Glycoprotein Ib-IX-mediated activation of integrin α iibβ 3:effects of receptor clustering and von Willebrand factor adhesion

Abstract

The interaction between the platelet glycoprotein (GP) Ib-IX complex and vonWillebrand factor (VWF) initiates both hemostasis and pathological thrombosis. This interaction is not only the first adhesive event of platelets at sites of vessel injury, but also facilitates fibrinogen binding to α IIbβ 3, which subsequently results in platelet aggregation. Since it has been suggested that GP Ib-IX clustering may promote platelet activation, we investigated the effect of such clustering on both VWF-GP Ib-IX and fibrinogen-α IIbβ 3 bonds using optical tweezers. In our system, fusion of tandem repeats of FK506-binding protein (FKBP) to the cytoplasmic tail of the GP IX subunit of the GP Ib-IX complex allowed subsequent receptor clustering within the plasma membrane by the bivalent, cellpermeant small molecule ligand AP20187. We measured binding forces between polystyrene beads coated with either plasma-derived VWF or the VWF A1 domain and GP Ib- IX(FKBP) 2, and those between fibrinogen-coated beads and α IIbβ 3 expressed on Chinese hamster ovary cells. The minimal detachment force between GP Ib-IX(FKBP) 2 and A1 or plasma-derived VWF doubled after AP20187 was added. The binding force between immobilized fibrinogen and α IIbβ 3 was not changed by the clustering agent; however, the strength of single fibrinogen-α IIbβ 3 bonds increased significantly after ligation of GP Ib-IX(FKBP) 2 by A1. These results demonstrate that GP Ib-IX clustering increases the overall strength of its interaction with VWF. Furthermore, signals from GP Ib-IX can activate α IIbβ 3, thereby increasing the strength of its interaction with fibrinogen. © 2003 International Society on Thrombosis and Haemostasis.