Small GTPase Rho Regulates Thrombin-Induced Platelet Aggregation

Biochemical and Biophysical Research Communications 280, 970 –975 (2001) doi:10.1006/bbrc.2001.4237, available online at http://www.idealibrary.com on

Small GTPase Rho Regulates Thrombin-Induced Platelet Aggregation Hiroaki Nishioka, 1 Hisanori Horiuchi, 2 Arata Tabuchi, Akira Yoshioka, Ryutaro Shirakawa, and Toru Kita Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Received December 27, 2000

Platelets play essential roles in hemostasis and thrombosis by aggregating with each other. However, the molecular mechanism governing platelet aggregation is not yet fully understood. Here, we established an assay system using platelets permeabilized with streptolysin-O to analyze mechanism of the thrombininduced aggregation, focusing upon a controversial issue in the field whether small GTPase Rho regulates the aggregation. Incubation of the permeabilized platelets with Rho GDP-dissociation inhibitor, an inhibitory regulator for Rho family GTPases, extracted Rho family proteins extensively from the plasma and intracellular membranes, and inhibited the thrombininduced aggregation. Incubation of the permeabilized platelets with botulinum exoenzyme C3, which specifically inhibits Rho function by ADP-ribosylating it, abolished the thrombin-induced aggregation. Thus, Rho is involved in thrombin-induced aggregation of platelets. © 2001 Academic Press Key Words: platelet; thrombin; aggregation; Rho; RhoGDI; C3.

Platelets play essential roles in hemostasis and thrombosis by aggregating with each other (1, 2). Platelet aggregation is mediated by integrin ␣IIb␤3, which becomes to bind both fibrinogen and von Willebrand factor upon platelet activation (1, 2). However, Abbreviations used: GDI, GDP-dissociation inhibitor; GST, glutathione-S transferase; TRAP; thrombin receptor-activating peptide; SDS–PAGE, sodium dodesylsulfate-polyacrylamide electrophoresis; BSA, bovine serum albumin; SLO, streptolysin-O; NAD; nicotinamide adenine dinucleotide; LDH; lactate dehydrogenase. 1 Present address: Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy. 2 To whom correspondence should be addressed at Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 606-8507, Japan. Fax: ⫹81-75-751-3574. E-mail: horiuchi@kuhp. kyoto-u.ac.jp.

0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

the molecular mechanism of the process is not yet fully understood. Rho family GTPases such as Rho, Rac, and Cdc42 have been shown to play important roles in cytoskeletal reorganization (3–5). Similar to the cases of other small GTPases, the function of Rho GTPases are regulated by their GDP/GTP cycle and GTP-bound active forms of Rho GTPases convey signals through interaction with their effector molecules (3–5). Furthermore, Rho GTPases have a unique regulator named Rho GDP-dissociation inhibitor (GDI) 1, which forms a stable complex with Rho GTPases in their inactive GDPbound forms, extracts them from the membranes, and inhibits the GDP/GTP exchange (6 – 8). Therefore, RhoGDI is supposed to be a negative regulator of Rho family GTPases (3–5). In platelets, at present, it remains controversial whether Rho is involved in the regulation of platelet aggregation (9, 10). Morii et al. demonstrated that Rho regulates platelet aggregation since a specific inhibitor of Rho, botulinum exoenzyme C3 which ADPribosylates Asn41 in the effector domain of Rho (11), blocked the thrombin-induced aggregation (9). Notably, they could demonstrate strong inhibition of the aggregation by C3 at 50 ␮g/ml specifically using a Tris-based buffer, whereas a Hepes-based buffer showed weak inhibition (9). On the other hand, Leng et al. recently demonstrated that C3 did not inhibit platelet aggregation induced by thrombin receptoractivating peptide (TRAP) (10) although they used extremely high concentrations (200 – 400 ␮g/ml) of C3. Leng et al. speculated that the inhibitory effect of C3 demonstrated by Morii et al. (9) could be due to nonspecific effects of the Tris-based buffer (10). Here, we established an aggregation assay system using permeabilized platelets, where they preserved the thrombin-induced aggregation activity even after incubation of them for 15 min with reagents to be tested. With this assay, we demonstrated that Rho is involved in the regulation of thrombin-induced platelet

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aggregation by showing that treatment of the platelets with RhoGDI or C3 in a Hepes-based buffer blocked the aggregation.

RESULTS

MATERIALS AND METHODS

SLO binds to membrane at 4°C and forms pores when incubated at higher temperatures (20). We took advantage of this property of SLO to specifically permeabilize the plasma membrane of platelets. The permeabilization was confirmed by measuring the loss of cytosolic LDH from the cells, which was subsequently detected in the supernatant after removal of the platelets by centrifugation. The amounts of LDH recovered in the supernatant increased in an SLO-concentrationdependent manner (data not shown). We used 0.1 ␮g/ml SLO for the aggregation assay, with 5–10% loss of LDH during the permeabilization procedure. The Rho small GTPase family is composed of several members including Cdc42, Rac1, and Rho. In platelets, approximately 20% of RhoA, 50% of Cdc42, and little of Rac1 were detected in the membrane fraction of platelets while most of Rab5, a member of the Rab GTPase family known to regulate intracellular transport (21, 22), was detected in the membrane fraction (23) (Fig. 1A). Incubation of the permeabilized platelets with RhoGDI extracted RhoA and Cdc42 from the permeabilized platelets in a concentration-dependent manner and almost completely with 10 ␮M RhoGDI (Fig. 1B), inferring that recombinant RhoGDI entered the permeabilized platelets, where it extracted the RhoGTPases from the membrane by forming complexes.

Materials and methods. E. coli, expressing glutathione S-transferase (GST)-tagged RhoGDI (7) and His-tagged RabGDI (12) were kindly provided by Dr. Y. Takai, Osaka Univ., Japan, and Dr. M. Zerial, EMBL, Heidelberg, Germany, respectively. GST-RhoGDI and His-RabGDI were affinity-purified using glutathione-sepharose (Amersham-Pharmacia) and Ni-agarose beads (Qiagen), respectively, according to the manufacturer’s instructions, followed by dialysis against Buffer A (50 mM Hepes/KOH pH 7.4, 0.42 mM NaH 2PO 4, 11.9 mM NaHCO 3, 1 mM MgCl 2, 2 mM EGTA, 100 mM KCl, and 1 mM glucose) containing 1 mM CaCl 2. Purified GSTRab3A (13) (a kind gift from Dr. Y. Takai) was dialyzed against the same buffer. Botulinum exoenzyme C3 (14) was provided by Dr. S. Narumiya, Kyoto Univ., Japan, and Y27632 (15) from Welfide Corp., Japan. An anti-Rab5 monoclonal antibody was a gift of Dr. M. Zerial and anti-Cdc42, -Rac1, -RhoA, and -Rab3A rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology. Horseradish peroxydase-labeled anti-rabbit and -mouse IgG monoclonal antibodies were from Amersham, which were used as secondary antibodies for immunoblotting visualized by enhanced chemiluminescence method (Amersham). Unless otherwise specified, all the chemicals were purchased from Sigma, except for streptolysin-O (SLO) which was from Dr. Bhakdi, Mainz Univ., Mainz, Germany. Lactate dehydrogenase (LDH) was quantified with LDH-cytotoxic test kit (Wako Chemical, Osaka, Japan). Protein concentrations were determined by the Bradford’s method (16) (Bio Rad) or densitometric scanning of the Coomassie blue-stained band in SDS–PAGE gels (17), using bovine serum albumin (BSA) as a standard. For separation of platelet cytosol from the membrane fraction, low speed supernatant of sonicated platelets were further centrifuged at 100,000g for 30 min. The supernatant was used as the cytosolic fraction and the pellet as the membrane fraction. Comparable amounts were analyzed by Western blot analysis. Aggregation assay using the permeabilized platelets. Washed human platelets from healthy donors were prepared as previously described (18), resuspended in ice-cold Buffer A containing 20 ng/ml prostaglandin E 1 and 2 mM CaCl 2, which gave a calculated free [Ca 2⫹] concentration at 20 ␮M (19), and kept at 4°C until use. Then the platelets were permeabilized with Streptolysin-O (SLO) basically as described (12, 20) with some modifications. The platelets were incubated with 0.1 ␮g/ml SLO for 10 min at 4°C, washed once to remove unbound SLO and resuspended at a density of 8 ⫻ 10 7/ml, quantified with a Coulter Counter, in an ice-cold Buffer A containing 1 mM CaCl 2 at 200 nM free [Ca 2⫹] (19). Then the treated platelets were incubated at 37°C for indicated periods (5–15 min) in the presence or absence of tested materials without stirring. Then, the permeabilized platelets were stimulated with indicated concentration of thrombin with stirring at 37°C and the aggregation was measured with a light transmission aggregometer, MCM HEMA TRACER 212 (MC Medical). The results shown were the representative of four independent experiments with similar results. ADP-ribosylation of Rho by C3 exoenzyme. Platelets permeabilized with 0.1 ␮g/ml SLO were incubated with [ 32P]nicotinamide adenine dinucleotide (NAD) in the presence of indicated concentrations of C3 at 37°C for 15 min. In another set of experiments, the permeabilized platelets were first incubated with C3 for 15 min in the absence of [ 32P]NAD followed by incubation with [ 32P]NAD and 53 ␮g/ml C3 for 15 min. The samples were analyzed by SDS–PAGE followed by autoradiography. The results shown were the representative of four independent experiments with similar results.

The Thrombin-Induced Aggregation Assay Using SLO-Permeabilized Platelets

RhoGDI Inhibited the Thrombin-Induced Platelet Aggregation Platelets permeabilized with 0.1 ␮g/ml SLO preserved the thrombin-induced aggregation activity even after preincubation at 37°C for 5–15 min (Figs. 1– 4). Incubation with 10 ␮M RhoGDI at 37°C for 5 min extracted most of RhoA and Cdc42 from the membrane (Fig. 1B), and caused complete inhibition of the 0.1 U/ml thrombin-induced aggregation (Fig. 1C). The inhibition was abolished when the RhoGDI sample was boiled at 95°C for 10 min (Fig. 1C), which could be due to the heat-sensitive nature of recombinant RhoGDI (6). On the other hand, 10 ␮M RabGDI, which is a regulator for the Rab GTPase family known to regulate vesicle transport, had no effect on the aggregation (Fig. 1C), suggesting that RabGTPases are not involved in the thrombin-induced platelet aggregation. GSTtagged Rab3A, a protein involved in neurotransmitter release and not detected in platelets (data not shown), at 10 ␮M had no effects on the aggregation (Fig. 1C). The results suggested that Rab3A was not involved in the aggregation and that the RhoGDI’s effect was not due to its tag, GST. Furthermore, RhoGDI at 10 ␮M had no effect on the aggregation of intact platelets

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FIG. 1. Incubation of permeabilized platelets with RhoGDI extracted RhoGTPases and inhibited the thrombin-induced aggregation. (A) Comparable amounts of the membrane and cytosolic fractions of the platelets were immunoblotted with anti-Rab5, -RhoA, -Rac1, and -Cdc42 antibodies. (B) After the permeabilized platelets were incubated with indicated concentrations of RhoGDI for 37°C for 5 min, the platelets were collected by centrifugation. The platelet-associated RhoA and Cdc42 were detected by immunoblotting. (C) The permeabilized platelets incubated with 10 ␮M RhoGDI, RhoGDI boiled at 95°C for 10 min, BSA, His-RabGDI, and GST-Rab3A at 37°C for 5 min were then stimulated with 0.1 U/ml thrombin and the aggregation was analyzed. The vertical bar indicates a 10% aggregation response.

(data not shown), suggesting that RhoGDI exerted its function from within platelets. These results suggested that Rho family protein(s) are involved in the regulation of platelet aggregation. Interestingly, under conditions where RhoGDI completely blocked the aggregation induced by 0.1 U/ml thrombin, the inhibition was partial when the RhoGDI-treated platelets were stimulated with a stronger stimulus of 0.5 U/ml thrombin (Fig. 2).

min in the absence of [ 32P]NAD, very little Rho in the platelets was further [ 32P]ADP-ribosylated by incubation with 53 ␮g/ml C3 and [ 32P]NAD (Fig. 3A, lane (II)). Judging from these results, most of Rho in the permeabilized platelets was supposed to be [ 32P]ADPribosylated after incubation with 10 and 50 ␮g/ml C3 at 37°C for 15 min (Fig. 3A). Under these conditions,

C3, a Rho Specific Inhibitor, Inhibited the ThrombinInduced Platelet Aggregation We next examined whether the C3 exoenzyme, a specific inhibitor of Rho (14), affects the thrombininduced aggregation of the permeabilized platelets. When the permeabilized platelets were incubated with C3 and [ 32P]NAD, a single band of [ 32P]ADPribosylated Rho was detected around 20 kD in the autoradiography of the sample (data not shown), as demonstrated previously (14). C3 ADP-ribosylated Rho in the permeabilized platelets in a time- (data not shown) and a concentration-dependent manner (Fig. 3A, lane (I)). Furthermore, after incubation of the permeabilized platelets with C3 at 10 and 50 ␮g/ml for 15

FIG. 2. Platelet aggregation induced by 0.1 U/ml thrombin was completely inhibited by the RhoGDI treatment, but that induced by 0.5 U/ml thrombin partially. The permeabilized platelets incubated with RhoGDI at 10 ␮M at 37°C for 5 min were then stimulated with 0.1 U/ml or 0.5 U/ml thrombin and the aggregation was analyzed. The vertical bar indicates a 10% aggregation response.

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inhibit the thrombin-induced aggregation both in intact platelets (data not shown) or SLO-treated permeabilized platelets (Fig. 4). Thus, Rho kinase/ROCK might not be involved in the thrombin-induced platelet aggregation. DISCUSSION

FIG. 3. Low, but not high, concentrations of C3 inhibited the thrombin-induced aggregation. (A) The permeabilized platelets were incubated with indicated concentrations of C3 with [ 32P]NAD at 37°C for 15 min (lane (I)) or with indicated concentrations of C3 for 15 min without [ 32P]NAD followed by incubation with [ 32P]NAD and 53 ␮g/ml C3 for 15 min (lane (II)). The samples were analyzed by SDS–PAGE followed by autoradiography and [ 32P]ADP-ribosylated Rho was shown. (B) The permeabilized platelets incubated with indicated concentrations of C3 at 37°C for 15 min were then stimulated with 0.1 U/ml thrombin and the aggregation was analyzed. The vertical bar indicates a 10% aggregation response.

treatment of the permeabilized platelets by 1 ␮g/ml C3, which ADP-ribosylated approximately 70% of Rho (Fig. 3A), partially inhibited the thrombin-induced aggregation (Fig. 3B). Then, treatment of them by 10 ␮g/ml C3 completely blocked it (Fig. 3B). These results suggested that Rho is involved in the thrombin-induced platelet aggregation. Most surprisingly, treatment of the permeabilized platelets by C3 at high concentrations (50 and 100 ␮g/ml) did not inhibit the aggregation (Fig. 3B), although C3 at 50 ␮g/ml ADP-ribosylated most of Rho as C3 at 10 ␮g/ml (Fig. 3A). In addition, C3 exoenzyme alone at 100 ␮g/ml did not cause the platelet aggregation without thrombin-stimulation (data not shown).

It is controversial whether small GTPase Rho regulates platelet aggregation (9, 10). Here, we established an in vitro aggregation assay using SLO-treated permeabilized platelets and demonstrated that small GTPase Rho is involved in the regulation of thrombininduced aggregation. In the assay, we were able to incubate the permeabilized platelets for 15 min at 37°C prior to thrombin stimulation without losing the aggregation activity. Although several aggregation assay systems utilizing permeabilized platelets have been established (26 –28), as far as we know, there have been no assays which allow such preincubation with exogenous reagents before stimulation. Therefore, our assay system has great advantages for analyzing the molecular mechanism of the platelet aggregation. The preincubation allowed exogenously-added recombinant RhoGDI and C3 to enter the permeabilized platelets and exert their functions. Most of membranebound RhoA and another Rho family member Cdc42 were extracted during preincubation with RhoGDI (Fig. 1B), indicating that the pores in the plasma membrane were large enough for protein transport across the plasma membrane. Furthermore, the RhoGDI treatment of the permeabilized platelets inhibited the thrombin-induced aggregation (Figs. 1C and 2), indicating that Rho family protein(s) regulates the aggregation. Previous experiments have examined the effects of the Rho by using a Rho specific inhibitor, C3 ADP-

A Rho-Kinase/ROCK Inhibitor Did Not Affect the Thrombin-Induced Platelet Aggregation So far, several molecules have been identified as effector molecules of Rho including Rho-kinase/ROCK (5, 24). Since Klages et al. have shown that a Rhokinase/ROCK inhibitor, Y27632, inhibited the thromboxane A2-induced platelet aggregation (25), we next examined whether Rho-kinase/ROCK was involved in the thrombin-induced platelet aggregation using Y27632. In our assay, Y27632 at 1–100 ␮M did not

FIG. 4. Y27632, a Rho-kinase/ROCK inhibitor, did not affect the thrombin-induced platelet aggregation. The permeabilized platelets incubated with indicated concentrations of Y27632 at 37°C for 15 min were then stimulated with 0.1 U/ml thrombin and the aggregation was analyzed. The vertical bar indicates a 10% aggregation response.

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ribosyltransferase (29 –31), on platelet aggregation (9, 10). Morii et al. have shown that treatment of platelets with 50 ␮g/ml C3 for 2 h at 37°C ADP-ribosylated less than 25% of Rho, resulting in blocking the thrombininduced aggregation in a Tris-based buffer (9). On the other hand, Leng et al. recently demonstrated that the C3-treatment at extremely high concentrations (200 – 400 ␮g/ml) in a Hepes-based buffer for 4 h at 37°C ADP-ribosylated more than 90% of Rho resulting in no inhibition of TRAP (thrombin receptor activated protein)-induced platelet aggregation (10). With the results, they speculated that the inhibitory effect of C3 shown by Morii et al. (9) could be due to the Tris-based buffer used in the study by Morii et al. (10). In the present study, we used a Hepes-based buffer and showed that the C3-treatment inhibited the thrombin-induced platelet aggregation, demonstrating that the inhibition was not due to the Tris-based buffer. Furthermore, we observed that C3 at low concentrations (1–10 ␮g/ml) efficiently ADP-ribosylated Rho and inhibited the thrombin-induced platelet aggregation in a concentration-dependent manner (Fig. 3B). The concentrations of C3 used in this work were much lower than those used previously (9, 10), probably because C3 easily entered the permeabilized platelets through the pores in the plasma membrane. C3 at 10 ␮g/ml caused almost complete ADP-ribosylation of Rho and inhibition of the thrombin-induced aggregation. The results strongly suggested that Rho is involved in the regulation of the aggregation. To our surprise, 50 –100 ␮g/ml C3 did not inhibit the aggregation (Fig. 3B) although almost complete ADPribosylation of Rho was observed by C3 at 50 ␮g/ml. Even though high concentrations of C3 did not inhibit platelet aggregation, this observation must be balanced with the clear concentration-dependent effect of C3 at lower concentrations. Klages et al. also investigated the effect of C3 on thromboxane A2-induced early activation of platelets and observed that platelet shape change and aggregation were affected by C3treatment (25). However, they did not make any conclusions concerning the involvement of Rho in the platelet aggregation (25), probably because they observed unexpected activation of platelets after incubation of platelets with a high concentration of C3 for long periods (25). Our observation that high concentrations of C3 did not inhibit the thrombin-induced aggregation could be due to such unexpected activation of platelets associated with high concentrations of C3. Since most of Rho was ADP-ribosylated by 50 ␮g/ml C3 to the similar level by 10 ␮g/ml C3 (Fig. 3A), the effect of the high concentration of C3 might not be mediated by Rho. However, C3 did not seem to cause platelet aggregation directly since C3 alone even at high concentrations could not induce the aggregation without thrombin stimulation (data not shown). Taken to-

gether, we concluded that Rho is involved in the regulation of the thrombin-induced platelet aggregation. Further investigation is required to determine whether other member(s) of Rho family such as Cdc42 is also involved in the regulation of the aggregation. So far, several molecules have been identified as effector molecules of Rho (5, 24). Among them, Klages et al. demonstrated that Rho kinase/ROCK could regulate thromboxane A2-induced platelet shape change and aggregation by experiments using its specific inhibitor Y27632 (25). Furthermore, Suzuki et al. have shown that Rho kinase/ROCK regulates myosinphosphatase activity in platelets (32). However, we observed that Y27632 did not inhibit the thrombininduced platelet aggregation (Fig. 4), suggesting that it is mediated by other effector molecule(s) of Rho. The discrepancy of Y27632’s effects in response to thromboxane A2 and thrombin could be due to distinct intracellular signaling pathways. For example, while the thromboxane A2 signal is mediated through Gq (25), the thrombin signal is through both Gi and Gq (2). Further investigation is required to elucidate how Rho regulates the thrombin-induced platelet aggregation. ACKNOWLEDGMENTS We are grateful to Dr. S. Narumiya and Dr. Y. Takai for valuable discussion and providing materials, to Dr. Y. Takai and Dr. H. McBride for critical reading of the manuscript, to Welfide Corp. for providing Y27632, and to Ms. T. Matsubara for technical assistance. This work was supported by Research Grants from Ministry of Education, Science, Sports, and Culture, Japan (Nos. 11158208, 11680629, and 10215208 to H.H. and 09281104, 09281103, 11694266, and 11307018 to T.K.), and partially Research Grants by grants from Tanabe Medical Frontier Conference, Takeda Science Foundation, and Study Group of Molecular Cardiology to H.H.

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