©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endogenous Cleavage of Phospholipase C-3 by Agonist-induced Activation of Calpain in Human Platelets (*)

(Received for publication, August 10, 1994; and in revised form, October 24, 1994)

Yoshiko Banno (1) Shigeru Nakashima (1) Takahisa Hachiya (2) Yoshinori Nozawa (1)(§)

From the  (1)Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500, Japan and the (2)Medical and Biological Laboratories Co., Ltd., Naka-ku, Nagoya 460, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two membrane-associated phosphoinositide-specific phospholipase Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were activated by brain G-protein beta subunits have been isolated from human platelets. The truncation of mPI-PLC-1 that was mediated by µ-calpain induced much higher activation by beta subunits (Banno, Y., Asano, T., and Nozawa, Y.(1994) FEBS Lett. 340, 185-188). On the basis of size and immunological cross-reactivity, mPI-PLC-1 (155 kDa) was PLC-beta3, and mPI-PLC-2 (100 kDa) was its truncated form. The cPI-PLC (140 kDa) was recognized by the antibody selective for internal sequences of PLC-beta3 but not by the antibody raised against its carboxyl terminus, indicating that it may be related to PLC-beta3.

Treatment of human platelets with A23187 and dibucaine, activators of calpain, caused cleavage of actin-binding protein and talin in a time-dependent manner. At the same time, decrease of PLC-beta3 (155 and 140 kDa) and concomitant increase of the 100-kDa product of cleavage were observed on immunoblots with the antibody to internal sequences of PLC-beta3. Furthermore, stimulation of platelets by natural agonists, thrombin and collagen, caused the cleavage of PLC-beta3 (155 and 140 kDa) and an increase of 100 kDa PLC-beta3 in a time- and dose-dependent manner. The cleavage of these PLC-beta3 enzymes was completely blocked by calpain inhibitor, calpeptin, indicating that the PLC-beta3 modification may be a consequence of platelet activation leading to activation of calpain. This is the first demonstration that PLC-beta3 is indeed cleaved by calpain upon platelet activation by physiological agonists. The cleavage of PLC-beta3 evoked by thrombin and collagen but not ADP was correlated with irreversible aggregation, suggesting that the PLC-beta3 modification may play a role in secondary irreversible aggregation in agonist-stimulated human platelets.


INTRODUCTION

Phosphoinositide-specific phospholipase C (PI-PLC) (^1)is an important signal-transducing enzyme to generate two second messengers, inositol trisphosphate and diacylglycerol(1, 2, 3) . There are nine distinct isozymes in total (beta1, beta2, beta3, beta4, 1, 2, 1, 2, and 3)(4, 5, 6, 7) . The PI-PLC isozymes are activated by differential mechanisms upon receptor stimulation. Tyrosine phosphorylation is thought to be implicated in the activation of PLC- isozymes(8, 9, 10) . Receptors containing seven membrane-spanning domains have been known to couple to effector enzymes via guanine nucleotide-binding protein (G-protein)(11) . Recent investigations provide evidence that receptor-mediated activation of PLC-beta isozymes is caused by two distinct mechanisms, one through alpha subunits of the G(q) family insensitive to pertussis toxin, and the other through the beta subunits of pertusis toxin-sensitive G-proteins(12, 13, 14, 15, 16, 17) . Both G(q)alpha and beta subunits activate PLC-beta isozymes to different extents. The alpha subunits of the G(q) family activate PLC-beta isozymes in the order of PLC-beta1 > PLC-beta3 > PLC-beta2, whereas beta subunits stimulate PLC-beta isozymes in the order of PLC-beta3 > PLC-beta2 > PLC-beta1(18, 19) .

It has been known that in human platelets multiple forms of PI-PLC (beta, 1, 2 and ) were present in cytosol and membrane fractions(20, 21) . We previously demonstrated that membrane-associated platelet PI-PLC activity was enhanced by either GTPS-activated G(i) or G(o) to nearly the same extent(22) . This (22) and other recent findings (23, 24) suggest that beta subunits of heterotrimeric G-proteins may be involved in the PI-PLC activation in human platelets. Furthermore, it was recently shown that truncated PLC-beta isozyme was found to be activated remarkably by G-protein beta subunits(25) . We have also demonstrated enhancement of the cleaved PLC-beta activity by beta subunits and suggested that truncation of the PLC-beta by µ-calpain greatly enhanced its activation by G-protein beta subunits in vitro(26) . In human platelets, agonist-induced activation of calpain (27, 28, 29) and limited proteolysis of some specific substrates have been known to occur in the course of a platelet activation(30, 31, 32, 33) .

In this study, we have demonstrated by using specific antibody that stimulation by physiological agonists of human platelets evoked the cleavage of PLC-beta3 to a 100-kDa truncated form via calpain activation. We then propose that the PLC-beta3 is an endogenous substrate for calpain in human platelets and that the limited proteolysis would be a prerequisite for activation by beta subunits. In addition, it has also been suggested that the PLC-beta3 modification may be implicated in secondary irreversible aggregation.


EXPERIMENTAL PROCEDURES

Materials

[^3H]Phosphatidylinostiol 4,5-bisphosphate (PIP(2), 8.8 Ci/mmol) was obtained from DuPont NEN. Thrombin was obtained from Mochida Pharmaceutical Company (Tokyo, Japan). A23187, collagen, and dibucaine were purchased from Sigma. E-64 was from Peptide Institute Inc. (Osaka, Japan). Calpeptin was kindly provided by Dr. Jun-ichi Kambayashi (Osaka University Medical School, Japan).

Assay for PI-PLC Activity and Activation by G-protein beta Subunits

PLC activity was determined as described previously, using [^3H]PIP(2) (20,000 dpm), PIP(2) (0.1 mM)/phosphatidylethanolamine (1.0 mM) as substrate(26) . The activation of PI-PLC by G-protein beta subunits was examined as described by Smrcka and Sternweis(19) . The reaction mixture (60 µl) contained 50 mM Na-Hepes (pH 7.2), 0.17 mM EDTA, 3 mM EGTA, 1 mM dithiothreitol, 17 mM NaCl, 0.7 mM KCl, 0.83 mM MgCl(2), 1.5 mg/ml bovine serum albumin, and 1.5 mM CaCl(2) to give 100 nM free Ca concentration. The bovine brain G-protein beta subunits (200 nM)were solubilized in 0.3% octyl beta-D-glucopyranoside and added in the reaction mixture (final concentration, 0.05% octyl beta-glucopyranoside and 0.01% cholate).

Isolation of PI-PLCs Activated by G-protein beta Subunits from Human Platelet Membrane and Cytosol

Membrane-associated PI-PLC

The cytosolic and membrane fractions were prepared from outdated human platelet concentrates as described previously(34) . The membrane-associated PI-PLC (mPI-PLC) enzymes were purified as described previously(34) . Briefly, the membrane fraction was suspended in buffer A (20 mM Tris-HCl, pH 7.4, 5 mM EGTA, 1 mM dithiothreitol, various protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml E-64) containing 1% sodium cholate, stirred for 2 h at 4 °C, and centrifuged at 100,000 times g for 60 min. The cholate extract was dialyzed against buffer B (20 mM Tris-HCl, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 1 mM EDTA, and various protease inhibitors) containing 0.5% cholate. The dialyzed solution was applied onto a Fast Q-Sepharose column (0.1 - 0.6 M). Two activity peaks were resolved. The main activity peak, which could be activated by 200 nM brain G-protein beta subunits, was eluted at 0.25-0.35 M NaCl, pooled, and dialyzed against buffer B. The dialysate was applied onto a heparin-Sepharose column and eluted with a linear NaCl gradient (0.1-0.7 M) in buffer B. Three activity peaks were resolved; the first peak eluted at 0.2-0.3 M NaCl, the second peak eluted at 0.35-0.45 M NaCl, and the third peak eluted at 0.5-0.65 M NaCl. The third peak, which was considerably activated by G-protein beta subunits, was pooled and concentrated in an Amicon concentrator. The solution was loaded on an Ultrogel AcA-44 column and eluted with buffer B containing 0.3 M NaCl. The activity fractions were pooled and dialyzed against buffer C (20 mM Tris-HCl, pH 7.4, and 1 mM dithiothreitol). The dialysate was applied onto a hydroxyapatite HCA-100S column and eluted with a linear gradient of potassium phosphate (0.1-0.5 M). The activity fractions eluted with 0.3-0.35 M potassium phosphate were pooled and applied onto a Mono Q HPLC column (HR 5/5) equilibrated with buffer C, which was then eluted at a flow rate of 1 ml/min with NaCl gradients from 0 to 0.2 M for 5 min, from 0.2 to 0.32 M for 30 min, and from 0.32 to 0.7 M for 5 min. Two activity peaks were resolved. A major activity peak activated by G-protein beta subunits was eluted at 0.23-0.26 M NaCl, and this activity fraction was concentrated and applied onto a Superose 12 column. The two activity peaks (mPI-PLC-1 and mPI-PLC-2) were resolved and separately pooled.

Cytosolic PI-PLC

The cytosolic fraction of human platelets was loaded onto a Fast Q-Sepharose column equilibrated with buffer A. After washing with the same buffer, elution was performed with a linear concentration gradient from 0.1 to 0.4 M NaCl in buffer A. A major activity peak, which was activated by G-protein beta subunits, was eluted between 0.25 and 0.35 M NaCl, pooled, and concentrated. The concentrated solution was applied onto an Ultrogel AcA-44 column equilibrated with buffer B containing 0.3 M NaCl and eluted with the same buffer. The activity fractions were collected and dialyzed against buffer B. The dialysate was applied onto heparin-agarose HPLC column and eluted with a linear NaCl gradient (0.1-0.8 M) for 40 min at a flow rate of 1 ml/min. The activity peak (cPI-PLC) was eluted between 0.4 and 0.7 M NaCl, pooled, and concentrated.

The G-protein beta subunits from bovine brain were purified as described previously(35) .

Protein concentrations were routinely determined with the Bio-Rad protein assay kit using bovine serum albumin as standard.

Platelet Preparation

Human blood was drawn by venepuncture into volume of 3.8% trisodium citrate and gently mixed. Platelet-rich plasma was prepared by centrifuging the whole blood at 200 times g for 20 min and aspirating platelet-rich plasma. Hirudin (0.1 unit/ml) and apyrase (2 units/ml) were added to platelet-rich plasma. The platelet-rich plasma was layered on 40% bovine serum albumin and spun at 800 times g for 20 min to form a soft platelet pellet. The pellet was resuspended in 1 ml of a modified Hepes-Tyrode buffer (129 mM NaCl, 8.9 mM NaHCO(3), 0.8 mM KH(2)PO(4), 0.8 mM MgCl(2), 5.6 mM dextrose, and 10 mM Hepes, pH 7.4). The platelet suspension was layered onto a Sepharose 2B gel column equilibrated with calcium-free modified Hepes-Tyrode buffer and eluted with the same buffer. Platelet concentration was adjusted to 5 times 10^8 cells/ml.

Gel Electrophoresis and Western Blotting

Platelet suspension was added to 1 mM calcium with or without calpeptin (30 µM) and incubated at 37 °C for 5 min. To activate calpain, platelets were treated with A23187 or dibucaine in Hepes-Tyrode buffer containing 1 mM calcium and 0.1% dimethyl sulfoxide. Platelets were stimulated with thrombin or collagen in Hepes-Tyrode buffer containing 1 mM calcium with stirring at 37 °C. Platelet stimulation was terminated by the addition of 3 times concentrated Laemmli's sample buffer (36) containing 10 mM EGTA and 10 mM EDTA. After boiling for 5 min, SDS electrophoresis was carried out with 6% polyacrylamide gel. To examine cleavage of actin-binding protein and talin, the gel was stained with Coomassie Brilliant Blue, destained, and dried. To examine cleavage of PLC-beta3, separated proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane. To block residual protein binding sites, nitrocellulose membrane was incubated in a Tris-buffered saline (TBS) buffer containing 0.05% Tween-20 and 5% casein. The blots were washed with TBST (TBS with 0.2% Tween-20) and incubated with antibodies. The primary antibody was removed and the blots were washed in TBST. To detect antibody reaction, the blots were incubated with peroxidase-conjugated second antibody, washed in TBST, and detected with ECL detection.

Antibodies

Antisera were raised against the peptides corresponding to the partial sequences of PLC-beta3 as reported previously(19, 37) . The antibody (Ab-beta3 M) to peptides corresponding to PLC-beta3 amino acid residues 550-561 (TDPKKPTTDEGT) and the antibody (Ab-beta3C) to residues 1202-1217 (HLSGADSESQEENTQL) were generated in rabbits by injection of the synthetic peptides that had been conjugated to keyhole limpet hemocyanin with glutaraldehyde. Antisera were purified by using CNBr-activated agarose gel bounded with synthetic peptides. Antibodies to the sequence common to X regions 338-347 (GCRCVELDCW) and the sequence common to Y regions 626-633 (LSRIYPKG) were prepared as described(37) . Polyclonal antibodies to PLC-beta1, PLC-beta2, and PLC-beta4 were obtained from Santa Cruz Biotechnology, Inc.


RESULTS

Western Blot Analysis of the Isolated Platelet PI-PLCs Activated by G-protein beta Subunits

Our previous studies demonstrated that a truncated PI-PLC isolated from the human platelet membrane fraction was activated by brain G-protein beta subunits to a much higher extent than the intact enzyme(26) . Furthermore, incubation of the intact PI-PLC with µ-calpain caused a marked enhancement of PIP(2)-hydrolyzing activity by beta subunits in a time- and dose-dependent manner, suggesting that the truncation of the intact PI-PLC may be mediated by µ-calpain. We also found that a cytosolic PI-PLC was also activated by beta subunits. To examine whether the truncation of the PI-PLC by calpain occurs in human platelets, as a first step we attempted to identify these isolated platelet PI-PLC enzymes. On the gel permeation chromatography of a Superose 12 column, the membrane-associated PI-PLC was resolved into two activity peaks: mPI-PLC-1 with higher molecular mass and mPI-PLC-2 with lower molecular mass. A cytosolic PI-PLC (cPI-PLC) that was activated by G-protein beta subunits was also partially purified. Three PI-PLC enzymes (mPI-PLC-1, mPI-PLC-2, and cPI-PLC) were increased in the PIP(2)-hydrolyzing activity by G-protein beta subunits. The mPI-PLC-2 was markedly activated by beta subunits (25-fold activation compared with 5.7-fold for mPI-PLC-1 and 6.0-fold for cPI-PLC). (^2)In order to identify these PI-PLC enzymes, they were subjected to Western blot analysis with various antibodies raised against PI-PLC isozymes, but they were not recognized with monoclonal antisera to PLC-1 and -1. The membrane-associated mPI-PLC-1, mPI-PLC-2, and cytosolic cPI-PLC were analyzed by Western blot with antibodies to other PI-PLC subtypes. The mPI-PLC-1 with 155-kDa molecular mass was found to be strongly immunoreactive with the antibody Ab-beta3C that recognizes the sequence corresponding to amino acid residues 1206-1217 in the COOH terminus of PLC-beta3 (Fig. 1A, lane 1) and also reacted with the antibody Ab-beta3M to the sequence of residues 550-561 located between the X and Y domain in PLC-beta3 (Fig. 1A, lane 2). The mPI-PLC-1 was also strongly reacted with an antibody to the conserved amino acid sequence in the Y domain of mammalian PI-PLCs (Ab-Y) (Fig. 1A, lane 3) and weakly reacted with an antibody to the conserved amino acid sequence in the X domain of mammalian PI-PLCs (Ab-X) (Fig. 1A, lane 4). The mPI-PLC-2 was immunoreactive with the Ab-beta3M of 100 kDa on the SDS-polyacrylamide gel (Fig. 1B, lane 2). It was also reactive with Ab-Y and Ab-X (Fig. 1B, lanes 3 and 4) but not with the Ab-beta3C (lane 1), indicating that the mPI-PLC-2 was a truncated form of the COOH-terminal region of intact platelet PLC-beta3. The platelet cytosolic cPI-PLC was strongly immunoreactive with Ab-beta3M with a molecular mass of 140 kDa on SDS-polyacrylamide gel (Fig. 1C, lane 2). However, it was not recognized by the Ab-beta3C (Fig. 1C, lane 1). The cPI-PLC was recognized with Ab-X but not with Ab-Y (Fig. 1C, lane 3 and 4). All three enzyme forms were not recognized by the antibody raised against PLC-beta1 (Fig. 1, A, B, and C, lane 5). These results indicated that mPI-PLC-1 (150-kDa enzyme) was immunoreactive with four antibodies (Ab-beta3C, Ab-beta3M, Ab-Y, and Ab-X); mPI-PLC-2 (100-kDa enzyme) reacted with Ab-beta3M, Ab-Y, and Ab-X but not with Ab-beta3C; and cPI-PLC (140-kDa enzyme) was recognized by Ab-beta3M and Ab-X but not by Ab-beta3C and Ab-Y.


Figure 1: Immunoblot analysis of isolated mPI-PLC-1, mPI-PLC-2, and cPI-PLC. The isolated mPI-PLC-1 (A), mPI-PLC-2 (B), and cPI-PLC (C) were subjected to SDS-6% polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with the various antibodies. Lane 1, anti-PLC-beta3 COOH-terminal domain (Ab-beta3C); lane 2, anti-PLC-beta3 peptides(550-561) (Ab-beta3M); lane 3, anti-PLC-Y domain (Ab-Y); lane 4, anti-PLC-X domain (Ab-X); lane 5, anti-PLC-beta1 (Ab-beta1). Arrows indicate molecular mass standards (from top): 205 kDa (myosin), 112 kDa (beta-galactosidase), 79.5 kDa (bovine serum albumin)



Limited Proteolysis of PLC-beta3 by Calpain Activation in Human Platelets

In order to know whether cleavage of PLC-beta3 occurs in response to calpain activation, we have used two pharmacological activators of calpain, A23187 and dibucaine. Human platelets were stimulated with A23187 in the presence and absence of extracellular calcium or a specific and membrane-permeable calpain inhibitor, calpeptin(38) . Fig. 2A shows that A23187 (1 µM) induced time-dependent cleavage of actin-binding protein and talin, which are known to be substrates for calpain in human platelets(27, 28) . The cleavage of these cytoskeletal proteins was completely inhibited by the removal of extracellular calcium (addition of 3 mM EGTA) or by the calpain inhibitor, 30 µM calpeptin, suggesting that the cleavage of these proteins was caused by calpain activation by A23187. At the same time, the 155-kDa species of PLC-beta3 decreased in a time- and dose-dependent manner when examined by the antibody to Ab-beta3C against the COOH terminus of PLC-beta3, with a concomitant increase of lower molecular mass band (100 kDa) immunoreactive with Ab-beta3M against the peptide between X and Y of PLC-beta3 (Fig. 2, B and C). Western blotting with Ab-beta3M showed that the 140-kDa band was also reduced time- and dose-dependently upon A23187 stimulation of platelets. The decreases of both the 155- and 140-kDa bands and production of the 100-kDa band caused by A23187 were completely blocked in the presence of EGTA (3 mM) or by pretreatment with 30 µM calpeptin. Cleavage by calpain rather than some other modification of PLC-beta3 was suggested by the fact that the lower band (100 kDa) was recognized by Ab-beta3M but not by Ab-beta3C.


Figure 2: A23187-induced cleavage of ABP, talin, and PLC-beta3. A, platelet suspension (5 times 10^8 cells/ml) was incubated with A23187 (1 µM) for the indicated periods with stirring. Calcium (1 mM), EGTA (3 mM), or calpeptin (30 µM) was added 5 min before the addition of A23187. Platelets were lysed by the addition of 3 times concentrated SDS sample buffer, containing 10 mM EGTA and 10 mM EDTA. Whole platelet lysates (10 µg of protein) were analyzed by SDS-PAGE (6%). The results of Coomassie Blue staining are shown. B, the platelet lysates from A were separated by SDS-PAGE, and proteins were transferred to nitrocellulose. Ab-beta3C and Ab-beta3M were used to detect PLC-beta3. C, platelets were stimulated with various concentrations of A23187 for 10 min and then lysed and analyzed for the cleavage of PLC-beta3 by Western blotting as in B.



Another calpain activator, dibucaine, also induced timedependent cleavages of actin-binding protein and talin (Fig. 3A) and also of PLC-beta3 (155 and 140 kDa) (Fig. 3, B and C). The PLC-beta3 cleavage induced by dibucaine (1 mM) was inhibited by 30 µM calpeptin, suggesting that the cleavage of PLC-beta3 induced by dibucaine was mediated through calpain activation. On the other hand, the presence of 3 mM EGTA could not completely block the cleavage of actin-binding protein, talin, and PLC-beta3 induced by dibucaine. This result was consistent with those of other studies, which showed that dibucaine is a direct activator of calpain(32) . The cleaved enzymes produced by dibucaine had a similar molecular mass (100 kDa) to that produced by A23187 and that the cleaved enzyme was recognized by Ab-beta3M but not by Ab-beta3C (Fig. 3, B and C).


Figure 3: Dibucaine-induced cleavages of ABP, taline, and PLC-beta3. A, platelets (5 times 10^8 cells/ml) were treated with dibucaine (1 mM) for the indicated periods in the presence of calcium (1 mM) or EGTA (3 mM) or after pretreatment of calpeptin (30 µM). Platelets were lysed and analyzed for cleavage of ABP and talin by Coomassie Blue staining. B, the platelet lysates of the same sample employed in A were analyzed by Western blotting as Fig. 2B. Ab-beta3C and Ab-beta3M were used to detect PLC-beta3. C, platelets were stimulated with various concentrations of dibucaine for 10 min and then lysed and analyzed for the cleavage of PLC-beta3 by using Ab-beta3C and Ab-BM.



Cleavage of PLC-beta3 in Agonist-stimulated Human Platelets

To examine whether cleavage of PLC-beta3 occurs in response to physiological agonists in human platelets, platelets stimulated by thrombin and collagen were subjected to SDS-PAGE and then analysis of immunoreactivity. Thrombin stimulation of human platelets caused PLC-beta3 cleavage in a time-dependent manner (Fig. 4). The PLC-beta3 cleavage in thrombin-stimulated platelets was completely blocked by depletion of extracellular calcium (addition of 3 mM EGTA) or by pretreatment with calpain inhibitor (30 µM calpeptin). The results suggest evidence that thrombin-induced PLC-beta3 cleavage was mediated through the activation of calpain. The level of the 100-kDa cleaved product was gradually increased during the 10 min after stimulation with a concurrent decrease of the 155- and 140-kDa protein bands, which were recognized by the Ab-beta3M, suggesting that the truncated 100-kDa PLC-beta3 was stable in platelets after stimulation with thrombin. This 100-kDa enzyme was recognized by Ab-X and Ab-Y as well as Ab-beta3M but not by Ab-beta3C, indicating that it arose as a result of cleavage at a single region between COOH-terminus and Y domain of PLC-beta3.


Figure 4: Thrombin-induced cleavage of PC-beta3. Platelets (5 times 10^8 cells/ml) in modified Hepes-Tyrode. buffer were treated with 1 unit/ml of thrombin for the indicated times with stirring. Calcium (1 mm), EGTA (3 mM), and calpeptin (30 µM) were added 5 min before stimulation. Total platelet lysates (10 µg of protein) were resolved by SDS-PAGE and immunoblotted with Ab-beta3C, Ab-beta3M, Ab-Y, and Ab-X.



Collagen, another agonist that causes activation of calpain(28) , also caused the proteolytic modification of PLC-beta3 in a time- and dose-dependent manner (Fig. 5, A and B); the 155- and 140-kDa PLC-beta3 were gradually decreased, and the 100-kDa form was concomitantly increased. The cleavage of the PLC-beta3 enzymes caused by collagen stimulation was also completely blocked by the addition of 3 mM EGTA and pretreatment with 30 µM calpeptin. These results again suggest that the truncation of PLC-beta3 was a consequence of platelet activation by which calpain was activated.


Figure 5: Collagen-induced cleavage of PLC-beta3. A, platelets (5 times 10^8 cells/mlin) in modified Hepes-Tyrode buffer were treated with 10 µg/ml of collagen for the indicated times with stirring. Calcium (1 mM), EGTA (3 mM), and calpeptin (30 µM) were added 5 min before stimulation. Total platelet lysates (10 µg of protein) were resolved by SDS-PAGE and immunoblotted with Ab-beta3C and Ab-beta3M. B, platelets (5 times 10^8 cells/ml) in modified Hepes-Tyrode buffer supplemented with 1 mM calcium were stimulated with various concentrations of collagen for 10 min and then lysed and analyzed for the cleavage of PLC-beta3 as in A.



As shown in Fig. 6, A and B, the cleavage of PLC-beta3 in response to various concentrations of thrombin was correlated with the extent of platelet aggregation. Platelets treated with thrombin (1 unit/ml), collagen (20 µg/ml), and A23187 (1 µM) caused irreversible aggregation (Fig. 7). On the other hand, platelet-activating factor caused reversible aggregation. When platelets were stimulated with weak agonists such as ADP in the absence of fibrinogen, they exhibited no aggregation. The platelet-activating factor and ADP did not evoke PLC-beta3 cleavage (data not shown). These results suggest that the PLC-beta3 cleavage may correlate to irreversible aggregation.


Figure 6: Cleavage of thrombin-induced PLC-beta3 and platelet aggregation. A, platelets (5 times 10^8 cells/ml) in modified Hepes-Tyrode buffer supplemented with 1 mM CaCl(2) were treated with various concentrations of thrombin for 10 min with stirring. Total platelet lysates (10 µg of protein) were resolved by SDS-PAGE and immunoblotted with Ab-beta3C and Ab-beta3M. B, platelet aggregations in A were measured in aggregometer and expressed as the percentage of maximum aggregation. Maximum aggregation is defined as aggregation seen 5 min after thrombin stimulation.




Figure 7: Platelet aggregation by various agonists. Platelets (5 times 10^8 cells/ml) in modified Hepes-Tyrode buffer supplemented with 1 mM CaCl(2) without fibrinogen were treated with various agonists (1 unit/ml thrombin, 20 µg/ml collagen, 1 µM A23187, 1 µM platelet-activating factor, 10 mM ADP) for 2 min. Aggregometer tracings are shown in ordinate displaying light transmission.




DISCUSSION

We have demonstrated here that agonist stimulation of human platelets induced cleavage of PLC-beta3 (155 and 140 kDa) to generate 100-kDa product as examined by immunoblot analysis. The cleavage of PLC-beta3 (155 and 140 kDa) was a consequence of platelet activation and resulted from an agonist-induced intracellular event that was concurrent with the activation of calpain. The truncated PLC-beta3 form (100 kDa) was isolated from platelet membrane fraction and was immunoreactive with Ab-beta3M, Ab-Y, and Ab-X but not with Ab-beta3C. Thus it was conceivable that a single cleavage of the PLC-beta3 occurred at the linkage between the carboxyl-terminal region and Y domain of the intact PLC-beta3. The truncated PLC-beta3 (100 kDa) was activated to a much higher extent by beta subunits of G-protein as compared with the intact PLC-beta3. The 155- and 140-kDa PLC-beta3 was detected in the resting human platelets by Ab-beta3M. The 155-kDa PLC-beta3 was isolated from the membrane fraction, and the 140-kDa enzyme was from the cytosolic fraction of human platelets. Agonist stimulation of human platelets caused cleavage of both PLC-beta3 enzymes at almost the same time, thereby producing 100-kDa enzyme. The cleavage was completely blocked by calpain inhibitor, calpeptin, suggesting that both enzymes share a sequence attacked by calpain. Although 140-kDa PLC-beta3 could be considered to be defective in the carboxyl-terminal region of 155-kDa PLC-beta3, there is no direct evidence that 140-kDa PLC-beta3 was indeed derived from 155-kDa enzyme by limited proteolysis. A recent study (39) has demonstrated that brain PLC-beta1 exists in two immunologically indistinguishable forms of 150 and 140 kDa, and analysis of PLC-beta1 genomic DNA indicated that PLC-beta1a (150 kDa) and PLC-beta1b (140 kDa) were derived from a single gene by alternative RNA splicing. These results could imply that the platelet 140-kDa PLC-beta3 may be generated by alternative splicing. However, the reason why the 140-kDa enzyme has lost the immunoreactivity to Ab-Y has remained unknown.

On the other hand, it has been reported that the PLC-beta1 was activated by the G(q)alpha class containing alphaq, alpha11, alpha16, and alpha14 (12, 13, 14) but that both PLC-beta2 and PLC-beta3 were efficiently activated by G-protein beta subunits(15, 16, 17, 18, 19) . Experiments using a series of specific deletions and truncations of PLC-beta1 have demonstrated that the region required for activation by G(q)alpha is localized to the sequence corresponding to residues 903-1142 of PLC-beta1(14) . The truncated 100-kDa PLC-beta1 produced by calpain could no longer be activated by G(q)alpha(40) . Furthermore, a 110-kDa PI-PLC purified from brain cytosol, a truncated form of PLC-beta3, was found to be markedly activated by beta subunits but not affected by G(q)alpha(25) . Lee et al.(41) showed that the PLC-beta2 mutants that lack the carboxyl-terminal were activated by beta subunits but not by G(q)alpha. It was also reported that PLC-beta3 but not PLC-beta1 was activated by alphaq/11 even in the presence of a saturating concentration of beta subunits(19) . These observations indicated that beta subunits could activate PLC-beta subtypes by interacting at a site different from that of G(q)alpha. In human platelets, G(i)2 is a major pertussis toxin substrate, whereas both G(i)1 and G(i)3 are much less so(42) . It is also shown that platelets express pertussis toxin-insensitive G-protein, G(q), which is coupled to the thromboxane A2 receptor(43) . Thus one would assume that G(i)2 may be responsible for the activation of PI-PLC in thrombin-stimulated platelets(44) . In Triton X-100 extraction of human platelets, PLC-beta2 and PLC-beta3 were immunodetectable by polyclonal antibodies to PLC-beta2 and PLC-beta3, respectively, but PLC-beta1 and PLCbeta4 were not undetectable. (^3)PLC-beta3 is reported to be activated by beta subunits of pertussis toxin-sensitive G-proteins as well as alpha subunit of the G(q) family(18, 19) . These results lead us to speculate that in thrombin-stimulated human platelets the beta subunits of G(i)2 may be involved in activation of PLC-beta2 and PLC-beta3.

Despite substantial evidence that human platelets contain high amounts of calpain (31, 46) and various substrates of calpain, such as platelet factor XIII(30) , actin binding protein, talin(47) , PI-PLC(48) , and protein kinase C(49) , were cleaved in vivo and in vitro, the physiological relevance of these cleavage events has remained obscure. Fox et al.(27, 28) demonstrated that agonist-induced calpain activation was involved in the shedding of procoagulant-rich microvesicles from aggregating platelets and also indicated that calpain activation was caused by signaling through the binding of integrin GpIIb-IIIa to adhesive ligand(50) . Recent studies have indicated that calpain activation and cleavage of specific endogenous protein substrates, such as pp60(32) and protein phosphotyrosine phosphatase 1B (33) were correlated with irreversible aggregation in human platelets. However, close association of calpain with platelet functions is still controversial, since it was shown from results using two inhibitors that calpain-mediated proteolysis in platelets is not an obligatory event leading to shape change, adhesion, aggregation, and 5-hydroxytryptamine release(51) .

As for PI-PLC, an earlier work has indicated that the higher molecular forms (400 and 270 kDa) of platelet cytosolic PI-PLC were converted into the 100-kDa form without substantial loss of activity by incubation with a calpain in vitro, suggesting that platelet PI-PLC enzymes were a substrate of calpain(48) . However, it was not shown whether PI-PLC(s) was endogenous substrate for activated calpain in platelets stimulated by natural agonists in vivo. There were some evidences suggesting activation of PI-PLC through agonist-induced calpain activation by using calpain inhibitors. Ishii et al.(52) demonstrated that thrombin-induced PI hydrolysis leading to inositol phosphate production as well as aggregation and secretion were inhibited by a membrane permeable thioprotease inhibitor, E-64d, suggesting that calpain, a major thiol protease of platelets, may participate in platelet functions through the activation of PI-PLC. Although these results provided a strong indication for a role of calpain in PI-PLC activation, the inhibitor was not specific for calpain in intact cells. On the other hand, Ariyoshi et al.(45) have observed in experiments using a specific potent calpain inhibitor, calpeptin, that its high concentration (300 µM) inhibited inositol 1,4,5-trisphosphate formation, aggregation, and [Ca](i) elevation in thrombin- or collagen-stimulated platelets, whereas its lower concentration (30 µM) did not affect these events. It was also shown that this amount of calpeptin (30 µM) was enough to inhibit the agonist-induced calpain activation.

Our studies demonstrated here that PLC-beta3 is an endogenous substrate for calpain in agonist-induced platelet activation and that cleavage of PLC-beta3 mediated by calpain activation occurs in the irreversible aggregation induced by potent agonists such as thrombin and collagen but not in reversible aggregation by weak agonists such as ADP in the absence of fibrinogen. The generation of the cleaved form of PLC-beta3 (100 kDa) induced by thrombin stimulation seems to correlate in time with calpain activation and irreversible aggregation, which occur 30 or 60 s after the addition of thrombin(50) . Thus, these observations lead us to consider that the modification of PLC-beta3 may play a role in the secondary irreversible aggregation of platelets.


FOOTNOTES

*
This study was supported by a grant-in-aid from the Ministry of Science, Culture, and Education of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 582-65-1241 (ext. 2228); Fax: 582-65-9002.

(^1)
The abbreviations used are: PI-PLC, phosphatidylinositol-specific phospholipase C; G-protein, GTP-binding protein; E-64d, (L-trans-epoxysuccinyl-leucylamide(3-methyl)butane) ethyl ester; TBS, Tris-buffered saline; TBST, Tris-buffered saline containing Tween-20; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; ABP, actin-binding protein; PIP(2), phosphatidylinositol 4,5-biphosphate; HPLC, high pressure liquid chromatography; mPI-PLC, membrane-associated PI-PLC; cPI-PLC, cytosolic PI-PLC.

(^2)
Y. Banno, T. Asano, and Y. Nozawa, unpublished observation.

(^3)
Y. Banno and Y. Nozawa, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. J. Kambayashi (Osaka University Medical School, Osaka, Japan) for the supply of calpain inhibitor, calpeptin, and Dr. S. Toyoshima (Japan Tobacco Inc., Pharmaceutical Basic Research Laboratories, Yokohama, Japan) for providing synthetic peptides of phospholipase C, X, and Y domains.


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