Dynamic force spectroscopy of glycoprotein Ib-IX and von Willebrand factor.
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 platelet-type von Willebrand disease, which results from gain-of-function (GOF) mutations in amino acid residues 233 or 239 of the GP Ibalpha 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 Ibalpha 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 Ibalpha 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(off)(0)) values for interactions involving mutant and wild-type GP Ib-IX receptors with A1 and found the A1/wild-type GP Ib-IX k(off)(0) value of 5.47 +/- 0.25 s(-1) to be significantly greater than the GOF k(off)(0) values and significantly less than the loss-of-function k(off)(0) 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 Ibalpha is altered.
Arya
Maneesh
M
Department of Bioengineering, Rice University, Houston, TX 77251, USA.
Kolomeisky
Anatoly B
AB
Romo
Gabriel M
GM
Cruz
Miguel A
MA
López
José A
JA
Anvari
Bahman
B
D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D016466:CHO Cells; D006224:Cricetinae; D006487:Hemostasis / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D055095:Optics and Photonics / Q000295:instrumentation*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis / Q000295:instrumentation*; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D046912:Multiprotein Complexes; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor