Maneesh Arya

Glycoprotein Ib–IX-mediated activation of integrin αIIbβ3: effects of receptor clustering and von Willebrand factor adhesion

Summary.  The interaction between the platelet glycoprotein (GP) Ib–IX complex and von Willebrand 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 αIIbβ3, 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–αIIbβ3 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, cell‐permeant 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 αIIbβ3 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 αIIbβ3 was not changed by the clustering agent; however, the strength of single fibrinogen–αIIbβ3 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 αIIbβ3, thereby increasing the strength of its interaction with fibrinogen.

Glycoprotein Ib-IX-mediated activation of integrin αIIbβ3: effects of receptor clustering and von Willebrand factor adhesion: Receptor clustering and VWF adhesion

Summary.  The interaction between the platelet glycoprotein (GP) Ib–IX complex and von Willebrand 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 αIIbβ3, 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–αIIbβ3 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, cell‐permeant 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 αIIbβ3 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 αIIbβ3 was not changed by the clustering agent; however, the strength of single fibrinogen–αIIbβ3 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 αIIbβ3, thereby increasing the strength of its interaction with fibrinogen.

Glycoprotein Ib-IX-mediated activation of integrin α iibβ 3

Summary.  The interaction between the platelet glycoprotein (GP) Ib–IX complex and von Willebrand 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 αIIbβ3, 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–αIIbβ3 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, cell‐permeant 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 αIIbβ3 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 αIIbβ3 was not changed by the clustering agent; however, the strength of single fibrinogen–αIIbβ3 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 αIIbβ3, thereby increasing the strength of its interaction with fibrinogen.

Dynamic Measurements of Forces between Thrombus-inducing Proteins Using Optical Tweezers

Both hemostasis and thrombosis occur as a result of platelet adhesion to the subendothelial matrix, platelet activation, and platelet aggregation. The first stage in hemostasis and thrombosis is the binding of the platelet membrane receptor, glycoprotein (GP) Ib-IX complex, to its ligand, von Willebrand factor (VWF), in the subendothelium. In particular, the A1 domain of VWF is responsible for binding GP Ib-IX. After immobilizing A1 on a 2.0 μm diameter polystyrene bead, we optically trapped the bead using a titanium-sapphire laser tuned to 830 nm. The A1-coated bead was then moved towards a transfected Chinese hamster ovary cell which expressed the GP Ib-IX complex, and allowed to adhere to the cell. We subsequently detached the cell from the bead at different constant loading rates, ranging over three orders of magnitude, by using a piezoelectrically-driven translational stage. Displacement of the bead was simultaneously monitored from the trapping center using a quadrant photodetector to determine the force required to detach A1 from GP Ib-IX. These dynamic measurements of unbinding force emphasize the important role that shear rate plays in the initial stage of thrombus formation.

Measurement of the bond strength between VWF A1 domain and clustered platelet glycoprotein Ib-IX using optical tweezers

Adhesion between the A1 domain of the plasma protein von Willebrand factor (VWF) and the platelet glycoprotein (GP) Ib-IX complex is the first stage in thrombus formation and ultimately leads to firm platelet adhesion. Studies have suggested that GP Ib-IX receptor clustering may modulate platelet adhesion and signaling. Therefore, we have investigated the importance of GP Ib-IX clustering in the bond strength between Al and GP Ib-IX. After immobilizing A1 on polystyrene beads, we optically trapped the bead using a laser tuned to 830 nm. The bead was then allowed to adhere for 10 seconds to a transfected Chinese hamster ovary cell which expressed GP Ib-IX receptors that could be conditionally clustered with a small molecule dimerizer. We obtained the force to detach A1 from GP Ib-IX by determining the minimum laser power required to separate the bead from the cell. When pulling the bound bead away from the cell, we found that the lowest strength between GP Ib-IX and A1 effectively doubled when the dimerizer was added. These results indicate that GP Ib-IX clustering can increase the bond strength between the receptor and its cognate ligand, suggesting a role for receptor clustering in thrombus formation.

Investigating intermolecular forces associated with thrombus initiation using optical tweezers

Thrombus formation occurs when a platelet membrane receptor, glycoprotein (GP) Ib-IX-V complex, binds to its ligand, von Willebrand factor (vWf), in the subendothelium or plasma. To determine which GP Ib-IX-V amino acid sequences are critical for bond formation, we have used optical tweezers to measure forces involved in the binding of vWf to GP Ib-IX-V variants. Inasmuch as GP Ib(alpha) subunit is the primary component in human GP Ib-IX-V complex that binds to vWf, and that canine GP Ib(alpha) , on the other hand, does not bind to human vWf, we progressively replaced human GP Ib(alpha) amino acid sequences with canine GP Ib(alpha) sequences to determine the sequences essential for vWf/GP Ib(alpha) binding. After measuring the adhesive forces between optically trapped, vWf-coated beads and GP Ib(alpha) variants expressed on mammalian cells, we determined that leucine- rich repeat 2 of GP Ib(alpha) was necessary for vWf/GP Ib-IX- V bond formation. We also found that deletion of the N- terminal flanking sequence and leucine-rich repeat 1 reduced adhesion strength to vWf but did not abolish binding. While divalent cations are known to influence binding of vWf, addition of 1mM CaCl2 had no effect on measured vWf/GP Ib(alpha) bond strengths.