J. Biochem. 104, 628-632 (1988)

Functionally Active Is Present in Human '

Koji Suzuki,2 Junji Nishioka, Tatsuya Hayashi, and Yoshitane Kosaka Department of Laboratory Medicine, Mie University School of Medicine, Tsu, Mie 514

Received for publication, April 27, 1988

We found functionally active thrombomodulin in human platelets (60±18 molecules per

). appeared not to be activated by with gel-filtered platelets. However, the activation of protein C by thrombin was accelerated by thrombin-stimulated and washed platelets. This cofactor activity of the platelets was neutralized by the anti-lung thrombomodulin-F(ab•L)2. From the Triton X-extract of platelets, thrombomodulin was partially purified by diisopropylphosphoryl-thrombin-agarose affinity chromatography. The Mr of the predominant platelet thrombomodulin was 78,000 before and 109,000 after reduction on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, values identical to those of placental thrombomodulin. The specific activity of the cofactor activity, apparent (0.4nM) for thrombin and Km, (0.67ƒÊM) for protein C of platelet throm bomodulin were also identical to those of placenta thrombomodulin. Thrombomodulin may play a role in activation of protein C on the surface of platelets.

Thrombomodulin is a vascular endothelial cell membrane

glycoprotein that serves as a receptor of thrombin (1). EXPERIMENTAL PROCEDURES Thrombin bound to thrombomodulin activates a vitamin K-dependent serine protease zymogen, protein C. Activat Materials•\Protein C (2), placenta thrombomodulin (13, ed protein C inactivates blood cofactors, Fac 14), thrombin (2,800U/mg) (15), and protein C inhibitor tors Va and VIIIa (2,3). Thus, thrombomodulin plays an (16) were purified from human materials. The Mr and important role in the regulation of intravascular coagula absorption coefficient used for the respective proteins were tion. as follows: protein C, 62,000, A1%280nm=13.7 (2); placenta The endothelium has been considered to play a role in the thrombomodulin, 78,000 (14), A1%280nm=14.0 (tentative regulation of the blood coagulation system. Heparin-like value); thrombin, 37,000, A1%280nm=18.3 (15). Triton X-100, substances (4), vitamin K-dependent (5), plas Lubrol PX, sodium dodecyl sulfate (SDS), benzamidine minogen activator (6), prostacyclin(7), and thrombo chloride, and Tween-20 were purchased from Nakarai modulin, are all important in the antithrombogenic nature Chem., Kyoto. BrCN-activated Sepharose 4B and Sepha of endothelium. The endothelium also promotes coagula rose 2B were obtained from Pharmacia. Bovine serum tion. The serves as a receptor of Factor VII/ albumin, pre-stained standard Mr markers for SDS VIIa (8). Receptors of Factor IX/IXa modulate the proco polyacrylamide gel electrophoresis, peroxidase -conjugated agulant activity of Factor IXa on the cell surface (9). anti-rabbit IgG-goat IgG and 4-chloro-1-naphthol were Platelets stimulated by agonists release many factors obtained from Bio-Rad. Anti-human platelet glycoprotein that promote coagulation (10). Platelets also provide a Ib-IgG was purchased from Cosmo Bio, Tokyo. Diiso surface to which both Factor Va and Factor Xa bind, and propylfluorophosphate (DFP) was purchased from Fluka. this binding is associated with an increase in the rate of Boc-Leu-Ser-Thr-Arg-MCA, a fluorogenic substrate for prothrombin activation (11). Recently, platelets also have activated protein C (17), was obtained from the Protein been shown to influence the regulation of coagulation. Research Foundation, Osaka. The synthetic thrombin in

Protein S on the platelet surface serves as a receptor of hibitor, (2R, 4R)-4-methyl-l-[N2-(3-methyl-1 ,2,3,4 activated protein C and promotes this enzyme-catalyzed tetrahydro-8-quinoline sulfonyl)-L-arginyl] -2-piperidine inactivation of Factor Va (12). carboxylic acid monohydrate (MD-805)(18) was provided In the of this similarity between endothelial cells by Mitsubishi Kasei, Tokyo . Diisopropylphosphoryl and platelets with respect to procoagulant and anti (DIP)-thrombin agarose was prepared as described (19). coagulant activity, we studied whether thrombomodulin is Anti-human lung thrombomodulin-rabbit F(ab')2 was pre present in platelets. pared (20) from the IgG as previously described (14). Assay of Thrombomodulin Activity•\Activity of solubi 1 This work was supported in part by Grants-in-Aid for Scientific lized thrombomodulin in the presence of detergent was Research (61571110, 62480433, and 63637001) from the Ministry of assayed as follows. To 100 ,u 1 of a solution consisting of 0.05 MT Education, Science and Culture of Japan. ris-HC1, 0.1 M NaCl, 2mM CaCl2, and 0 .1% bovine 2 To whom correspondence should be addressed . serum albumin at pH 7.5, were added 10 pl of thrombin (5 Abbreviations: SDS, sodium dodecyl sulfate: DFP, diisopropylfluoro ƒÊ g/ml), 10ƒÊl of protein C (200ƒÊg/ml), and 10ƒÊl of the phosphate; DIP, diisopropylphosphoryl; Boc, t-butoxycarbonyl; MCA, 4-methylcoumarylamide; MD-805, (2R, 4R)-4-methyl- sample containing thrombomodulin. The mixture was 1[N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl] incubated at 37•Ž for 30min, and the reaction was stopped 2-piperidinecarboxylic acid monohydrate. by the addition of 50ƒÊ1 of 100ƒÊM MD-805. The amount of

628 J. Biochem . Platelet Thrombomodulin 629

activated protein C formed was assayed with 2 ml of 200 reproducibility test and 4.6% (n=10) in an inter-assay ƒÊ M Boc-Leu-Ser-Thr-Arg-MCA in a buffer consisting of reproducibility test. In a recovery test of purified placental 0.05M Tris-HCl and 0.1M CsCl at pH 8.0. Fluorescence of thrombomodulin after addition to human plasma, the the released aminomethylcoumarin was determined with a recovery rate was 94-102%. fluorospectrophotometer at 380 rim (excitation) and 440 Binding Assay of Thrombomodulin to Thrombin•\The run (emission). One picomole of purified human activated apparent dissociation constant (Kd,) of thrombomodulin for protein C has been determined to release 29pmol of thrombin was determined to measure the amount of throm aminomethylcoumarin/min in the presence of 0.1M CsCl, bomodulin bound to thrombin which had been fixed to pH 8.0 (19). microplate wells as follows. Flat-bottomed microplate Activity of platelet thrombomodulin was assayed as wells were coated with thrombin (5ƒÊg/100ƒÊl) in 0.1 M follows. First, thrombin-stimulated platelets were pre phosphate buffer, pH 7.5, by overnight incubation. After pared. Briefly, normal human blood obtained in 5mM the wells were blocked with 150ƒÊl of 10% bovine serum EDTA was centrifuged at 1,000 x g for 10 min at 20•Ž. The albumin for 2 h, 100ƒÊ1 of various concentration of throm supernatant, collected as platelet-rich plasma, was then bomodulin in phosphate-buffered saline containing 0.1% gel-filtered at 20•Ž on a Sepharose 2B column equilibrated bovine serum albumin and 0.05% Lubrol PX at pH 7.0 was in a buffer consisting of 0.05M Tris-HC1 and 0.1M NaCl at added and incubated for 2 h. The wells were washed three pH 7.5 (Tris-buffered saline). The gel-filtered platelets times with phosphate-buffered saline containing 0.5% were incubated at 37•Ž for 60 min after the addition of bovine serum albumin and 0.02% Tween-20, then 100ƒÊ1 of thrombin (0.5ƒÊg/ml final) and CaCl2 (5mM final). The the anti-thrombomodulin-F(abƒÊ) (0.2ƒÊg/ml) coupled with platelets were pelleted by centrifugation at 2,000 X g for 15 ƒÀ-D-galactosidase was added and incubated for 1 h. After min at 20•Ž, and the pellet was resuspended in the the wells were washed with phosphate-buffered saline, then Tris-buffered saline containing 2mM EDTA. These 100ƒÊl of freshly prepared 4-methyl-ƒÀ-D-galactoside (0.1 platelets were washed twice by the same procedure as mg/ml) in the same buffer was added. After a 30 min described above and then suspended in Tris-buffered saline incubation, fluorescence of each well was determined as containing 5mM CaCl2. A typical assay procedure of described above. platelet thrombomodulin activity was as follows. To 250,u l Partial Purification of Platelet Thrombomodulin of the platelets (1•~106 cells/ƒÊ1) in the Tris-buffered saline •\ Platelet thrombomodulin was partially purified by the containning 5mM CaC12 were added 5,u 1 of thrombin (20ƒÊ/ method reported for the purification of human (14) and ml) and 50ƒÊl of protein C (200ƒÊg/ml), and the mixture bovine (19) lung thrombomodulin. In brief, the platelet was incubated at 37•Ž. At intervals, 100ƒÊ1 of 50ƒÊM pellet obtained by centrifugation at 2,000•~g for 15 min MD-805 in Tris-buffered saline was added to terminate from 50 packs of platelet-rich plasma (it contained about

protein C activation. The activity of activated protein C in 2•~1012 platelets), which was provided by a local blood the reaction mixture was then measured using 1.5 ml of bank, was washed three times with 200 ml of a solution 200ƒÊM Boc-Leu-Ser-Thr-Arg-MCA in buffer consisting of containing 0.02 M Tris-HCl, 0.1M NaCl, 1mM EDTA, 1 0.05M Tris-HCl and 0.1M CsCl at pH 8.0. mM benzamidine, and 1 mM DFP at pH 7.0. The pellet was Enzyme Immunoassay of Thrombomodulin-Throm suspended in 30 ml of a buffer containing 0.02 M Tris-HCl, bomodulin was antigenically determined by the sandwich 0.25 M sucrose, 0.5 mM CaC12, 1 M benzamidine, 1 mM type enzyme immunoassay using anti-human lung throm DFP, 0.02% sodium azide, and 0.5% Triton X-100 at pH bomodulin-rabbit F(ab•L) coupled with ƒÀ-D-galactosidase 7.5, and sonicated at 40 watts for 30 s on ice three times. that had been prepared as described (20). Assays were The suspension was centrifuged at 12,000•~g for 45 min at performed at room temperature. Flat-bottomed microplate 4•Ž to obtain a supernatant containing cell membrane wells were coated with the F(ab•L)2 (0.5ƒÊg/100ƒÊl) in 0.1M proteins. This extraction from the pellets was repeated sodium bicarbonate buffer at pH 9.2. After a 1 h incubation, twice more. Finally 100 ml of membrane extract was unbound materials were removed by washing with the same obtained. This extract was then applied to a column (2.5 buffer. The wells were blocked with 200ƒÊl of 10% normal •~ 8cm) of DIP-thrombin-agarose equilibrated with a buffer rabbit serum for 2 h, then washed three times with containing 0.02M Tris-HCl, 0.1M NaCl, 0.5mM CaCl2, 1 mM benzamidine, and 0.05% Lubrol PX at pH 7.5, at 20•Ž. phosphate-buffered saline consisting of 0.01M sodium phosphate, 0.1M NaCl and 1mM MgCl2 (pH 7.0) contain The column was washed with 200 ml of the equilibration ing 0.1% bovine serum albumin and 0.05% Tween-20. buffer, and the bound proteins were eluted with a buffer Then, 100,u l of the sample solution, diluted appropriately consisting of 0.02 M Tris-HC1, 1M NaCl, 0.1mM EDTA, 1 in phosphate-buffered saline containing 0.1% bovine serum mM benzamidine, 0.1mM DFP, and 0.05% Lubrol PX at albumin, 0.5% gelatin, and 0.05% Tween-20, was added pH 7.5. The fractions containing the thrombomodulin were and incubated for 2 h. After being washed three times, the pooled, and concentrated using a Millipore Immersible wells were incubated for another hour after the addition of CX-30. 100ƒÊl of the F(ab•L) (lƒÊg/ml) coupled with ƒÀ-D-galacto Immunoblot of Thrombomodulin-SDS-polyacrylamide sidase. The wells were washed and 100 u l of freshly gel electrophoresis was performed by the method of Blobel and Dobberstein (21) using 5-15% gradient slab gels. The prepared 4-methyl umbelliferyl-ƒÀ-D-galactoside (0.1mg/ ml) in phosphate-buffered saline was added. After 30 min, sample of partially purified platelet thrombomodulin, the fluorescence of each well was determined at 360nm placental thrombomodulin and pre-stained standard markers were electrophoresed with and without 2 (excitation) and 450 nm (emission) using a Fluororeader - mercaptoethanol. Immunoblot analysis was performed by (Dynatech Laboratories). By this enzyme immunoassay, thrombomodulin in the range of 0.25 to 100ng/ml could be the method of Burnette (22). The thrombomodulin on the nitrocellulose membrane was detected by treatment first measured. The C.V. was 3.7% (n=10) in an intra-assay

Vol. 104, No. 4, 1988 630 K. Suzuki et al.

with the anti-thrombomodulin-rabbit F(ab•L)2, next with protein C was not activated even by increasing the platelet peroxidase-conjugated anti-rabbit IgG-goat IgG, and then count (data not shown). As D'Angelo et al.(23) showed that with the substrate for peroxidase, 4-chloro-1-naphthol. an inhibitor of tissue plasminogen activator, which was The Mr values of the prestained markers were corrected released from platelets by thrombin stimulation, neutral based on the Mr of the standard proteins (Pharmacia low Mr izes activated protein C and thrombin, the platelets were calibration ). treated with thrombin and then washed well to remove the inhibitor before the addition of protein C. Figure 1 shows RESULTS that the amount of activated protein C increased in the presence of the thrombin-stimulated and washed platelets Activity of Platelet Thrombomodulin-First, we deter (open circles) depending on the incubation time. Without mined the cofactor activity of the gel-filtered platelets for protein C or the thrombin-stimulated platelets, the ami thrombin-catalyzed activation of protein C. However, dolytic activity of the reaction mixture could not be detected in the presence of the thrombin inhibitor, MD 805. The amidolytic activity was neutralized by purified protein C inhibitor in the presence of dextran sulfate (data not shown). The activator activity of the thrombin stimulated platelets was decreased by incubation with the anti-thrombomodulin F(ab•L)2. The protein C activation activity of the thrombin-stimulated platelets was further increased by addition of thrombin to the reaction mixture. This cofactor activity of the thrombin-stimulated platelets depended on the platelet count (data not shown). The protein C activation activity in the reaction mixture could be detected with more than 7.5•~104 platelets/ml when

TABLE I. Specific activities of platelet and placental throm bomodulin. Thrombomodulin activity (1 unit) corresponds to the amount of activated protein C formed (1pmol/min/ml) under the Fig. 1. Time-dependent activation of protein C by thrombin conditions described in "EXPERIMENTAL PROCEDURES." Con stimulated and washed platelets. The reaction mixture (in 305ƒÊl) centration of thrombomodulin antigen was determined by enzyme consisted of: o, protein C (550nM)+thrombin (9.5nM)+thrombin immunoassay. stimulated platelets (2.5•~108 cells); •›, protein C (550nM) +thrombin-stimulated platelets (2.5•~108 cells); •œ, protein C (550 nM)+thrombin (9.5nM); •£, protein C (550nM) +thrombin (9.5 nM) +thrombin-stimulated platelets (2.5•~108 cells) which had been treated with anti -thrombomodulin F(ab•L)2, (50ƒÊg/ml) for 60 min; • , thrombin (9.5nM)+thrombin-stimulated platelets (2.5•~108 cells). APC indicates activated protein C. The conditions are described in detail in "EXPERIMENTAL PROCEDURES."

Fig. 3. Activation of protein C by thrombin in the presence of platelet or placental thrombomodulin. To 200ƒÊ1 of 0.05M Fig. 2. Immunoblot analysis of thrombomodulin. Samples in Tris-HCl, 0.1M NaCl, 2mM CaCl2, and 0.1% bovine serum albumin, lanes 1 to 3 were unreduced and those in lanes 4 to 6 were reduced pH 7.5, were added 50,u l of platelet thrombomodulin (4.7ƒÊg/ml) prior to SDS-polyacrylamide gel electrophoresis. Lanes 1 and 4, (•›) or placental thrombomodulin (6.8ƒÊg/ml) (•œ), 3ƒÊ1 of thrombin placental thrombomodulin (2ƒÊg); lanes 2 and 5, platelet throm (60ƒÊg/ml) and 30ƒÊ1 of various concentrations of protein C. The bomodulin (0.5ƒÊg); lanes 3 and 6, pre-stained standard markers with conditions are described in detail in "EXPERIMENTAL PROCE Mr indicating from top to bottom, 109,000, 87,000, 49,000, and DURES." APC indicates activated protein C. Insert shows a double 34,000. The method is described in "EXPERIMENTAL PROCE reciprocal plot of the same data to obtain the apparent Km of DURES." thrombin-thrombomodulin complex for protein C.

J. Biochem . Platelet Thrombomodulin 631

bomodulin, activation of various concentrations of protein C by thrombin in the presence of platelet or placental thrombomodulin was determined. From the values in Fig. 3, the apparent Km values for protein C of the platelet and placental proteins were obtained as 0.67 and 0.75ƒÊM, respectively. Figure 4 shows the binding of platelet and placental thrombomodulin to thrombin fixed to microplate wells. Platelet thrombomodulin had an apparent Kd of 0.4nM for thrombin, which was the same as that of placental throm bomodulin.

DISCUSSION

We found that human platelets contain functionally active thrombomodulin. The reason why thrombomodulin has not been identified so far in platelets may be because the Fig. 4. Binding of thrombomodulin to thrombin fixed to the microplate wells. •›, placental thrombomodulin; •œ, platelet throm amount of thrombomodulin in platelets is small and the bomodulin. The assay procedure is as follows: 100ƒÊl of the indicated activated protein C formed is rapidly neutralized by tissue concentrations of thrombomodulin (abscissa) was incubated with the plasminogen activator inhibitor released from the throm thrombin-coated microplate wells. The wells were washed, then 100 bin-stimulated platelets (23). We have overcome these ƒÊl of anti-thrombomodulin-F(ab•L) (0.2ƒÊg/ml) coupled with ƒÀ-D-galactosidase was added, and the bound F(ab•L) was determined by problems by using an enzyme immunoassay with high sensitivity and by removing the inhibitor by washing the the addition of 100ƒÊl of 4-methyl-ƒÀ-D-galactoside (0.1mg/ml). The conditions are described in detail in "EXPERIMENTAL PROCE platelets. With our enzyme immunoassay, approximately DURES." Insert shows a double-reciprocal plot of the same data to 0.78•}0.24ng of thrombomodulin was determined in 108 obtain the apparent Kd of thrombomodulin for thrombin. platelets, and the plasma concentration of soluble throm bomodulin was 13.7•}3.0ng per ml (24). This value is considerably lower than the value (292•}60ng/ml plasma) thrombin was added and with more than 3•~105 platelets/ reported by Ishii and Majerus (25). However, recently Ishii ml in the absence of additional thrombin. and coworkers obtained a value of 21.9•}8.8ng protein per Properties of Platelet Thrombomodulin-We determined ml plasma by enzyme immunoassay using monoclonal the thrombomodulin in the Triton X 100 extract of human anti-human thrombomodulin-IgGs (26). Thus, based on the platelets and 0.78•}0.24ng protein was detected per 1 amounts of thrombomodulin in platelets and plasma, 11 •~108 platelets (mean•}SD, n=8). Assuming that throm 19% of the total thrombomodulin in plasma is estimated to bomodulin was extracted completely from platelets, 60•} exist in platelets. 18 molecules were estimated to exist per platelet. This We found that the platelet thrombomodulin had the same Mr and kinetic properties as the cellular thrombomodulin protein was partially purified from the Triton X-100 extract by DIP-thrombin-agarose chromatography. The isolated from lung and placenta (1, 13, 14, 19). Ishii and concentration of the platelet thrombomodulin in this Majerus reported the presence of the smaller Mr soluble forms of thrombomodulin (Mr=63,000 and 54,000), dif preparation was approximately 10% of the total protein. The platelet thrombomodulin was then compared with the ferent from the cellular form with Mr = 78, 000, in human

purified placental protein. plasma and urine (25). They showed that the apparent Kd As shown in Fig. 2, without reduction, the platelet of the plasma thrombomodulin for thrombin was three or thrombomodulin appeared to be a single chain with Mr fourfold larger than that of the cellular form. The platelet 78,000, as did placental thrombomodulin. After reduction thrombomodulin, which was distinct from the plasma in addition to the predominant protein with Mr 109,000, forms in its Mr and the Kd for thrombin, is presumably two minor components with Mr 94,000 and 82,000 were synthesized in the megakaryocyte and stored in the detected, as with placental thrombomodulin. These minor platelet, as are (27) and protein S components would be degraded forms, still linked with (28). Recently, Ogura et al. (29) detected immunoreactive disulfide bonds, of the intact thrombomodulin. The ob protein on the surface of a megakaryoblastic cell line and served Mr change of the platelet thrombomodulin before platelets with monoclonal anti-thrombomodulin-IgG. The localization of thrombomodulin in or on intact platelets and and after reduction was identical to that of lung thrombo modulin (14). Analysis using anti-thrombomodulin F(ab•L) 2, megakaryocytes is under investigation. As thrombomodulin has a high affinity for thrombin, it both under nonreducing and reducing conditions, revealed no bands with the same Mr as anti-human platelet glyco might serve as a thrombin-binding protein on platelets. The glycoprotein Ib with Mr 185,000 (30), glycoprotein V with protein Ib-IgG (data not shown). The cofactor activity for thrombin-catalyzed activation Mr 75,000 (31), Mr 74,000 protein (32), and complexed of protein C and the thrombomodulin antigen concentration proteins with Mr 900,000 (33) have been reported to be thrombin-binding proteins on the platelet. Glycoprotein Ib were then examined using the partially purified platelet apparently differs from thrombomodulin in terms of the Mr thrombomodulin fraction. Table I shows that the specific and immunological properties. The glycoprotein V also activity of platelet thrombomodulin was the same as that of differs, since this protein was released from platelets by the placental thrombomodulin. To obtain the apparent Michaelis constant (Km) for protein C of platelet throm thrombin (31), but platelet thrombomodulin was not (data

Vol. 104, No. 4, 1988 632 K. Suzuki et al.

not shown). It is unclear which of the membrane proteins Stern, D. (1987) J. Biol. Chem. 262, 6023-6031 identified so far corresponds to thrombomodulin. Based on 10. Ludlam, C.A. (1987) in Haemostasis and Thrombosis (Bloom, A. & Thomas, D., eds.) pp. 933-952, Churchill Livingstone the results of enzyme immunoassay, each platelet was 11. Tracy, P., Nesheim, M., & Mann, K. (1981) J. Biol. Chem. 256, estimated to contain 60•}18 thrombomodulin molecules, 743-751 which is considerably fewer than the number of high 12. Suzuki, K., Nishioka, J., Matsuda, M., Murayama, H., & affinity thrombin-binding sites reported previously (300 Hashimoto, S. (1984) J. Biochem. 96, 455-460 - 400 molecules per platelet). Based on the Kd value for 13. Salem, H.H., Maruyama, I., Ishii, H., & Majerus, P.W. (1984) J. thrombin, thrombomodulin might be a protein involved in Biol. Chem. 259,12246-12251 high-affinity binding sites for thrombin. It is not known 14. Suzuki, K., Kusumoto, H., Deyashiki, Y., Nishioka, J., whether platelet thrombomodulin participates in platelet Maruyama, I., Zushi, M., Kawahara, S., Honda, G., Yamamoto, S., & Horiguchi, S. (1987) EMBO J. 6, 1891-1897 activation by thrombin. 15. Lundblad, R.L., Uhteg, L.C., Vogel, C.N., Kingdon, H.S., & The average content of thrombomodulin is 40,000 Mann, K.G. (1975) Biochem. Biophys. Res. Commun. 66, 482 50,000 molecules per endothelial cell. Thus, the total - 489 amount of endothelial thrombomodulin in an adult man was 16. Suzuki, K., Nishioka, J., & Hashimoto, S. (1983) J. Biol. Chem. estimated to be about 300mg (25), while the amount of the 258,163-168 17. Ohno, Y., Kato, H., Morita, T., Iwanaga, S., Takada, K., & platelet thrombomodulin in whole blood of an adult man (70 Sakakibara, S. (1980) J. Biochem. 90, 1387-1395 kg body weight, 3•}105 platelets/mm3) was estimated to be 18. Okamoto, S., Hijikata, A., Kikumoto, R., Tonomura, S., Hara, about 13ƒÊg in total. In view of this quantitative difference, H., Ninomiya, K., Maruyama, A., Sugano, M., & Tamao, Y. endothelial cell thrombomodulin is considered to contribute (1981) Biochem. Biophys. Res. Commun. 101, 440-445 to the regulation of coagulation for circulating blood. The 19. Suzuki, K., Kusumoto, H., & Hashimoto, S. (1986) Biochim. platelet thrombomodulin may participate in the regulation Biophys. Acta 882, 343-352 of activation of and prothrombin on platelets 20. Yoshitake, S., Hamaguchi, Y., & Ishikawa, E. (1979) Scand. J. which are aggregated at a localized area of injury or Immunol. 10, 81-86 21. Blobel, G.B. & Dobberstein, B. (1975) J. Cell Biol. 17, 835-851 inflammation on the vessel wall. The activated protein C 22. Burnette, W.N. (1981) Anal. Biochem. 112, 195-203 formed by the thrombin-thrombomodulin complex on the 23. D'Angelo, A., Lockhart, M.S., D'Angelo, S.V., & Taylor, F.B., platelets may inactivate Factors VIIIa and Va in concert Jr. (1987) Blood 69, 231-237 with protein S on the surface of the platelets. 24. Nishioka, J. & Suzuki, K. (1987) The 10th Congress of the Japanese Society on Thrombosis and Hemostasis, Maebashi, p. 51 (abstract) REFERENCES 25. Ishii, H. & Majerus, P.W. (1985) J. Clin. Invest. 76, 2178-2181 26. Nagashima, K., Kazama, M., Tahara, C., Nakamura, K., Abe, T., 1. Esmon, N.L., Owen, W.G., & Esmon, C.T. (1982) J Biol. Chem. Nakano, M., Tsubouchi, J., & Ishii, H. (1987) The 10th Congress 257,859-864 of the Japanese Society on Thrombosis and Hemostasis, 2. Suzuki, K., Stenflo, J., Dahlback, B., & Teodorsson, G.(1983) J. Maebashi, p. 129 (abstract) Biol. Chem. 258,1914-1920 27. Sporn, L.A., Chavin, S.I., Marder, V.J., & Wagner, D.D. (1985) 3. Vehar, G.A. & Davie, E.W. (1980) Biochemistry 19, 401-410 J. Clin. Invest. 76,1102-1106 4. Marcum, J., McKenny, J., & Rosenberg, R. (1984) J. Clin. Invest. 28. Ogura, M., Tanabe, N., Nishioka, J., Suzuki, K., & Saito, H. 74,341-350 (1987) Blood 70, 301-306 5. Stern, D., Brett, J., Harris, K., & Nawroth, P. (1986) J. Cell Biol. 29. Ogura, M., Takamatsu, J., Tanabe, N., Saito, H., Nagura, H., & 102,1971-1978 Maruyama, I. (1988) Acta Haematol. J. 51, 477 (abstract) 6. Loskutoff, D. & Edington, T.(1977) Proc. Natl. Acad. Sci. U.S. 30. Ganguly, P. & Gould, N.L. (1979) Br. J. Haematol. 42,137-145 74,3903-3907 31. Berndt, M.C. & Phillips, D.R. (1981) J. Biol. Chem. 256 7. Weksler, B., Marcus, A., &Jaffe, E. (1977) Proc. Natl. Acad. Sci. , 59-6532 . Chelladurai, M., Fossett, N.G., & Ganguly, P. (1983) J. Biol. U.S. 74, 3922-3936 Chem. 258, 1407-1410 8. Stern, D., Nawroth, P., Handley, D., & Kisiel, W. (1985) Proc. 33. Harmon, J.T. & Jamieson, G.A. (1985) Biochemistry 24 Natl. Acad. Sci. U.S. 82, 2523-2527 , 58-64 9. Rimon, S., Melamed, R., Savion, N., Scott, T., Nawroth, P., &

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