1. by Kesheng Dai, Richard Bodnar, Michael C. Berndt, and Xiaoping Du
A critical role for ζ protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications
by Kesheng Dai, Richard Bodnar, Michael C. Berndt, and Xiaoping Du
Blood
Volume 106(6):
September 15, 2005
©2005 by American Society of Hematology

2. Disruption of 14-3-3ζ binding to GPIb-IX by mutagenesis and a novel inhibitor.
Disruption of ζ binding to GPIb-IX by mutagenesis and a novel inhibitor. (A) Schematic depicting GPIb-IX and the reported ζ binding sites (black boxes). Also shown is the GPIbα ζ binding sequence from which a novel membrane-permeable inhibitor of ζ-GPIbα interaction, MPαC, is derived. (B) CHO cells expressing wild-type, GPIb-IX (1b9), or GPIb-IX mutants bearing ζ binding disrupting mutations, S609A (GPIbα Ser609 to alanine mutation), S166A (GPIbβ Ser166 to alanine mutation), and S609A/S166A double mutations, were solubilized in Triton X-100 containing lysis buffer and immunoprecipitated with the anti-GPIbα antibody LJ-P3 (P3), or control IgG. The immunoprecipitates were immunoblotted with anti-GPIbα and anti ζ antibodies. (C) Platelets were preincubated with MPαC, control peptides (MαC, MPαCsc, or MαCsc), or DMSO. Platelets were then solubilized and immunoprecipitated with an anti-GPIbα antibody (P3). Immunoprecipitates were immunoblotted with anti-GPIbα and anti ζ antibodies.
Kesheng Dai et al. Blood 2005;106:
©2005 by American Society of Hematology

3. The effect of an inhibitor of ζ-GPIb-IX interaction, MPαC, on ristocetin-induced platelet aggregation.
The effect of an inhibitor of ζ-GPIb-IX interaction, MPαC, on ristocetin-induced platelet aggregation. (A) PRP was preincubated with DMSO as control or with increasing concentrations of the myristoylated ζ inhibitor peptide for 5 minutes, and stimulated with ristocetin to induce platelet aggregation. (B) Ristocetin-induced platelet aggregation in the presence of 100 μM myristoylated ζ inhibitor peptides; MPαC; control peptides MαC, MPαCsc, or MαCsc; or DMSO. (C) Ristocetin-induced platelet aggregation in the presence of nonmyristoylated peptide, PαC (phosphorylated) or αC (nonphosphorylated) with sequence identical to MPαC, or myristic anhydride (MA).
Kesheng Dai et al. Blood 2005;106:
©2005 by American Society of Hematology

4. MPαC specifically inhibits VWF- and GPIb-IX-dependent platelet agglutination.
MPαC specifically inhibits VWF- and GPIb-IX-dependent platelet agglutination. (A,B) PRP was preincubated with myristoylated peptides MPαC, MαC, MPαCsc, or MαCsc, or vehicle (DMSO) together with 1 mM of the integrin inhibitor RGDS. Ristocetin (1.25 mg/mL) was added to induce GPIb-IX-specific platelet agglutination. Typical agglutination traces are shown in panel A, and quantitative data from 4 experiments are shown in panel B (mean ± SD). (C-E) PRP was preincubated with MPαC; MαC or MαCsc; or DMSO, then stimulated with ADP (C), collagen (D), or the thromboxane A2 analog, U46619 (E) to induce platelet aggregation.
Kesheng Dai et al. Blood 2005;106:
©2005 by American Society of Hematology

5. Effects of MPαC on VWF binding to GPIb-IX-and VWF-mediated platelet adhesion.
Effects of MPαC on VWF binding to GPIb-IX-and VWF-mediated platelet adhesion. (A) Washed human platelets were preincubated with MPαC or control peptides MαC or MαCsc, or DMSO and incubated with ristocetin in the presence (VWF) or absence (control) of VWF. VWF binding was detected using FITC-labeled anti-VWF antibody and flow cytometry. (B) Quantitative data from 3 experiments (mean ± SD). VWF binding index equals total fluorescence/background fluorescence-1. (C) Platelets were preincubated with MPαC or control peptides MαC or MαCsc, or DMSO, and then perfused through VWF-coated capillary tubes. Numbers of adherent platelets were counted at 10 randomly selected time frames and locations (mean ± SD).
Kesheng Dai et al. Blood 2005;106:
©2005 by American Society of Hematology

6. The effect of disruption of the ζ binding sites in GPIbα and GPIbβ by mutagenesis on the VWF binding function.
The effect of disruption of the ζ binding sites in GPIbα and GPIbβ by mutagenesis on the VWF binding function. (A) CHO cells expressing wild-type GPIb-IX (1b9), S609A, S166A, or S166A/S609A mutants were incubated with human VWF in the presence of ristocetin or with ristocetin alone (controls). VWF binding to cells was analyzed by flow cytometry using FITC-labeled anti-VWF antibody, SZ29. The same cells were also analyzed for surface expression of GPIb-IX by flow cytometry using an anti-GPIbα antibody SZ2 followed by a FITC-labeled goat anti-mouse IgG. Results from a typical experiment are shown in panel A. Quantitative data are shown in panel B, in which fluorescence intensity of VWF binding (mean ± SD, 6 experiments) was corrected for the relative level of GPIb-IX expression. FL indicates fluorescence.
Kesheng Dai et al. Blood 2005;106:
©2005 by American Society of Hematology

7. The effects of disruption of ζ binding to GPIbα on cell adhesion to VWF and on PKA inhibitor-induced GPIb-IX activation.
The effects of disruption of ζ binding to GPIbα on cell adhesion to VWF and on PKA inhibitor-induced GPIb-IX activation. (A) 1b9, S166A, S166/609A, or S609A cell lines were perfused through VWF-coated capillaries at 150 s-1. Transient adhesion (rolling) of these cells was recorded. The number of rolling cells was counted in 30 randomly selected fields of 0.25 mm2 and at randomly selected time points. The results shown are the mean ± SD of cell number/mm2. (B,C) S166A cells (B) or S166A/S609A cells (C) were preincubated with MPαC; control peptides MαC, MPαCsc, MαCsc; or DMSO, and then perfused into VWF-coated glass capillaries. Numbers of adherent cells were counted at 10 randomly selected time frames and locations (mean ± SD). (D) CHO cells expressing wild-type GPIb-IX (1b9) and mutant GPIb-IX with deletion of the GPIbα C-terminal binding site (Δ591 and Δ605) were preincubated without or with 100 μM PKI for 15 minutes at 22°C. The cells were then incubated with VWF and ristocetin at 22°C for 30 minutes. VWF binding was detected using the FITC-labeled anti-VWF antibody, SZ29, and flow cytometry. Nonspecific fluorescence was determined by incubating the cells with ristocetin alone. Effects of PKI on enhancing VWF binding in the indicated cell lines were quantified and expressed as the percentage increase in the fluorescence intensity of VWF binding compared with the fluorescence intensity of VWF binding in the absence of PKI. Shown in the figure are the results from 3 separate experiments (mean ± SD).
Kesheng Dai et al. Blood 2005;106:
©2005 by American Society of Hematology

8. A new toggle switch model for 14-3-3ζ-dependent regulation of VWF binding function of GPIb-IX.
A new toggle switch model for ζ-dependent regulation of VWF binding function of GPIb-IX. Intracellular cAMP levels regulate VWF-dependent platelet adhesion by controlling the ζ binding states in a way similar to a toggle switch. Disruption of ζ interaction with GPIbα inhibits activation of VWF binding function of GPIb-IX and thus platelet adhesion. GP indicates glycoprotein.
Kesheng Dai et al. Blood 2005;106:
©2005 by American Society of Hematology