THROMBOSIS RESEARCH 67; 687-696,1992 0049-3848192 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

PURIFICATION WITH THE

OF -BIT, RAT AND MOUSE PROTEIN USE OF MONOCLONAL ANTIBODY TO HUMAN PROTEIN C, PCol

C

Michio Kimura, Kayoko Kurosawa-Ohsawa, Mikiko Takahashi, Masayoshi Koyama, Shigeaki Tanaka and Tetsuro Matsuishi Pharma Research Laboratories, Hoechst Japan Limited, 1-3-2 Minamidai, Kawagoe, Saitama 350, JAPAN

(Received 2.3.1992; accepted in revised form 23.7.1992 by Editor A. Takada)

specific Ca++-dependent monoclonal antibody to ycarboxyglutamic acid (Gla) domain of protein C was produced. It did not cross-react to the other vitamin Kdependent plasma proteins but to protein C of the other species. Using this monoclonal antibody, PCOl, rabbit (6Opg) and mouse (4Opg) protein Cs were (17Opg), rat isolated from 100 ml of their plasma by affinity Al1 of these protein Cs were two chain chromatography. form linked by disulfide bond as wel1 as human protein C Rat and activated by thrombin-thrombomodulin complex. and mouse protein Cs showed similar characters to human protein C. On the other hand rabbit protein C had different Mr of heavy and light chains and showed lower anticoagulant activity compared with human protein C.

ODUCTIOI Protein C is a vitamin K-dependent serine protease precursor It is related to anticoagulant system in blood coagulation (1,Z). then complex thrombin-thrombomodulin (3) and activated by Protein C has inactivates Factor Va (4) and Factor VIIIa (5). (6), bovine (7), canine (8) and mouse been isolated from human Al1 of these protein Cs were purified as two chain plasma (9). zymogen linked by a single disulfide bond.

Keywords:

Rabbit protein C, Rat protein C, Mouse protein Monoclonal antibody, Purification, Coagulation

687

C,

688

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PROTEIN C AFFINITY PURIFICATION

C from to purify protein In the present work, we aimed rabbit,rat and mouse plasma by immunoaffinity chromatography using which recognized Gla domain of human a monoclonal antibody, protein C calcium-dependently. By immunoaffinity chromatography, we could develop a simple method for purification of protein C The purified protein Cs were compared with from blood plasma. human protein C as to the activation and the biological activity.

Human protein C, protein S, protein Z, prothrombin, Factor 1X (University of and Factor X were kindly provided by Dr. W. Kisiel Human Factor VII and human a-thrombin were purified New Mexico). according to the methods of Broze and Majerus (10) and Kawabata et al. Protein C activator was purified from (ll), respectively. lyophilized venom of Agkistrodon contortrix contortrix (Sigma) according to the method described (12). Monoclonal antibody to human protein C, PCOl, was prepared as described previously (9). PCOl-Sepharose 4B was made by coupling PCOl to Activated CHSepharose 4B (Pharmacia) according to the manufactural manual. The followings were purchased; rabbit, rat and mouse blood plasmas from Nihon SLC, Hamamatsu, Japan, protein C deficient plasma, Pathromtin and human antithrombin 111 (AT 111) from Behringwerke, (H-D-Lys-Z-Pro-Arg-pNA) from West Germany, Chromozym PCa Boehringer Mannheim, rabbit thrombomodulin from West Germany, American Diagnostica, Heparin from Green Cross, Tokyo. SDS-polyacryl_amide

gel

electroohoresis

(SDS-PAGE)

and

Western

SDS-PAGE was performed according to the method of Laemmli (13) using 12.5 30 gel. Western blotting was carried out by the method of Burnette (14) using PCOl and peroxidase-labeled rabbit antimouse immunoglobulins antibody. ion of raut and mouse wrotein C One hundred ml of citrate plasma containing 10 mM benzamidineHCl and 8 ml of 1 M BaC were mixed and stirred at 4 'C for 1 h. The precipitate was collected by centrifugation at 5,000 rpm for 10 min and washed with 15 ml of 0.15 M NaCl, 5 mM BaC containing 10 mM benzamidine-HCl. Then the precipitate was suspended in 8 ml of 20 mM Tris-HCl, pH 7.2, and 3 ml of 50 % saturated ammonium sulfate was added. After stirring for 1 h the suspension was centrifuged and the supernatant was dialysed against 2 L of 0.15 M NaCl, 20 mM Tris-HCl, pH 7.2, containing 10 mM benzamidine-HCl. After adding of CaC12 up to 5 mM, the dialyzate was applied to PCOl-Sepharose 4B column equilibrated with 20 mM Tris-HCl, pH 7.2, containing 0.15 M NaCl and 5 mM CaC12. Elution was performed with 5 mM EDTA in 20 mM Tris-HCl, pH 7.2, 0.15 M NaCl. Next, the eluate was applied to DEAE-HPLC column (8.2 x 75 mm, Waters) equilibrated with 20 mM Tris-HCl, pH 8.0, and protein C was eluted with linear gradient of NaCl from 0 to 0.5 M. ion of wrotein C bv wrotein C activator Protein C (2 pg/ml) and protein C activator

(0.15 pg/ml)

were

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689

mixed in 20 mM Tris-HCl, pH 8 .O, 0.15 M NaCl and incubated at 37 "C for 10 min (human and rabbit protein C) oz for 40 min (rat and mouse protein C). Activation was terminated by addition of 0.1 vol. of 10 % SDS, 20 % 2-mercaptoethanol and heating at 80 "C for 5 min. Then samples were subjected to SDS-PAGE. Assav for amidolvtic activitv of orotein C Activation by protein C activator: Protein C (2pg/ml) and protein C activator (0.2 pg/ml) were mixed in 20 mM Tris-HCl, pH 8.0, containing 2 mM EDTA and 0.05 % bovine serum albumin and incubated at 37 'C for 0, 3, 2, 4, 8, 16, 32 and 64 min. At the time indicated a portion (40 pl) was taken and mixed in 400 ~1 of 0.8 mM Chromozym PCa in 20 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl at 37 "C. After incubation for 10 min the reaction was terminated with 400 /.llof 50 % acetic acid and the absorbance at 405 nm was measured. Activation by thrombin-thrombomodulin complex: Protein C (1.9 (O.'Ï pg/ml) was incubated at 37 'C with complex of human thrombin pg/ml) and rabbit thrombomodulin (7.5 U/ml) in 320 pl of 50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 0.01 % Tween 80, 5 mM CaC12. At the time indicated, samples were withdrawn and 40 /ll of the transferred to reaction tubes containing 50 CL1 of 2.0 U/ml AT111 and 2.5 U/ml heparin and incubated at 37 'C for 5 min to terminate the reaction. Then amidolytic activity was measured as described above.

for

anticoaaulanteofn

I

.

C Protein C was activated by protein C activator as described Fifty pl The reaction was stopped by cooling at 0°C. previously. of human protein C deficient plasma and 50 pl of Pathromtin was Then 100 pl of activated mixed and incubated at 37 'C for 2 min. and incubated for 1 protein C was added at various concentration min. Clotting time was measured after addition of 50 ~1 of CaC12.

Assav

Determination of wrotein concentration Protein concentrations of purified with a BCA protein assay kit (PIERCE) as the standerd.

protein Cs were determined using bovine serum albumin

PESULTS antibody, PCOl, In this purification we used the monoclonal antibodies prepared in our anti-human which is one of the laboratory. PCOl recognized the Gla domain of protein C in the The cross-reactivity of PCOl to presence of calcium ion (9,15). other vitamin K-dependent plasma proteins was examined by Western PCOl bound light chain of protein C Blotting analysis (Fig. 1). indicating that the antibody only in the presence of Ca++, recognizes the calcium-dependent conformation in the Gla domain of protein C. In order to examine the reactivity of PCOl to rabbit, rat and blotting analysis was performed to the mouse protein C, Western barium citrate adsorpted samples of rabbit, rat and mouse plasma There was a PCOl bound material in each sample with (Fig. 2). These bands seems to be light chains of the M,=20,000-30,000. protein C was very large The light chain of rabbit protein Cs.

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PROTEIN C AFFINITY PURIFICATION

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(4 f f

130K 75K

f

SOK

f

39K

f

27K

f

17K

II

VII

IX

x

PC

PS PZ

II

VII

IX

x

PC

PS PZ

of monoclonal antibody, Fig. 1 Cross-reactivity PCOl, to vitamin K-dependent plasma proteins. Human prothrombin (II), Factor VII (VII), Factor 1X (1X), Factor X (X), protein C and protein Z (Z) were subjected to S (PS) (PC), protein SDS-PAGE (A) and immuno:blotting analysis (B) with PCOl.

94K

-

67K

-

43K

-

29K

-

20K

-

Hm

1234

Fig. 2 Detection of Protein Cs on rabbit, rat and mouse blood plasma. Barium citrate adsorption was performed with 1 ml of plasma as described in MATERIALS AND METHODS. The precipitate wes suspended with 100 % saturated ammonium Pl of 30 sulfate and centrifuged. The supernatant was dialyzed in 20 mM Tris-HCl, pH 7.2. The dialyzed sample was subjected to immunoblotting analysis using PCOl. The samples from human (l), rabbit (2), rat (3), and mouse (4) plasma were applied to SDS-polyacrylamide gel with the volume of 2.5 )11, 2.5 pl, 5 pl and 10 pl, respectively.

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PROTEIN

C AFFINITY

PURIFICATION

691

compared with that of human, and rat mouse protein C. Single chain form of protein C was observed only on human sample with M,=63,000. On mouse sample, PCOl reacted several proteins with Mr=30,000, 70,000 and 90,000. It seems that these proteins originate from mouse immuno-globulins, because rabbit immunoblobulins to mouse immuno-globulins were used for this analysis as second antibody. This result suggests that PCOl reacts specifically to protein C in rabbit, rat and mouse plasma. Rabbit, rat and mouse protein C were purified from 100 ml of blood plasma by the procedure described in MATERIALS AND METHODS; first step was barium-citrate adsorption, second step was affinity chromatography by PCOl and the final step was anion exchange chromatography. Rabbit, rat and mouse protein C were obtained 170 Figure 3 shows SDS-PAGE of pg, 60 pg and 40 pg, respectively. purified protein Cs under reduced and nonreduced conditions. These protein Cs consisted of two chains linked by disulfide bond NO single chain form as wel1 as human and bovine protein C (6,7). protein C, which is found in human plasma about 10-20 0, was Heavy and light chains of rat and purified from these plasma. mouse protein Cs were similar molecular weight to those of human heavy Rabbit protein C, however, consisted of smaller protein C. than light chain (Mr=32,000) chain larger ( Mr=36,500 ) and human protein C (heavy; Mr=z38,OOO, light; ïYr=25,OOO).

B

43K

+

20K

+

C

rl, +

_

+

-

Purified Fig. 3 SDS-PAGE of purified protein Cs. C (C) were subjected rat (B), and mouse protein (-) 2-mercaptoethanol. with (+) or without

+

-

rabbit (A), to SDS-PAGE

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67K

43K

28K

1

2

3

4

5

6

7

8

pattern of protein Cs on SDS-PAGE. Human Fig. 4 Activation protein C (7), were (3), rat (5), and mouse (l), rabbit activated by protein C activator as described in MATERIALS AND protein C of METHODS and subjected to SDS-PAGE ; activated human, rabbit, rat and mouse are 2, 4, 6 and 8, respectively.

Rabbit, rat and mouse protein Cs were activated by protein C activator of snake venom or thrombin-thrombomodulin complex as Figure 4 shows SDS-PAGE analysis of wel1 as human protein C. activated protein Cs by protein C activator. The heavy chains of these protein Cs became slightly smaller by releasing the activation peptide after activation as wel1 as human protein C. Time course of activation of these protein Cs was examined by measuring the amidolytic activity on synthetic chromogenic substrate (Fig. 5A). Rabbit protein C was activated by protein C activator at similar rate to human protein C but its activated form had very low amidolytic activity compared with human protein C. On the other hand rat and mouse protein Cs were activated slower than human and rabbit protein Cs but their activated forms had comparable amidolytic activities to activated human protein C. On this condition, it was necessary for rat and mouse protein C to be incubated about eight-fold longer than human and rabbit protein C for full activation. Activation profile was also examined using thrombin-thrombomodulin complex (Fig. 5B). The profile was similar to that using protein C activator of snake venom. These results suggest that the structure of the activation site of rabbit protein C is similar to that of human protein C but the rabbit activated protein C has different substrate specificities from human activated protein C or low amidolytic acivity. In

PROTEIN C AFFINITY PURIFICATION

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693

0.6

ln z a

0.6 UI

z

0.4

a

0.4

0.2

0.2

m

I

0.0

0.0 0

20

60

40

Time

0

20

(min)

40

Time

60

(min)

Fig. 5 Activation of protein Cs by protein C activator and thrombin-thrombomodulin complex. Human ( 0 ), rabbit ( m ), and mouse protien C ( 0 ) were activated by rat ( 0 ), protein C activator (A) and thrombin-thrombomodulin complex (B) for 2, 4, 8, 16, 32, 64 min and amidolytic activities were measured as described in MATERIALS AND METHODS.

ol 0

2

4

Protein

6

8

10

C (pglml)

protein Cs. activity of Anticoagulant Fig. 6 Anticoagulant activities of human ( 0 1, rabbit ( ?? ), and mouse protein C ( 0 ) were measured rat ( 0 ), with human protein C deficient plasma as described in MATERIALS

AND METHODS.

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PROTEIN C AFFINITY PURIFICATION

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and mouse protein C have a little different contrast, rat structure at their activation site from human protein C but their activated forms have similar activity to human activated protein The isolated rat protein C was already activated about 10 % C. thrombinactivator or by protein C before activation The cause of the activation is unclear. thrombomodulin complex. The anticoagulant activities of the protein Cs were also measured rat and Human, to human protein C deficient plasma (Fig. 6). mouse activated protein C had similar anticoagulant activity while than the rabbit activated protein C showed very lower activity others.

Piscussiorl protein C has been purified by In several laboratories, immunoaffinity chromatography using monoclonal antibodies against monoclonal human protein C (16,17). Especially Ca ++-dependent which recognize Gla domain or epidermal growth factor antibodies, (EGF)-like domain of protein C, are very useful tooi for affinity These antibodies, however, are limited in use due purification. to other to their specificities. That is, some cross-react are too vitamin K-dependent plasma proteins and the others specific to recognize protein Cs of other species. Our monoclonal antibody, PCOl, presented here does not cross-react to other human vitamin K-dependent plasma proteins (Fig. 1); it is nevertheless reported, react to rabbit, rat and mouse protein C. As previously PCOl recognizes Gla domain of protein C in the presence of calcium These results suggest that there is a Ca++-induced ion (9,15). unique structure in Gla domain of protein C, which is common among various from that of other vitamin Kspecies but different dependent plasma proteins. Though species specificity of protein C in coagulation and fibrinolysis system has been reported (18,19), it has not been described from molecular aspects because of the difficulty of the purification from smal1 amount of blood plasma. This problem has been overcome by use of the monoclonal antibody, PCOl, and enough amount of protein Cs has been purified from rabbit, rat and mouse blood plasma for the analysis of their activation mode. In the comparison of the molecular weight, the activation and the activities, rabbit protein C was different from human protein C. The light chain of rabbit protein C was extremely larger than that of human protein C, which is likely to be due to the attachment of carbohydrate chains because there are four N-linked carbohydrate chains in human protein C (20,21). Another possible explanation is that rabbit light chain may include one more domain such as EGF-like domain or large insertion sequence, 50-60 amino acids, in addition of Gla domain and two EGF-like domains. Rabbit activated protein C had both low amidolytic activity to synthetic substrate and low anticoagulant activity to human protein C deficient plasma. These low activities may be associated with the large light chain of rabbit protein C, if a Ca++-inducing conformational change of the light chain affects the serine protease part of protein C as previously described (22). We have shown that protein C is purified easily by PCOlimmunoaffinity chromatography from smal1 amount of several species plasma. This monoclonal antibody, PCOl, may be a tool of utility

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PROTEIN C AFFINITY PURIFICATION

for a genera1 detection as wel1 as purification.

of protein

We wish to thank supporting the work.

Drs.

E.

C in plasma

Konz

and

of various

H.

B.

species

for

Maruyama

1. STENFLO, J. A new vitamin K dependent protein: purification from bovine plasma and preliminary characterization. J. Biol. Chem. 251, 355-363, 1976. 2. ESMON, C. T. Protein-C: Biochemistry, implication. Bloed 62, 1155-1158, 1983.

physiology,

3. ESMON, N. L., OWEN, W. G., and ESMON, C. T. membrane-bound cofactor for thrombin-catalyzed protein C. J. Biol. Chem. 257, 859-864, 1982. 4. SUZUKI, K., STENFLO, J., DAHLBACH, Inactivation of human coagulation factor J. Biol. Chem. 258, 1914-1920, 1983.

and clinical

Isolation of a activation of

B., and TEODORSSON, V by activated protein

B. C.

and properties of 5. VEHAR, G. A., and DAVIE, E. W. Preparation bovine factor VIII (Antihemophilic Factor). Biochemistry 19, 401410, 1980. 6. KISIEL, 1979.

W.

Human

protein

C.

J.

Clin.

Invest.

64,

761-769,

7. KISIEL, W., CANFIELD, W. M., ERICSSON, L. H., and DAVIE, E. W. of bovine plasma protein C following Anticoagulant properties activation by thrombin. Biochemistry 16, 5824-5831, 1977. 8. COMP, P. C., JACOCKS, Activation of protein C 1982.

R. M., FERRELL, G. L., and ESMON, C. T. in vivo. J. Clin. Invest. 70, 127-134,

K., KIMURA, M., KUME-IWAKI, A., TANAKA, T., 9. KUROSAWA-OHSAWA, Anti-protein C monoclonal antibody induces and TANAKA, S. thrombus in mice. Blood 75, 2156-2163, 1990. 10. BROZE Jr., G. J., and MAJERUS, Methods Enzymol. 80, 228-237, 1981. ll. KAWABATA, S., MORITA, enzymatic Differente in staphylocoagulase complex 1073-1078, 1985.

P.

w.

Human

factor

VII.

IGARASHI, H. IWANAGA, S., and T., a-thrombinbetween properties Biochem. 97, and free a-thrombin. J.

of a protein C 12. KISIEL, W., CHOI, E., and KONDO, S. Isolation activator from Southern Copperhead Venom. Biochem. Biophys. Res. 917-922, 1987. Commun. 143,

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13. LAEMMLI, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 681-682, 1970. 14. BURNETTE, W. N. "Western Blotting": Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195-203, 1981. 15. TAKAHASHI, M., KIMURA, M., KUROSAWA-OHSAWA, K., KUME-IWAKI, A ., TANAKA, S., and MATSUISHI, T. Epitopes of anti-human protein C monoclonal antibodies. Seikagaku (in Japanese) 62, 715, 1990. 16. NAKAMURA, S. and SAKATA, Y. Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calcium ion complex. Biochem. Biophys. Acts 925, 8593, 1987. 17. REGNAULT, V., RIVAT, C., PFISTER, M., and STOLTZ, J-F. Monoclonal antibodies against human plasma protein C and their use for immunoaffinity chromatography. Thromb. Res. 63, 629-640, 1991. 18. MARCINAK, E. Inhibitor of human blood coagulation elicited by thrombin. J. Lab. Clin. Med. 79, 924-930, 1972. 19. WEISTEIN, R. E. and WALKER, F. J. Species specificity of the fibrinolytic effects of activated protein C. Thromb. Res. 63, 123-131, 1991. 20. FOSTER, D., and DAVIE, E. W. Characterization of a cDNA cording for human protein C. Proc. Natl. Acad. Sci. USA 81, 47664770, 1984. 21. MILETICH, J. P. and BROZE Jr., G. J. PProtein glycosylated at asparagine 329. J. Biol. Chem. 265, 1990.

C is not 1394-1404,

22. ÖHLIN, A-K., BJÖRK, I., and STENFLO, J. Proteolytic formation of protein C containing the y-carboxyglutamic acid rich domain and the EGF-like region. Biochemistry 29, 644-651, 1990.

Purification of rabbit, rat and mouse protein C with the use of monoclonal antibody to human protein C, PC01.

Ca(++)-dependent monoclonal antibody specific to gamma-carboxyglutamic acid (Gla) domain of protein C was produced. It did not cross-react to the othe...
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