Cytoplasmic Truncation of Glycoprotein Ibα Weakens Its Interaction with von Willebrand Factor and Impairs Cell Adhesion

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Abstract
The interaction of the platelet glycoprotein (GP) Ib−IX−V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib−IX−V complex, the recognition of VWF appears to be mediated entirely by GP Ibα, the largest of four GP Ib−IX−V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibα in the GP Ib−IX−VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibβ, GP IX, and either wild-type GP Ibα or GP Ibα mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibα polypeptides with truncations of 6−82 amino acids rolled faster than cells expressing wild-type GP Ibα. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibα with longer truncations. The rolling velocity of cells expressing truncated GP Ibα mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibα−VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibα−VWF bonds.