Dynamic Force Spectroscopy of Glycoprotein Ib-IX and von Willebrand Factor (original) (raw)

The first stage in hemostasis is the binding of the platelet membrane receptor glycoprotein (GP) Ib-IX complex to the A1 domain of von Willebrand factor in the subendothelium. A bleeding disorder associated with this interaction is platelettype von Willebrand disease, which results from gain-of-function (GOF) mutations in amino acid residues 233 or 239 of the GP Iba subunit of GP Ib-IX. Using optical tweezers and a quadrant photodetector, we investigated the binding of A1 to GOF and loss-of-function mutants of GP Iba with mutations in the region containing the two known naturally occurring mutations. By dynamically measuring unbinding force profiles at loading rates ranging from 200-20,000 pN/s, we found that the bond strengths between A1 and GP Iba GOF mutants (233, 235, 237, and 239) were significantly greater than the A1/wild-type GP Ib-IX bond at all loading rates examined (p , 0.05). In addition, mutants 231 and 232 exhibited significantly lower bond strengths with A1 than the wild-type receptors (p , 0.05). We computed unloaded dissociation rate constant (k 0 off ) values for interactions involving mutant and wild-type GP Ib-IX receptors with A1 and found the A1/wild-type GP Ib-IX k 0 off value of 5.47 6 0.25 s ÿ1 to be significantly greater than the GOF k 0 off values and significantly less than the loss-of-function k 0 off values. Our data illustrate the importance of the bond kinetics associated with the VWF/GP Ib-IX interaction in hemostasis and also demonstrate the drastic changes in binding that can occur when only a single amino acid of GP Iba is altered.