Role of 14-3-3ζ in platelet glycoprotein Ibα-von Willebrand factor interaction-induced signaling

Abstract

The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture initiates platelet transient adhesion to the injured vessel wall, which triggers intracellular signaling cascades leading to platelet activation and thrombus formation. 14-3-3ζ has been verified to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX. However, the data regarding the role of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling still remain controversial. In the present study, the data indicate that the S609A mutation replacing Ser(609) of GPIbα with alanine (S609A) significantly prevented the association of 14-3-3ζ with GPIbα before and after the VWF binding to GPIbα. GPIb-IX-VWF interaction-induced activations of Src family kinases and protein kinase C were clearly reduced in S609A mutation. Furthermore, S609A mutation significantly inhibited GPIb-IX-VWF interaction-induced elevation of cytoplasmic Ca(2+) levels in flow cytometry analysis. Taken together, these data indicate that the association of 14-3-3ζ with the cytoplasmic domain of GPIbα plays an important role in GPIb-IX-VWF interaction-induced signaling.

Keywords: 14-3-3ζ; glycoprotein (GP) Ib-IX; platelets; von Willebrand factor (VWF).

Figure 1

The S609A mutation disrupts the interaction of 14-3-3ζ with GPIbα before and after von Willebrand factor (VWF) binding. (a) 1b9 or S609A cells were stimulated by ristocetin in the presence or absence of VWF and solubilized in lysis buffer. The lysates were incubated with SZ2, and then precipitated with protein G beads. The precipitates were subjected to Western blot with SZ2 and anti-14-3-3ζ antibody, respectively. The immunoblot is representative of 3 independent experiments; (b) Quantitative data from 3 independent experiments (mean ± SD) are shown. The relative binding index of 14-3-3ζ equals arbitrary quantitation of 14-3-3ζ/GPIbα of treated cells divided by 14-3-3ζ/GPIbα of 1b9 cells stimulated by ristocetin in the absence of VWF.

Figure 2

The S609A mutation inhibits activation of Src family kinases. (a) 1b9 or S609A cells were stimulated by ristocetin in the presence or absence of VWF, then solubilized and subjected to Western blot analysis with anti-Src and anti-phospho-Src family (pTyr416) antibody, respectively. The immunoblot is representative of 3 different experiments; (b) Quantitative data from 3 different experiments (mean ± SD) are demonstrated. The relative activation index of Src equals arbitrary quantitation of phospho-Src (pTyr416)/arbitrary quantitation of total Src.

Figure 3

The S609A mutation blocks PKC activation. (a) 1b9 or S609A cells were stimulated by ristocetin in the presence or absence of VWF, and then were subjected to Western blot analysis with anti-PKC and anti-phospho-PKC (pSer660) antibody. Actin levels demonstrate similar loading. The immunoblot is representative of 3 different experiments; (b) Quantitative data from 3 independent experiments (mean ± SD) are shown. The relative activation index of PKC equals arbitrary quantitation of phospho-PKC (pSer660)/arbitrary quantitation of total PKC.

Figure 4

The S609A mutation inhibits elevation of intracellular Ca²⁺. (a, b) 1b9 or S609A cells were incubated with 8 μM Fluo-3/AM for 30 min at 37 °C in the dark. The external Ca²⁺ was adjusted to 1 mM, and then cells were stimulated by ristocetin in the presence or absence of VWF for 10 min at RT, and analyzed by flow cytometry. The Fluo-3/AM-loaded cells were also pre-treated with BAPTA-AM at 37 °C for 20 min before ristocetin/VWF treatment, and then intracellular Ca²⁺ levels were measured by flow cytometry. Representative histograms of Fluo3-fluorescence of cells are shown (a). The geometric mean fluorescence intensity of Fluo-3/AM binding is demonstrated (mean ± SD) (n = 3) (b).