title: Shear-dependent rolling on von Willebrand factor of mammalian cells expressing the platelet glycoprotein Ib-IX-V complex.
abstract: Mural thrombi form on exposed arterial subendothelium by a two-step process of platelet adhesion and aggregation. At high shear stresses such as are found in stenotic arteries, both steps are mediated by von Willebrand factor (vWF). Platelets initially adhere on vWF affixed to the subendothelial matrix through the glycoprotein (GP) Ib-IX-V complex. To examine the role of the GP Ib-IX-V complex under dynamic conditions, we modeled initial platelet adhesion at shear stresses ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Mammalian cells expressing the full complex tethered and rolled on the vWF substrate, whereas control cells did not. The rolling was completely inhibited by the monoclonal GP Ib antibody, AK2, or the vWF antibody, 5D2, both shown previously to block vWF-dependent platelet aggregation. Other GP Ib antibodies, WM23 and SZ2, did not significantly change the number or mean velocity of rolling cells. At low levels of GP Ib surface expression, cells expressing the full complex rolled slower than cells expressing the complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells into the chamber did not significantly change the number or the mean velocity of rolling cells. Inhibiting sulfation of the tyrosine residues within the GP Ib subunit reduced the number but did not change the mean velocity of the rolling cells. Our results indicate that, under the conditions of these experiments, bonds between vWF and GP Ib constantly form and break under fluid shear stress. Additionally, our results suggest that GP Ib-IX-V complexes behave like selectin receptors in their ability to mediate smooth rolling while cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood vessel wall. The system described provides a valuable approach for investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.
authors.affiliation: Cox Laboratory for Biomedical Engineering, Rice University, Houston, TX, USA.
authors.forename: B J
authors.initials: BJ
authors.lastname: Fredrickson
authors.forename: J F
authors.initials: JF
authors.lastname: Dong
authors.forename: L V
authors.initials: LV
authors.lastname: McIntire
authors.forename: J A
authors.initials: JA
authors.lastname: López
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016466:CHO Cells; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000166:cytology; D005434:Flow Cytometry; D018056:Hemorheology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007739:L Cells; D046911:Macromolecular Substances; D008322:Mammals; D051379:Mice; D010973:Platelet Adhesiveness / Q000502:physiology*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013314:Stress, Mechanical; D013329:Structure-Activity Relationship; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000502:physiology; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; C005662:tyrosine O-sulfate; D014443:Tyrosine