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.