Scholarly article on topic 'Inhibitory effects of total saponin from Korean Red Ginseng on [Ca2+]i mobilization through phosphorylation of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and inositol 1,4,5-trisphosphate receptor type I in human platelets'

Inhibitory effects of total saponin from Korean Red Ginseng on [Ca2+]i mobilization through phosphorylation of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and inositol 1,4,5-trisphosphate receptor type I in human platelets Academic research paper on "Chemical sciences"

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Keywords
{Ca2+-mobilization / "inositol 1 / 4 / 5-trisphosphate receptor type I (Ser1756) phosphorylation" / " Panax ginseng " / "protein kinase A catalytic subunit (Thr197) phosphorylation" / "total saponin from Korean Red Ginseng"}

Abstract of research paper on Chemical sciences, author of scientific article — Jung-Hae Shin, Hyuk-Woo Kwon, Hyun-Jeong Cho, Man Hee Rhee, Hwa-Jin Park

Abstract Background Intracellular Ca2+([Ca2+]i) is a platelet aggregation-inducing molecule. Therefore, understanding the inhibitory mechanism of [Ca2+]i mobilization is very important to evaluate the antiplatelet effect of a substance. This study was carried out to understand the Ca2+-antagonistic effect of total saponin from Korean Red Ginseng (KRG-TS). Methods We investigated the Ca2+-antagonistic effect of KRG-TS on cyclic nucleotides-associated phosphorylation of inositol 1,4,5-trisphosphate receptor type I (IP3RI) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation. Results The inhibition of [Ca2+]i mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-Br-cAMPS), which was more stronger than the inhibition by a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) (Thr197) by KRG-TS. The phosphorylation of IP3RI (Ser1756) by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS compared with that by Rp-8-Br-cGMPS. These results suggest that the inhibitory effect of [Ca2+]i mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. KRG-TS also inhibited the release of adenosine triphosphate and serotonin. In addition, only G-Rg3 of protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation. Conclusion These results strongly indicate that KRG-TS is a potent beneficial compound that inhibits [Ca2+]i mobilization in thrombin–platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease.

Academic research paper on topic "Inhibitory effects of total saponin from Korean Red Ginseng on [Ca2+]i mobilization through phosphorylation of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and inositol 1,4,5-trisphosphate receptor type I in human platelets"

Accepted Manuscript

Inhibitory effects of [Ca ]i mobilization by total saponin from Korean red ginseng via phosphorylation of PKA catalytic subunit and IP3RI in human platelets

Jung-Hae Shin, Hyuk-Woo Kwon, Hyun-Jeong Cho, Man Hee Rhee, Hwa-Jin Park

PII: S1226-8453(15)00031-7

DOI: 10.1016/j.jgr.2015.03.006

Reference: JGR 93

To appear in: Journal of Ginseng Research

Received Date: 9 December 2014 Revised Date: 16 March 2015 Accepted Date: 17 March 2015

Please cite this article as: Shin J-H, Kwon H-W, Cho H-J, Rhee MH, Park H-J, Inhibitory effects 2+

of [Ca ]i mobilization by total saponin from Korean red ginseng via phosphorylation of PKA catalytic subunit and IP3RI in human platelets, Journal of Ginseng Research (2015), doi: 10.1016/ j.jgr.2015.03.006.

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1 Inhibitory effects of [Ca2+]i mobilization by total saponin from Korean red ginseng via

2 phosphorylation of PKA catalytic subunit and IP3RI in human platelets

4 Jung-Hae Shin", Hyuk-Woo Kwon1f, Hyun-Jeong Cho2, Man Hee Rhee3 and Hwa-Jin Park1'*

6 Department of Biomedical Laboratory Science, College of Biomedical Science and Engineering, Inje

7 University, 197, Inje-ro, Gimhae, Gyungnam 621-749, Republic of Korea

8 2Department of Biomedical Laboratory Science, College of Medical Science, Konyang University, 685,

9 Gasuwon-dong, Seo-gu, Daejeon 302-718, Republic of Korea

10 3Laboratory of Veterinary Physiology & Signaling, College of Veterinary Medicine, Kyungpook

11 National University, Daegu 702-701, Republic of Korea

15 Correspondence to: Hwa-Jin Park, Department of Biomedical Laboratory Science, College of

16 Biomedical Science and Engineering, Inje University, 197, Inje-ro, Gimhae, Gyungnam 621-749,

17 Republic of Korea

18 Tel: +82-55-320-3538, Fax: +82-55-334-3426;

19 E-mail: mlsj park@inj e.ac.kr

21 Jung-Hae Shin and Hyuk-Woo Kwon contributed equally to this work.

23 Running title: Inhibition of [Ca2+]i mobilization by KRG-TS

Abstract

Background: Intracellular Ca ([Ca ]i) is platelet aggregation-inducing molecule. Therefore,

understanding the inhibitory mechanism of [Ca ]i mobilization is very important to evaluate

the antiplatelet effect of a substance. This study was carried out to understand the Ca -antagonistic effect of total saponin from Korean red ginseng (KRG-TS).

Method: We investigated the effect of KRG-TS on Ca -antagonistic cyclic nucleotides-associated the phosphorylation of inositol 1, 4, 5-trisphosphate receptor type I (IP3RI) and cAMP-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation.

Results: The inhibition [Ca ]i mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-Br-cAMPS), which was more strong than that by a cGMP-dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) (Thr197) by KRG-TS. The IP3RI (Ser1756) phosphorylation

by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS as compared with that by Rp-8-

Br-cGMPS. These results suggest that the inhibitory effect of [Ca ]i mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. In addition, KRG-TS inhibited the release of ATP and serotonin. In addition, G-Rg3 only of

44 protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation.

46 Conclusion: These results strongly indicate that KRG-TS is a potent beneficial compound

47 that inhibits [Ca ]i mobilization in thrombin-platelet interactions, which may result in the

48 prevention of platelet aggregation-mediated thrombotic disease.

51 Key words: Panax ginseng, total saponin from Korean red ginseng, Ca -mobilization,

52 PKAc (Thr197) phosphorylation, IP3RI (Ser1756) phosphorylation.

Introduction

Platelet aggregation is absolutely essential for the formation of a hemostatic plug when normal blood vessels are injured. However, the interactions between platelets and thrombin can also cause circulatory disorders, such as thrombosis, atherosclerosis, and myocardial infarction [1]. Accordingly, inhibition of the platelet-thrombin interaction might be a promising approach for the prevention of thrombosis. It is known that thrombin, a platelet agonist, stimulates platelet aggregation by binding to the Gq-coupled proteinase-activated receptor, which involves in activating phospholipase C-P (PLC-P). Activated PLC-P

hydrolyzes phosphatidylinositol 4, 5-bisphosphate (PIP2) to inositol 1, 4, 5-trisphosphate (IP3)

and diacylglycerol (DG) [2-5]. Moreover, IP3 mobilizes cytosol free Ca ([Ca ]i) from

dense tubular system by binding to IP3 receptor type I (IP3RI). The Increased [Ca2+]i activates

both the Ca /calmodulin-dependent phosphorylation of myosin light chain (20 kDa) and the

DG-dependent phosphorylation of pleckstrin (47 kDa) to induce granule secretion (i.e. ATP,

secretion), and platelet aggregation [6, 7]. The Ca -antagonistic effects of cAMP and cGMP are mediated via cAMP- and cGMP-dependent protein kinase (PKA, PKG), which phosphorylates substrate protein, IP3RI. IP3RI phosphorylation involves in inhibition of

[Ca ]i mobilization [8-10]. Therefore, phosphorylating IP3RI is very useful for evaluating the

Ca -antagonistic effect of substances or compounds.

Ginseng, the root of Panax ginseng Meyer, has been used frequently in traditional oriental

medicine, and is known to have various pharmacological activities such as anti-inflammatory

action, anti-oxidation, antitumor, anti-diabetes, and anti-hepatotoxicity [11, 12]. In recent, it

is reported that Korean red ginseng has an effect on cardiovascular disease, which is

characterized with regard to reduction of blood pressure and arterial stiffness by inhibition of

Rho kinase [13], anti-coagulation by prolong of prothrombin and activated partial

thromboplastin time [14], endothelium relaxation by nitric oxide-cGMP pathway [15], and

inhibition of hypercholesterolemia-induced platelet aggregation [16]. In our previous report,

we demonstrated that total saponin from Korean red ginseng (KRG-TS) is a beneficial

traditional oriental medicine in platelet-mediated thrombotic disease via suppression of

cyclooxygenase-1 (COX-1) and thromboxane A2 synthase (TXAS) to inhibit production of

thromboxane A2 (TXA2) [17]. In addition, KRG-TS was involved in increase of cAMP level

and subsequent reduction of [Ca ]i mobilization in thrombin-induced rat platelet aggregation

[18]. With regard to the effects of ginsenosides on platelet aggregation, it is well known that

ginsenoside Rg3 (G-Rg3) and its chemical derivatives (dihydroxyginsenoside Rg3, ginsenoside Rp1) have antiplatelet effects by regulating the aggregation-inhibiting molecule

(cAMP), and -stimulating molecules (extracellular kinase 2, tyrosine kinase-dependent

phosphoproteins, TXA2, intracellular Ca , integrin aIIb/p3, and so on) [19-21]. In this study,

to understand the inhibitory action mode of [Ca2 ]i mobilization by KRG-TS [18], we

investigated whether KRG-TS involves in phosphorylation of IP3RI, and which cyclic

nucleotide of cAMP and cGMP participates to IP3RI phosphorylation to attenuate [Ca ]i mobilization in thrombin-induced human platelet aggregation. In addition, in this study, we showed that G-Rg3 only of protopanaxadiol in KRG-TS inhibited, but all of the protopanaxatriol in KRG-TS did not thrombin-induced platelet aggregation.

Materials and Methods Materials

KRG-TS was obtained from R&D Headquarter, Korea Ginseng Corporation (Daejeon, Korea). Thrombin was purchased from Chrono-Log Co. (Havertown, PA, USA). ATP assay kit was purchased from Biomedical Research Service Center (Buffalo, NY, USA). Serotonin ELISA kit was purchased from Labor Diagnostika Nord GmbH & CO. (Nordhorn, Germany). Fura 2-AM, and other reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Anti-phosphor-IP3-receptor type I (Ser1756), anti-phosphor-PKA-C (Thr197), anti-rabbit IgG-horseradish peroxidase conjugate (HRP), and lysis buffer were obtained from Cell Signaling (Beverly, MA, USA). Polyvinylidene difluoride (PVDF) membrane was from GE Healthcare (Piseataway, New Jersey, USA). Enhanced chemiluminesence solution (ECL) was from GE Healthcare (Chalfont St, Giles, Buckinghamshire, UK). Protopanaxadiol saponin rG-Ra1, G-Rb1, G-Rb2, G-Rb3, G-Rc, G-Rd, G-Rg3 (20R), G-Rg3 (20S), and G-Rh2 (20R)1, and Protopanaxatriol saponin [G-Re, G-Rf, G-Rg1, G-Rg2 (20R) and G-Rh1 (20S)1 were

purchased from Ambo Institute (Daejon, Korea).

119 Preparation of washed human platelets

120 Human platelet-rich plasma (PRP) anti-coagulated with acid-citrate-dextrose solution (0.8%

121 citric acid, 2.2% sodium citrate, 2.45% glucose) were obtained from Korean Red Cross Blood

122 Center (Changwon, Korea). PRP was centrifuged for 10 min at 125 xg to remove a little red

123 blood cells, and was centrifuged for 10 min at 1,300 xg to obtain the platelet pellets. The

124 platelets were washed twice with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM

125 NaHCO3, 0.36 mM NaH2PO4, 5.5 mM glucose, and 1 mM EDTA, pH 6.5). The washed

126 platelets were then resuspended in suspension buffer (138 mM NaCl, 2.7 mM KCl, 12 mM

127 NaHCO3, 0.36 mM NaH2PO4, 0.49 mM MgCl2, 5.5 mM glucose, 0.25% gelatin, pH 6.9) to a

128 final concentration of 5x10 /mL. All of the above procedures were carried out at 25°C to

129 avoid platelet aggregation from any effect of low temperatures. The Korea National Institute

130 for Bioethics Policy Public Institutional Review Board (Seoul, Korea) approved these

131 experiments (PIRB12-072).

133 Measurement of platelet aggregation

134 Washed platelets (10 /mL) were preincubated for 3 min at 37°C in the presence of 2 mM

135 exogenous CaCl2 with or without substances, then stimulated with thrombin (0.05 U/mL) for

136 5 min. Aggregation was monitored using an aggregometer (Chrono-Log, Corporation) at a

137 constant stirring speed of 1,000 rpm. Each aggregation rate was calculated as an increase in

138 light transmission. The suspension buffer was used as the reference (transmission 0). KRG-

139 TS was dissolved in platelet suspension buffer (pH 6.9).

141 Determination of cytosolic-free Ca2+ ([Ca2+]i)

142 PRP was incubated with 5 pM Fura 2-AM at 37°C for 60 min. Because Fura 2-AM is

143 light sensitive, the tube containing the PRP was covered with aluminum foil during loading.

144 The Fura 2-loaded washed platelets were prepared using the procedure described above and

145 10 platelets/mL were preincubated for 3 min at 37°C with or without KRG-TS in the

146 presence of 2 mM CaCl2, then stimulated with thrombin (0.05 U/mL) for 5 min for evaluation

147 of [Ca ]i. Fura 2 fluorescence was measured with a spectrofluorometer (SFM 25; Bio-Teck

148 Instrument, Italy) with an excitation wavelength that was changed every 0.5 sec from 340 to

380 nm; the emission wavelength was set at 510 nm. The [Ca ]i values were calculated using the method of Schaeffer [22].

Determination of ATP, and serotonin release

Washed platelets (10 /mL) were preincubated for 3 min at 37°C with or without various substances in the presence of 2 mM CaCl2, then stimulated with thrombin (0.05 U/mL). The reaction was terminated, and centrifuged and supernatant were used for the assay of ATP and serotonin release. ATP release was measured in a luminometer (GloMax 20/20, Promega, Madison, USA) using an ATP assay kit. Serotonin release was measured with a Synergy HT multi-Model Microplate Reader (BioTek Instruments, Winoosku, VT., USA) using serotonin ELISA kit.

Western blot for analysis of PKA catalytic subunit (PKAc)-, and IP3RI-phosphorylation

Washed platelets (10 /mL) were preincubated with or without KRG-TS in the presence of

2 mM CaCl2 for 3min and then stimulated with thrombin (0.05 U/mL) for 5 min at 37°C. The

reactions were terminated by adding an equal volume (250 pL) of lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM serine/threonine phosphatase inhibitor P-glycerophosphate, 1 mM ATPase, alkaline and acid phosphatase, and protein phosphotyrosine phosphatase inhibitor Na3VO4, 1 pg/mL serine and cysteine protease inhibitor leupeptin, and 1 mM serine protease and acetylcholinesterase inhibitor phenylmethanesulfonyl fluoride, pH 7.5). Platelet lysates containing the same protein (15 pg) were used for analysis. Protein concentrations were measured by using bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, USA). The effects of substances on PKAc-, and IP3RI-phosphorylations were analyzed by western blotting. A 6-8% SDS-PAGE was used for electrophoresis and a PVDF membrane was used for protein transfer from the gel. The dilutions for anti- phosphor-IP3RI (Ser1756), anti-phosphor-PKAc (Thr197) and anti-rabbit IgG-HRP were 1:1000, 1:1000 and 1:10000, respectively. The membranes were visualized using ECL. Blots were analyzed by using the Quantity One, Ver. 4.5 (BioRad, Hercules, CA, USA).

Statistical analyses

181 The experimental results are expressed as the mean ± S.D. accompanied by the number of

182 observations. Data were assessed by analysis of variance (ANOVA). If this analysis indicated

183 significant differences among the group means, then each group was compared by the

184 Newman-Keuls method. Statistical analysis was performed according to the SPSS 21.0.0

185 (SPSS, Chicago, IL, USA). P<0.05 was considered to be statistically significant.

186 Results

188 Effects of KRG-TS on thrombin-induced human platelet aggregation

189 The concentration of thrombin-induced maximal human platelet aggregation was

190 approximately 0.05 U/mL (Fig. 1A). Therefore, thrombin 0.05 U/mL was used as the human

191 platelet agonist in this study. The light transmission in response to various concentrations of

192 KRG-TS (25, 50, 100, 150 ^g/mL) in intact platelets was 1.3 ± 0.6 % (at 25 ^g/mL of KRG-

193 TS), 1.3 ± 0.6 % (at 50 ^g/mL of KRG-TS), 1.3 ± 0.6 % (at 100 ^g/mL of KRG-TS) and 1.3

194 ± 0.6 % (at 150 p,g/mL of KRG-TS), respectively, which was not significant different from

195 that (1.0 ± 0.0 %) in resting platelets (Fig. 1B-a~1B-e). These mean that KRG-TS alone did

196 not effect on platelet aggregation as compared with that in intact platelet without KRG-TS.

197 When washed human platelets (10 /mL) were activated with thrombin, the aggregation rate

198 was increased up to 87.8 ± 5.7 %. However, various concentrations of KRG-TS (25 to 150

199 p,g/mL) significantly reduced thrombin-stimulated platelet aggregation in a dose-dependent

200 manner (Fig. 1C), and it's the half-maximal inhibitory concentration (IC50) was

201 approximately 45 p,g/ml (Fig. 1D). This IC50 is low as compared with that (81.1 p,g/mL) in rat

platelets [18]. In addition, 150 pg/mL of KRG-TS inhibited to 95.9 % thrombin-induced human platelet aggregation (87.8 ± 5.7 %) by thrombin.

Effects of KRG-TS on thrombin-induced human platelet aggregation in the presence of

PKA- or PKG inhibitor

Because [Ca ]i is essential to platelet aggregation, we investigated the effect of KRG-TS

on thrombin-induced platelet aggregation in the presence of PKA- or PKG-inhibitor,

intracellular Ca antagonistic compounds. As shown in Fig. 2A, thrombin increased light transmission (Fig. 2A-a), but KRG-TS decreased thrombin-induced light transmission (Fig. 2A-d). PKA inhibitor Rp-8-Br-cAMPS (Fig. 2A-b, Fig. 2B), and PKG inhibitor Rp-8-Br-cGMPS (Fig. 2A-c, Fig 2B) increased KRG-TS-attenuated light transmission in thrombin-activated platelets. The inhibitory degree (Fig. 2A-b, Fig. 2B) by PKA inhibitor was more strong than that (Fig. 2A-c, Fig 2B) by PKG inhibitor. These results suggest that the

inhibition of thrombin-induced human platelet aggregation by KRG-TS is greatly dependent

2+ 2+ on cAMP-dependent Ca2+ antagonistic condition than cGMP-dependent Ca2+-antagonistic

condition.

Effects of KRG-TS on [Ca2+]i mobilization.

Next, we investigated the effect of KRG-TS on Ca antagonistic activity. As shown in Fig. 3A, Thrombin increased [Ca2+]i level from 101.2 ± 0.5 nM, the basal level, to 590.6 ± 18.3 nM (Fig. 3A, small table). However, This was significantly decreased by various

concentrations (25 to 150 yg/mL) of KRG-TS in a dose-dependent manner (Fig. 3A). If the

inhibition of [Ca ]i mobilization by KRG-TS is resulted from IP3R phosphorylation via the cAMP/PKA or cGMP/PKG pathway, the KRG-TS-reduced [Ca2+]i level would be increased by inhibitors of PKA or PKG. Accordingly, we investigated the effects of PKA inhibitor Rp-8-Br-cAMPS and PKG inhibitor Rp-8-Br-cGMPS on KRG-TS-reduced [Ca2+]i mobilization. As shown in Fig. 3B, the PKA inhibitor Rp-8-Br-cAMPS (50 to 250 [M) dose dependently increased the [Ca2+]i level as compared with that (110.9 ± 1.9 nM) by KRG-TS (150 [g/mL)

in the thrombin-induced platelet aggregation (Fig. 3B). On the other hand, PKG inhibitor Rp-

8-Br-cGMPS (50 to 250 [M) very weakly increased the [Ca ]i level as compared with that (110.9 ± 1.9 nM) by KRG-TS (150 [g/mL) in thrombin-induced platelet aggregation (Fig.

3C). As shown in Table 1, Rp-8-Br-cAMPS (250 [M) increased the [Ca2+]i level to 155.1 %,

and Rp-8-Br-cGMPS (250 [M) increased the [Ca2+]i level to 29.4 %. These mean that the

reduction of [Ca ]i mobilization (Fig. 3A) by KRG-TS may be more dependent on the pathway of cAMP/PKA than cGMP/PKG. We, therefore, investigated the effect of KRG-TS on phosphorylation of PKA catalytic subunit (PKAc) as an indicator of cAMP/PKA activation.

Effects of KRG-TS on PKAc (Thr197) phosphorylation

It is known that the phosphorylation by cAMP binding to PKAc (Thr ) is an essential step to cause biological functions by cAMP/PKA pathway [23]. As shown in Fig. 4, KRG-TS

dose dependently phosphorylated Thr in PKAc (42 kDa), and abruptly increased the ratio

of phosphorylated PKAc (Thr ) to P-actin in thrombin-induced platelet aggregation (Fig. 4, lane 4, 5). PKA inhibitor Rp-8-Br-cAMPS strongly decreased KRG-TS-elevated

phosphorylation of PKAc (Thr ) (Fig. 4, lane 6), which means that the phosphorylation of PKAc (Thr197) by KRG-TS depends on cAMP/PKA pathway. This also is support from the fact that PKA activator pCPT-cAMP increased the phosphorylation of PKAc (Thr197) (Fig. 4, lane 7).

Effects of KRG-TS on inositol 1, 4, 5-trisphosphate receptor type I (IP3RI) (Ser1756) phosphorylation in the presence of PKA inhibitor, and PKG inhibitor

Next, we investigated the IP3RI phosphorylation associated PKAc (Thr )

1756 1756

phosphorylation. The phosphorylation of IP3RI (Ser ) and the ratio of p-IP3RI (Ser ) to P-actin were increased under of PKA activator pCPT-cAMP (Fig. 5, lane 7) in thrombin-induced human platelet aggregation. These mean that cAMP/PKA involves in IP3RI (Ser1756) phosphorylation. As shown in Fig. 5, lane 3 and 4, the phosphorylation (p-IP3RI) of IP3RI

1756 1756

(Ser ), and the ratio of p-IP3RI (Ser1756) to P -actin were dose dependently increased in the

presence of both thrombin and KRG-TS. To investigate whether cAMP/PKAc (Thr197) phosphorylation by KRG-TS (Fig. 4, lane 5) involved in IP3RI (Ser1756) phosphorylation (Fig. 5, lane 4), we used PKA inhibitor Rp-8-Br-cAMPS (Fig. 4, lane 6) that inhibited KRG-TS-

induced PKAc (Thr ) phosphorylation. As shown in Fig. 5, lane 5, PKA inhibitor Rp-8-Br-cAMPS potently decreased the p-IP3RI (Ser1756), and the ratio of p- IP3RI (Ser1756) to P-actin achieved by both thrombin and KRG-TS. These results mean that the increase of p-IP3RI (Ser1756) by KRG-TS is dependent on cAMP/PKAc (Thr197). PKG inhibitor Rp-8-Br-cGMPS also decreased KRG-TS-induced p-IP3RI (Ser1756) (Fig. 5, lane 6). But its inhibitory degree

(22.6 %) was lower as compared with that (44.3 %) by PKA inhibitor Rp-8-Br-cAMPS (Table 2). These mean that the increase of p-IP3RI (Ser1756) by KRG-TS was also resulted from cAMP/PKA-pathway than cGMP/PKG pathway.

Effect of KRG-TS on ATP and serotonin release

Intracellular [Ca ]i mobilization is known to involve in ATP and serotonin release from

dense body of platelets [24]. Because KRG-TS inhibited thrombin-elevated [Ca ]i mobilization (Fig. 3A), we investigated whether KRG-TS involves in inhibition of ATP and

serotonin release. As shown in Fig. 6, KRG-TS dose dependently inhibited thrombin-induced

ATP (Fig. 6A) and serotonin release (Fig. 6C). Because the inhibition of [Ca ]i mobilization by KRG-TS was dependent on cAMP/PKA-, and cGMP/PKG pathway, next, we investigated whether the inhibition of ATP and serotonin release by KRG-TS was resulted from cAMP/PKA-, and cGMP/PKG-pathway. PKA inhibitor Rp-8-Br-cAMPS (Fig. 6B), and PKG inhibitor Rp-8-Br-cGMPS (Fig. 6D) increased KRG-TS-decreased ATP and serotonin release. These mean that the inhibition of ATP, and serotonin release by KRG-TS (Fig. 6A, 6C) is dependent on cAMP/PKA-, and cGMP/PKG-pathway. These are supported also from the

phenomena that PKA activator pCPT-cAMP (Fig. 6B), and PKG activator 8-Br-cGMP (Fig. 6D) inhibit thrombin-induced ATP and serotonin release.

Effects of ginsenosides in KRG-TS on thrombin-induced platelet aggregation

In our previous report [18], we reported that five ginsenosides (G-Rb1, G-Rb2, G-Rc, G-Rd, and G-Rg3) as 20 (S)-protopanaxadiol saponin (PPDS), three ginsenosides (G-Re, G-Rg1 and G-Rg2) as 20 (S)-protopanaxatriol saponin (PPTS). Besides these ginsenosides, it is known that G-Ra1, G-Rb3, and G-Rh2 as protopanaxadiol saponin, and G-Rf and G-Rh1 as protpanaxatriol saponin are also contained in Panax ginseng [25]. Here, to investigate which ginsenoside of KRG-TS was involved in inhibition of thrombin-induced human platelet aggregation, we investigated the effect of ginsenosides in KRG-TS and other ginsenosides on thrombin-induced human platelet aggregation. In this study, G-Ra1, G-Rb1, G-Rb2, G-Rb3, G-Rc, G-Rd, and G-Rh2 (20R) of PPDS did not inhibit thrombin-induced human platelet aggregation, but G-Rg3 (20R, 20S) only dose dependently inhibited thrombin-induced human platelet aggregation (Table 3). In addition, the all [G-Re, G-Rf, G-Rg1, G-Rg2 (20R), and G-Rh1 (20S)] of PPTS did not inhibit thrombin-induced human platelet aggregation (Table 4).

Discussion

Although KRG-TS elevated cAMP only in thrombin-induced human platelets [17], to confirm which cAMP/PKA- or cGMP/PKG-pathway contributed to the inhibition of platelet aggregation by KRG-TS, we investigated the effect of PKA inhibitor and PKG inhibitor on thrombin-induced human platelet aggregation in the presence of KRG-TS. As the results, PKA inhibitor Rp-8-Br-cAMPS and PKG inhibitor Rp-8-Br-cGMPS increased KRG-TS-decreased light transmission in thrombin-induced human platelet aggregation. However, the elevation of light transmission by PKA inhibitor was more strong than that by PKG-inhibitor in KRG-TS-inhibited thrombin-induced human platelet aggregation. These results mean that

cAMP/PKA pathway was mainly contributed to the inhibition of thrombin-induced platelet

aggregation, and [Ca ]i mobilization by KRG-TS. Of several aggregation-inducing

molecules (i.e. Ca , TXA2) are commonly generated by agonists (i.e. thrombin, collagen, and

2+ 2+ ADP). IP3 mobilizes [Ca ]i, and subsequently activates Ca -dependent phospholipase-C or -

A2 to separate TXA2 precursor arachidonic acid (20:4) from glycerophospholipids, and TXA2

is produced from 20:4 via activation of COX-1/TXAS. TXA2 produces IP3 to mobilize [Ca ]i via G-protein coupled receptor/PLC-P pathway, and constricts vessel tract [5, 26-28], which

enforces thrombus formation. Therefore, the inhibition of [Ca ]i mobilization by IP3 and TXA2 production by COX-1/TXAS is very important to evaluate an antiplatelet effect of a substance. In our previous report [17], it is confirmed that KRG-TS inhibits TXA2 production

by attenuating COX-1 and TXAS activities. Even though KRG-TS inhibited thrombin-

2+ 2+ induced [Ca ]i mobilization, its the inhibitory mechanism is unknown. The Ca -antagonistic

reaction by cAMP and cGMP is mediated by PKA/IP3RI phosphorylation pathway, and

PKG/IP3RI phosphorylation pathway. If KRG-TS that elevated the level of cAMP [18]

stimulates IP3RI (Ser1756) phosphorylation in thrombin-activated human platelets, this is a

clear evidence that KRG-TS inhibits [Ca2+]i mobilization via cAMP/PKA/IP3RI (Ser1756)

phosphorylation pathway. In this report, we confirmed that KRG-TS inhibited [Ca ]i mobilization via IP3RI (Ser1756) phosphorylation by cAMP/PKAc, which is supported from the result that cAMP inhibitor Rp-8-Br-cAMPS inhibited KRG-TS-elevated the

1756 197

phosphorylation of both IP3RI (Ser1756) and PKAc (Thr197) in thrombin-induced human

platelet aggregation, if not so, cAMP inhibitor Rp-8-Br-cAMPS would not increase KRG-TS-

decreased [Ca ]i mobilization in thrombin-induced human platelet aggregation. It is known that IP3 induces serotonin release from platelet dense body, which means that IP3 involves in

serotonin release by elevating [Ca ]i via IP3RI [29]. This reflects that KRG-TS may involve in inhibition of serotonin release by phosphorylating IP3RI (Ser1756).

A lot of agonists such as collagen, thrombin and ADP mobilize [Ca ]i to phosphorylate

Ca /calmodulin-dependent myosin light chain (20 kDa), which involves in granule secretion

such as ATP and serotonin [6, 7], and platelet aggregation. It is thought that the inhibition of

ATP and serotonin secretion by KRG-TS is resulted from the elevation of Ca -antagonistic

molecule cAMP and subsequent the inhibition of [Ca ]i mobilization, which also is

supported from the facts that KRG-TS stimulated the phosphorylation of both PKAc (Thr ) and IP3RI (Ser1756).

Platelet aggregation is generated at site of vascular wall injury, and is involved in the formation of thrombus. During the formation of thrombus, platelets release cell growth proteins such as platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) in a-granule [30, 31]. It is well established that PDGF and VEGF induce the proliferation of fibroblast, vascular smooth cells, and epithelial cells, and subsequently enhance the rate of atherosclerosis lesion progression [32-36]. The progression of atherosclerosis is strongly induced by inflammatory cell such as monocyte/macrophage, and

neutrophil [37]. Although KRG-TS has antiplatelet effects, if KRG-TS does not inhibit

inflammation by leukocyte, the progression of atherosclerosis lesion would be generated at

site of vascular wall injury, and a question for antiplatelet effects of KRG-TS might be raised.

Byeon et al. reported that saponin fraction inhibits lipopolysaccharide (LPS)-induced

inflammation [381, and it is well reviewed that ginsenosides have anti-inflammatory effects

by inhibiting the production of various pro-inflammatory mediators (i.e. PGE2, NO) [39]. In

recent, it is reported that Korean red ginseng saponin fraction down-regulates LPS-induced

pro-inflammatory mediators (i.e. NO, interluukin-1P) [40]. Considering these three previous

reports [38-40], it is thought that KRG-TS may have antithrombotic-, and antiatherosclerotic-

effects without generation of inflammation and progression of atherosclerotic lesion at site of

vascular wall injury. Therefore, KRG-TS is highlighted as a non-toxic antiplatelet compound,

and could be clinically applied to the prevention of platelet-mediated thrombosis. This is

supported from report that Korean red ginseng has protective effects on rat carotid artery

thrombosis in vivo [41], and both ginseng and ginsenoside are very beneficial candidator for

prevention of cardiovascular disease [42].

With regard to the antiplatelet effects of ginsenosides in KRG-TS, G-Rg3 (20R, 20S) only

inhibited thrombin-induced platelet aggregation. This is in accord with the reports that G-Rg3 (20R, 20S) inhibited arachidonic acid- or U46619-induced platelet aggregation [43], and its analogue (G-Rp1, dihydroxy G-Rg3) inhibited collagen- or thrombin-induced platelet aggregation [19, 20]. In another reports, it is known that G-Rg1 (MW. 800.94) and G-Rg2 (MW. 781.01) inhibit various agonists (i.e. thrombin, collagen, ADP)-induced platelet aggregation [44-46], but their inhibitory concentrations were 1 mg/mL (1.2 mM) to 4 mg/mL (5 mM) of G-Rg1 [44, 45], and were 1 mg/mL (1.3 mM) of G-Rg2 [45, 46]. These inhibitory concentrations (1.2~5 mM) of G-Rg1 or G-Rg2 [44-46] are very high as compared with that G-Rg3 and its analogues (G-Rp1, dihydroxy G-Rg3) have antiplatelet effect at concentration of micromole unit [19, 20, 43]. Therefore, it is thought that the antiplatelet effects by G-Rg1 and G-Rg2 should be reevaluated. Because G-Rg3 only in KRG-TS inhibited thrombin-

induced human platelet aggregation, it is thought that G-Rg3 in KRG-TS might be

contributed to the inhibition of platelet aggregation, and [Ca ]i mobilization by KRG-TS,

which is also proved in support of report that G-Rg3 elevated Ca -antagonistic cAMP level [20].

Thrombosis is resulted mainly from the irreversible aggregation which is intimately

related with the serotonin released from agonists (i.e. collagen, thrombin, etc.)-activated

platelets [47-49]. In addition, the released serotonin involves in causing migraine [50-52].

In conclusion, the most important result of this study is that KRG-TS significantly phosphorylates IP3RI (Ser1756) to inhibit thrombin-induced [Ca2 ] mobilization, which contributed to attenuating the release of ATP and serotonin. Therefore, our results suggest

that KRG-TS may be a physiologically effective negative regulator during platelet

aggregation, a cause of thrombosis, atherosclerosis, and myocardial infarction. Inhibitory

effect of serotonin by KRG-TS may be also evaluated as anti-migraine substance.

Acknowledgements

This study was supported by a grant (NRF-2011-0012143 to Hwa-Jin Park) from the Basic

Science Research Program via the National Research Foundation of Korea(NRF)funded by

the Ministry of Education, Science and Technology, Korea

Conflict of interest

The authors declare no conflict of interest.

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523 Table 1. Effects of Rp-8-Br-cAMPS and Rp-8-Br-cGMPS on [Ca2+]i mobilization

[Ca2+] (nM) Increase (%)

KRG-TS (150 ^g/ml) + Thrombin (0.05 U/mL) 110.9 ± 1.9

KRG-TS (150 ^g/ml) + Rp-8-Br-cAMPS (250 ^M) + Thrombin (0.05 U/mL) 238.6 ± 5.9 155.11)

KRG-TS (150 ^g/ml) + Rp-8-Br-cGMPS (250 ^M) + Thrombin (0.05 U/mL) 143.6 ± 2.8 29.42)

524 Data were from Fig. 3B, C. 1) Increase (%) = [(KRG-TS + Thrombin + Rp-8-Br-cAMPS) - (KRG-TS

525 + Thrombin)] / (KRG-TS + Thrombin) x 100, 2) Increase (%) = [(KRG-TS + Thrombin + Rp-8-Br-

526 cGMPS) - (KRG-TS + Thrombin)] / (KRG-TS + Thrombin) x 100.

545 Table 2. Changes of p-^R/p-actin ratio

_p-IP3R/|3-actin_A(%)

Thrombin (0.05 U/mL) 2.7 ± 0.1

KRG-TS (150 [xg/mL) 267.6 ±7.3 9847.21' + Thrombin (0.05 U/mL)

KRGjTS (150 (xg/mL) 148.9 ± 2.1 - -44.32'

+ Thrombin (0.05 U/mL) + Rp-8-Br-cAMPS (250 |xM)

KRGjTS (150 (xg/mL) 196.2 ± 8.5 - -26.63'

+ Thrombin (0.05 U/mL) + Rp-8-Br-cGMPS (250 |xM)

547 Data were from Fig. 5. 1) A (%) = IYKRG-TS + Thrombin) - Thrombin] / Thrombin x 100, 2) A (%) =

548 IYKRG-TS + Thrombin + Rp-8-Br-cAMPS) - (KRG-TS + Thrombin)] / (KRG-TS + Thrombin) x 100, 3)

549 A (%) = IYKRG-TS + Thrombin + Rp-8-Br-cGMPS) - (KRG-TS + Thrombin)] / (KRG-TS + Thrombin)

550 x 100.

567 Table 3. Effects of Protopanaxadiol on thrombin-induced human platelet aggregation

Treatment Aggregation (%) Treatment Aggregation (%)

Thrombin (0.05 U/mL) 86.7 ± 1.5

Ginsenoside Rai Ginsenoside Rd

50 uM 83.3 ± 1.2 50 uM 85.0 ± 3.0

100 uM 83.0 ± 1.7 100 uM 84.0 ± 1.7

200 uM 83.0 ± 1.0 200 uM 86.3 ± 1.5

300 uM 86.7 ± 1.5 300 uM 84.3 ± 4.0

Ginsenoside Rbi Ginsenoside Rg3 (20R)

50 uM 83.0 ± 1.7 50 uM 86.7 ± 1.5

100 uM 84.3 ± 0.6 100 uM 86.0 ± 2.0

200 uM 85.3 ± 3.1 200 uM 75.7 ± 3.2

300 uM 83.0 ± 3.5 300 uM 62.0 ± 2.0

Ginsenoside Rb2 Ginsenoside Rg3 (20S)

50 uM 86.7 ± 1.5 50 uM 79.7 ± 2.1*

100 uM 84.7 ± 0.6 100 uM 39.7 ± 2.1**

200 uM 86.3 ± 2.1 200 uM 9.7 ± 2.1**

300 uM 84.7 ± 0.6 300 uM 3.7 ± 1.5**

Ginsenoside Rb3 Ginsenoside Rh2 (20R)

50 uM 82.3 ± 3.5 50 uM 83.3 ± 1.2

100 uM 85.7 ± 1.2 100 uM 84.7 ± 0.6

200 uM 83.7 ± 1.5 200 uM 85.3 ± 3.1

300 uM 84.0 ± 3.6 300 uM 81.0 ± 1.7

Ginsenoside Rc

50 uM 84.7 ± 3.2

100 uM 84.7 ± 0.6

200 uM 86.3 ± 2.1

300 uM 84.0 ± 2.6

Results are expressed as % of aggregation induced by thrombin. The data are expressed as the mean ± S.D. (n=4). p<0.05, p<0.001 versus the thrombin-stimulated human platelets.

579 Table 4. Effects of Protopanaxatriol on thrombin-induced human platelet aggregation

Treatment Aggregation (%) Treatment Aggregation (%)

Thrombin (0.05 U/mL) 86.7 ± 1.5

Ginsenoside Re Ginsenoside Rg2 (20R)

50 uM 82.7 ± 1.2 50 uM 84.0 ± 2.6

100 uM 82.3 ± 1.5 100 uM 87.0 ± 1.0

200 uM 85.3 ± 2.5 200 uM 85.3 ± 2.9

300 uM 84.7 ± 0.6 300 uM 86.0 ± 2.6

Ginsenoside Rf Ginsenoside Rh1 (20S)

50 uM 82.7 ± 2.1 50 uM 84.3 ± 1.5

100 uM 86.7 ± 1.5 100 uM 83.3 ± 2.1

200 uM 84.7 ± 0.6 200 uM 82.7 ± 1.2

300 uM 83.0 ± 1.0 300 uM 85.3 ± 2.5

Ginsenoside Rg1

50 uM 84.3 ± 2.1

100 uM 83.0 ± 1.7

200 uM 84.7 ± 4.0

300 uM 85.0 ± 1.0

581 Results are expressed as % of aggregation induced by thrombin. The data are expressed as the

582 mean ± S.D. (n=4).

FIGURE LEGENDS

Fig. 1. Effects of KRG-TS on thrombin-induced human platelet aggregation. (A) The concentration threshold of thrombin on human platelet aggregation. (B) The effects of KRG-TS on resting human platelets. (C) Effects of KRG-TS on thrombin-induced human platelet aggregation. (D) The IC50 value of KRG-TS in thrombin stimulated human platelet aggregation. Measurement of platelet aggregation was carried out as described in "Materials and Methods" section. The rate of inhibition by KRG-TS was recorded as the percentage of the thrombin induced aggregation rate. The IC50 value of KRG-TS was calculated according

to the 4-parameter log fit method. The data are expressed as the mean ± S.D.

(n=4). p<0.05 versus the thrombin-induced human platelet aggregation.

Fig. 2. Effects of KRG-TS on thrombin-induced human platelet aggregation in the presence of PKA- or PKG-inhibitor (A) Effects of KRG-TS, PKA inhibitor, or PKG-inhibitor on thrombin-elevated light transmission. a) Thrombin (0.05 U/mL); b) Thrombin (0.05 U/mL) + KRG-TS (150 pg/mL) + Rp-8-Br-cAMPS (250 pM); c) Thrombin (0.05 U/mL) + KRG-TS (150 pg/mL) + Rp-8-Br-cGMPS (250 pM); d) Thrombin (0.05 U/mL) + KRG-TS (150

pg/mL) (B) Effects of KRG-TS, PKA inhibitor, or PKG inhibitor on thrombin-induced human platelet aggregation. Washed human platelets (10 /mL) were preincubated with KRG-TS (150 pg/mL) in the presence of A-Kinase inhibitor (Rp-8-Br-cAMPS) or G-kinase

inhibitor (Rp-8-Br-cGMPS) for 3 min at 37°C, and then thrombin (0.05 U/mL) was added.

The data are expressed as the mean ± S.D. (n=4). p<0.05 versus the thrombin-stimulated human platelets, 'p<0.05 versus the thrombin-stimulated platelets in the presence of KRG-TS (150 ug/mL).

Fig. 3. Effects of KRG-TS on thrombin-induced [Ca ]i mobilization. (A) Inhibitory effects of KRG-TS on thrombin-induced [Ca2+]i mobilization. (B) Effects of KRG-TS on [Ca2+]i

mobilization in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS). (C) Effect of KRG-TS

2+ 2+ on [Ca ]i mobilization in the presence of G-kinase inhibitor (Rp-8-Br-cGMPS). [Ca ]i was

determined as described in "Materials and Methods" section. The data are expressed as the

mean ± S.D. (n=4). p<0.05 versus the thrombin-stimulated human platelets, p<0.05 versus

the thrombin-stimulated human platelets in the presence of KRG-TS (150 ug/mL), #p<0.05

versus the thrombin-stimulated human platelets in the presence of KRG-TS (150 ug/mL).

Fig. 4. Effect of KRG-TS on PKAc phosphorylation.

Lane 1, Intact platelets (base); Lane 2, Thrombin (0.05 U/mL); Lane 3, Thrombin (0.05 U/mL) + KRG-TS (50 |g/mL); Lane 4, Thrombin (0.05 U/mL) + KRG-TS (100 |g/mL); Lane 5, Thrombin (0.05 U/mL) + KRG-TS (150 |g/mL); Lane 6, Thrombin (0.05 U/mL) + KRG-TS (150 |g/mL) + Rp-8-Br-cAMPS (250 |M); Lane 7, Thrombin (0.05 U/mL) +

pCPT-cAMP (1 mM). Western blotting was performed as described in "Materials and

Methods" section. The data are expressed as the mean ± S.D. (n=4). p<0.05 versus the thrombin-stimulated human platelets, 'p<0.05 versus the thrombin-stimulated human platelets in the presence of KRG-TS (150 |g/mL).

Fig. 5. Effect of KRG-TS on inositol 1, 4, 5-trisphosphate receptor type I (IP3RI) (Ser1756) phosphorylation. Lane 1, Intact platelets (base); Lane 2, Thrombin (0.05 U/mL); Lane 3, Thrombin (0.05 U/mL) + KRG-TS (100 |g/mL); Lane 4, Thrombin (0.05 U/mL) + KRG-TS (150 |g/mL); Lane 5, Thrombin (0.05 U/mL) + KRG-TS (150 |g/mL) + Rp-8-Br-cAMPS (250 |M); Lane 6, Thrombin (0.05 U/mL) + KRG-TS (150 |g/mL) + Rp-8-Br-cGMPS (250

645 |uM); Lane 7, Thrombin (0.05 U/mL) + pCPT-cAMP (1 mM); Lane 8, Thrombin (0.05 U/mL)

646 + 8-Br-cGMP (1 mM). Western blotting was performed as described in "Materials and

647 Methods" section. The data are expressed as the mean ± S.D. (n=4). p<0.05 versus the

648 thrombin-stimulated platelets, '//<0.05 versus the thrombin-stimulated human platelets in the

649 presence of KRG-TS (150 ug/mL).

651 Fig. 6. Effects of KRG-TS on ATP and serotonin release. (A) Effects of KRG-TS on ATP

652 release in thrombin-activated platelets. (B) Effects of KRG-TS on ATP release in the

653 presence of PKA- or PKG-inhibitor. (C) Effects of KRG-TS on serotonin release. (D) Effects

654 of KRG-TS on serotonin release in the presence of PKA- or PKG-inhibitor. Data are

655 expressed as mean ± S.D. (n=4). /<0.05 versus the thrombin-stimulated human platelets,

656 '/<0.05 versus the thrombin-stimulated human platelets in the presence of KRG-TS (150

657 ug/mL).

100 90 SO 70 60 50 40 30 20 10 0

0.00625

0.0125

Thrombin (11/ml,)

661 662

680 681 682

686 Fig. 1

a. Intact

b. KRG-TS (25 ng/mL) c. KRG-TS (50 ng/mL)

^d. KRG-TS (100 jig/mL) e. KRG-TS (150 ng/mL)

Fig. 1

LOO 90 80 70 60 50 40 30 20 10

Thrombin (0.05 U/mL) KRG-TS (ng/mL)

Fig. 1

t OX OJD S3

100 90 80 70 60 50 40 30 20 10 0

ICso = 45 jig/mL

.......I

......I

KRG-TS (mg/niL)

Fig. 2 A

!/l V3

<fl S es

0 10 20 30 40 50 60 70 80 90 100

Time (min)

Fig. 2

90 80 70 60 50 40 30 20 10 0

Thrombin (0.05 U/mL) KRG-TS (ng/mL) Rp 8 Br cAMPS (250 ji.Yl) Rp-8-Br-cGMPS (250 jiM)

500 ■

— 300

200 ■

[CaJ+]j (nNl) Inhibition (%)

Thrombin (0,05 U/mL) KRG-TS (150 (ig/mL) + Thrombin (0,05 U/ml) 590.6 ± 18.3 110,9 ± 1.9 0 3i,:

Thrombin (0.05 U/mL) KRG-TS (ng/mL)

+ + + + + 25 50 100 150

811 812

820 821 822

350 -i

Thrombin (0.05 U/rnL) KRG-TS (ng/mL) Rp-8 Br-cAMPS (jiM)

150 50

150 150

150 250

Si 150

Thrombin (0.05 U/mL) KRG-TS (jig/mL) Rp-8-Br-cGMPS (jJ.Vl)

880 881 882

886 887

150 250

Fig. 4

J3 —

400.0 350.0 300.0 250.0 200.0 150.0 100.0 50.0 0.0

p-PKAc (Thr197) P-actin

Thrombin (0.05 IJ/mL) KRG-TS (ng/mL) Rp 8 Br-cAMPS (250 nM) pCPT-cAMP (1 mM)

3 4 5 6 7

m - — 42 kDa

—43 kDa + +

Fig. 5

p IP3RI (Ser1™) (i-actin

Thrombin (0.05 U/mL) KRG-TS (ng/mL) Rp-8-Br-cAMPS (250 (jM) Rp 8 Br cGMPS (250 (iM) pCPT cAMP (1 mM) 8-Br-cGMP (1 mM)

4.0 3.5 3.0 2.5 2.0

1.5 L.O

0.5 0.0

2 3 4 5

+ + + + 100 150 150

+ + 150

320 kDa 43 kDa

Fig. 6

Thrombin (0.05 U/mL) KRG-TS (ng/ml)

Fig. 6

Thrombin (0.05 U/mL) KRG-TS (fig/mL) Rp-8-Br-cAMPS (250 jiM) Rp-8-Br-cGMPS (250 (iM) pCPT-cAMP (1 niM) 8-Br-cGMP (1 mM)

■ 1 1

150 150

Fig. 6

Thrombin (0.05 U/mL) KRG-TS (ng/mL)

+ + + + 25 50 100 150

999 1000 1001 1002

1010 1011 1012

1018 1019

1020 1021

Fig. 6

Thrombin (0.05 U/mL) KRG-TS (fig/mL) Rp-8-Br-cAMPS (250 fiM) Rp-8-Br-cGMPS (250 pM) pCPT-cAMP (1 mM) 8-Br-cGMP (1 mM)

■ I n 11

+ + + + + + 150 150 150