title: Kinetics of GPIbalpha-vWF-A1 tether bond under flow: effect of GPIbalpha mutations on the association and dissociation rates.
abstract: The interaction between platelet glycoprotein (GP) Ib-IX-V complex and von Willebrand factor (vWF) is the first step of the hemostatic response to vessel injury. In platelet-type von Willebrand disease, two mutations, G233V and M239V, have been described within the Cys209-Cys248 disulfide loop of GPIbalpha that compromise hemostasis by increasing the affinity for vWF. We have earlier shown that converting other residues in this region to valine alters the affinity of GPIbalpha for vWF, with mutations K237V and Q232V, respectively, showing the greatest increase and decrease in affinity. Here, we investigated further the effect of these two mutations on the kinetics of the GPIbalpha interaction with the vWF-A1 domain under dynamic flow conditions. We measured the cellular on- and off-rate constants of Chinese hamster ovary cells expressing GPIb-IX complexes containing wild-type or mutant GPIbalpha interacting with vWF-A1-coated surfaces at different shear stresses. We found that the gain-of-function mutant, K237V, rolled very slowly and continuously on vWF-A1 surface while the loss-of-function mutant, Q232V, showed fast, saltatory movement compared to the wild-type (WT). The off-rate constants, calculated based on the analysis of lifetimes of transient tethers formed on surfaces coated with limiting densities of vWF-A1, revealed that the Q232V and K237V dissociated 1.25-fold faster and 2.2-fold slower than the WT. The cellular on-rate constant of WT, measured in terms of tethering frequency, was threefold more and threefold less than Q232V and K237V, respectively. Thus, the gain- and loss-of-function mutations in GPIbalpha affect both the association and dissociation kinetics of the GPIbalpha-vWF-A1 bond. These findings are in contrast to the functionally similar selectin bonds where some of the mutations have been reported to affect only the dissociation rate.
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas, USA.
authors.forename: R Anand
authors.initials: RA
authors.lastname: Kumar
authors.forename: Jing-fei
authors.initials: JF
authors.lastname: Dong
authors.forename: Jenny A
authors.initials: JA
authors.lastname: Thaggard
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Cruz
authors.forename: José A
authors.initials: JA
authors.lastname: López
authors.forename: Larry V
authors.initials: LV
authors.lastname: McIntire
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D016466:CHO Cells; D002455:Cell Division; D006224:Cricetinae; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004220:Disulfides; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; C032193:glycoprotein receptor GPIb-IX; D014841:von Willebrand Factor