Inhibition by endothelial cells of platelet aggregating activity of thrombin - role of thrombomodulin

THROMBOSIS RESEARCH 50; 647-656, 1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

INHIBITION BY ENDOTHELIAL CELLS OF PLATELET AGGREGATING ACTIVITY OF THROMBIN - ROLE OF THROMBOMODULIN

M. Murata, Y. Ikeda, Y. Araki, H. Murakami, K. Sato, M. Yamamoto*, K. Watanabe*, Y. Ando*, T. Igawa**, and I. Maruyama*** Department of Haematology and *Department of Laboratory Medicine, Keio University, Tokyo, **Tokushima Research Institute, Otsuka Pharmaceutical Co. Ltd., and ***Department of Internal Medicine, Kagoshima University, Kagoshima, Japan. (Received 4.6.1987; Accepted in revised form 16.3.1988 by Editor S. Okamoto) ABSTRACT Cultured human umbilical vein endothelial cells inhibited the platelet aggregating activity of thrombin in the absence of plasma. Using a new method in which thrombin-induced platelet aggregation was measured in the presence of endothelial cells, we showed that aspirin-treated endothelial cells inhibited platelet aggregating activity of thrombin in an incubation time- and cell numberdependent manner. This inhibitory effect of endothelial cells was partially blocked by the pretreatment of endothelial cells with monoclonal anti-thrombomodulin IgG(anti-TMIgG). It is suggested that (1) endothelial cells play a role in the clearance of thrombin by binding and inactivating this enzyme and that (2) thrombomodulin on endothelial cells, apart from its role for protein C activation, may be involved in this endothelial function.

INTRODUCTION It has been well known that vascular endothelialcells have anti-thrombogenie properties. They produce prostaglandin Iz(PGIz), a potent inhibitor of platelet aggregation (1) and also synthesize and secrete tissue plasminogen activator (2), an important enzyme of fibrin-dependent fibrinolysis. In addition, endothelial cells are able to take up and inactivate thrombin (3-7). However, the exact mechanism of thrombin inactivation by endothelial cells is not completely clear.

Key words : endothelial cells, thrombomodulin, platelet aggregation, thrombin. 647

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thrombomodulin and glycosaminoglycans on the surface of endoRecently, thelial cells have been suggested to play some roles in this inactivation. Purified thrombomodulin was shown to bind thrombin and to inhibit its procoagulant and platelet activating activity (8,9). Whether thrombomodulin on the surface of the cells can inhibit the action of thrombin on both platelets and fibrinogen is, however, not clear. We have developed a new method in which endothelial cells are cultured in a glass cuvette and the platelet aggregating activity of thrombin is measured in the presence of endothelial cells using a conventional aggregometer. Using this method, we have clearly demonstrated that endothelial cells play a role in the clearance of thrombin and that thrombomodulin may be involved in this endothelial function.

MATERIALS Culture of endothelial

AND METHODS

cells

Human umbilical vein endothelial cells were isolated and culturedaccordBriefly,priing to the method of Jaffe et al. (10) with some modifications. mary cultures were initiated from 0.25% trypsin digests of the luminal surface Cells were cultured in modified medium 199 (Nissui Pharmaof the vessels. ceutical Co., Japan) containing 20% fetal calf serum (Gibco Laboratories, U.S.A.), 30 ug/ml of endothelial cell growth factor (Collaborative Research Inc., U.S.A.), and 12 U/ml of heparin (Shimizu Pharmaceutical Co., Japan) in 5% con, 95% air at 37°C. Confluent monolayers were subcultured by brief exposure to 0.05% trypsin-0.02% EDTA. Cells at second passage were harvested by trypsin-EDTA and resuspended in the medium described above but except for heparin. Two hundred microliters of cell suspensions containing 0.5-2~10~ endothelial cells were transferred into a cuvette with a diameter of 5 mm for The surface of cuvette was precoated with platelet aggregation studies. human fibronectin (Collaborative Research Inc., U.S.A.) at a concentration of Cuvettes were kept in oblique position and were incubated for 2 2 ng/ml. days. Endothelial cells could attach to the lateral lumen of the cuvettes and grow confluently (Figure 1). Preparation

of washed platelets

Blood was obtained from healthy volunteers and immediately mixed with 3.1% sodium citrate (9:l). Platelet rich plasma (PRP) was obtained by centrifugation at 50 g for 15 min at 22°C. PRP thus prepared was centrifuged at The resultant pellets were washed once with HEPES-Tyrode 2000 g for 10 min. buffer containing 10 mM HEPES, 0.1% glucose, pH 7.4, and were resuspended in the same buffer at a concentration of 6 x 105/ul. Experimentalprocedures Endothelial cell monolayers cultured in fibronectin-coated glass cuvettes were first incubated with 1 mM aspirin (ASA) for 60 min followed by washing In some experiments, they were further incubated with medium containing once. mouse monoclonal anti-human thrombomodulin IgG (anti-TMIgG) or normal mouse IgG at a concentration of 1 ng/ml for 30 min. Medium was removed, and monolayers were washed with phosphate buffered saline pH 7.4. One hundred microliters of human a-thrombin (a generous gift of Dr. J.W.Fenton) at concentrations of 0.025-0.1 U/ml were added to the cuvette and incubated at 37'C for

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One hundred microliters of washed human platelet various time intervals. suspensions (6x105/~1) were then added and the change cf light transmission was recorded continuously by the aggregometer (Science Inc., U.S.A.). It was confirmed that there was virtually no difference in light transmission between platelet suspensions in endothelial cell-cultured cuvettes This and in control cuvettes where endothelial cells were not cultured. indicated that culture of endothelial cells in a glass cuvette itself did not Furthermore, no obvious detachinfluence the change of light transmission. ment of endothelial cells was demonstrated during the experimental procedures.

RESULTS

FIGURE 1 Phase Contrast Micrograph of Endothelial Cells Cultured on the Lateral Lumen of a Cuvette

Effect of ASA on PGI, synthesis by endothelial

cells

It is known that thrombin stimulates the PGI2 synthesis of endothelial cells. Whether endothelial cells cultured in cuvettes in our experiments can synthesize PGI2 when stimulated with thrombin and, if so, whether ASA can inhibit thrombin-induced PG12 synthesis of endothelial cells were investigated (Table 1). In non-stimulated endothelial cells, 0.35 ng/104 cells of 6-keto PGFl, (measured by radioimmunoassay) accumulated in the culture medium in 5 min. When endothelial cells were stimulated with a-thrombin (0.05-0.5 U/ml), the amount of 6-keto PGFl, was increased to 0.8-0.95 ng/104 cells/5 min. Endothelial cells pretreated with 1 mM ASA, however, could not synthesize PG12 even after stimulated with thrombin. In control glass cuvettes where endothelial cells were not cultured, a-thrombin caused full aggregation of washed platelets at a concentration of 0.025 U/ml, while aggregation was completely inhibited when o-thrombin was added to platelet suspension in endothelial cell attached cuvettes. Sub-

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TABLE 1 Synthesis of PGI2 by Endothelial

Cells

6-keto PGFl, (ng/104 cells/5 min) EC treated with EC treated with buffer alone ASA (1 mM)

U-thrombin (U/ml) 0

not detected

0.35f0.08

0.05

not detected

o.YOf0.18

0.10

not detected

0.80M.07

0.50

not detected

0.95kO.16

Endothelial cells (EC) cultured in glass cuvettes were treated with either ASA (1 mM) or buffer for GO min. After washing once, HEPES-Tyrode buffer was added followed by stimulation with a-thrombin at various concentrations. After 5 min, aliquots were removed for measurement of The results are expressed 6-keto PGFl, by radioimmunoassay. as meanfSD (n=6).

sequently, thrombin-induced platelet aggregation was measured in the presence of endothelial cells treated with 1 mM ASA. ASA-treated endothelial cells showed no inhibitory effect on thrombin-induced platelet aggregation indicating that inhibitory effect of the endothelial cells on platelet aggregation was due to PGI2 (Figure 2). PLT min non-treated E.C.

ASA - treated E.C.

% light transmission

FIGURE 2 Thrombin-induced Platelet Aggregation in the Presence of Non-treated or ASA-treated Endothelial Cells Washed platelets were added to the cuvettes where cultured endothelial cells (5x104/cuvette) were treated with ASA or buffer. Thrombin (0.025 U/ml) was added thereafter.

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Inactivation

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of thrombin by endothelial

651

cells

When the same concentration of a-thrombin (0.025 U/ml) was first incubated with ASA-treated endothelial cells for 5 min and washed platelet suspensions were then added to thrombin-endothelial mixtures, aggregation was comTherefore, this inhibition of aggregation was not ascribed pletely inhibited. to PGI2, but to other endothelial factors. In a preliminary experiment, we have added an antagonist of platelet activating factor (PAF), CV3988, to culture medium just before thrombin stimulation because endothelial cells are known to synthesize PAF upon stimulation with thrombin. However, addition of CV3988 at a concentration of 10b4M did Therefore, PAF inhibitor was omitted in not affect the extent of aggregation. the following experiments. For endothelial cells to fully exhibit its inhibitory effect on thrombininduced platelet aggregation, it was necessary to incubate thrombin with endoThe effect of incubation time on the inhibithelial cells for a few minutes. tion of aggregation by endothelial cells was shown in Figure 3.

PLT min. 5 min.

% light transmission

FIGURE 3 Inhibition of Thrombin-induced Platelet Aggregation by ASA-treated Endothelial Cells a-thrombin (0.025 U/ml) was incubated with ASA-treated endothelial cells (5x104/cuvette) for various time interThen washed platelets were added. vals indicated.

In subsequent experiments, a-thrombin at various concentrations was first incubated with endothelial cells (5x104/cuvette)for 5 min and platelet susInhibition of thrombin-induced platelet aggregation by pensions were added. endothelial cells was shown only when thrombin concentration was low (0.0250.05 U/ml). When thrombin concentration was over 0.1 U/ml, no apparent inhibition was observed as shown in Table 2.

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TABLE 2 Inhibition of Thrombin-induced Platelet Aggregation by ASA-treated Endothelial Cells cr-thrombin

% aggregation at 3 minutes EC(-)

ASA-treated EC

0.025

70

0

0.05

75

35

0.1

75

75

(U/ml)

a-thrombin at various concentrations indicated was incubated with endothelial cells (5xlO'/cuvette) for 5 min, and platelet suspension was then added. Thrombin-induced platelet aggregation was also measured in the absence of endothelial cells.

The effect of the numbers of endothelial cells on the inhibition of platelet aggregation was shown in Table 3. ct-thrombinwas first incubated with various numbers of endothelial cells for 5 min and platelet suspensions were then added. The more endothelial cells were cultured in cuvettes, the more pronounced inhibition of platelet aggregation was observed.

TABLE 3 Effect of Endothelial Cell Number on Thrombin-induced Platelet Aggregation number of EC ( /cuvette)

% aggregation at 3 min a-thrombin (U/ml) 0.025

0

0.04

0.06

70

70

75

5 x lo4

0

60

70

1 x lo5

0

5

40

2 x lo5

0

0

20

cl-thrombinwas incubated with various numbers of endothelial cells for 5 min, and platelets were then added.

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Effect of anti-TMIgG on the ability of endothelial cells to inhibit thrombin-induced platelet aggregation In the next series of experiments, endothelial cells were pretreated with mouse monoclonal anti-TMIgG or normal mouse IgG for 30 min (Figure 4). In the absence of endothelial cells, maximal aggregation was 70% at a thrombin concentration of 0.025 U/ml. When thrombin at the same concentration was preincubated with normal IgG-treated endothelial cells, aggregation was completely inhibited. On the other hand, aggregation was partially inhibited when thrombin was preincubated with anti-TMIgG-treated endothelial cells. These data suggest that thrombomodulin on endothelial cell surface partially regulates the platelet aggregating activity of thrombin.

PLT

normal IgG treated E.C.

anti-TM treated

IgG E.C.

%’ light transmission

FIGURE 4 Effect of Anti-TMIgG on the Ability of Endothelial Cells to Inhibit Thrombin-induced Platelet Aggregation Endothelial cells (5x104/cuvette) were treated with 1 gig/mlof anti-TMIgG or 1 ug/ml of normal IgG for 30 min, washed, and incubated with cl-thrombin(0.025 U/ml) for 5 min. Then, platelet suspensions were added.

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DISCUSSION It is well known that endothelial cells possess the antithrombotic properties. Interaction of thrombin with endothelial cells is one aspect to In this paper, experiments have been designed to clarify these properties. the role of endothelial cells in thrombin-induced platelet activation. Our method has certain advantages over conventional platelet aggregation studies since aggregation can be monitored in the presence of endothelial Endothelial cells were easily cultured on a glass cuvette with morphocells. logy and function well comparable to those cultured on standard culturedishes. Endothelial cells were able to synthesize PGI? upon stimulation with thrombin, while ASA-treated endothelial cells were not. Endothelial cells are now considered to exhibit its inhibitory effect on thrombin-induced platelet activation by two different mechanisms, that is, synthesis of PGI2, a potent inhibitor of platelet aggregation, and endothelial cell-mediated thrombin inactivation possibly through thrombomodulin and glycosaminoglycans. Treatment of endothelial cells by ASA enabled us to assess the importance of the latter mechanism in inhibition of thrombin-induced aggre, gation by endothelial cells. It was clearly shown that a-thrombin lost its platelet-activating activity after being incubated with ASA-treated endoInhibition of thrombin-induced platelet thelial cells for a few minutes. aggregation by ASA-treated endothelial cells was dependent upon incubation time and the number of endothelial cells. When the concentration of thrombin was over 0.1 U/ml, 5~10~ of ASA-treated endothelial cells failed to inhibit platelet aggregation completely. It has been shown that cultured human umbilical vein endothelial cells are capable of binding thrombin (3,4). Lollar et al. (5) suggested that thrombin generated in the circulation bound to active-site-independent, high affinity binding sites on the endothelial cell surface. Using segments of porcine aorta, Swedenborg et al. (6,7) showed that in the absence of plasma, the amidolytic activity of thrombin, taken up on the endothelium, disappeared only slowly. They concluded that endothelium itself seemed to slowly inhibit bound thrombin in the absence of plasma. These studies were performed, however, at relatively high concentration of thrombin (2.5-25 U/ml), because no method was available to measure the enzymatic activity of thrombin at concentration lower than 0.1 U/ml. Therefore the physiological importance of these observation is still unclear. Clearly, our method has advantages since the activity of low concentration of After thrombin at a low thrombin can be measured by means of aggregometry. concentration was incubated with endothelial cells, the activity of thrombin This observation seems in good was diminished as early as in 3 minutes. agreement with the results obtained by Awbrey et al. (3). The ratio of endothelial cell number to platelet number in our experiments is about l:lOOO, which seems to be similar to that in a large vessel in vivo. This may suggest that even on a large vessel (where surface vs volume ratio is low compared with the ratio in arterioles or capillaries), endothelium may contribute to regulate the activity of thrombin in circulation. Thrombomodulin is a surface protein on endothelial cells which binds thrombin and thereby increases its ability to activate protein C (11). It has been identified on the endothelium of human arteries, veins, capillaries,

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lymphatics, and syncytiotrophoblast of placenta (12). It is also present in human plasma and urine (13). Esmon et al. demonstrated that thrombomodulin isolated from rabbit lung inhibited the ability of thrombin to clot fibrinogen, to activate factor V and to activate platelets (8,9). On the other hand, Salem et al. isolated thrombomodulin from human placenta (14), which, however, showed little inhibitory effect on the procoagulant activity of thrombin (15). The effect of thrombomodulin on endothelial cells (but not in isolated form) on the activity of thrombin has not been examined in detail. Whether or not the endothelium plays some roles for inactivation of thrombin was, therefore, a matter of great interest. We found that anti-TMIgG diminished the inhibitory effect of endothelial cells on platelet aggregating,activity of thrombin. It is therefore suggested that thrombomodulin on the cell surface takes part in the endothelial regulation of platelet activation. Maruyama et al. (16) has reported that about 50-60% of thrombin binding sites on endothelial cells are thrombomodulin. The fact that anti-TMIgG did not completely abolish the ability of endothelial cells to inactivate thrombin is consistant with their results. Other substances such as glycosaminoglycans may also play some roles in binding and inactivating thrombin. In our experiments, however, inactivation of thrombin by endothelial cells was investigated in the absence of plasma. Further investigations are, therefore, needed to clarify the importance of thrombomodulin in vivo.

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9. ESMON, N.L., CARROLL, R.C. and ESMON, C.T. ability of thrombin to activate platelets. 12242, 1983.

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Thrombomodulin blocks the J. Biol. Chem. 258. 12238-

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