14-3-3ζ has been confirmed to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX [12]. However, the role of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling still remains controversial. We had established two CHO cell lines expressing wild type GPIb-IX (1b9) and mutant GPIb-IX replacing Ser⁶⁰⁹ of GPIbα with alanine (S609A) [12]. In order to investigate the role of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling, firstly, the VWF binding functions of 1b9 and S609A were assessed by flow cytometry. Consistent with the previous report [12], a certain level of VWF binding to S609A or 1b9 was detected in the present study, and there was no statistical difference in the VWF binding function between 1b9 and S609A cells (Figure S1). Then, the associations of 14-3-3ζ with GPIbα were examined in 1b9 and S609A cells by coimmunoprecipitation analysis before and after VWF binding. GPIb-IX-expressing CHO cells were firstly stimulated by ristocetin, which can induce the association of VWF with GPIbα in the presence or absence of VWF, and then were solubilized in cell lysis buffer. The lysates were immunoprecipitated with SZ2 and protein G-conjugated sepharose 4B beads, and then analyzed by SDS-PAGE and Western blot with an antibody SZ2 that specifically recognizes GPIbα and anti-14-3-3ζ antibody, respectively. As demonstrated in Figure 1, the association of 14-3-3ζ with GPIbα was obviously reduced in S609A cells both before and after VWF binding.

The interaction of GPIbα with VWF triggers activation of mutiple protein kinases [2], such as one or more Src family kinases [18,19]. In addition, phosphorylation of tyrosine 416 (pTyr416) in the activation loop of Src upregulates its enzymatic activity. Thus, to investigate the role of 14-3-3ζ in GPIbα-VWF interaction-induced signaling, the activation of Src family kinases was analyzed. 1b9 and S609A were stimulated by ristocetin in the presence or absence of VWF, and then were lysed and subjected to SDS-PAGE and Western blot analysis with anti-Src and anti-phospho-Src family (pTyr416), respectively. The results showed that the S609A mutation resulted in inhibited activation of Src family kinases (Figure 2).

The interaction of GPIbα with VWF induces activation of PKC [2], which is represented as serine 660-phosphorylated PKC (pSer660). To investigate whether 14-3-3ζ is involved in activation of PKC elicited by the interaction of GPIb-IX with VWF, S609A and 1b9 cells were stimulated with ristocetin in the presence or absence of VWF, and then were subjected to PKC activation analysis. As shown in Figure 3, the S609A mutation significantly inhibited activation of PKC, indicating that the association of 14-3-3ζ with GPIbα is indispensable for GPIb-IX-VWF interaction-induced PKC activation.

The interaction of GPIb-IX with VWF triggers elevation of cytoplasmic Ca²⁺ concentrations [5,20]. Thus, the roles of 14-3-3ζ in GPIb-IX-dependent elevation of the intracellular Ca²⁺ level were investigated. As shown in Figure 4, the intracellular Ca²⁺ was significantly reduced by a membrane-permeable Ca²⁺ chelator BAPTA-AM in ristocetin-induced GPIb-IX-expressing cells, indicating that the rise in intracellular Ca²⁺ is not an artifact. Compared with wild type GPIb-IX, the S609A mutation dramatically reduced the elevation of the cytoplasmic Ca²⁺ levels in CHO cells.

Monoclonal antibodies SZ29 against VWF [23] and SZ2 against GPIbα [24] were described previously. Purified human VWF and botrocetin were generous gifts from Xiaoping Du (University of Illinois, Chicago, IL, USA). Ristocetin and aprotinin were purchased from Sigma (St. Louis, MO, USA). Non-essential amino acids, penicillin and streptomycin, l-glutamine, l-trans-Epoxysuccinyl-leucylamido (4-guanidino) butane (E64) were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). Fluo-3/AM was purchased from Invitrogen Molecular Probes (Eugene, OR, USA). 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Goat anti-mouse immunoglobulin (IgG) conjugated with horseradish peroxidase (GAM-HRP), goat anti-rabbit immunoglobulin (IgG) conjugated with horseradish peroxidase (GAR-HRP), and FITC (fluorescein isothiocyanate)-conjugated goat anti-mouse IgG (FITC-GAM) were purchased from Biosource (Camarillo, CA, USA). Anti-phospho-Src family (pTyr416) rabbit polyclonal antibody was from Cell Signaling Technology (Beverly, MA, USA). Anti-Src mouse monoclonal antibody was from Upstate Biotechnology (Lake Placid, NY, USA). Anti-PKC mouse monoclonal antibody sc-17804 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-PKC (pSer660) rabbit polyclonal antibody was from BioVision (Mountain View, CA, USA (CATALOG#: 3451-100)).

CHO cells expressing recombinant wild-type GPIb-IX (1b9), GPIb-IX mutants (S609A) with a serine-to-alanine point mutation at Ser⁶⁰⁹ in GPIbα have been described previously [12].

1b9 or S609A cells (2 × 10⁶/mL) were stimulated by ristocetin (1.25 mg/mL) in the presence or absence of VWF (35 μg/mL) for 30 min at room temperature (RT). Pre-treated GPIb-IX-expressing cells were subjected to VWF binding analysis as described previously [12–14].

For GPIb-IX and 14-3-3ζ association assay, GPIb-IX-expressing CHO cells including 1b9 and S609A (2 × 10⁶/mL) were firstly stimulated by ristocetin (1.25 mg/mL) in the presence or absence of VWF (35 μg/mL) for 30 min at RT, and then were solubilized in an equal volume of 2× cell lysis buffer (2% Triton X-100, 0.1 M Tris, 0.01 M EGTA, and 0.15 M NaCl, 1 mM dithiothreitol, pH 7.4) containing 0.1 mM E64 and 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were immunoprecipitated with SZ2 and protein G-conjugated sepharose 4B beads, and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and immunoblotted with SZ2 and anti-14-3-3ζ antibody, respectively [12,14].

For Src and PKC analysis, 1b9 or S609A cells (2 × 10⁶/mL) were stimulated by ristocetin (1.25 mg/mL) in the presence or absence of VWF (35 μg/mL) for 30 min at RT. Pre-treated GPIb-IX-expressing cells were solubilized in the same cell lysis buffer and subjected to Western blot analysis under reducing conditions with anti-Src, anti-phospho-Src family (pTyr416), anti-PKC and anti-phospho-PKC (pSer660), respectively.

Intracellular Ca²⁺ concentrations were detected with the Ca²⁺-sensitive fluorochrome Fluo-3/acetoxymethyl ester (Fluo-3/AM) by flow cytometric analysis [25]. Briefly, GPIb-IX-expressing cells were incubated with 8 μM Fluo-3/AM for 30 min at 37 °C in the dark. After washing once, cells were resuspended at a concentration of 2 × 10⁶/mL. The external Ca²⁺ was adjusted to 1 mM, and then GPIb-IX-expressing cells were stimulated by ristocetin (1.25 mg/mL) in the presence or absence of VWF (35 μg/mL) for 30 min at RT, and analyzed by flow cytometry. In some experiments, Fluo-3/AM-loaded GPIb-IX-expressing cells were pre-treated with BAPTA-AM (10 μM) at 37 °C for 20 min before ristocetin/VWF treatment, and then intracellular Ca²⁺ levels were measured by flow cytometry.