Monoclonal antibodies to prothrombin

THROMBOSIS RESEARCH 47; 501-510, 1987 0049-3848187 $3.00 + .OO Printed in the USA. Pergamon Journals Ltd. All rights reserved.

MONOCLONAL

Medical

ANTIBODIES

TO PROTHROMBIN

Om P. Malhotra and Oscar Sudilovsky Research Service, Veterans Administration Medical Center Institute of Pathology, Case Western Reserve University Cleveland, Ohio 44106 USA

and

(Received 25.4.1987; Accepted in original form 23.5.1987 by Editor W.H. Seegers)

ABSTRACT Hybridoma technology was used for the production of murine monoclonal Hybrid cell cultures were assayed antibodies to bovine normal prothrombin. both in the absence and presence of for the production of antibodies, calcium ions, by Enzyme-Linked Immunosorbent Assay (ELISA). Antibodyproducing cell lines were cloned two times and grown as ascites tumors. Monoclonal antibodies (McAb), isolated by affinity chromatography (Protein A-Sepharose), were tested for their affinity for normal (IO-Gla) and dicoumarol-induced abnormal prothrombins containing 2, 5, 7, 8 and 9 ‘6A total of 24 McAb were obtained and the carboxyglutamyl (Cla) residues. immunoglobulins were of the IgCl subclass. Nine of the twenty-four McAb did not require Ca2+ for the formation of Ag-Ab complexes, and reacted equally with normal and Cla-deficient prothrombins. These antibodies had affinity for prethrombinl (Pl) but not for the Cla-containing prothrombin fragment1 (Fl) portion of the molecule. In contrast, the 15 Ca*+-dependent McAb reacted with Fl but not with Pl. They discriminated the abnormal prothrombins based upon their Gla content. For example, though all the Ca2+-dependent McAb formed Ag-Ab complexes with 9-, essentially none formed with 5- or less&la prothrombins. [Some reacted equally with 9- and IO-Gla (normal) prothrombin while others had only 25% of normal affinity for 9-Gla isomer]. Only four and twelve of the 15 McAb had some affinity respectively. for 7-and S&la variants These results show that antibodies which react with the Ca 2+ -stabilized conformation of prothrombin are not specific for normal prothrombin, as has been reported in the literature.

INTRODUCTION Prothrombin is a single-chain glycoprotein with a molecular mass of 72,000 daltons. It contains 582 amino acids, ten of which are ‘d-carboxyglutamyl (Cla) residues present within the first 33 residues of the NH2-terminal portion of the normal Key words:

Bovine prothrombin, dicoumarol-induced, monoclonal Ca*+-dependent antibodies, immuno-cross-reactivity

501

antibodies,

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MONOCLONAL ANTIBODIES TO PROTHROMBIN

Vol. 47. No. 5

molecule. The Gla residues arise fron vitamin K-dependent carboxylation of ten specific glutamyl residues which occurs primarily at the luminal side of the rough endoplasmic reticulum (l-2). These residues enable the molecule to bind Ca*+ and undergo a conformational transition(s) that has been monitored by circular dichroism, the quenching of intrinsic fluorescence (3-7) and immunochemical techniques (g-10). This Ca*+-stabilized structure is essential for the Ca*+-mediated binding of prothrombin to phospholipid (6). This binding brings the zymogen into juxtaposition with the prothrombinase complex [consisting of Factor X, (a serine protease), Factor V (a protein cofactor), negatively-charged phospholipid vesicles (or platelets) and Ca ?+I, thereby markedly decreasing the Km for prothrombin activation (11-I 3). During vitamin K-deficiency, either natural or induced by the vitamin Kantagonists dicoumarol or warfarin, carboxylation of the glutamyl residues is inhibited; therefore, abnormal forms of prothrombin molecules deficient in Gla residues, as well as in functional activity, circulate in the blood of human (14-16) and bovine (17-20) species. Warfarin is a widely-used anticoagulant and has played a central role in the prophylaxis of thromboembolic disease for over four decades. Although warfarin is highly efficacious, its toxic to therapeutic ratio is narrow. The dosage of warfarin, therefore, must be carefully titrated by monitoring the prothrombin time. Despite accurate measurements of prothrombin time, however, 10 to 20% of patients treated with warfarin develop either a bleeding or a thromboembolic complication due to Although warfarin is an effective excessive or inadequate warfarin therapy (21-26). antithrombic agent, its attendant risks create confusion as to proper dosage, monitoring procedures and indications for therapeutic use. For these reasons, we directed our attention to understanding the mechanism of action of warfarin. During 1970/71 (17), we successfully isolated dicoumarol-induced atypical prothrombins and found that the total plasma prothrombin (normal plus atypical) concentration does not decrease during dicoumarol treatment (19). A year later (1972), we reported the existence of multiple As a matter of fact, we have isolated and forms of variant prothrombins (18,19). purified a battery of variant prothrombins containing 0, 1, 2, 5, 7 (27-30), 6, 8, and 9 Cla residues (31,32) from bovine species. Immunoassays have been used to quantitate protein antigen in plasma. Polyclonal antibodies reacting to the entire protein can determine the total antigen concentration but do not provide information concerning its functional properties. Thus, we adapted hybridoma technology (33), which can be used effectively to develop immunochemical reagents which interact with a single antigenic site. We have raised both polyclonal and In this monoclonal (McAb) antibodies against normal (IO-Gla) prothrombin. We give particular communication, we describe the characteristics of 24 McAb. emphasis to those monoclonals which form antigen-antibody (Ag-Ab) complexes with normal (IO-Cla) prothrombin and discuss the extent of their interaction with Gladeficient prothrombins.

MATERIALS

AND METHODS

Proteins:

normal

Highly purified preparation of normal (IO-Cla) bovine plasma, as described previously (34).

prothrombin was obtained from To insure maximum purity, the

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47,

No.

preparation reacted

5

MONOCLONAL

was primarily

subsequent

subjected with

ANTIBODIES

to immunoaffinity normal

immunoaffinity

prothrombin.

TO PROTHROMBIN

chromatography, This purified

503

utilizing McAb which material was used for

experiments.

Partially-carboxylated prothrombins containing 2-, 5- and 7-Gla residues were isolated and purified as described previously (27-29). During the DEAE-cellulose chromatography of prothrombin preparations, from dicoumarol-treated plasma which adsorbed to barium citrate, the ascending portion of the prothrombin peak contained 7-, 8-, 9- and some IO-Cla prothrombin isomers, while the descending portion contained 9-, Each of the 7-, X-, 9- and lo-Gla lo- and some 8-Gla prothrombin isomers. prothrombins, free from each other, were isolated by immunoaffinity chromatography. For this purpose, Ca2+-dependent McAb raised against normal prothrombin (35) and coupled to Affi-Gel 10 (Bio-Rad) were used. Prethrombinl (Pl) and prothrombin fragment1 normal prothrombin with thrombin, and then subjecting cellulose chromatography and gel filtration (36). Immunization

(Fl) were obtained by digesting the reaction products to DEAE-

and cell fusion:

BALB/c female mice were injected subcutaneously with 500 ,ug of normal prothrombin in 0.2 ml saline, emulsified with an equal volume of Freund’s complete adjuvant. The injections were given at three to four sites, in the back near the legs and at the base of the tail. After approximately ten days, a drop of blood was taken to test for the presence of specific antibodies. Four weeks later, animals giving the best antiserum were injected intraperitoneally with an additional 100 Ag of normal prothrombin in 0.2 ml saline (without adjuvant). A final boost of 1OOyg of antigen in saline was given after another 2 weeks and 4 days prior to cell fusion. The spleens from the immunized mice were aseptically removed and sin le cell suspensions were prepared. Spleen lymphocyte (107) and SP2/0 myeloma cell (10 !I) were fused in the presence of 50% polyethylene glycol (PEG 1500), essentially following the method of Galfre and Milstein (33). Antibody-positive clones were expanded in tissue culture flasks to obtain sufficient cells for storage in liquid nitrogen and for growth as ascites tumors, the source of high titers of antibodies. Approximately 2 to 4 x 106 hybridoma cells were injected intraperitoneally into the mice. Seven days prior to this inoculation, each mouse had been injected intraperitoneally with 0.3 ml of sterile pristane oil. Eight to twelve days after the cell injection, ascitic fluid was obtained by the insertion of a sterile lo-gauge needle into the peritoneal cavity. Each mouse was tapped two to three times. The immunoglobulin from these fluids was isolated by centrifugation and affinity chromatography on protein A Sepharose (37). Enzyme-linked

immunosorbent

assay @LISA):

Normal and Cla-deficient prothrombins (5 Ag per ml) were coated onto 96-well flat-bottomed polystyrene microtitre plates (Linbro, Falow Laboratories). To coat the plates, 100,ul of antigen, in 0.05M Tris HCI (pH 8.9) containing 0.14M NaCl and either 2.5 mM CaC12 or 2.0 mM EDTA, was added to each well, covered with Saran Wrap and incubated overnight at 4oC. The assays were conducted at 370C with 1.5h incubations for each step. After each step, the reagents were washed off with Tween-saline (0.5 ml Tween-20, 8.77 g NaCI, 0.02 g NaN3 and 2.5 mM CaC12). The extra binding sites on the plates were blocked by adding to each 200~11 of 1% bovine serum albumin (BSA) in 0.03

504

MONOCLONAL ANTIBODIES TO PROTHROMBIN

Vol. 47, No. 5

M Tris-HCI buffer (pH 7.4) containing 0.1 M NaCI, Tween-20 and 2.5 mM CaC12 (solution A). One hundred,ul of hybridoma supernate (or purified IgG from ascitic fluid and appropriately diluted with solution A) was added to each well to allow the primary antibody to bind to the antigen. A secondary antibody, 1:lOOO diluted goat anti-mouse IgG alkaline phosphatase conjugate (Sigma), was allowed to bind to the primary antibody bound to the antigen. The alkaline phosphatase was detected by adding 100 ~1 of pnitrophenyl phosphate, 0.9 mg per ml of 0.1 M diethanolamine, pH 9.8, containing 0.36 mM MgCI2. The yellow-colored p-nitrophenol formed was measured by a micro-ELISA plate reader at 405 nm. The control value was subtracted. [CaC12 was either not added or was replaced by EDTA to test whether or not the Ag-Ab reaction w:.s calcium iondependent.]

RESULTS Twenty-four hybridoma cell lines were obtained after cloning and subcloning. Immunochemical characterization of the McAb from the ascitic fluid showed that 15 of the 24 McAb required calcium ions for their interaction with (normal) prothrombin. Determination of their cross-reactivity with the Cla-deficient variants by enzyme immunoassay (EIA) showed that the antibodies which did not require calcium ions for their interaction with (normal) prothrombin were equally reactive with 0- to 9-Gla prothrombins. These antibodies did not have affinity for prothrombin fragment1 (Fl), However, they did form molecule. the Gla-containing portion of the prothrombin antigen-antibody (Ag-A b) complexes with prethrombinl, which contains the thrombin potential of the molecule. In contrast, calcium-dependent antibodies were reactive with Fl but not with PI. Moreover, they discriminated Gla-deficient prothrombins from normal; however, they had some affinity for Gla-rich variant prothrombins but none for those with few Gla residues per molecule. For example, though all of the 15 calcium-dependent McAb formed antigen-antibody (Ag-Ab) complexes with 9-Gla, none formed complexes with 5or less-Gla prothrombin. Only four and twelve of the 15 McAb had some affinity for 7and 8-Gla prothrombin isomers, respectively. Relative affinity of nine representative calcium-dependent antibodies with Gla-deficient prothrombins, as determined by The data show that the reactivity of the antibodies ELISA, is given in Table I. decreased markedly in moving from 9- to 8-Gla variant prothrombin. On the other hand, some (5 of 15) McAb (e.g. E4, G8, F7, Fg) had more or less the same affinity for The remaining ten McAb, however, had 9- as they had for IO&la (normal) prothrombin. significantly less binding capacity for 9-Gla prothrombin, but the decrease in moving from IO- to 9-Gla prothrombin was not as sharp as it was between the 9- and 8-Gla interaction of McAb H2 with lo-, 9- and 8- Gla variants (Table 1). For example, prothrombins revealed absorbances 0.97, 0.51 and 0.019, respectively. Based upon the antigenic specificity of calcium-dependent [anti(normal) Category I antibodies prothrombin] antibodies, they were categorized as follows: reacted primarily with IO-Gla and some with 9-Gla but not with the 8-Gla isomer (McAb E7 and G6). Category II antibodies were similar to Category I antibodies except that they also reacted, though minimally, with the S-Gla variant (McAb 87 and H2). Category III antibodies reacted with 9- and IO-Gla equally, and less with 8-Gla (McAb F7, G8, and E4). Category IV antibodies reacted with 9- and IO-Gla equally, and less with 8- and 7-Gla, but not with 5- or less-Gla (McAb F9). Category V antibodies reacted primarily with lo-, to a lesser degree with 9-, still less with 8- and little with 7-Gla prothrombin (McAb F8).

Affinity

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MONOCLONAL ANTIBODIES TO PROTHROMBIN

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TABLE I of calcium ion-dependent anti- (normal) prothrombin and Gla-deficient prothrombin by ELISA (Absorbance

McAb with normal at 405 nm)

Prothrombin

McAb

IO-Gla

9-Cla

8-Gla

7-Gla

5-Cla

2-Gla

c6

1.01

0.57

0.00

0.00

0.00

0.00

E7

0.58

0.26

0.00

0.00

0.00

0.00

I-I2

0.97

0.51

0.019

0.00

0.00

0.00

B7

0.86

0.487

0.014

0.00

0.00

0.00

E4

1.18

1.13

0.031

0.00

0.00

0.00

G8

1.04

0.92

0.023

0.00

0.00

0.00

P7

1.18

1.053

0.064

0.00

0.00

0.00

P9

1.2

1.08

0.076

0.018

0.00

0.00

F8

0.836

0.46

0.142

0.044

0.00

0.00

Reactivity of antibodies, which react with Ca2+-stabilized conformation of normal prothrombin, with partially carboxylated prothrombins. Prothrombins, at a concentration of 5 &ml, were used to coat the walls of the microtitre plate wells. Average values (absorbance at 405 nm) of assays were made in duplicate.

The per cent binding capacity of the 9-Cla variant with two calcium-dependent McAb, viz. G6 and E7, shows that it has about 25% of normal affinity. The average absorbances observed for 9-Cla at a concentration of 2.0pg/ml was 0.76 for G6 and 0.575 for E7 while for IO-Gla (normal) prothrombin at a concentration 0.5j_rg/ml absorbances were 0.767 for G6 and 0.616 for E7 McAb (Table II).

Relative

affinity

TABLE 11 of lo- and 9-Gla prothrombin with Ca2+-dependent do not react with 8- (or less-) Gla isomers.

McAb which

Prothrombin 9-Gla,&ml

IO-Gla,yg/ml

McAb

1.0

1.5

2.0

5.0

0.5

1.5

G6

0.34

0.60

0.76

1.85

0.77

1.32

E7

0.20

0.37

0.58

1.94

0.62

1.32

The relative affinity was determined assays were made in quadruplicate.

by ELISA.

Average

values (absorbance

at 405nm) of

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MONOCLONAL ANTIBODIES TO PROTHROMBIN

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DISCUSSION Anti- (normal) prothrombin polyclonal Ab, which react with the Ca2+-stabilized conformation of (normal) prothrombin, are reported to be specific for normal molecules because of their inability to form Ag-Ab complexes with abnormal (Gla-deficient) prothrombins. This is supposedly true for both the human (38,39) and bovine (g-10) none of our Ca *+-dependent species. In contrast, McAb (raised against normal bovine prothrombin) is specific for the normal molecules. All of these McAb cross-reacted with 9-, while some also cross-reacted with S- and 7-Gla variants. Our studies also show that if all the Ca *+-dependent McAb were to be pooled and tested, the composite results should reveal no (or minimal) affinity for 5- or less&la variants; some for 7-, more for S- and most for lo-Cla (normal) prothrombin. We, in fact, have found that this trend does exist with the Ca*+-dependent polyclonal antibodies, even after removal of the Ab which cross-reacted with 2-Gla isomer. The average absorbances (with the fractionated Ab) were 1.98 for IO-Gla; 1.57 for 9-Cla; 0.3 for 8-Cla; 0.052 for 7-Gla; 0.038 for 5-Gla and 0.00 for 2-Gla prothrombin. The antibodies, which were bound to 2-Gla Affi-gel (Bio-Rad) and then eluted by EDTA, bound to IO- and 9-Gla prothrombin more or less equivalently. Binding to the other The Gla-deficient variants decreased progressively with the loss of Cla-residues. average absorbances for IO-, 9-, 8-, 7-, 5- and 2-Gla prothrombin isomers were 1.467, 1.377, 0.316, 0.128, 0.087 and 0.049, respectively (Unpublished data). In contrast to the published reports, our data with both monoclonal and polyclonal Ca*+-dependent antibodies unequivocally indicate that they are not specific for normal This discrepancy can occur when working with the abnormal (IO-Gla) prothrombin. prothrombin preparation isolated from the barium citrate-treated plasma. Since 6- to the preparation isolated from the IO-Cla prothrombin adsorbs onto barium citrate, according to my (OPM) expertise with the bovine barium citrate-treated plasma, system, should contain a mixture of 0- to 5-Gla variants, primarily consisting of 0-, IThese variant prothrombins, of course, do not form Ag-Ab and 2-Cla isomers. complexes with the antibodies directed against the Ca*+-stabilized conformation of prothrombin. Calcium ion-dependent antibodies specific to the Cla-rich domain of normal prothrombin and antibodies specific to the acarboxy prothrombin have been considered In fact, use of to measure normal and abnormal prothrombins, respectively. conformation-immunospecific antibodies isolated against normal and abnormal (human) prothrombin have shown that abnormal isomers appear in a variety of hepatic and nutritional disorders. These antibodies have been reported to be useful in the screening of hepatic dysfunction, the identification of patients with severe liver disease (38) and the detection of the transition from chronic active hepatitis to hepatoma (39). Those for specific to abnormal prothrombin, however, are not useful to assess the potential bleeding and/or thromboembolic complications with warfarin therapy (40). Instead, only normal prothrombin antigen, which correlates with biological coagulant activity (41), should be considered. all of these elegant studies were performed with normal Unfortunately, prothrombin or with abnormal prothrombin preparations with restricted heterogeneity, For this reason, the total (normal plus Glalacking some of the Gla-deficient isomers. deficient) amount of prothrombin was found to be 20% less than that observed with Echis carinatus, which converts normal and abnormal prothrombin to thrombin (38). The 20% deficiency apparently resulted from the lack of antibodies to the other GlaFurthermore, the cross-reactivity of Ca*+-dependent deficient prothrombins.

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507

MONOCLONAL ANTIBODIES TO PROTHROMBIN

antibodies with 9- to 7-Gla was not taken into consideration. Our studies, on the other hand, indicate that knowledge of the whole spectrum of Cla-deficient isomers is essential for the development of clinically superior immunoassays for the determination of prothrombins, normal and abnormal.

ACKNOWLEDGEMENTS This work was supported by the Medical Research Service of the Veterans Administration and by U.S. National Institute of Health Grants CA 35362. The authors are grateful to Dr. Jack Pensky for reviewing the manuscript. The technical assistance of Mrs. Peggy Mendelson and Mrs. Eulalia C. Sudilovsky is greatly appreciated.

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