BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
45, 41-47
(1991)
Platelet Aggregation Induced by Platelet-Activating Factor Is Suppressed by Cystine Protease Inhibitor HIDEYUKI TAKANO, HIDEAKI KAWAGUCHI, Department
of
Cardiovasculur
Medicine, Sapporo
KENJI IIZUKA, AND HISAKAZU YASUDA
Hokkaido 060, Japan
University,
School
of Medicine,
Received April 30, 1990; and in revised form July 16. 1990
Platelet-activating factor (PAF), I-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (l-3), a mediator of anaphylaxis and inflammation (4-7), is released from leukocytes after immunologic stimulation (8). Our series of experiments have revealed that PAF directly acts on Swiss mouse 3T3 fibroblasts, releasing arachidonic acid from their membrane (9), and stimulates the release of prostacyclin from aortic smooth muscle cells (10). In renal epithelial cells PAF stimulates prostaglandin E2 (PGEJ release and increases calcium membrane permeability (11). We also reported that PAF has an effect on the stimulation of angiotensin converting enzyme (12). PAF is involved in several important cellular events. For example, it causes the aggregation and degranulation of platelets (4,8,13) and stimulates arachidonic acid release and subsequent thromboxane A2 synthesis (14). PAF, a powerful vasodilator, also possesses marked hypotensive activity when given to one-kidney, one-clip hypertensive rats (15,16). Therefore, PAF antagonist or inhibitor may have an important role in the prevention of PAFmediated pathological events. The problem in using PAF as antihypertensive drug is its effects on platelet aggregation. This study was undertaken to examine the effect of NCO-700, calcium-activated neutral protease (CANP), and cathepsin B inhibitor (17) on platelet aggregation induced by PAF. We attempted to clarify the mechanism of platelet aggregation induced by PAF. MATERIALS AND METHODS Materials
Platelet-activating factor (1-U-hexadecyl-2-acetyl-phosphocholine) and leupeptin were purchased from Sigma Chemical Company, St. Louis, Missouri. NCO700 (bis[ethyl (2R, 3R)-3-l(S)-1-methyl-1-[4-2, 3. 4-trimethoxyphenyl methyl)-piperazine-l-ylcarbonyl]butylcarbonyl]oxiran-2carboxylatelsulfate), a cystine protease and calcium-activated neutral protease inhibitor, was obtained from Nippon Chemiphar. CANP specific inhibitors I and II were obtained from Nakarai. 41 0885-4505/91
$3.00
Copyright D 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
42
TAKANO
ET AL.
Preparation of Washed Platelets The entire preparation of washed platelets was performed at room temperature using plastic containers and pipettes. Nine volumes of blood were obtained from the central ear artery of a rbbit directly into 1 vol of acid citrate dextrose (ACD) (18). The washed platelets were prepared as described earlier (19). Briefly, the blood was centrifuged at 170g for 10 min, and the top two-thirds of the plateletrich plasma (PRP) was carefully removed. The PRP suspension was centrifuged at 1OOOg for 10 min. The upper platelet-poor plasma (PPP) layer was removed and discarded. The remaining platelets were resuspended in Tyrode’s solution, pH 6.5, containing 0.1 mM EGTA. After centrifugation at 750 g for 10 min, the upper portion of the Tyrode’s solution was removed after which the platelets were resuspended in the same solution and sedimented to the bottom of the tube by centrifugation at 1OOOgfor 10 min. The platelet pellet was resuspended in an appropriate volume of the same solution, and the volume was adjusted to 1.25 x lo6 platelets/pi. The platelets were stored in a sealed plastic container at room temperature until used. Platelet Aggregation Aggregation of platelets was studied turbidometrically at 37” C with continuous recording of light transmission using an aggregometer (Hematracer-1, NKK, Japan). Forty microliters of the washed platelet suspension was added to an aggregometer cuvette together with 155 ~1 of prewarmed, 37°C Tyrode’s 10 mM Hepes solution, pH 7.2, containing 1.3 mM CaCl,. The platelets were stirred at 1000 r-pm. NCO-700 in volumes of 5 ~1 was then added to the platelet suspension, resulting in a final concentration of 2.5 x lO’/pl. After preincubation with NCO700 (O-20 min), PAF was added to the platelet suspension and allowed to react (unless otherwise mentioned, the final concentration of PAF was 10 nM). Preliminary experiments determined that the reproducible concentration of platelet aggregation induced by PAF was lo-* M. Labeling
and Serotonin
Release
Labeling and [‘4C]serotonin viously described (14).
and release of serotonin
were carried out as pre-
Statistical Analysis Triplicate samples were analyzed in all experiments. Results are expressed as means +- SE. Student’s t test was used to analyze the results, with the level of significance at P < 0.05. RESULTS
Inhibition of Platelet Aggregation by NCO-700 Platelet aggregation induced by PAF was inhibited in the presence of NCO700. As shown in Fig. 1, in the presence of 2.5 x 10-j M NCO-700, even after 20 min preincubation with NCO-700, aggregation was reversible. Irreversible inhibition was achieved by 20 min preincubation at 10e4 M NCO-700. With 4 x
PAF-INDUCED
PLATELET
AGGREGATION
IDOoM
400uM
'25oM
43
FIG. 1. Effect of NCO-700 on platelet aggregation induced by PAF (lo-” M). Platelets were preincubated with 2.5 x lo-‘. 10m4, or 4 X 10m4M NCO-700 for the indicated period as described under Materials and Methods.
10m4 M NCO-700, a 5-min preincubation time was sufficient to obtain completely irreversible inhibition. This inhibition was dependent on the preincubation time with NCO-700 (Fig. 2) Even at lower concentration (25 PM, platelet aggregation was significantly inhibited at 3 min preincubation (P < 0.001 compared to without preincubation). However, in the presence of low3 M NCO-700, complete inhibition was observed without preincubation (Fig. 3A). Furthermore, the inhibition still occurred when NCO-700 was added to the incubation mixture together with PAF, and NCO-700 could also inhibit platelet aggregation shortly after the aggregation started (Fig. 3B); however, it could not reverse platelet aggregation after the aggregation was completed (Fig. 3C). PAF-induced platelet aggregation was also inhibited by leupeptin (Fig. 4). But CANP inhibitors I and II did not inhibit platelet aggregation (Fig. 5). They had no effect on platelet aggregation after 20 min preincubation (data not shown). Effect of NCO-700 on Indomethacin-Treated
When platelets were preincubated stimulation with PAF, the inhibition
Preincubation
Platelets
with 10e4 M NCO-700 for 3 min before of platelet aggregation was approximately
Time
(ml nl
2. Effect of preincubation time with NCO-700 inhibitor on platelet aggregation induced by PAF. Platelet aggregation was determined as described under Materials and Methods. (0) 2.5 x lo-’ M. (A) 1O-4 M, to) 4 X 10m4 M. FIG.
44
TAKANO
A
B
N g
o-
-L : -10 20. p
ET AL.
C
D
N
-P
v) ._
/----
P
4 \
/’
5c 40. IL I- 60. .c m J 80.
i
1
:,I
i L
FIG. 3. Platelet aggregation induced by PAF and NCO-700. Platelet aggregation was determined as described under Materials and Methods in the presence of PAF (lo-* M) and IO-’ M NCO-700. (A) lo-’ MNCO-~I)O was added to platelets together with IO-’ M PAF. (B) NCO-700 was added to platelets shortly after the addition of PAF (10m8 M). (C) NCO-700 was added to platelets after the complete aggregation induced by PAF (lo-’ M). (D) Control platelet aggregation induced by PAF (lo-’ M).
20%. Indomethacin had little inhibitory effect on PAF-induced aggregation (P < 0.001, compared with NCO-700). However, when platelets were preincubated with 2 x IO-’ M indomethacin and 10e4 M NCO-700 for 3 min, the inhibition rate of PAF-induced platelet aggregation increased to 40% (P < 0.001, compared with NCO-700) (Fig. 6). These results indicate that PAF-induced platelet aggregation was caused by both thromboxane A2 and NCO-700-sensitive protease in our incubation system. Effect of NCO-700 on Figure 7 shows the PAF. Twenty minutes complete inhibition of
7Ic L L
Serotonin Secretion release of serotonin from platelets stimulated with IO-’ M of preincubation with NCO-700 ( 10e4 M) resulted in almost serotonin release; however, without prior incubation with 01
PlilM
1mM
Control
FIG. 4. Effect of leupeptin on platelet aggregation induced by PAF. Platelet aggregation was determined as described under Materials and Methods in the presence of 10m8M PAF and the indicated concentrations of leupeptin. Platelet aggregation was determined without preincubation with leupeptin.
PAF-INDUCED
PLATELET
C(l) : Calpain C(ll) : Calpain
AGGREGATION
Inhibitor Inhibitor
45
I II
FIG. 5. Effect of CANP inhibitors 1 and II on platelet aggregation induced by PAF. Platelet aggregation was determined as described under Materials and Methods in the presence of 10m8 M PAF and the indicated concentrations of CANP inhibitor I or II. Platelet aggregation was determined without preincubation with CANP inhibitors.
NCO-700 (10e4 M) serotonin release was inhibited by only 60%. After this preincubation period, platelet aggregation was inhibited more than 80%. DISCUSSION In our study, NCO-700 inhibited platelet aggregation induced by PAF. This inhibitory effect on PAF-induced platelet aggregation was augmented further by the addition of indomethacin. These results showed that PAF-induced platelet aggregation was partly caused by thromboxane A2. This result supports previous reports (14,20). The inhibitory effect of NCO-700 on PAF-induced platelet aggregation was accompanied by a suppression of serotonin release (degranulation) which stimulated platelet aggregation. Data on the inhibition of platelet aggregation, induced by PAF, support the hypothesis that NCO-700-sensitive protease(s), CANP, and/or cathepsin B might be involved in platelet aggregation. Platelet aggregation induced by several agents (collagen, thrombin, ADP, and
8
60
T
c
ItI
.-0 40 .9.c 20 c
0
FIG. 6. Effect of indomethacin aggregation induced by PAF (IO-* (IDM, 2 X lW5 M).
NC0
IDM
IC
-
GM and NCO-700 on platelet aggregation induced by PAF. Platelet M) was inhibited by NCO-700 (NCO, 1O-4 M) or/and indomethacin
46
TAKANO
ET AL. 100
40
9 ,L :
20 0 FIG. 7. Effect of NCO-700 on serotonin secretion. Serotonin secretion from platelets treated with lo-* M PAF was determined after the indicated periods of preincubation with NCO-700. Inhibition rates of platelet aggregation (A) and serotonin secretion (0).
epinephrine) has been reported to be inhibited by protease inhibitors (21). In another study, leupeptin, a cystine protease inhibitor, prevented platelet activation induced by thrombin and trypsin (22). In our experiment it also inhibit PAF-induced platelet aggregation. Serine protease inhibitor is known to suppress platelet aggregation induced by PAF (21). On the other hand, it has been reported that calcium-dependent proteolysis occurs during plaelet aggregation. Several platelet proteins are known to be sensitive to proteolysis induced by calciumactivated neutral protease (23). Thus, it is possible that CANP may contribute to platelet activation by PAF. But CANP specific inhibitors I and II did not inhibit platelet aggregation induced by PAF in the present study. Our experiments indicate that the inhibitory effect of NCO-700 on platelet aggregation induced by PAF does not depend on CANP inhibition. From this study we conclude that NCO-700 is an effective agent in the prevention of platelet aggregation induced by PAF. Whether NCO-700 has any effect on the hypotensive action of PAF remains to be proven in vivo. If NCO-700 does not alter the hypotensive effect of PAF, the combination of PAF and NCO700 may be useful in hypertension therapy. The Experiments on the effect of NCO-700 on PAF in vivo are underway. SUMMARY
The effect of NCO-700, a cystine protease inhibitor, on platelet-activating factor-induced platelet aggregation was determined. A newly synthesized cystine protease inhibitor (calcium-activated neutral protease and cathepsin B inhibitor), NCO-700 (bis[ethyl (2R, 3R)-3-[(S)-methyl-l-[4-(2,3,Ctrimethoxyphenylmethyl) piperazine-l-ylcarbonyl]butylcarbonyl]oxiran-2-carboxylate]sulfate), inhibited platelet-activating factor-induced platelet aggregation. The inhibition was dependent on the preincubation time with NCO-700 and on the concentration of the inhibitor. The release of serotonin was also inhibited almost completely by the 20-min preincubation with 1O-4 M NCO-700. Leupeptin also inhibited plateletactivating facor-induced platelet aggregation. But calcium-activated neutral pro-
PAF-INDUCED
PLATELET
AGGREGATION
47
tease inhibitor did not inhibit it. These observations suggest that NCO-700sensitive protease such as cystine protease may contribute to platelet aggregation induced by platelet-activating factor. ACKNOWLEDGMENTS We thank Dr. Y. Makita and Miss M. Shouki for their technical assistance. This study was supported by a Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (01870041. 02404042, and 02454250).
REFERENCES 1. Demopoulos, C. L.. Pinckard, R. N.. and Hanahan, D. J., J. Biol. Chem. 254, 9355 (1979). 2. Blank, M. L., Snyder, F., Byers. L. W., Brook, B.. and Muirhead, E. E.. Biochem. Biophys. Res. Commun. 90, 1194 (1979). 3. Hanahan, D. J., Demopoulos, C. A., Leihr, J., and Pinckard, R. N.. J. Biol. Chem. 255, 5514 (1980). 4. Benveniste, J., Cochrane, C. J., and Henson, P. M., J. Exp. Med. 136, 1356 (1972). 5. O’Flaherty. J. T., Wykle, R. L., and Miller, C. H., Amer. J. P&ho/. 103, 70 (1981). 6. Chilton, F. H., O’Flaherty, J. T.. Walsh, C. E.. Thomas, M. J., Wykle, R. L., and Waite, B. M., J. Biol. Chem. 257, 5402 (1982). 7. Goetzl, E. J., Derian, C. K.. Tauber, A. I., and Valone. F. H., Biochem. Biophys. Res. Commun. 94, 881 (1980). 8. Henson, P. M., J. C/in. Invest. 6, 481 (1977). 9. Kawaguchi, H., and Yasuda, H., FEBS Lett. 176, 93 (1984). 10. Kawaguchi, H., and Yasuda. H., Hypertension 8, 192 (1986). 11. Kawaguchi, H., and Yasuda, H., Biochim. Biophys. Acta 875, 525 (1986). 12. Kawaguchi. H., and Yasuda, H., FEBS Lett. 221, 305 (1987). 13. Macmanus, L. M., Shaw, J. O., and Pinckard, R. N., J. Immunol. 125, 1950 (1980). 14. Shaw, J. 0.. Klusick, S. J., and Hanahan. D. J., Biochim. Biophys. Acta 663, 222 (1981). 15. Blank, M. L., Snyder, F., Byers, L. W., Brooks, B., and Muirhead, E. E., Biochem. Biophys. Res. Commun. 90, 1194 (1979). 16. Prewitt, R. L., Leach, B. E.. Byers. L. W., Brook, B., Lands, W. E. M.. and Muirhead, E. E., Hypertension 1, 299 (1979). 17. Toyo-oka, T., Kamishiro, T., Hara, T., Nakamuru, N., Kitahara, M.. and Masaki, T.. ArzneimForsch. 36, 190 (1983). 18. Kawaguchi, H., Ishibashi, T., and Imai, Y., Lipids 17, 577 (1982). 19. Kawaguchi, H., Ishibashi, T., and Imai, Y.. Lipids 16, 37 (1981). 20. Rao, G. H. R., Schmid H. H. 0.. Reddy. K. R., and White, J. G., Biochim. Biophys. Acta 715, 205 (1982). 21. Sugatani, J., Miwa, M., and Hanahan, D. J., J. Biol. Chem. 262, 5740 (1987). 22. Ruggiero, M., and Lapetina, E. G., Biochem. Biophys. Res. Commun. 131, 1198 (1985). 23. Fox, J. E. B., Reynolds, C. C., and Phillips, D. R., J. Biol. Chem. 258, 9973 (1983).
MEDICINE
AND
METABOLIC
BIOLOGY
45, 41-47
(1991)
Platelet Aggregation Induced by Platelet-Activating Factor Is Suppressed by Cystine Protease Inhibitor HIDEYUKI TAKANO, HIDEAKI KAWAGUCHI, Department
of
Cardiovasculur
Medicine, Sapporo
KENJI IIZUKA, AND HISAKAZU YASUDA
Hokkaido 060, Japan
University,
School
of Medicine,
Received April 30, 1990; and in revised form July 16. 1990
Platelet-activating factor (PAF), I-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (l-3), a mediator of anaphylaxis and inflammation (4-7), is released from leukocytes after immunologic stimulation (8). Our series of experiments have revealed that PAF directly acts on Swiss mouse 3T3 fibroblasts, releasing arachidonic acid from their membrane (9), and stimulates the release of prostacyclin from aortic smooth muscle cells (10). In renal epithelial cells PAF stimulates prostaglandin E2 (PGEJ release and increases calcium membrane permeability (11). We also reported that PAF has an effect on the stimulation of angiotensin converting enzyme (12). PAF is involved in several important cellular events. For example, it causes the aggregation and degranulation of platelets (4,8,13) and stimulates arachidonic acid release and subsequent thromboxane A2 synthesis (14). PAF, a powerful vasodilator, also possesses marked hypotensive activity when given to one-kidney, one-clip hypertensive rats (15,16). Therefore, PAF antagonist or inhibitor may have an important role in the prevention of PAFmediated pathological events. The problem in using PAF as antihypertensive drug is its effects on platelet aggregation. This study was undertaken to examine the effect of NCO-700, calcium-activated neutral protease (CANP), and cathepsin B inhibitor (17) on platelet aggregation induced by PAF. We attempted to clarify the mechanism of platelet aggregation induced by PAF. MATERIALS AND METHODS Materials
Platelet-activating factor (1-U-hexadecyl-2-acetyl-phosphocholine) and leupeptin were purchased from Sigma Chemical Company, St. Louis, Missouri. NCO700 (bis[ethyl (2R, 3R)-3-l(S)-1-methyl-1-[4-2, 3. 4-trimethoxyphenyl methyl)-piperazine-l-ylcarbonyl]butylcarbonyl]oxiran-2carboxylatelsulfate), a cystine protease and calcium-activated neutral protease inhibitor, was obtained from Nippon Chemiphar. CANP specific inhibitors I and II were obtained from Nakarai. 41 0885-4505/91
$3.00
Copyright D 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
42
TAKANO
ET AL.
Preparation of Washed Platelets The entire preparation of washed platelets was performed at room temperature using plastic containers and pipettes. Nine volumes of blood were obtained from the central ear artery of a rbbit directly into 1 vol of acid citrate dextrose (ACD) (18). The washed platelets were prepared as described earlier (19). Briefly, the blood was centrifuged at 170g for 10 min, and the top two-thirds of the plateletrich plasma (PRP) was carefully removed. The PRP suspension was centrifuged at 1OOOg for 10 min. The upper platelet-poor plasma (PPP) layer was removed and discarded. The remaining platelets were resuspended in Tyrode’s solution, pH 6.5, containing 0.1 mM EGTA. After centrifugation at 750 g for 10 min, the upper portion of the Tyrode’s solution was removed after which the platelets were resuspended in the same solution and sedimented to the bottom of the tube by centrifugation at 1OOOgfor 10 min. The platelet pellet was resuspended in an appropriate volume of the same solution, and the volume was adjusted to 1.25 x lo6 platelets/pi. The platelets were stored in a sealed plastic container at room temperature until used. Platelet Aggregation Aggregation of platelets was studied turbidometrically at 37” C with continuous recording of light transmission using an aggregometer (Hematracer-1, NKK, Japan). Forty microliters of the washed platelet suspension was added to an aggregometer cuvette together with 155 ~1 of prewarmed, 37°C Tyrode’s 10 mM Hepes solution, pH 7.2, containing 1.3 mM CaCl,. The platelets were stirred at 1000 r-pm. NCO-700 in volumes of 5 ~1 was then added to the platelet suspension, resulting in a final concentration of 2.5 x lO’/pl. After preincubation with NCO700 (O-20 min), PAF was added to the platelet suspension and allowed to react (unless otherwise mentioned, the final concentration of PAF was 10 nM). Preliminary experiments determined that the reproducible concentration of platelet aggregation induced by PAF was lo-* M. Labeling
and Serotonin
Release
Labeling and [‘4C]serotonin viously described (14).
and release of serotonin
were carried out as pre-
Statistical Analysis Triplicate samples were analyzed in all experiments. Results are expressed as means +- SE. Student’s t test was used to analyze the results, with the level of significance at P < 0.05. RESULTS
Inhibition of Platelet Aggregation by NCO-700 Platelet aggregation induced by PAF was inhibited in the presence of NCO700. As shown in Fig. 1, in the presence of 2.5 x 10-j M NCO-700, even after 20 min preincubation with NCO-700, aggregation was reversible. Irreversible inhibition was achieved by 20 min preincubation at 10e4 M NCO-700. With 4 x
PAF-INDUCED
PLATELET
AGGREGATION
IDOoM
400uM
'25oM
43
FIG. 1. Effect of NCO-700 on platelet aggregation induced by PAF (lo-” M). Platelets were preincubated with 2.5 x lo-‘. 10m4, or 4 X 10m4M NCO-700 for the indicated period as described under Materials and Methods.
10m4 M NCO-700, a 5-min preincubation time was sufficient to obtain completely irreversible inhibition. This inhibition was dependent on the preincubation time with NCO-700 (Fig. 2) Even at lower concentration (25 PM, platelet aggregation was significantly inhibited at 3 min preincubation (P < 0.001 compared to without preincubation). However, in the presence of low3 M NCO-700, complete inhibition was observed without preincubation (Fig. 3A). Furthermore, the inhibition still occurred when NCO-700 was added to the incubation mixture together with PAF, and NCO-700 could also inhibit platelet aggregation shortly after the aggregation started (Fig. 3B); however, it could not reverse platelet aggregation after the aggregation was completed (Fig. 3C). PAF-induced platelet aggregation was also inhibited by leupeptin (Fig. 4). But CANP inhibitors I and II did not inhibit platelet aggregation (Fig. 5). They had no effect on platelet aggregation after 20 min preincubation (data not shown). Effect of NCO-700 on Indomethacin-Treated
When platelets were preincubated stimulation with PAF, the inhibition
Preincubation
Platelets
with 10e4 M NCO-700 for 3 min before of platelet aggregation was approximately
Time
(ml nl
2. Effect of preincubation time with NCO-700 inhibitor on platelet aggregation induced by PAF. Platelet aggregation was determined as described under Materials and Methods. (0) 2.5 x lo-’ M. (A) 1O-4 M, to) 4 X 10m4 M. FIG.
44
TAKANO
A
B
N g
o-
-L : -10 20. p
ET AL.
C
D
N
-P
v) ._
/----
P
4 \
/’
5c 40. IL I- 60. .c m J 80.
i
1
:,I
i L
FIG. 3. Platelet aggregation induced by PAF and NCO-700. Platelet aggregation was determined as described under Materials and Methods in the presence of PAF (lo-* M) and IO-’ M NCO-700. (A) lo-’ MNCO-~I)O was added to platelets together with IO-’ M PAF. (B) NCO-700 was added to platelets shortly after the addition of PAF (10m8 M). (C) NCO-700 was added to platelets after the complete aggregation induced by PAF (lo-’ M). (D) Control platelet aggregation induced by PAF (lo-’ M).
20%. Indomethacin had little inhibitory effect on PAF-induced aggregation (P < 0.001, compared with NCO-700). However, when platelets were preincubated with 2 x IO-’ M indomethacin and 10e4 M NCO-700 for 3 min, the inhibition rate of PAF-induced platelet aggregation increased to 40% (P < 0.001, compared with NCO-700) (Fig. 6). These results indicate that PAF-induced platelet aggregation was caused by both thromboxane A2 and NCO-700-sensitive protease in our incubation system. Effect of NCO-700 on Figure 7 shows the PAF. Twenty minutes complete inhibition of
7Ic L L
Serotonin Secretion release of serotonin from platelets stimulated with IO-’ M of preincubation with NCO-700 ( 10e4 M) resulted in almost serotonin release; however, without prior incubation with 01
PlilM
1mM
Control
FIG. 4. Effect of leupeptin on platelet aggregation induced by PAF. Platelet aggregation was determined as described under Materials and Methods in the presence of 10m8M PAF and the indicated concentrations of leupeptin. Platelet aggregation was determined without preincubation with leupeptin.
PAF-INDUCED
PLATELET
C(l) : Calpain C(ll) : Calpain
AGGREGATION
Inhibitor Inhibitor
45
I II
FIG. 5. Effect of CANP inhibitors 1 and II on platelet aggregation induced by PAF. Platelet aggregation was determined as described under Materials and Methods in the presence of 10m8 M PAF and the indicated concentrations of CANP inhibitor I or II. Platelet aggregation was determined without preincubation with CANP inhibitors.
NCO-700 (10e4 M) serotonin release was inhibited by only 60%. After this preincubation period, platelet aggregation was inhibited more than 80%. DISCUSSION In our study, NCO-700 inhibited platelet aggregation induced by PAF. This inhibitory effect on PAF-induced platelet aggregation was augmented further by the addition of indomethacin. These results showed that PAF-induced platelet aggregation was partly caused by thromboxane A2. This result supports previous reports (14,20). The inhibitory effect of NCO-700 on PAF-induced platelet aggregation was accompanied by a suppression of serotonin release (degranulation) which stimulated platelet aggregation. Data on the inhibition of platelet aggregation, induced by PAF, support the hypothesis that NCO-700-sensitive protease(s), CANP, and/or cathepsin B might be involved in platelet aggregation. Platelet aggregation induced by several agents (collagen, thrombin, ADP, and
8
60
T
c
ItI
.-0 40 .9.c 20 c
0
FIG. 6. Effect of indomethacin aggregation induced by PAF (IO-* (IDM, 2 X lW5 M).
NC0
IDM
IC
-
GM and NCO-700 on platelet aggregation induced by PAF. Platelet M) was inhibited by NCO-700 (NCO, 1O-4 M) or/and indomethacin
46
TAKANO
ET AL. 100
40
9 ,L :
20 0 FIG. 7. Effect of NCO-700 on serotonin secretion. Serotonin secretion from platelets treated with lo-* M PAF was determined after the indicated periods of preincubation with NCO-700. Inhibition rates of platelet aggregation (A) and serotonin secretion (0).
epinephrine) has been reported to be inhibited by protease inhibitors (21). In another study, leupeptin, a cystine protease inhibitor, prevented platelet activation induced by thrombin and trypsin (22). In our experiment it also inhibit PAF-induced platelet aggregation. Serine protease inhibitor is known to suppress platelet aggregation induced by PAF (21). On the other hand, it has been reported that calcium-dependent proteolysis occurs during plaelet aggregation. Several platelet proteins are known to be sensitive to proteolysis induced by calciumactivated neutral protease (23). Thus, it is possible that CANP may contribute to platelet activation by PAF. But CANP specific inhibitors I and II did not inhibit platelet aggregation induced by PAF in the present study. Our experiments indicate that the inhibitory effect of NCO-700 on platelet aggregation induced by PAF does not depend on CANP inhibition. From this study we conclude that NCO-700 is an effective agent in the prevention of platelet aggregation induced by PAF. Whether NCO-700 has any effect on the hypotensive action of PAF remains to be proven in vivo. If NCO-700 does not alter the hypotensive effect of PAF, the combination of PAF and NCO700 may be useful in hypertension therapy. The Experiments on the effect of NCO-700 on PAF in vivo are underway. SUMMARY
The effect of NCO-700, a cystine protease inhibitor, on platelet-activating factor-induced platelet aggregation was determined. A newly synthesized cystine protease inhibitor (calcium-activated neutral protease and cathepsin B inhibitor), NCO-700 (bis[ethyl (2R, 3R)-3-[(S)-methyl-l-[4-(2,3,Ctrimethoxyphenylmethyl) piperazine-l-ylcarbonyl]butylcarbonyl]oxiran-2-carboxylate]sulfate), inhibited platelet-activating factor-induced platelet aggregation. The inhibition was dependent on the preincubation time with NCO-700 and on the concentration of the inhibitor. The release of serotonin was also inhibited almost completely by the 20-min preincubation with 1O-4 M NCO-700. Leupeptin also inhibited plateletactivating facor-induced platelet aggregation. But calcium-activated neutral pro-
PAF-INDUCED
PLATELET
AGGREGATION
47
tease inhibitor did not inhibit it. These observations suggest that NCO-700sensitive protease such as cystine protease may contribute to platelet aggregation induced by platelet-activating factor. ACKNOWLEDGMENTS We thank Dr. Y. Makita and Miss M. Shouki for their technical assistance. This study was supported by a Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (01870041. 02404042, and 02454250).
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