J. Biochem. 108, 874-878 (1990)

Monoclonal Antibodies for Human Thrombomodulin Which Recognize Binding Sites for Thrombin and Protein C1 Tatsuya Hayashi and Koji Suzuki2 Division of Enzyme Cytology, Institute for Enzyme Research, University of Tokushima, Tokushima, Tokushima 770

Monoclonal antibodies for human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C, were prepared and their epitopes characterized. All six antibodies (MFTM-l-MFTM-6) bound to an elastase-digested active fragment of thrombomodulin, which contains six consecutive EGF domains. Binding of thrombomodulin to these antibodies did not depend on Ca2+ concentration. MFTM-4, MFTM-5, and MFTM-6 strongly inhibited protein C activation by thrombin and thrombomodulin. MFTM-4 and MFTM-5 inhibited thrombin binding to fixed thrombomodulin and bound to a recombinant mutant EGF456 protein, which contained the fourth, fifth, and sixth EGF domains of thrombomodulin. However, MFTM-6 did not inhibit thrombin binding to thrombomodulin and did not bind to EGF456 protein. Binding of thrombomodulin to fixed MFTM-4 or MFTM-5 was competitively inhibited by a recombinant mutant EGF45 protein which contained the fourth andfifthEGF domains and also by a mutant EGF56 protein which contained the fifth and sixth EGF-domains. These results suggest that epitopes of MFTM-4 and MFTM-5 are located in the fifth EGF domain of thrombomodulin. Thus, the binding site for thrombin is located in the fifth EGF domain. These results also suggest that an epitope for MFTM-6 is located at a region near the binding site for y-carboxyglutamic acid residues of protein C via Ca2+ on thrombomodulin.

Thrombomodulin is a high-affinity thrombin receptor (iCi = 0.48 nM) on vascular endothelial cells and placental syncytiotrophoblasts (1-3). Thrombin bound to thrombomodulin specifically activates a vitamin K-dependent serine protease zymogen, protein C (2). Activated protein C proteolytically inactivates blood coagulation cofactors, Factor Va and Factor Villa (4-6). Thrombomodulin also inhibits procoagulant activities of thrombin, such as fibrinogen clotting, Factor V activation (7) and platelet activation (8). Thus, thrombomodulin plays an important role in the regulation of intravascular blood coagulation. Previously, we determined the nucleotide sequence of human thrombomodulin cDNA (9). The amino acid sequence, deduced from the nucleotide sequence, indicated that mature thrombomodulin is composed offivetentative domains; an NH2-terminal domain, a domain containing six consecutive EGF domains, an O-glycosylation site-rich domain, a transmembrane domain, and a cytoplasmic domain. Kurosawa et al suggested that the thrombinbinding site on thrombomodulin is contained in a cyanogen bromide-digested 10-kDa fragment which also contained the fifth and sixth EGF domains. However, this fragment did not show cofactor activity for protein C activation (10, 1

This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and by grants from the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and the Inamori Foundation. 1 To whom correspondence should be addressed. Abbreviations: EGF, epidermal growth factor; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Gla, y-carboxyglutamic acid; MCA, 4-methylcoumarinyl-7-amide; BOC, f-butoxycarbonyl; pNA, p-nitroaniline; BSA, bovine serum albumin; TBS, Tris-buffered saline; ELJSA, enzyme-linked immunosorbent assay.

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22). Kurosawa et al. (10) also suggested that y-carboxyglutamic acid (Gla) residues of protein C interact with an elastase-digested active fragment of thrombomodulin via Ca2+. We reported that a region containing the fourth, fifth, and sixth EGF domains was essential for efficient protein C activation (12, 13). In the present study, we prepared and characterized monoclonal antibodies for human thrombomodulin, and localized the binding sites for thrombin and protein C within thrombomodulin using these antibodies to elucidate the molecular mechanism of the activation of protein C by thrombin-thrombomodulin complex. EXPERIMENTAL PROCEDURES Materials—Protein C (5), thrombin (14), and antithrombin HI (15) were purified from human materials. Thrombomodulin was purified from human placenta (26). The Mr and absorption coefficients used for the respective proteins were as follows: protein C: 62,000, 4 ^ = 1 3 . 7 (7); thrombin: 37,000, Ajgo = 20.0 (14); placental thrombomodulin: 78,000, Awa= 14.0 (27). Maxisorp II microwell plates were purchased from NUNC. PVDF membranes for Western blotting were obtained from Millipore. Lubrol-PX, sodium dodecyl sulfate (SDS), Tween-20 and o-phenylenediamine were purchased from Nacalai Tesque, Kyoto. BrCNactivated Sepharose 4B, Protein A-Sepharose CL-4B were obtained from Pharmacia. Gelatin, prestained M markers for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), peroxidase-conjugated anti-rabbit IgG-goat IgG, peroxidase-conjugated anti-mouse IgG-goat IgG, 4-chloro-lnaphthol, and Chelex 100 were obtained from Bio-Rad. Peroxidase-conjugated streptavidin was purchased from J. Biochem.

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Received for publication, June 7, 1990

Functional Domains of Thrombomodidin

Vol. 108, No. 5, 1990

of the freshly prepared substrate solution (2.5 mg/ml o-phenylenediamine and 0.03% H2O2 in 0.1 M phosphate buffer, pH 5.4) was then added to each well. The enzyme reaction was terminated after 30 min by adding 50 //I of 4.5 M sulfuric acid. The absorbance of each well was determined at 490 run using a Dynatech MR-600 Microplate Reader. In the case of fixed monoclonal antibodies, flat-bottomed wells of microwell plates were coated with monoclonal anti-thrombomodulin IgG (3//g/ml) in 0.1 M sodium bicarbonate buffer, pH 9.2. After a 2-h incubation, unbound material was removed by washing with the same buffer. The wells were blocked with 150 fi\ of TBS containing 10% BSA for 2 h, then washed three times with TBS containing 0.5% BSA and 0.05% Tween 20. Then 100//I of various concentrations of thrombomodulin was added to the wells and the plates were incubated for 2 h. After three washings with the same buffer, polyclonal anti-thrombomodulin rabbit IgG or biotinylated monoclonal anti-thrombomodulin murine IgG was added and the plates were further incubated for 3 h. After washing with the same buffer, polyclonal anti-thrombomodulin rabbit IgG was detected with peroxidase-conjugated anti-rabbit IgG-goat IgG and biotinylated monoclonal antibody was detected with peroxidase-conjugated streptavidin. Measurements of peroxidase activity were performed as described above. When the effect of Ca2+ concentration on the binding of thrombomodulin to monoclonal antibody was to be determined, all buffer solutions were treated with Chelex 100 to eliminate metal ions. Then, various Ca2+ concentrations were adjusted by the addition of CaCl2 solution. Effect of Antibodies on Cofactor Activity of Thrombomodulin for Protein C Activation—Thrombomodulin was assayed for its ability to accelerate the thrombin-catalyzed activation of protein C(13). Thrombomodulin was incubated with each monoclonal antibody in 100 )A of TBS at 4*C overnight. Thereafter, 50 ^1 of TBS containing 0.3% BSA and 10 mM CaCl2, 10 //I of thrombin (2 fig/m\), and 15 //I of protein C (100/*g/ml) were added to the incubation mixture and incubated at 37'C for 1 h. The reaction was stopped by the addition of antithrombin HI (40 ng/ra\ final) and heparin (1 unit/ml final). The amount of activated protein C formed was assayed using 2 ml of 200 /x M Boc-Leu-Ser-Thr-Arg-MCA in a buffer consisting of 0.05 M Tris-HCl and 0.1 M CsCl, pH 8.0. The fluorescence of the released aminomethylcoumarin was determined by using a fluorospectrophotometer with excitation at 380 nm and emission at 440 nm. One pmol of activated protein C released 29 pmol of aminomethylcoumarin/min under these conditions (23). Effects of Monoclonal Antibodies on Thrombin Binding to Thrombomodulin—Wells of microwell plates were coated with 100//I of thrombomodulin (3//g/ml) in 0.1 M sodium bicarbonate buffer, pH 9.2, for 2 h. The wells were blocked with 150//I of TBS containing 10% BSA for 1 h, then 100//I of various concentrations of monoclonal antibody diluted appropriately with TBS containing 0.5% BSA and 0.05% Tween 20 was added and the plates were incubated for 2 h. After three washings with TBS containing 0.5% BSA and 0.05% Tween 20, 100 ^1 of thrombin in TBS containing 0.5% BSA was added to each well, and the plates were further incubated for 30 min. After three washings, 100 //I of 200 ^M peptide substrate S-2238 was

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Amersham International, U.K. Boc-Leu-Ser-Thr-ArgMCA, a fluorogenic substrate for activated protein C, was obtained from the Protein Research Foundation, Osaka. A chromogenic substrate for thrombin, H-D-Phe-Pip-ArgpNA (S-2238) was obtained from Kabi, Sweden. Bovine serum albumin (BSA) and 2,6,10,14-tetramethylpentadecane (pristane) were purchased from Sigma. All other chemicals and reagents obtained were of the best grade commercially available. Preparation of Monoclonal Antibodies to Human Thrombomodulin—Monoclonal antibodies to human thrombomodulin were prepared by the method of Oi and Herzenberg (18). Anti-thrombomodulin antibody-producing cell lines, screened by solid-phase enzyme-linked immunosorbent assay (ELJSA) (19), were recloned by the limiting dilution method. Finally, each clone was injected intraperitoneally into BALB/c mice that had been pretreated with pristane. Monoclonal antibodies were purified from the mouse ascites fluid by affinity chromatography using protein A-Sepharose CL-4B. Preparation of Recombinant Mutant Thromobomodutin Proteins—Recombinant mutant proteins of human thrombomodulin were prepared by site-directed deletion mutagenesis (20) using thrombomodulin cDNA and an expression plasmid pSV2 vector as described previously (13). Next, pSV2TMEGF456 plasmids containing the fourth, fifth, and sixth EGF domains, pSV2TMEGF45 plasmids containing the fourth and fifth EGF domains and pSV2TMEGF56 plasmids containing the fifth and sixth EGF domains were constructed and transfected into COS-1 cells. Recombinant mutant EGF456, EGF45, and EGF56 proteins expressed by COS-1 cells were partially purified from culture medium by affinity chromatography using anti-human thrombomodulin rabbit IgG-coupled Sepharose 4B. SDS-PAGE—This was performed by the method of Blobel and Dobberstein using 7.5 to 15.0% polyacrylamide gradient slab gels (21). Western Blotting Analysis—Western blotting after SDS-PAGE of thrombomodulin, elastase-treated thrombomodulin, and/or EGF456 protein was carried out according to the method of Burnette (22) using PVDF membrane. After electrophoretic transfer, the binding of murine monoclonal antibodies or rabbit polyclonal antibodies to proteins immobilized on PVDF membranes was detected using peroxidase-conjugated anti-mouse IgG-goat IgG or peroxidase-conjugated anti-rabbit IgG-goat IgG, and 4chloro -1 - naphthol. Solid-Phase ELJSA—Assays were performed at room temperature. In the case of fixed thrombomodulin, flatbottomed wells of microwell plates were coated with 100 //1 of thrombomodulin (3 //g/ml) in 0.1 M sodium bicarbonate buffer, pH 9.2. After a 2-h incubation, unbound material was removed by washing with the same buffer. The wells were blocked with 150 //I of 0.05 M Tris-HCl, 0.1 M NaCl, pH 7.5 (TBS) containing 10% BSA for 2 h, then washed three times with TBS containing 0.5% BSA and 0.05% Tween 20. Then 100 //I of monoclonal antibody at various concentrations was added to the wells and the plates were incubated for 2 h. After another three washings with the same buffer, 100^1 of peroxidase-conjugated anti-mouse IgG-goat IgG in the same buffer was added to wells and the plates were incubated for 2 h. After three washings, 100 //I

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T. Hayashi and K. Suzuki

RESULTS

Characterization of Monoclonal Antibodies—Six monoclonal antibodies for human thrombomodulin, designated MFTM-l-MFTM-6, were cloned. MFTM-4 belonged to the subclass IgG2a/k and the five other antibodies to IgGl/kThe results from Western blotting analysis are summarized in Table I. All antibodies bound to nonreduced thrombomoduUn, and only MFTM-4 and MFTM-5 bound to reduced thrombomodulin. Furthermore all antibodies bound to an elastase-digested active fragment of thrombomodulin (10), and as shown in Fig. 1, only MFTM-4 and TABLE I. Binding of monoclonal antibodies to elastase-dlgested thrombomodulin and recombinant EGF456 protein. Binding of monoclonal antibodies to various thrombomodulin (TM) proteins was analyzed by Western blotting as described in "EXPERIMENTAL PROCEDURES.' The binding ability of each antibody to TM protein is indicated as follows: + +, strongly binding; +, weakly binding; —, no binding. Antibody

Nonreduced TM

Reduced TM

Elastase-digested TM fragment

MFTM-5 bound to a recombinant EGF456 protein which contained the fourth, fifth, and sixth EGF domains of thrombomoduUn. The binding ability of thrombomodulin to fixed antibodies was examined over a wide range of Ca2+ concentrations. However, the interactions between thrombomodulin and all antibodies were not affected by Ca2+ concentration (data not shown). Competition between Antibodies for Bidning to Thrombomodulin—Competition between antibodies for binding to thrombomodulin was examined by solid-phase ELISA. As summarized in Table II, MFTM-1 competed with all other antibodies, MFTM-4 and MFTM-5 competed with each other, and MFTM-2, MFTM-3, and MFTM-6 competed among themselves. Effects of Monoclonal Antibodies on ThrombomodulinDependent Protein C Activation—Figure 2 shows that MFTM-4, MFTM-5, and MFTM-6 strongly inhibited protein C activation by thrombin in the presence of thrombomodulin, and MFTM-1, MFTM-2, and MFTM-3 moderTABLE n. Competition between monoclonal antibodies for binding to thrombomodulin. After 100//I of each monoclonal antibody (3 //g/ml) had been fixed to wells of a microwell plate, 100 til of thrombomodulin (12.5 ng/ml) was added and the plate was incubated for 2 h. After three washings, various biotinylated monoclonal antibodies were added. After 2 h incubation, biotinylated monoclonal antibody bound to thrombomodulin was determined using peroxidase-conjugated streptavidin as described in "EXPERIMENTAL PROCEDURES." Fixed Binding of biotinylated antibody antibody MFTM-1 MFTM-2 MFTM-3 MFTM-4 MFTM-5 MFTM-6 0.128 0.111