The Von Willebrand Factor D'D3 Domains Modulate the Interacton of Platelet Receptor Glycoprotein Ib with the VWF-A1 Domain Under Conditions of Shear Stress

Abstract 2212

The VWF A1 domain contains the binding site for the platelet receptor glycoprotein (GP)Ib receptor, and this interaction forms the initial tethering event to capture platelets at sites of injury under high shear stress. The VWF-A1:GPIbα interaction has a high association and dissociation rate and as such platelets translocate along the VWF surface until stable attachment can occur. It has been previously shown that under static conditions the VWF D'D3 domains shield the A1 from interaction with GPIbα, however it has not been established if this occurs under conditions of shear stress. To this end we constructed VWF fragments spanning the D'-A3 domains and the A1A2A3 domains. These were expressed and purified and the ability of these constructs to interact with platelets was analysed in an in vitro flow assays system. Purified and fluorescently labelled platelets resuspended in plasma free blood were perfused over immobilised VWF at a range of shear rates (400–2000s⁻¹), and real time movies of the interactions captured. Interestingly, platelets translocated slower across VWF-A1A2A3, with lower rolling velocities compared to VWF-D'A3 at all shear rates tested. Significantly the dissociation rate constant (k_off) of the transient tethers formed between GPIbα and VWF was similar for both constructs, indicating that that the observed difference in platelet rolling velocity is not due to an alteration in bond lifetime. Frame-by-frame analysis of platelet translocation demonstrated that platelets paused more often on VWF lacking the D'D3 domains and therefore translocated a shorter distance in a set time when compared to VWF D'A3 resulting in decreased rolling velocities. We hypothesise that the accessibility of the GPIb binding site in the A1 domain is increased in the absence of the D'D3 domains, resulting in more interactions. Together these data demonstrate the D'D3 domains are able to shield the A1 domain under shear stress conditions.