Antithrombin III modulates the effect of thrombin on the metabolism of glycosaminoglycans in cultured endothelial cells

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

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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

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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.

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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
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AT III AND GAG OF ENDOTHELIUM

TABLE I Characterization

of 35S-G.4G in the Endothelial Cell Layer or with Thrombin and .4T III.

Heparan

sulfate”’ (%I

Treated

Others” (%)

Control

76 f4

28 t-r 2

Thrombin

75 +4

29 t 1

77 + 3

28 t 3

Thrombin

plus .4T III

with Thrombin

Confluent cultures of endothelial cells were incubated for 23 h at 37 ‘C with 1.0 WIH U/ml thrombin combined with or without 30 Inh.mV/ml AT IIT in the presence of 370 kBq/ml [36S]sulfate, and the 35S-GAG in the cell layer was characterized. \‘alues are means + SE of 5 samples. “‘3”S-G.4G resistant to chondroitin XBC lyase ” 35S-GAG resistant to nitrous acid

0

500

1000

ATIll (m Inh.U/ml)

FIG. 2. Effect of AT III on the accumulation of 35S-G.4G in the endothelial cell layer. Confluent cultures of endothelial cells were incubated for 24 h at 37 “C with 40, 100, 500, or 1,000 Inh.mU/ml .4T III in the presence of 370 kBq/ml [35S]sulfate. Values are means + SE of -! samples.

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AT III AND GAG OF ENDOTHELIUM

TABLE II Effect

of Pretreatment with AT III or HC II on Thrombin-Induced 36S-G.JIG in the Endothelial Cell Layer.

3zS-GAG

Control Thrombin

AT III

1,390 + 47

1,441 + 105 853 + 77**

Confluent cultures of endothelial cells were Inh.mU/ml AT III or 40 Inh.mU/ml HC II for 4 h the cell layer was treated with 1.0 NIH U/ml IcBq/ml [3SS]sulfate for 24 h. Values are means different from control, **P
in

(dpm)

CMF-PBS

758 + 57***

Decrease

HC II

1,365 + 39 807 + 4.5**

incubated with CMF-PBS or 40 at 37 “C. After the pretreatment, thrombin in the presence of 370 2 SE of 4 samples. Significantly

TABLE III Interaction

of Thrombin with AT III on the Incorporation of [35S]Srtlfate [3H]Glucosamine into GAG in the Endothelial Cell Layer.

[“5S]Sulfate (dpml

Control

1,041 + 77*

Thrombin Thrombin

plus .4T III

[ 3H]Glucosamine (dpml

and

3SS/3H

3,070 + 280X

0.343 + 0.024

722 + 40

2,105 + 243

0.345 + 0.038

1,152 + 89**

3,994 + 406

0.302 + 0.046

Confluent cultures of endothelial cells were incubated for 24 h at 37 “C with 1.0 NIH U/ml thrombin combined with or without 40 Inh.mU/ml AT III in the presence of both [35S]sulfate and [3H]glucosamine. Values are means + SE of 4 samples. Significantly different from thrombin treatment, *P
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Effect of pretreatment with AT III on the accumulation of 3JS-GAG in the cell layer Since AT III is postulated to be interact with thrombin on the surface of endothelium (solid phase), effect of pretreatment of endothelial cells with AT III on thrombin-induced decrease in 3LSS-GAGin the cell layer was investigated. As shown in Table II, a pretreatment with neither AT III nor HC II prevented the suppressive effect of thrombin. Polysaccharide chain formation and sulfation It is possible that the recovery of endothelial cell 3”S-G.4G content by AT II? may result from an increase in the sulfation of polysaccharide in the process of GAG in the cell layer was labeled GAG synthesis. To examine this possibility, with both [“=S]sulfate and [3H]glucosamine ss a marker of sulfation and polysaccharide chain formation, respectively, and the ratio of the radioactivity of 36S to that of 3H was calculated (Table III). Although thrombin decreased the Treatment incorporation of both 36S and =H, AT III prevented both decrease. with neither thrombin nor thrombin and AT III changed the ratio of 3sS to 3H. Interaction between thrombin and AT III on the release of 35S-GAG Since it was reported that thrombin stimulates the release of 2sS-G.4G from cultured endothelial cells (12), the effect of AT III on this thrombin action ~~1s esamined (Table TV). The accumulation of JzS-G.4G in both cell layer and medium was significantly decreased by thrombin, however, the percentage of the released 3sS-GAG was significantly increased by the protease. This suggests that thrombin stimulated the release of 3cS-GAG from the cell layer and the decreased accumulation of ==S-GAG in th e medium was regarded as a reflection of thrombin-induced decrease in the production of “JS-GXG. AT III prevented the

TABLE IV Interaction

of Thrombin with AT III on the Release of ‘3sS-G.4G from the Endothelial Cell Layer.

3=S-GAG released

JSS-G.4G remained

(dpm)

(dpm)

Control

369 + 15

1,331 + 51

Thrombin

287 5 16*

Thrombin plus 4T III

283 + 33

723 t 22*** 1,266 + 85

35S-GAG release

("/.I _~_____ 21.7 + 0.6 28.4 t 0.9*** 19.1 + 1.6

Confluent cultures of endothelial cells were incubated for 21 h at 37 “C with 1.0 NIH U/ml thrombin combined with or without 40 Tnh.MU/ml AT III in the presence of 370 kBq/ml [35S]sulfate. The cultures were then incubated for 3 h in the fresh medium in the absence of thrombin and AT III. Values are means f_ SE of -t samples. Significantly different from control, *P
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AT III AND GAG OF ENDOTHELIUM

stimulatory effect of thrombin GAG content in the cell layer.

on the release

of 35S-GAG with a recover

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of z5S-

DISCUSSION We have previously reported that thrombin has a suppressive effect on the production of GAG by cultured endothelial cells (4). In that report, we showed that gabexate mesilate, a serine protease inhibitor, prevented the thrombin effect, suggesting that AT III and/or HC II, which are endogenous serine protease inhibitors and inhibit thrombin, may modulate the thrombin action in viva. Thrombin-induced decrease in GAG content of endothelial cell layer is postulated to be a risk factor of thrombosis because GAG of endothelium contains anticoagulant heparan sulfate (13). Since thrombosis in patients with deficiency of either AT III or HC II were reported (14-16), it is important to clarify which of the inhibitors is a potent modulator of thrombin effect on endothelial cell GAG production. In the present study, we found that AT III prevented thrombin-induced decrease in 35S-GAG of the cell layer whereas HC II did not eshibit such a preventive effect (Fig. 1). In addition, the percentage of heparan sulfate was not changed by treatment with both thrombin and AT III, in other words, thrombin did not decrease the content of heparan sulfate of the cell layer in the presence of _4T III (Table I). Thus, we postulated that XT TIT rather than HC TI may modulate the thrombin-induced decrease in the anticoagulant activity on the surface of endothelium in viva. Since AT III did not alter the accumulation of %=S-GAG in the cell layer individually (Fig. 2), the preventive effect of AT III on the GAG production would result from an inhibition of thrombin. McGuire and Tollefsen (17) demonstrated that HC IT was activated bp fibroblasts and vascular smooth muscle cells but not *4 significant amount of dermatsn sulfate in these cells by endothelial cells. would be responsible for the activation. Tn contrast, heparan sulfate molecules purified from cloned bovine aortic endothelial cells have been shown to activate 1T TIT (13). TILE., it is likely that AT ITI inhibited thrombin using heparan sulfate produced by endothelial cells, resulting in a prevention of the decrease in GAG content. It is not simple whether AT IIT inhibited thrombin in the solid phase or in the liquid phase. We showed that a pretreatment of endothelial cells with AT III did not prevent t.he thrombin-induced decrease in 35S-GAG accumlllation in the cell layer (Table II). From this result, it appeared that AT III inhibited thrombin in the liquid phase rather than in the solid phase. However, we cannot exclude the possibility of the interaction of AT TIT with thrombin on the cell surface, since OLII' observation may be due t.o the low surface (2 cm2) of the cell layer in our system. However, it may safely be said that AT III can prevent the thrombin suppression of GAG production even if endothelial cell layer does not preserve a large amount of GAG. We showed that, AT III prevented thrombin-induced decrease in the incorporation of [3H]glucosamine as well as [ 35S]sulfate into GAG of endothelial cell layer (Table TII). This suggests that the prevention by AT III occurred in the polysaccharide chain formation as well as in the sulfation. On the other hand, thrombin stimulates the release of GAG from .endothelial cells (Et), suggesting that thrombin-stimulated endothelial cells may increase a local anticoagulant activity in the liquid phase in the early satge of thrombin exposure. It was shown

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715

that AT III prevented the stimulatory effect of thrombin on 35S-GAG release (Table IV). The effect of thrombin on the GAG metabolism overall disappeared by AT III. The present study provided an implication of one mechanism of thrombosis in The AT III deficiency would induce a decrease patients with AT III deficiency. in the anticoagulant activity on the surface of endothelium through thrombininduced decrease in anticoagulant heparan sulfate. This abnormality would facilitate the conversion of fibrinogen to fibrin by thrombin on the surface of Our data suggest one of the physiologic effect of AT III as 3 endothelium. modulator of thrombin action.

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