THROMBOSIS RESEARCH 62; 707-716,199l 0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
ANTITHROMBIN III MODULATES THE EFFECT OF THROMBIN ON THE METABOLISM OF GLYCOSAMINOGLYCANS IN CULTURED ENDOTHELIAL CELLS
.4kai’.2, Toshiyuki.RajiZ*3, Yumiko Hayakawa2, Tomohiro Hayashi”, and Nobuo S3kur3q3wAz
Takuya
medicine, ’Department of Neurosurgery and 2Department of Clinical Laboratory Faculty Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, 3Department of Environmental Science, Faculty of Toyama 930-01, Japan, Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920- 11, Japan
(Received 21.11.1990; accepted in original form 11.3.1991 by Editor O.N. Ulutin)
ABSTRACT We previously reported that a treatment of cultures of endothelial cells from bovine aorta with thrombin resulted in 3 less accumulation of glycosaminoglycans (G.4G) in the cell layer. In the present study, we found that thrombin-induced decrease in the accumulation of [“5S]sulfate-labeled GBG (35S-G.4G) such 3s heparan sulfate was prevented by antithrombin III (AT III) but not by heparin cofactor II effect on the ‘=S(HC TT). However, .4T III did not show 3 significant GAG accumulation individually. Pretreatment of the cell layer with neither .4T III nor HC II showed any preventive effect. Tihen GAG in the cell layer was labeled with both [35S)sulfate and [3H]glucosamine, neither thrombin nor a combination of thrombin with AT III changed the ratio of the radioactivity of 36S to that of 3H. .4lthough thrombin stimulated the release of 35S-GAG from the cell layer, -\T IIT completely prevented the stimulatory effect. In conclusion, it was suggested that .4T III may inhibit the thrombin action on GAG metabolism of endothelial cells to prevent thrombosis in \?lro.
INTRODUCTION Thrombin has several physiologic effects function including a stimulation of synthesis plasminogen activator (2) and its inhibitor (3).
Tiey words : _intithrombin III, Endothelial cofactor II, Thrombin, Thrombosis.
707
cells,
on vascular endothelial cell of prostacyclin (l), tissue Recently we have found that
Glycosaminoglycans,
Heparin
AT III AND GAG OF ENDOTHELIUM
Vol. 62, No. 6
thrombin decreases glycosaminoglycans (GAG) content of the cell layer of cultured bovine aortic endothelial cells (4). Since the GAG of endothelial cells contains anticoagulant heparan sulfate (5) and are postulated to be involved in the anticoagulant property of endothelium, our finding may imply a possible mechanism of thrombin-induced thrombogenesis. On the other hand, human plasma contains two heparin-dependent inhibitor of thrombin; one is antithrombin III (AT III) and the other is heparin cofactor II (HCII) (6). AT III and HC II each inhibit thrombin by formation of stable complexes with the protease (7,8). In the previous study, we showed that gabexate mesilate which is 3 serine protease inhibitor, prevented the thrombin suppression of GAG production by endothelial cells (4). This result suggested t.hat AT III and/or HC II may modulate the suppressive effect of thrombin on GAG production by the cells in vivo. In the present study, we investigated the effect of AT III and HC II on thrombin-induced decrease in the GPIG accumulation in cultured endothelial cell layer. MATERIALS AND METHODS Materials. Regents and chemicals were obtained from following sources : Bovine thrombin from Sigma (St. Louis, MO, US.4); human AT III and pronase from Boehringer Mannheim (Germany); RPM1 1640 medium and ASF 301 medium from Nissui Pharmaceutical Co.,Ltd. (Tokyo, Japan) and .4jinomoto Co.,Ltd. (Tokyo, Japan), respectively; fetal bovine serum (FBS) from Filtron (.4ustralia); N3r[36S]OJ (18.7 GBq/mmol) and D-[1,6-3H(N)]glucos3mine from New England Nuclear (Boston, MA, USA); chondroitin ABC lyase from Seikagaku kogyo Co.,Ltd. (Tokyo, .Jap3n); culture dishes and culture plates from Costar (Cambridge, M.4, USA). Endothelial cell culture. Endothelial cells were isolated from bovine aorta by scraping the surface of the intima. The cells were cultured with RPM1 1640 medium supplemented with 10% FBS in 100 mm dishes until confluent in 5% COa in air in a humid atmosphere at 37 “C. The cells were then transferred into 21-well culture plates and cultured until confluent. The confluent cell layer was principally used in the present study. Purification and adjustment purified from normal human 3djustment of the activity of ously (IO), using 3 synthetic
of the activity of HC II to that of AT III. HC II was plasma by the method of Pamagishi et al. (9). The HC II to that of AT III was made as described previsubstrate S-2238.
Treatments. After preparation of the confluent cell layer, the medium was discarded and the cell layer was washed twice with serum-free _4SF 301 medium. The cell layer was then incubated for 24 h in 0.5 ml of the fresh ASF 301 medium with 370 lrBq/ml [3sS]sulf3te. In another experiment, the cell layer was labeled with both 370 kBq/ml [35S]sulfate and 370 kBq/ml [3H]glucosamine. An aqueous solution of thrombin (1.0 NIH U/ml), AT III (1.0 Inh.U/ml and below), or HC II (20 or 40 Inh.mU/ml) was added to the medium. After incubation, the medium was discarded or harvested and the cell layer ~3s washed twice with phosphatebuffered saline without Ca and Mg (CMF-PBS). The cell layer was then incubated with 0.25 ml of CMF-PBS containing 0.25% trypsin and 0.05% EDT.4 for 5 min at 37 “C to disperse the cells. The trypsinized cell suspension was collected and
Vol. 62, No. 6
AT III AND GAG OF ENDOTHELIUM
709
the well was washed with 0.25 ml of CMF-PBS: The wash was combined with the cell suspension and centrifuged at 3,000 rpm for 5 min to obtain the supernatant (“trypsinate fraction”). The trypsinate fraction was used for the following analyses of GAG. Quantitative analysis of GAG. The GAG labeled with [35Slsulfate (35S-GAG) or with both [3sS]sulfate and [3H]glucosamine was quantitatively analyzed by the method of Wasteson et 31. (11). The trypsinate fraction was incubated with 3 mg/ml pronase at 50 “C for 3 h. The digest was incubated with 4 mg/ml carrier chondroitin sulfate and 0.5% cetylpyridinium chloride (CPC) at 37 OC for 1 h, and centrifuged at 3,000 rpm for 15 min to obtain the precipitat,ed GAG-CPC comples. The precipitate was dissolved in 0.1 ml of 1 N NaCl and re-precipitated by addition of 1.4 ml of 80% aqueous ethanol. The suspension was centrifuged at 3,000 rpm for 15 min and the precipitate was obtained. The precipitate was dissolved in 0.4 ml of distilled water and the radioactivity was determined by liquid scintillation counter. Characterization of “5S-GAG. The 3JS-GAG obtained from the cell layer in 6-well culture plates was characterized. The trypsinate from cultures treated with 1.0 r-:/ml thrombin or with both 1.0 [J/ml thrombin and -10 mU/ml XT III was incuhated for 3 h with 3 mg/ml pronase at 50 “C. The pronase digest was boiled for 3 min. After cooling to room temperature, the digest ~3s incubated for 4 h with 5 chondroitin ABC lyase at 37 OC or with 240 mlrl nitrous acid in 10% acetic acid nt room temperature. The J’S-GAG resistant to each treatment was precipitated by CPC. Heparan sulfate (%) was calculated by dividin g the radioactivity (dpm) of chondroitin ABC lyase-resistant 35S-GAG by that (dpm) of total 35S-GAG. The other GAG (%) w3s calclllated by dividing the radioactivity (dpm) of nitrcus acid-resistant 35S-GAG by that (dpm) of total 3ES-G.~G. Pretreatment with AT III or HC II. The confluent cells xere incubated at 37 “C in serum-free &SF 301 medium in the presence of -10 mU/ml AT III or 40 mu/ml HC I! for 4 h in 2-kwell culture plates. After incubation, the medium was discarded and the cell layer was washed txice with fresh ASF 301 medium. The cultures were treated with 1.0 U/ml thrombin in 0.5 ml of serum-free ASF 301 medium in the presence of 370 kBq/ml [“‘S]sulfate for 2-l h at 37 “C, and the radioactivity incorporated into GAG in the cell layer was measured. Release of 35S-GAG. The confluent cells were incubated at 37 ‘C with 30 mU/ml XT III combined with 1.0 U/ml thrombin in the presence of 370 lrBq/mI [‘5S]sulfate in serum-free ASF 301 medium in 23-well culture plates for 21 h. After incubation, the medium wa s discarded and the cell layer was washed twice with serum-free ASF 301 medium. The cultures were then incubated in 0.5 ml of fresh serum-free ASF 301 medium without thrombin and AT III for 3 h at 37 “C. The 35S-GAG released into the medium and that remained in the cell layer were precipitated with CPC and the incorporated radioactivity ~3s measured. Statistical analysis. The significance of difference between values was est,imated bg Student’s t test; P ?-alues of less than 0.05 were considered to indicate statistical significant difference.
710
AT III AND GAG OF ENDOTHELIUM
Vol. 62, No. 6
RESULTS Interaction of thrombin with AT III and HC II on the accumulation of “‘S-GAG in the cell layer The effect of AT III and HC II on thrombin-induced decrease in 3sS-G.4G in the cell layer is shown in Fig. 1. AT III at 20 and 30 mu/ml significantly prevented the suppressive effect of thrombin, while HC II did not eshibit such a preventive effect. Characterization of =“IS-GAG To investigate the interaction GAG in the cell layer, the ““S-GAG other GAG. .4s shown in Table I, each component in the presence or
of AT III with thrombin ?n the composition of was characterized as heparan sulfate and the thrombin did not changed the percentage of absence of AT III.
Individual effect of AT III Fig. 2 shows the individual effect of AT III on the accumulation of “‘S-GAG in the cell layer. Although AT III at 1,000 mU/ml tended to decrease the =“S-GAG ,accumulation, no significant change w,as observed at tested concentrations.
3? g
‘,*
x 2. E 8
*+*
ATM
0
; q
***
HCll
1.
”
01 ’ 0
20
40
ATM or HCII (m Inh.U/mi)
FIG. 1. Effect of AT III and HC II on thrombin-induced decrease in the accumulation of “%-GAG in the endothelial cell layer. Confluent cultures of endothelial cells were incubated for 24 h at 37 “C with 1.0 NH U/ml thrombin combined with XT TIT or HC II in the presence of 370 kBq/ml [=“S]sulfate. Values are means + SE of 1 samples. Significantly different from thrombin treatment, *** P
ANTITHROMBIN III MODULATES THE EFFECT OF THROMBIN ON THE METABOLISM OF GLYCOSAMINOGLYCANS IN CULTURED ENDOTHELIAL CELLS
.4kai’.2, Toshiyuki.RajiZ*3, Yumiko Hayakawa2, Tomohiro Hayashi”, and Nobuo S3kur3q3wAz
Takuya
medicine, ’Department of Neurosurgery and 2Department of Clinical Laboratory Faculty Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, 3Department of Environmental Science, Faculty of Toyama 930-01, Japan, Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920- 11, Japan
(Received 21.11.1990; accepted in original form 11.3.1991 by Editor O.N. Ulutin)
ABSTRACT We previously reported that a treatment of cultures of endothelial cells from bovine aorta with thrombin resulted in 3 less accumulation of glycosaminoglycans (G.4G) in the cell layer. In the present study, we found that thrombin-induced decrease in the accumulation of [“5S]sulfate-labeled GBG (35S-G.4G) such 3s heparan sulfate was prevented by antithrombin III (AT III) but not by heparin cofactor II effect on the ‘=S(HC TT). However, .4T III did not show 3 significant GAG accumulation individually. Pretreatment of the cell layer with neither .4T III nor HC II showed any preventive effect. Tihen GAG in the cell layer was labeled with both [35S)sulfate and [3H]glucosamine, neither thrombin nor a combination of thrombin with AT III changed the ratio of the radioactivity of 36S to that of 3H. .4lthough thrombin stimulated the release of 35S-GAG from the cell layer, -\T IIT completely prevented the stimulatory effect. In conclusion, it was suggested that .4T III may inhibit the thrombin action on GAG metabolism of endothelial cells to prevent thrombosis in \?lro.
INTRODUCTION Thrombin has several physiologic effects function including a stimulation of synthesis plasminogen activator (2) and its inhibitor (3).
Tiey words : _intithrombin III, Endothelial cofactor II, Thrombin, Thrombosis.
707
cells,
on vascular endothelial cell of prostacyclin (l), tissue Recently we have found that
Glycosaminoglycans,
Heparin
AT III AND GAG OF ENDOTHELIUM
Vol. 62, No. 6
thrombin decreases glycosaminoglycans (GAG) content of the cell layer of cultured bovine aortic endothelial cells (4). Since the GAG of endothelial cells contains anticoagulant heparan sulfate (5) and are postulated to be involved in the anticoagulant property of endothelium, our finding may imply a possible mechanism of thrombin-induced thrombogenesis. On the other hand, human plasma contains two heparin-dependent inhibitor of thrombin; one is antithrombin III (AT III) and the other is heparin cofactor II (HCII) (6). AT III and HC II each inhibit thrombin by formation of stable complexes with the protease (7,8). In the previous study, we showed that gabexate mesilate which is 3 serine protease inhibitor, prevented the thrombin suppression of GAG production by endothelial cells (4). This result suggested t.hat AT III and/or HC II may modulate the suppressive effect of thrombin on GAG production by the cells in vivo. In the present study, we investigated the effect of AT III and HC II on thrombin-induced decrease in the GPIG accumulation in cultured endothelial cell layer. MATERIALS AND METHODS Materials. Regents and chemicals were obtained from following sources : Bovine thrombin from Sigma (St. Louis, MO, US.4); human AT III and pronase from Boehringer Mannheim (Germany); RPM1 1640 medium and ASF 301 medium from Nissui Pharmaceutical Co.,Ltd. (Tokyo, Japan) and .4jinomoto Co.,Ltd. (Tokyo, Japan), respectively; fetal bovine serum (FBS) from Filtron (.4ustralia); N3r[36S]OJ (18.7 GBq/mmol) and D-[1,6-3H(N)]glucos3mine from New England Nuclear (Boston, MA, USA); chondroitin ABC lyase from Seikagaku kogyo Co.,Ltd. (Tokyo, .Jap3n); culture dishes and culture plates from Costar (Cambridge, M.4, USA). Endothelial cell culture. Endothelial cells were isolated from bovine aorta by scraping the surface of the intima. The cells were cultured with RPM1 1640 medium supplemented with 10% FBS in 100 mm dishes until confluent in 5% COa in air in a humid atmosphere at 37 “C. The cells were then transferred into 21-well culture plates and cultured until confluent. The confluent cell layer was principally used in the present study. Purification and adjustment purified from normal human 3djustment of the activity of ously (IO), using 3 synthetic
of the activity of HC II to that of AT III. HC II was plasma by the method of Pamagishi et al. (9). The HC II to that of AT III was made as described previsubstrate S-2238.
Treatments. After preparation of the confluent cell layer, the medium was discarded and the cell layer was washed twice with serum-free _4SF 301 medium. The cell layer was then incubated for 24 h in 0.5 ml of the fresh ASF 301 medium with 370 lrBq/ml [3sS]sulf3te. In another experiment, the cell layer was labeled with both 370 kBq/ml [35S]sulfate and 370 kBq/ml [3H]glucosamine. An aqueous solution of thrombin (1.0 NIH U/ml), AT III (1.0 Inh.U/ml and below), or HC II (20 or 40 Inh.mU/ml) was added to the medium. After incubation, the medium was discarded or harvested and the cell layer ~3s washed twice with phosphatebuffered saline without Ca and Mg (CMF-PBS). The cell layer was then incubated with 0.25 ml of CMF-PBS containing 0.25% trypsin and 0.05% EDT.4 for 5 min at 37 “C to disperse the cells. The trypsinized cell suspension was collected and
Vol. 62, No. 6
AT III AND GAG OF ENDOTHELIUM
709
the well was washed with 0.25 ml of CMF-PBS: The wash was combined with the cell suspension and centrifuged at 3,000 rpm for 5 min to obtain the supernatant (“trypsinate fraction”). The trypsinate fraction was used for the following analyses of GAG. Quantitative analysis of GAG. The GAG labeled with [35Slsulfate (35S-GAG) or with both [3sS]sulfate and [3H]glucosamine was quantitatively analyzed by the method of Wasteson et 31. (11). The trypsinate fraction was incubated with 3 mg/ml pronase at 50 “C for 3 h. The digest was incubated with 4 mg/ml carrier chondroitin sulfate and 0.5% cetylpyridinium chloride (CPC) at 37 OC for 1 h, and centrifuged at 3,000 rpm for 15 min to obtain the precipitat,ed GAG-CPC comples. The precipitate was dissolved in 0.1 ml of 1 N NaCl and re-precipitated by addition of 1.4 ml of 80% aqueous ethanol. The suspension was centrifuged at 3,000 rpm for 15 min and the precipitate was obtained. The precipitate was dissolved in 0.4 ml of distilled water and the radioactivity was determined by liquid scintillation counter. Characterization of “5S-GAG. The 3JS-GAG obtained from the cell layer in 6-well culture plates was characterized. The trypsinate from cultures treated with 1.0 r-:/ml thrombin or with both 1.0 [J/ml thrombin and -10 mU/ml XT III was incuhated for 3 h with 3 mg/ml pronase at 50 “C. The pronase digest was boiled for 3 min. After cooling to room temperature, the digest ~3s incubated for 4 h with 5 chondroitin ABC lyase at 37 OC or with 240 mlrl nitrous acid in 10% acetic acid nt room temperature. The J’S-GAG resistant to each treatment was precipitated by CPC. Heparan sulfate (%) was calculated by dividin g the radioactivity (dpm) of chondroitin ABC lyase-resistant 35S-GAG by that (dpm) of total 35S-GAG. The other GAG (%) w3s calclllated by dividing the radioactivity (dpm) of nitrcus acid-resistant 35S-GAG by that (dpm) of total 3ES-G.~G. Pretreatment with AT III or HC II. The confluent cells xere incubated at 37 “C in serum-free &SF 301 medium in the presence of -10 mU/ml AT III or 40 mu/ml HC I! for 4 h in 2-kwell culture plates. After incubation, the medium was discarded and the cell layer was washed txice with fresh ASF 301 medium. The cultures were treated with 1.0 U/ml thrombin in 0.5 ml of serum-free ASF 301 medium in the presence of 370 kBq/ml [“‘S]sulfate for 2-l h at 37 “C, and the radioactivity incorporated into GAG in the cell layer was measured. Release of 35S-GAG. The confluent cells were incubated at 37 ‘C with 30 mU/ml XT III combined with 1.0 U/ml thrombin in the presence of 370 lrBq/mI [‘5S]sulfate in serum-free ASF 301 medium in 23-well culture plates for 21 h. After incubation, the medium wa s discarded and the cell layer was washed twice with serum-free ASF 301 medium. The cultures were then incubated in 0.5 ml of fresh serum-free ASF 301 medium without thrombin and AT III for 3 h at 37 “C. The 35S-GAG released into the medium and that remained in the cell layer were precipitated with CPC and the incorporated radioactivity ~3s measured. Statistical analysis. The significance of difference between values was est,imated bg Student’s t test; P ?-alues of less than 0.05 were considered to indicate statistical significant difference.
710
AT III AND GAG OF ENDOTHELIUM
Vol. 62, No. 6
RESULTS Interaction of thrombin with AT III and HC II on the accumulation of “‘S-GAG in the cell layer The effect of AT III and HC II on thrombin-induced decrease in 3sS-G.4G in the cell layer is shown in Fig. 1. AT III at 20 and 30 mu/ml significantly prevented the suppressive effect of thrombin, while HC II did not eshibit such a preventive effect. Characterization of =“IS-GAG To investigate the interaction GAG in the cell layer, the ““S-GAG other GAG. .4s shown in Table I, each component in the presence or
of AT III with thrombin ?n the composition of was characterized as heparan sulfate and the thrombin did not changed the percentage of absence of AT III.
Individual effect of AT III Fig. 2 shows the individual effect of AT III on the accumulation of “‘S-GAG in the cell layer. Although AT III at 1,000 mU/ml tended to decrease the =“S-GAG ,accumulation, no significant change w,as observed at tested concentrations.
3? g
‘,*
x 2. E 8
*+*
ATM
0
; q
***
HCll
1.
”
01 ’ 0
20
40
ATM or HCII (m Inh.U/mi)
FIG. 1. Effect of AT III and HC II on thrombin-induced decrease in the accumulation of “%-GAG in the endothelial cell layer. Confluent cultures of endothelial cells were incubated for 24 h at 37 “C with 1.0 NH U/ml thrombin combined with XT TIT or HC II in the presence of 370 kBq/ml [=“S]sulfate. Values are means + SE of 1 samples. Significantly different from thrombin treatment, *** P
