Role of 14-3-3ζ in Platelet Glycoprotein Ibα-von Willebrand Factor Interaction-Induced Signaling

Weilin Zhang,Juan Du,Rong Yan,Lili Zhao,Jun Liu,Kesheng Dai

doi:10.3390/ijms13055364

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 Ser609 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 Ca2+ 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.

Highlights

The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet transient adhesion [1,2,3], and simultaneously triggers intracellular signaling cascades [4], such as activation of multiple protein kinases, elevation of intracellular Ca2+ levels, and phosphatidylserine (PS) exposure, leading to integrin αIIbβ3 activation and integrin-dependent platelet stable adhesion and thrombus formation [5]
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 Ser609 of GPIbα with alanine (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)

Summary

Introduction

The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet transient adhesion [1,2,3], and simultaneously triggers intracellular signaling cascades [4], such as activation of multiple protein kinases, elevation of intracellular Ca2+ levels, and phosphatidylserine (PS) exposure, leading to integrin αIIbβ activation and integrin-dependent platelet stable adhesion and thrombus formation [5]. Several typical events have been confirmed to play key roles in GPIbα-VWF interaction-induced platelet signaling, the molecule that initiates the GPIbα-VWF interaction-induced signaling leading to platelet activation remains unknown. Several intracellular molecules that have been confirmed to interact with the cytoplasmic domain of the GPIb-IX complex are involved in platelet activation. Calmodulin binds directly to the juxtamembrane cytoplasmic sequences of GPIbβ and GPV in resting platelets, but it dissociates from GPIb-IX when platelets are activated [10]. Phosphoinositide 3-kinase (PI3-kinase) interacts with the cytoplasmic domain of GPIbα and is associated with GPIb-IX-mediated platelet functions [11]. The interaction of 14-3-3ζ with the cytoplasmic domains of GPIb-IX plays a key role in the VWF binding function of GPIb-IX and subsequent platelet activation [12,13,14]. It has been reported that deletion of the 14-3-3ζ binding site in the C-terminal cytoplasmic domain of GPIbα inhibited

Methods

Results

Conclusion