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

    The role of intraplatelet reactive oxygen species in the regulation of platelet glycoprotein Ibα ectodomain shedding Pingping Zhang, Juan Du, Lili Zhao, Xiujuan Wang, Yiwen Zhang, Rong Yan, Jin Dai, Guanglei Liu, Feng Zhang, Kesheng Dai PII: DOI: Reference:

S0049-3848(13)00448-9 doi: 10.1016/j.thromres.2013.09.034 TR 5227

To appear in:

Thrombosis Research

Received date: Revised date: Accepted date:

26 June 2013 10 September 2013 24 September 2013

Please cite this article as: Zhang Pingping, Du Juan, Zhao Lili, Wang Xiujuan, Zhang Yiwen, Yan Rong, Dai Jin, Liu Guanglei, Zhang Feng, Dai Kesheng, The role of intraplatelet reactive oxygen species in the regulation of platelet glycoprotein Ibα ectodomain shedding, Thrombosis Research (2013), doi: 10.1016/j.thromres.2013.09.034

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Original Articles

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

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Pingping Zhang1,2*, Juan Du1*, Lili Zhao1, Xiujuan Wang1, Yiwen Zhang1, Rong Yan1, Jin Dai3, Guanglei

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Liu1, Feng Zhang2, and Kesheng Dai1

Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Suzhou, China;

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Department of Hematology, the First Affiliated Hospital of Bengbu medical college,

The School of Life Sciences, Peking University, Beijing, China.

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Bengbu, China;

*

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Ward count: 3182

These authors contributed equally to this work.

Kesheng Dai

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Correspondence to:

Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Suzhou 215006, China Tel.: 0086-512-67781370, Fax: 0086-512-67781370, E-Mail: [email protected]. Abstract Glycoprotein (GP) Ibα ectodomain shedding has become a generally accepted negative regulatory

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mechanism of platelet function. Stimulation of platelet with either physiological or chemical compound

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results in GPIbα ectodomain shedding in vitro and in vivo, the mechanism, however, is not totally

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understood. Here we show, collagen, thrombin, and calcium ionophore A23187 induce reactive oxygen species (ROS) generation, and simultaneously incur GPIbα ectodomain shedding. ROS

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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

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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

ROS-mediated GPIbα shedding.

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Keywords

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A23187-induced GPIbα ectodomain shedding. Calpain is an up-stream regulator that regulates

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Abbreviations

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Platelets; glycoprotein Ibα; shedding; reactive oxygen species (ROS); calpain

GP, glycoprotein; ROS, reactive oxygen species; NAC, N-acetylcysteine; DTT, dithiothreitol; MDL, carbobenzoxy-valinyl-phenylalaninal; VWF, von Willebrand factor; GC, glycocalicin; W7, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide; NEM, N-ethylmaleimide; PMA, phorbol 12-myristate-13-acetate; ADAM17, a disintegrin and metalloproteinase 17; DCFH-DA, 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate; FITC, fluorescein isothiocyanate; DMSO, dimethyl sulfoxide; CI I, calpain inhibitors I; ACD, acid-citratedextrose; RT, room temperature; DCFH, non-fluorescent compound dichlorofluorescein; DCF, dichlorofluorescein; ANOVA, one-way analysis of variance. Introduction

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Glycoprotein (GP) Ibα contains binding sites for von Willebrand factor (VWF), α-thrombin, P-selectin,

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and Mac-1 at the extracellular N-terminal 282 residues [1, 2]. Particularly, the interaction of GPIbα with

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VWF exposed at the injured vessel wall initiates platelet adhesion, and simultaneously triggers intracellular signaling leading to integrin activation and platelet thrombus formation [1-3]. Therefore,

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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

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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

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(PMA), or N-ethylmaleimide (NEM) [4-8]. We reported recently that the interaction of GPIbα with VWF, prolong inhibition of PKA, and hyperthermia also result in GPIbα ectodomain shedding [9]. Bergmeier

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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

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GPIbα shedding [10]. However, the regulatory mechanism of ADAM17-mediated GPIbα shedding is not totally understood.

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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

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observed in platelets stimulated with physiological or chemical compounds, such as collagen [15],

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thrombin [16], and thromboxane A2 analog U46619 [11]. However, the direct relation between

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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

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A23187-induced GPIbα ectodomain shedding. The data also indicate that calpain is an up-stream

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regulator of ROS generation, and regulates ROS-mediated GPIbα shedding. Materials and Methods Reagents

Monoclonal antibody SZ2 against GPIbα was generous gift from Prof. Changgeng Ruan (Soochow

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University, Suzhou, China). N-acetyl-L-cysteine (NAC), Dithiothreitol (DTT), ROS assay kit, 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), fluorescein isothiocyanate

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(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),

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calpain inhibitors I (CI I), calcium ionosphere A23187, thrombin, collagen (Chrono-Log), and

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carbobenzoxy-valinyl-phenylalaninal (MDL) were purchased from Sigma (St. louis, Missouri, USA).

Preparation of washed platelets For studies involving human subjects, approval was obtained from Soochow University institutional review board. Informed consent was provided according to the Declaration of Helsinki. Washed platelets were prepared as described previously. Briefly, fresh blood from healthy volunteers was anti-coagulated with 1/7 volume of acid-citratedextrose (ACD, 2.5% trisodium citrate, 2.0% D-glucose, 1.5% citric acid). After centrifugation, isolated platelets were washed twice with CGS buffer (123 mM NaCl, 33 mM D-glucose, 13 mM trisodium citrate, pH 6.5) and resuspended in modified Tyrode's buffer (2.5 mM Hepes, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 1 mM CaCl2, 1 mM MgCl2, 5.5 mM D-glucose, pH 7.4, MTB) to a final concentration of 3 × 108/ml. Washed platelets were incubated at

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room temperature (RT) for 1-2 hours (h) to recover to resting state.

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Western blot analysis of GPIbα ectodomain shedding

Washed platelets (3 × 108/ml) were pre-incubated with or without NAC (10 mM), DTT (3 mM), or

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vehicle control (DMSO) at RT for 15 minutes (min), and then were incubated with A23187 (5 μM), thrombin (1 U/ml), collagen (5 ug/ml), or dibucaine (1 mM) at 37 ℃ (or at RT for dibucaine) for different

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time (A23187 for 40 min, dibucaine for 15 min, thrombin and collagen for 30 min to get a optimal effect). The concentration of vehicle (DMSO) in each sample was not more than 1%. Samples were centrifuged at 1500 g for 5 min to harvest the supernatants. Each supernatant was added with one-fourth volume of 5 × SDS sample buffer, resolved in 8% SDS-PAGE, and immunoblotted with the

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anti-GPIbα N-terminal antibody SZ2.

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GPIbα ectodomain shedding detection by flow cytometry Washed platelets were pre-treated with or without NAC (10 mM), DTT (3 mM), or vehicle control

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(DMSO) at RT for 15 min, and then incubated with A23187 (5 µM), collagen (5 ug/ml), and dibucaine (1 mM) at RT or 37℃ for different time. 50 ul platelet suspension was incubated with SZ2 (5 μg/ml) or

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mouse IgG (5 μg/ml) at RT for 30 min. After washing, platelets were further incubated with FITC-labeled goat-anti mouse IgG antibody in the dark at RT for 30min, and then analyzed by flow cytometry.

ROS detection assay Platelets were isolated from whole blood obtained from healthy volunteers as described previously. ROS levels in platelets were examined using ROS assay kit according to the manufacture’s instruction. Briefly, washed platelets were treated with DCFH-DA (20 μM) at 37℃ for 45 min in the dark, and washed once with MTB. (DCFH-DA diffuses across cell membranes, and is hydrolysed by non-specific cellular esterases to form non-fluorescent compound dichlorofluorescein (DCFH), which is trapped

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predominantly within the cell. In the presence of ROS, DCFH rapidly undergoes one-electron oxidation

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to form a highly fluorescent compound dichlorofluorescein (DCF)). The pre-treated samples were

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incubated with or without NAC (10 mM), DTT (3 mM), calpain inhibitor I (100 μM), or MDL (100 μM) at 37℃ for 15 min, and then were further incubated with A23187 (5 μM), thrombin (1U), collagen (5 ug/ml)

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and dibucaine (1 mM), or vehicle at 37℃ for different time (collagen and thrombin for 30 min, A23187

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for 40 min). DCF fluorescence of treated-platelets was analyzed by flow cytometry.

Statistical analysis

All experiments were performed at least in triplicate, and data are presented as means ± SD. Data were analyzed using one-way analysis of variance (ANOVA) with Dunnett test. For comparison

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between two groups, a student paried t-test was used. P-value less than 0.05 were considered statistically significant.

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Results

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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

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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 induced ROS generations in platelets. Simultaneously, collagen, thrombin, and A23187 induced GPIbα ectodomain shedding was tested by flow cytometry or western blot with anti-GPIbα N-terminal antibody (SZ2). As shown in Fig. 1C and D, collagen and A23187 reduced GPIbα surface expression. Since stimulation of platelets with thrombin results in GPIbα internalization, thrombin induced GPIbα ectodomain shedding was detected by the appearance of GC in the supernatant.

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Thrombin-stimulated platelets were precipitated, and the proteolytic GPIbα extracellular fragment (GC)

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was detected in the supernatant (Fig. 1E). These data indicate that stimulations of platelets with

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physiological stimulus collagen and thrombin, or A23187 incur ROS generation, simultaneously result in GPIbα ectodomain shedding.

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ROS scavengers reduce collagen, thrombin, and A23187 induced ROS production and GPIbα

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ectodomain shedding

Next, we try to investigate the role of ROS in collagen, thrombin, and A23187 induced GPIbα ectodomain shedding. Firstly, the effects of two general accepted ROS scavengers, NAC and DTT, on collagen, thrombin, and A23187 induced ROS generation were tested. Washed platelets were treated with nonspecific ROS scavengers NAC and DTT, and then ROS levels were measured in collagen,

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thrombin, and A23187 stimulated platelets. As shown in Fig. 2A-C, NAC and DTT significantly reduced agonist induced intracellular ROS production. Then, we tested collagen, thrombin, and A23187

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induced GPIbα ectodomain shedding in platelets pretreated with NAC and DTT. As shown in Fig. 2D-F,

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agonists-induced GPIbα ectodomain shedding was abolished by NAC or DTT. These data indicate the role of ROS in A23187, collagen, and thrombin induced GPIbα ectodomain shedding.

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Calpain activator induced ROS generation and GPIbα ectodomain shedding Calpains are calcium-dependent thiol proteases highly expressed in human platelets, and play regulatory roles in both early events of platelet activation, spreading, and the late events of platelet-mediated fibrin clot retraction [10,18,19]. In our previous report [10], calpain was confirmed to play an important role in thrombin, or A23187 induced ADAM17-dependent GPIbα ectodomain shedding. In order to investigate the relationship between calpain and ROS in GPIbα shedding, dibucaine, a recognized activator of calpain, was selected to incubate with platelets, and then subjected to analysis of ROS generation. As shown in Fig. 3A,B, dibucaine obviously induced ROS generation in platelets, and simultaneously, dibucaine incurred GPIbα shedding (Fig. 3C-E). These

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data suggest the association between calpain activation and ROS generation during GPIbα shedding.

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ROS scavengers suppress ROS generation and GPIbα ectodomain shedding induced by calpain activator

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To further characterize the role of ROS in calpain-mediated GPIbα shedding, washed platelets were pre-incubated with ROS scavengers NAC or DTT, and then were incubated with dibucaine. As shown

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in Fig. 4A, NAC and DTT both reduced dibucaine-induced ROS production. Furthermore, NAC and DTT significantly reduced dibucaine-induced GPIbα ectodomain shedding (Fig. 4B-D). These data not only indicate the role of ROS in calpain-mediated GPIbα shedding, but also suggest that calpain is an up-stream regulator of ROS generation, and regulates ROS-mediated GPIbα shedding.

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Calpain inhibitors reduce A23187, thrombin, and dibucaine induced ROS generation

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In order to further confirm the role of calpain in ROS production in platelets, washed platelets were pre-incubated with calpain inhibitors, and then incubated with thrombin, A23187, and dibucaine. Firstly,

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calpain inhibitors CI I and MDL reduced dibucaine-induced ROS production (Fig. 5A), further clarify and characterize the role of calpain in ROS generation. Then, as shown in Fig. 5B-C, calpain inhibitors

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CI I or MDL inhibited ROS generations induced by thrombin and A23187. These data indicate that platelet agonists incur calpain activation resulting in ROS production in platelets. 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 [4-8], and ROS production could be detected in platelets

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stimulated with these agonists [15,16], there is not evidence indicate the direct linkage between ROS

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generation and GPIbα shedding in collagen and thrombin-stimulated platelets. Thus, firstly, we

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confirmed physiological stimulus collagen and thrombin, and chemical compound A23187 incurs ROS generation, simultaneously results in GPIbα shedding. Then, by using two ROS scavengers NAC and

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DTT, we verified the crucial role for ROS in collagen, thrombin, and A23187 induced GPIbα shedding. Though there is report that H2O2 could activate ADAM17 leading to GPIbα shedding [14], the evidence

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that ROS generation aroused by physiological stimulus results in GPIbα shedding remains deficiency. Thus, the current observation has important pathophysiological implications which suggest that physiological stimulations of platelets with collagen and thrombin lead to ROS-dependent GPIbα ectodomain shedding, which result in platelet dysfunction.

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Calpain, activated by both physiological and chemical agonists, has been confirmed to involve in the regulation of platelet activation, spreading, and platelet-mediated fibrin clot retraction in vivo and in

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vitro [10,18,19]. In our previous study [10]，we have verified that calpain plays an important role in thrombin, collagen, and A23187 induced ADAM17-dependent GPIbα ectodomain shedding. To further

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elucidate the regulatory mechanism of GPIbα shedding, the relationship between calpain and ROS in GPIbα shedding was investigated. The data demonstrate calpain activation results in ROS generation,

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and ROS scavenges inhibits calpain activator-induced GPIbα shedding in platelets. The data clearly indicate the role of ROS in calpain-mediated GPIbα shedding, furthermore, the data also suggest that calpain is an up-stream regulator of ROS generation and regulates ROS-mediated GPIbα shedding. Interestingly, we have reported previously that calpain inhibitors failed to block H2O2-induced GPIbα shedding [10], which further confirm that calpain is an up-stream regulator of ROS generation. Taken together, these findings draft an important regulatory pathway leading to GPIbα ectodomain shedding and platelet dysfunction. When platelets are stimulated with physiological or chemical stimulus in vivo or in vitro, calpain is activated simultaneously. Calpain activation leads to ROS generation which results in GPIbα ectodomain shedding. Alternatively, other physiological stimulations,

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such as activation of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase,

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generate ROS directly, also lead to GPIbα ectodomain shedding. As for mechanisms of

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ROS-dependent ADAM17 activation and GPIbα ectodomain shedding, ROS-related protein disulfide oxidoreductase or protein-disulphide isomerase activity might involve in the regulation of ADAM17

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activation.

In conclusion, the data indicate that ROS, regulated by calpain, plays a key role in collagen,

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thrombin, and A23187 induced GPIbα ectodomain shedding. These findings will help to understand the negative-regulatory mechanisms of platelet function, and may suggest a novel strategy to design new class of anti-platelet drug. Acknowledgements

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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

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(2012CB526600 to K. D), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), 2012 Jiangsu Provincial Special Program of Medical Science (BL2012005),

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Jiangsu Province’s Key Medical Center (ZX201102), and Jiangsu Province’s Outstanding Medical Academic Leader Program (K.D.).

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Conflict of interest statement

The authors declare that they have no conflict of interest. References [1] Du X. Signaling and regulation of the platelet glycoprotein Ib-IX-V complex. Curr Opin Hematol 2007;14:262-9. [2] Kroll MH, Harris TS, Moake JL, Handin RI, Schafer AI. von Willebrand factor binding to platelet GpIb initiates signals for platelet activation. J Clin Invest 1991;88:1568-73.

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Fig. 1. Collagen, thrombin, and A23187 induce ROS generation and GPIbα ectodomain shedding.

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Washed platelets were treated with or without DCFH-DA (20 μM), and further incubated with collagen (5 ug/ml), thrombin (1U), A23187 (5 μM), or vehicle. (A,B) DCF fluorescence of treated-platelets was

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analyzed by flow cytometry. Representative flow cytometric histograms are shown (A). ROS levels (DCF fluorescence) were presented as means ± SD from three independent experiments (B). (C,D) The pre-treated platelets were analysed for GPIbα surface expression by SZ2 and flow cytometry. Representative flow cytometric histograms are shown (C). Fluorescence intensities of GPIbα surface

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expression were presented as means ± SD from three independent experiments (D). (E) Washed platelets were incubated with thrombin (1U) or vehicle (control). The samples were centrifuged and the

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supernatants were resolved on an 8% SDS-PAGE and probed with SZ2. Results are representative of three separate experiments with different donors. *P < 0.05, compared with control.

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Fig. 2. ROS scavengers inhibit collagen, thrombin, and A23187-induced ROS generation and GPIbα shedding. Platelets were treated with or without DCFH-DA, and were further incubated with or without

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ROS inhibitors NAC (10 mM), DTT (3 mM), or vehicle (DMSO). The pre-treated platelets were stimulated with collagen (5 ug/ml), thrombin (1 U), or A23187 (5 μM). (A,B,C) Platelets pre-treated with DCFH-DA were subjected to ROS level analysis. ROS level (DCF fluorescence) were presented as means ± SD from three independent experiments. (D,E,F) Platelets pre-treated without DCFH-DA were analysed for GPIbα surface expression. Fluorescence intensities of GPIbα surface expression were presented as means ± SD from three independent experiments (D,E). Thrombin treated platelets were centrifuged and the supernatants were resolved on SDS-PAGE and probed with SZ2 (F). Results are representative of three separate experiments with different donors. *P < 0.05, compared with control.

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Fig. 3. Dibucaine induces both ROS generation and GPIbα ectodomain shedding. (A,B) Platelets were

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pre-treated with DCFH-DA (20 μM), and were further stimulated with dibucaine(1 mM) or vehicle

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(DMSO), and then were subjected to ROS level analysis. Representative flow cytometric histograms are shown (A). ROS levels (DCF fluorescence) were presented as means ± SD from three

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independent experiments (B). (C-E) Washed platelets were incubated with dibucaine (1 mM) or vehicle (DMSO), then analysed for GPIbα surface expression by flow cytometry and western blot.

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Representative flow cytometric histograms are shown (C). Fluorescence intensities of GPIbα surface expression were presented as means ± SD from three independent experiments (D). Pre-treated platelets were centrifuged and the supernatant was resolved on SDS-PAGE and probed with SZ2 (E). Results are representative of three separate experiments with different donors. *P < 0.05, compared

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with control.

Fig. 4. ROS scavengers inhibit dibucaine-induced ROS generation and GPIbα ectodomain shedding.

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(A) Platelets were pre-treated with DCFH-DA (20 μM), and were incubated with or without ROS inhibitors NAC (10 mM), DTT (3 mM), or vehicle (DMSO). Then the platelets were further treated with

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dibucaine (1 mM) and subjected to ROS level analysis. ROS levels (DCF fluorescence) were presented as means ± SD from three independent experiments. (B-D) Platelets were pre-incubated

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with ROS inhibitors NAC (10 mM), DTT (3 mM), or vehicle (DMSO), and then were incubated with or without (control) dibucaine (1 mM). Pre-treated platelets were analyzed for GPIbα surface expression. Representative flow cytometric histograms are shown (B). GPIbα surface expressions are presented as means ± SD from three independent experiments (C). Pre-treated platelets were centrifuged and the supernatants were resolved on SDS-PAGE and probed with SZ2 (D). *P < 0.05, compared with control. Fig. 5. Calpain inhibitors reduce dibucaine, thrombin, and A23187-induced ROS generation. Platelets were treated with DCFH-DA (20 μM), and were further incubated with or without calpain inhibitors C I I (100 μM), MDL (100 μM), or vehicle. The pre-treated samples were further incubated with dibucaine

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(A), thrombin (B), and A23187 (C,D), and subjected to ROS level analysis. ROS levels (DCF

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fluorescence) were presented as means ± SD from three independent experiments with different

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donors. *P < 0.05, compared with control.

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