THROMBOSIS RESEARCH 64; 321-330,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.
INHIBITORY EFFECT OF HEPARIN COFACTOR II ON THROMBIN-STIMULATED PROSTACYCLIN PRODUCTIONBY CDLTDREDVASCULAR SMOOTHMUSCLE CELLS
Tomohiro Hqyashi’, Toshiyuki i$iji2, Yumiko Hayakawa’, Kenji Niiya , Michiko Sakamoto and Nobuo Sakuragawa’ ‘Department of Clinical Laboratory Medicine, Faculty of Medicine, Toyama Medical and Phasmaceutical 2630 Sugitani, University, Department Toyama 930-01, Japan, of Environmental Science, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-11. Japan
(Received
23.8.1991;
accepted
in revised form 26.8.1991
by Editor V.V. Kakkar)
ABSTRACT We investigated the effect of heparin cofactor II (HCII) on thrombin-induced prostacyclin (PG12) production by A10 cells, an established cell line of vascular smooth muscle cells from murine aorta. Confluent monolayers of A10 cells were incubated with 0.1 NIH U/ml of thrombin for 30 min in the presence of antithrombin III (ATIII) or HCII, and PG12 production by cells was measured by the radioimmunoassay as 6-keto-prostaglandin F the stable metabolite of PG12. AT111 at 40 mInh.U ? k; and more significantly inhibited thrombin-induced PG12 production but HCII at the same doses did only by A10 cells, slightly. However, when A10 cells were preincubated with HCII for 30 min before exposure to thrombin, the PG12 was production markedly inhibited. The medium conditioned by A10 cells for 30 min did not enhance the inhibitory effect of HCII on thrombin-induced PGI2 production by the cells. On the other hand, A10 cells synthesized both dermatan sulfate and heparan sulfate which activating are capable of HCII. From these results, it was suggested that HCII would be activated by glycosaminoglycans (GAGS) such as dermatan sulfate of the layers and could cell inhibit thrombin-induced PG12 production. HCII may be a modulator of thrombin on the physiological functions of vascular smooth muscle cells, reacting to the cell surface GAGS. Key words: Heparin cofactor cells, Thrombin, Prostacyclin.
II, Vascular Dermatan sulfate. 321
smooth
muscle
cells,
A10
322
HCII AND SMOOTH MUSCLE CELL
Vol. 64, No. 3
INTRODUCTION Heparin cofactor II (HCII) is a 65,600-dalton glycoprotein in human plasma that inactivates thrombin by forming an equimolar complex with the protease in a fashion similar to that of antithrombin III (ATIII). Heparin accelerates the rate of thrombin inactivation by either HCII or AT111 more than l,OOOfold, while dermatan sulfate facilitates that only by HCII (1). Dermatan sulfate is a glycosaminoglycan significantly synthesized by smooth muscle cells and fibroblasts. suggesting that HCII is an extravascular modulator of thrombin in the coagulation process Tollefsen et al. showed that HCII was activated and (2). accelerated the complex formation with thrombin by fibroblasts and vascular smooth muscle cells (3). Thrombin, one of serine proteases, has a number of biological activities. Thrombin is a key enzyme of blood coagulation that activates coagulation factors V, VII, VIII and converts fibrinogen to fibrin, and stimulates XIII, platelet The protease can also serve as an anticoagulant aggregation. factor by activating protein C in the presence of thrombomodulin In addition to these functions in hemostasis, thrombin has (4). a mitogenic activity for fibroblasts (5), a chemotactic activity for monocytes and macrophage-derived cells (6), and some stimulatory effects on endothelial cells and smooth muscle cells. It has been known that thrombin stimulates the release of prostacyclin (PGI ) which is an inhibitor of platelet aggregation from vascular endot h elial cells and smooth muscle cells (7, 8). reported that HCII inhibits thrombinRecently, we have induced of prostacyclin (9) and tissue plasminogen release activator (10) from cultured endothelial cells in the presence of These data suggested that the interaction of dermatan sulfate. thrombin with HCII in the presence of extravascular dermatan sulfate may result in an easy aggregation of platelet and fibrin clot formation during hemostasis. The present study was designed to clarify the interaction of HCII with cultured vascular smooth muscle cells and the effect of HCII on thrombin-stimulated PGI2 production by the cells in order to investigate the physiological role of HCII and glycosaminoglycans of vascular smooth muscle cells.
MATJXTALSANDMETEODS Pure human thrombin and trypsin were purchased from Antithrombin III was from Sigma (St Louis, MO, U.S.A.). Chondroitinase ABC, GmbH (Germany). Mannheim Boehringer chondroitinase B and pronase were from Seikagaku Kogyo Co., Ltd. kits Na2[i5S1’Ihe 6-keto-prostaglandin Fha radioimmunoassay (Tokyo, and Japan (12.84 GBq/mmol) were pure ased from Amersham (Boston, MA, International plc 4(IJ.K.) and New England Nuclear RPM1 1640 medium and fetal bovine serum respectively. U.S.A.), were obtained from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) The culture dishes and and Filtron (Australia). respectively. were from Costar (Cambridge, MA, U.S.A.). A plates culture synthetic substrate of thrombin, S-2238, was from KabiVitrum AB
Materials
Vol. 64, No. 3
323
HCII AND SMOOTH MUSCLE CELL
A10 cells, an established cell line (Sweden). muscle cells from murine aorta, were purchased Type Culture Collection (Rockville, MD, U.S.A.).
of
vascular from the
smooth American
HCII was purified from normal human plasma Preparation of HCIC Only one band was seen by the method of Yamagishi et al. (11). in SDS-PAGE analysis at the final step of the purification. The activity of HCII was defined as an inhibitory unit (1nh.U) for thrombin, and adjusted to that of antithrombin III by the method of Bartl and Lill using a synthetic substrate S-2238 (9.12). A10 cells were maintained in RPM1 1640 medium Cell culture supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin and 100 ug/ml streptomycin at 37 ‘C in a humidified atmosphere of 5% CO2 in air. Confluent monolayers of A10 cells in 24-well culture Treatment plates were washed twice with reaction buffer (pH 7.4) containing 11 mM HEPES, 137 mM NaCl, 4 mM KCl, 3 mM CaC12, 1 mM MgC12, and The cell layers were then incubated for 25 min in 11 mM glucose. 0.5 ml of the reaction buffer containing thrombin in the absence or presence of heparin cofactor II or antithrombin III. After incubation, the buffer was harvested and used for the assay of 6-keto-prostaglandin F a. In another experiment, washed cell hor 18 $15 with layers were incubated fresh serum-free RPM1 1640 Slsulfate. medium containing 370 KBq/ml [ After incubation, the medium was discarded and incubated again with 0.5 ml of serumfree RPM1 1640 medium. The medium was collected and then the cell layer was incubated with 0.5 ml of phosphate buffered saline containing 0.25% trypsin and 0.05% EDTA for 5 min. This preparation of the cell layer includes GAG derived from cell e and that from the solubilized matri f5 (13). The content zqrfgg ( Slsulfate-labeled glycosaminoglycans S-GAG) both in the culture supernatant and in the cell layer was measured by the method of Wasteson et al (14) as follows. Samples were incubated with 3 mg/ml pronase at 50 ‘C for 3 hr. The pronase digest was mixed with 4 carrier chondroitin sulfate and 0.5% mg/ml cetylpyridinium chloride (CPC). After incubation at 37 ‘C -for 1 hr, the mixture was centrifuged at 3,000 rpm for 15 min to obtain the precipitated GAG-CPC complex. The precipitate was dissolved in 0.1 ml of 4 N NaCl and re-precipitated by addition of 1.4 ml of 80% aqueous ethanol, and then centrifuged at 3,000 rpm for 15 min. The precipitate was dissolved in 0.4 ml of distilled water and the radioactivity was measured by liquid scintillation counting. Characterization of glycosaminoglycans of A10 cells The 35S-GAG both in the harvested medium and in the cell layer after treatment with 3 mg/ml pronase was degraded by glucosidase ( 5 U/ml of chondroite se ABC or 0.5 U/ml of chondroitinase B) -for 4 hr at 37 ‘C. The S-GAG resistant to each enzyme treatment was d as described above and the radioactivity was measured. ;,“p? S-GAG resistant to the degradation by chondroitinase ABC was regarded as heparan sulfate, and that sensitive to chondroitinase B was regarded as dermatan sulfate. The release of GAG (%) was calculated by dividing the radioactivity in the
324
HCII AND SMOOTH MUSCLE CELL
Vol. 64, No. 3
medium by the total 35S-GAG in both cell layer and the medium. Assay of PG12 The content. of PG12 in the reaction buffer was measured by radioimmunoassay for 6-keto-prostaglandin Fla, the stable metabolite of PG12. In some experiments, the percent increase of control was calculated by applying the following formula: % increase of control= HCII treated group - control group Thrombin treated group - control group x 100 (“a) Results Statistical analysis significance by Student’s t-test.
were
analyzed
for
statistical
RESULTS
Effect ~-
of thrombin -on the production
-of prostacyclin
Fig. 1 shows the effect of thrombin PG12 by A10 cells. Thrombin at 0.1 significantly stimulated the production and reached dependent manner almost Therefore, in following experiments, the by 0.1 NIH U/ml thrombin for 30 min.
!I B i$ 200 4 d
0
on the production of NIH U/ml or more of PG12 in a doseplateau by 15 min. cells were stimulated
a)
400 E
by A10 cells ---
) 0
.Ol .1
.5
1
Thrombin W/ml)
0
30
60
incubation Time (min)
Effect of thronbin on PG12 production by A10 cells. Confluent monolayers of A10 cells were incubated with thrombin at was measured 37 'C for 5, 15, 30 and 60 min and PG12 production a) Dose-dependent effect by radioimmunoassay for 6-keto-PGFla. Time of thrombin stimulation (incubation time: 30 min). b) 0, control ; 0, course of thrombin-induced PG12 production. 0.1 U/ml thrombin; A, 0.5 U/ml thrombin; 0. 1.0 U/ml thrombin. Each point represents the mean + S.E. of 4 samples.
FIG.1
Inhibitory production
325
HCII AND SMOOTH MUSCLE CELL
Vol. 64, No. 3
effect Q-ells. --
of
AT111 and _---
HCII
on
thrombin-induced
E2 -
the effect of AT111 and HCII on the First, we examined AT111 at 40 thrombin-stimulated production of PGI by A10 cells. of PGI2 in a mInh.U/ml or more greatly diminished t2ne production dose dependent manner (Fig. 2). In contrast, HCII at the same doses showed only a slight inhibition.
0 0
20
4080
0
HCII(mu/ml)
FIG. 2 --
Effect production.
of EC11 and 0, without
ATIII
on
20
40
AT III (mUhI)
thrombin-induced
thrombin ; 0, 0.1 Each point represents the mean + S.E. of 4 samples. +*p
INHIBITORY EFFECT OF HEPARIN COFACTOR II ON THROMBIN-STIMULATED PROSTACYCLIN PRODUCTIONBY CDLTDREDVASCULAR SMOOTHMUSCLE CELLS
Tomohiro Hqyashi’, Toshiyuki i$iji2, Yumiko Hayakawa’, Kenji Niiya , Michiko Sakamoto and Nobuo Sakuragawa’ ‘Department of Clinical Laboratory Medicine, Faculty of Medicine, Toyama Medical and Phasmaceutical 2630 Sugitani, University, Department Toyama 930-01, Japan, of Environmental Science, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-11. Japan
(Received
23.8.1991;
accepted
in revised form 26.8.1991
by Editor V.V. Kakkar)
ABSTRACT We investigated the effect of heparin cofactor II (HCII) on thrombin-induced prostacyclin (PG12) production by A10 cells, an established cell line of vascular smooth muscle cells from murine aorta. Confluent monolayers of A10 cells were incubated with 0.1 NIH U/ml of thrombin for 30 min in the presence of antithrombin III (ATIII) or HCII, and PG12 production by cells was measured by the radioimmunoassay as 6-keto-prostaglandin F the stable metabolite of PG12. AT111 at 40 mInh.U ? k; and more significantly inhibited thrombin-induced PG12 production but HCII at the same doses did only by A10 cells, slightly. However, when A10 cells were preincubated with HCII for 30 min before exposure to thrombin, the PG12 was production markedly inhibited. The medium conditioned by A10 cells for 30 min did not enhance the inhibitory effect of HCII on thrombin-induced PGI2 production by the cells. On the other hand, A10 cells synthesized both dermatan sulfate and heparan sulfate which activating are capable of HCII. From these results, it was suggested that HCII would be activated by glycosaminoglycans (GAGS) such as dermatan sulfate of the layers and could cell inhibit thrombin-induced PG12 production. HCII may be a modulator of thrombin on the physiological functions of vascular smooth muscle cells, reacting to the cell surface GAGS. Key words: Heparin cofactor cells, Thrombin, Prostacyclin.
II, Vascular Dermatan sulfate. 321
smooth
muscle
cells,
A10
322
HCII AND SMOOTH MUSCLE CELL
Vol. 64, No. 3
INTRODUCTION Heparin cofactor II (HCII) is a 65,600-dalton glycoprotein in human plasma that inactivates thrombin by forming an equimolar complex with the protease in a fashion similar to that of antithrombin III (ATIII). Heparin accelerates the rate of thrombin inactivation by either HCII or AT111 more than l,OOOfold, while dermatan sulfate facilitates that only by HCII (1). Dermatan sulfate is a glycosaminoglycan significantly synthesized by smooth muscle cells and fibroblasts. suggesting that HCII is an extravascular modulator of thrombin in the coagulation process Tollefsen et al. showed that HCII was activated and (2). accelerated the complex formation with thrombin by fibroblasts and vascular smooth muscle cells (3). Thrombin, one of serine proteases, has a number of biological activities. Thrombin is a key enzyme of blood coagulation that activates coagulation factors V, VII, VIII and converts fibrinogen to fibrin, and stimulates XIII, platelet The protease can also serve as an anticoagulant aggregation. factor by activating protein C in the presence of thrombomodulin In addition to these functions in hemostasis, thrombin has (4). a mitogenic activity for fibroblasts (5), a chemotactic activity for monocytes and macrophage-derived cells (6), and some stimulatory effects on endothelial cells and smooth muscle cells. It has been known that thrombin stimulates the release of prostacyclin (PGI ) which is an inhibitor of platelet aggregation from vascular endot h elial cells and smooth muscle cells (7, 8). reported that HCII inhibits thrombinRecently, we have induced of prostacyclin (9) and tissue plasminogen release activator (10) from cultured endothelial cells in the presence of These data suggested that the interaction of dermatan sulfate. thrombin with HCII in the presence of extravascular dermatan sulfate may result in an easy aggregation of platelet and fibrin clot formation during hemostasis. The present study was designed to clarify the interaction of HCII with cultured vascular smooth muscle cells and the effect of HCII on thrombin-stimulated PGI2 production by the cells in order to investigate the physiological role of HCII and glycosaminoglycans of vascular smooth muscle cells.
MATJXTALSANDMETEODS Pure human thrombin and trypsin were purchased from Antithrombin III was from Sigma (St Louis, MO, U.S.A.). Chondroitinase ABC, GmbH (Germany). Mannheim Boehringer chondroitinase B and pronase were from Seikagaku Kogyo Co., Ltd. kits Na2[i5S1’Ihe 6-keto-prostaglandin Fha radioimmunoassay (Tokyo, and Japan (12.84 GBq/mmol) were pure ased from Amersham (Boston, MA, International plc 4(IJ.K.) and New England Nuclear RPM1 1640 medium and fetal bovine serum respectively. U.S.A.), were obtained from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) The culture dishes and and Filtron (Australia). respectively. were from Costar (Cambridge, MA, U.S.A.). A plates culture synthetic substrate of thrombin, S-2238, was from KabiVitrum AB
Materials
Vol. 64, No. 3
323
HCII AND SMOOTH MUSCLE CELL
A10 cells, an established cell line (Sweden). muscle cells from murine aorta, were purchased Type Culture Collection (Rockville, MD, U.S.A.).
of
vascular from the
smooth American
HCII was purified from normal human plasma Preparation of HCIC Only one band was seen by the method of Yamagishi et al. (11). in SDS-PAGE analysis at the final step of the purification. The activity of HCII was defined as an inhibitory unit (1nh.U) for thrombin, and adjusted to that of antithrombin III by the method of Bartl and Lill using a synthetic substrate S-2238 (9.12). A10 cells were maintained in RPM1 1640 medium Cell culture supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin and 100 ug/ml streptomycin at 37 ‘C in a humidified atmosphere of 5% CO2 in air. Confluent monolayers of A10 cells in 24-well culture Treatment plates were washed twice with reaction buffer (pH 7.4) containing 11 mM HEPES, 137 mM NaCl, 4 mM KCl, 3 mM CaC12, 1 mM MgC12, and The cell layers were then incubated for 25 min in 11 mM glucose. 0.5 ml of the reaction buffer containing thrombin in the absence or presence of heparin cofactor II or antithrombin III. After incubation, the buffer was harvested and used for the assay of 6-keto-prostaglandin F a. In another experiment, washed cell hor 18 $15 with layers were incubated fresh serum-free RPM1 1640 Slsulfate. medium containing 370 KBq/ml [ After incubation, the medium was discarded and incubated again with 0.5 ml of serumfree RPM1 1640 medium. The medium was collected and then the cell layer was incubated with 0.5 ml of phosphate buffered saline containing 0.25% trypsin and 0.05% EDTA for 5 min. This preparation of the cell layer includes GAG derived from cell e and that from the solubilized matri f5 (13). The content zqrfgg ( Slsulfate-labeled glycosaminoglycans S-GAG) both in the culture supernatant and in the cell layer was measured by the method of Wasteson et al (14) as follows. Samples were incubated with 3 mg/ml pronase at 50 ‘C for 3 hr. The pronase digest was mixed with 4 carrier chondroitin sulfate and 0.5% mg/ml cetylpyridinium chloride (CPC). After incubation at 37 ‘C -for 1 hr, the mixture was centrifuged at 3,000 rpm for 15 min to obtain the precipitated GAG-CPC complex. The precipitate was dissolved in 0.1 ml of 4 N NaCl and re-precipitated by addition of 1.4 ml of 80% aqueous ethanol, and then centrifuged at 3,000 rpm for 15 min. The precipitate was dissolved in 0.4 ml of distilled water and the radioactivity was measured by liquid scintillation counting. Characterization of glycosaminoglycans of A10 cells The 35S-GAG both in the harvested medium and in the cell layer after treatment with 3 mg/ml pronase was degraded by glucosidase ( 5 U/ml of chondroite se ABC or 0.5 U/ml of chondroitinase B) -for 4 hr at 37 ‘C. The S-GAG resistant to each enzyme treatment was d as described above and the radioactivity was measured. ;,“p? S-GAG resistant to the degradation by chondroitinase ABC was regarded as heparan sulfate, and that sensitive to chondroitinase B was regarded as dermatan sulfate. The release of GAG (%) was calculated by dividing the radioactivity in the
324
HCII AND SMOOTH MUSCLE CELL
Vol. 64, No. 3
medium by the total 35S-GAG in both cell layer and the medium. Assay of PG12 The content. of PG12 in the reaction buffer was measured by radioimmunoassay for 6-keto-prostaglandin Fla, the stable metabolite of PG12. In some experiments, the percent increase of control was calculated by applying the following formula: % increase of control= HCII treated group - control group Thrombin treated group - control group x 100 (“a) Results Statistical analysis significance by Student’s t-test.
were
analyzed
for
statistical
RESULTS
Effect ~-
of thrombin -on the production
-of prostacyclin
Fig. 1 shows the effect of thrombin PG12 by A10 cells. Thrombin at 0.1 significantly stimulated the production and reached dependent manner almost Therefore, in following experiments, the by 0.1 NIH U/ml thrombin for 30 min.
!I B i$ 200 4 d
0
on the production of NIH U/ml or more of PG12 in a doseplateau by 15 min. cells were stimulated
a)
400 E
by A10 cells ---
) 0
.Ol .1
.5
1
Thrombin W/ml)
0
30
60
incubation Time (min)
Effect of thronbin on PG12 production by A10 cells. Confluent monolayers of A10 cells were incubated with thrombin at was measured 37 'C for 5, 15, 30 and 60 min and PG12 production a) Dose-dependent effect by radioimmunoassay for 6-keto-PGFla. Time of thrombin stimulation (incubation time: 30 min). b) 0, control ; 0, course of thrombin-induced PG12 production. 0.1 U/ml thrombin; A, 0.5 U/ml thrombin; 0. 1.0 U/ml thrombin. Each point represents the mean + S.E. of 4 samples.
FIG.1
Inhibitory production
325
HCII AND SMOOTH MUSCLE CELL
Vol. 64, No. 3
effect Q-ells. --
of
AT111 and _---
HCII
on
thrombin-induced
E2 -
the effect of AT111 and HCII on the First, we examined AT111 at 40 thrombin-stimulated production of PGI by A10 cells. of PGI2 in a mInh.U/ml or more greatly diminished t2ne production dose dependent manner (Fig. 2). In contrast, HCII at the same doses showed only a slight inhibition.
0 0
20
4080
0
HCII(mu/ml)
FIG. 2 --
Effect production.
of EC11 and 0, without
ATIII
on
20
40
AT III (mUhI)
thrombin-induced
thrombin ; 0, 0.1 Each point represents the mean + S.E. of 4 samples. +*p
