Robert K
authors.initials: RK
authors.lastname: Andrews
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D006224:Cricetinae; D003435:Crotalid Venoms / Q000378:metabolism; D004285:Dogs; D018698:Glutamic Acid / Q000378:metabolism; D006388:Hemagglutinins / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D000090263:Leucine-Rich Repeat Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D019038:Platelet Glycoprotein GPIb-IX Complex; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D033081:Shear Strength; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D003435:Crotalid Venoms; D006388:Hemagglutinins; D000090263:Leucine-Rich Repeat Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D011506:Proteins; D011993:Recombinant Fusion Proteins; C094990:adhesion receptor; D014841:von Willebrand Factor; D018698:Glutamic Acid; C030961:botrocetin; D007930:Leucine