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.
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.
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