Studies of platelet glycoprotein Ib-IX-V adhesion tovon Willebrand factor using optical tweezers
Doctor of Philosophy
The first stage in hemostasis and thrombosis is the binding of the platelet membrane receptor, glycoprotein (GP) Ib-IX, to its ligand, von Willebrand factor (VWF), in the subendothelium. Using an optical tweezers system, we have measured the binding strength of: (1) VWF variants (unusually large VWF (ULVWF), plasma VWF, and isolated VWF A1 domain) to GP Ib-IX receptors; (2) GP Ib-IX variants (receptors with human-canine chimeric sequences in the leucine-rich domain, and truncated receptors) to plasma VWF; (3) plasma VWF to gain-of-function (GOF) and loss-of-function (LOF) mutated GP Ib-IX receptors; (4) VWF Al domain to clustered GP Ib-IX receptors; and (5) ULVWF multimers to GP Ib-IX in the presence of the VWF cleaving protease (ADAMTS-13) and other modulators. In addition, we dynamically measured unbinding force profiles between A1 and GP Ib-IX at loading rates ranging from 200--20,000 pN/s and examined the relationship between bond rupture force and loading rate. In the absence of shear stress, ULVWF multimers formed spontaneous high-strength bonds with GP Ib-IX, while plasma VWF required exogenous modulators. The strength of individual bonds formed with GP Ib-IX was similar for both ULVWF and the isolated A1-domain and greater than those of plasma VWF induced by an exogenous modulator. We observed ULVWF/GP Ib-IX binding was completely abolished with the addition of either ADAMTS-13 or plasma cryosupernatant. We found that the putative single bond strengths between A1 and GP Ibalpha GOF mutants were significantly greater than the A1/wild-type GP Ib-IX bond at all loading rates examined. In addition, the GP Ibalpha LOF mutants exhibited significantly lower putative single bond strengths with A1 than the wild-type receptors. The experiments involving the adhesion of either chimeric or truncated GP Ib-IX receptors to VWF illustrated the importance of various regions of the glycoprotein in properly binding VWF. The minimal detachment force between GP Ib-IX and A1 or plasma VWF doubled after an exogenous clustering agent was added, thereby demonstrating the importance of avidity, as opposed to affinity, modulation. Finally, we observed a linear relationship between the rupture force and the logarithm of the loading rate, consistent with the Bell Model.
Biomedical engineering; Biophysics