Elsevier

Thrombosis Research

Volume 132, Issue 6, December 2013, Pages 696-701
Thrombosis Research

Regular Article
The role of intraplatelet reactive oxygen species in the regulation of platelet glycoprotein Ibα ectodomain shedding

https://doi.org/10.1016/j.thromres.2013.09.034Get rights and content

Abstract

Glycoprotein (GP) Ibα ectodomain shedding has become a generally accepted negative regulatory mechanism of platelet function. Stimulation of platelet with either physiological or chemical compound results in GPIbα ectodomain shedding in vitro and in vivo, the mechanism, however, is not totally understood. Here we show, collagen, thrombin, and calcium ionophore A23187 induce reactive oxygen species (ROS) generation, and simultaneously incur GPIbα ectodomain shedding. ROS scavengers N-acetylcysteine (NAC) and dithiothreitol (DTT) abolish not only collagen, thrombin, and A23187 induced ROS production, but also GPIbα ectodomain shedding. Interestingly, a recognized calpain activator, dibucaine, induces both ROS production and GPIbα shedding, which are also obviously reduced by NAC and DTT. Furthermore, calpain inhibitors calpain inhibitor I and carbobenzoxy-valinyl-phenylalaninal, obviously reduce dibucaine, thrombin, and A23187-induced ROS generation. These data indicate that ROS plays a key role in collagen, thrombin, and A23187-induced GPIbα ectodomain shedding. Calpain is an up-stream regulator that regulates ROS-mediated GPIbα shedding.

Introduction

Glycoprotein (GP) Ibα contains binding sites for von Willebrand factor (VWF), α-thrombin, P-selectin, and Mac-1 at the extracellular N-terminal 282 residues [1], [2]. Particularly, the interaction of GPIbα with VWF exposed at the injured vessel wall initiates platelet adhesion, and simultaneously triggers intracellular signaling leading to integrin activation and platelet thrombus formation [1], [2], [3]. Therefore, GPIbα ectodomain shedding, which down-regulates the surface expression of the functional receptor and results in the generation of glycocalicin (GC), has important implications for thrombosis and hemostasis.

GPIbα ectodomain shedding has been reported to occur commonly in platelets stimulated by chemical or physiological agonists, such as α-thrombin, calcium ionophore A23187, calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), phorbol 12-myristate-13-acetate (PMA), or N-ethylmaleimide (NEM) [4], [5], [6], [7], [8]. We reported recently that the interaction of GPIbα with VWF, prolong inhibition of PKA, and hyperthermia also result in GPIbα ectodomain shedding [9]. Bergmeier W and coworkers have confirmed that a disintegrin and metalloproteinase 17 (ADAM17) is responsible for GPIbα ectodomain shedding [4]. In our previously study, calpain played key role in agonist-induced GPIbα shedding [10]. However, the regulatory mechanism of ADAM17-mediated GPIbα shedding is not totally understood.

Accumulating evidences have suggested the role of reactive oxygen species (ROS) in platelet reactivity in both physiologic and pathologic conditions. The generation of intraplatelet ROS was involved in the regulation of αIIbβ3 activation, granule secretion, and platelet shape change [11]. In lipopolysaccharide (LPS)-treated rats, ROS directly or by affecting the redox state of the animals, modulates both non-activated and thrombin-activated platelet adhesion to fibrinogen [12]. ROS was though to selectively regulate biochemical steps in platelet activation, and the distinct source(s) of ROS and discrete redox-sensitive pathway(s) may control platelet activation in response to GPVI or thrombin stimulation [13]. Particularly, incubation of washed murine platelets with H2O2 activated ADAM17 resulting in its target receptor shedding in platelets [14]. The generation of ROS has been observed in platelets stimulated with physiological or chemical compounds, such as collagen [15], thrombin [16], and thromboxane A2 analog U46619 [11]. However, the direct relation between physiological stimulation-induced ROS generation and GPIbα shedding still remains to be clarified.

In the current study, we demonstrate that ROS plays a key role in collagen, thrombin, and A23187-induced GPIbα ectodomain shedding. The data also indicate that calpain is an up-stream regulator of ROS generation, and regulates ROS-mediated GPIbα shedding.

Section snippets

Reagents

Monoclonal antibody SZ2 against GPIbα was generous gift from Prof. Changgeng Ruan (Soochow University, Suzhou, China). N-acetyl-L-cysteine (NAC), Dithiothreitol (DTT), ROS assay kit, 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG, HRP-conjugated goat anti-mouse IgG were purchased from Beyotime institute of Biotechnology (Beyotime, Haimen, China). Dibucaine, dimethyl sulfoxide (DMSO), calpain inhibitors I (CI I),

Collagen, Thrombin, and A23187 Induce ROS Generation and GPIbα Ectodomain Shedding

It has been reported previously that the generation of ROS could be induced in platelets stimulated with collagen and thrombin [17]. We have reported that thrombin induced platelet GPIbα ectodomain shedding [10]. In order to investigate the direct linkage between ROS generation and GPIbα ectodomain shedding, washed platelets were exposed to collagen, thrombin, and A23187, and then the generations of ROS were tested by flow cytometry. As shown in Fig. 1A and B, collagen, thrombin, and A23187

Discussion

GPIbα ectodomain shedding, leading to platelet dysfunction, has become one of the general accepted negative regulations of platelet function. However, the regulatory mechanism of GPIbα ectodomain shedding is not totally understood. In the current observation, the data show that ROS plays a key role in chemical or physiological agonists induced GPIbα shedding.

Although, in separate studies, it has been confirmed that physiological agonists collagen and thrombin induce GPIbα ectodomain shedding [4]

Conflict of Interest Statement

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (81130008 to K. D., 81200343 to R. Y.), National Key Basic Research Program of China (2012CB526600 to K. D.), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), 2012 Jiangsu Provincial Special Program of Medical Science (BL2012005), Jiangsu Province’s Key Medical Center (ZX201102), and Jiangsu Province’s Outstanding Medical Academic Leader Program (K. D.).

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