THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 19, Issue of September 5, pp. 10453-10457,1985 Printed in U. S. A.

Protein C in Bovine Plasma after Warfarin Treatment PURIFICATION, PARTIAL CHARACTERIZATION, AND P-HYDROXYASPARTIC ACID CONTENT*

(Received for publication, February 15, 1985)

Teruko Sugos, Ulla Persson, and Johan StenfloQ From the Department of Clinical Chemistry, University of Lund, Malm6 General Hospital, S-214 01 Malmo, Sweden

Protein C was purified from the plasma of a cow treated with the vitamin K antagonist warfarin. The purified protein appeared not to bind Ca2+ ions in contrast to protein C from an untreated animal. The y- carboxyglutamic acid content of the abnormal protein C was reduced to approximately 10% of normal, whereas the B-hydroxyaspartic acid content was only slightly decreased, suggesting that vitamin K is not involved in the postribosomal hydroxylation of the aspartic acid residue in position 71 of the light chain of protein C. The abnormal and normal proteins were activated at the same rates by thrombin, but normal protein C was more rapidly activated by the thrombin- thrombomodulin complex. Compared to normal protein C, the abnormal one had virtually no anticoagulant activity.

Protein C is a vitamin K-dependent regulator of blood coagulation which is activated to a serine protease by throm- bin (1-3). It contains y-carboxyglutamic acid and was recently found to contain approximately 1 residue of erythro-P-hy- droxyaspartic acid. Other vitamin K-dependent plasma pro- teins (4,5) also contain this “new” amino acid. Thus, we have found that factors IX and X contain from 0.3 to 1.0 P- hydroxyaspartic acid residues (6). In bovine protein C, P- hydroxyaspartic acid has been demonstrated at position 71 of the light chain (4). Protein S , another vitamin K-dependent plasma protein which serves as a cofactor in factor V. inacti- vation (7,8), contains 2-3 residues of P-hydroxyaspartic acid/ mol of protein (6). However, neither human nor bovine pro- thrombin contain the modified amino acid (6). The p-hydrox- yaspartic acid residues in factors VII, IX, X, and protein C appear in regions that show a pronounced sequences similarity (9, 10).

In human and bovine factor IX and in human protein C , it has been demonstrated that a cloned cDNA codes for aspartic acid in the position corresponding to the P-hydroxyaspartic acid residue (11-13). Therefore, the amino acid is formed by a post-translational hydroxylation. We have recently dem- onstrated that the P-hydroxyaspartic acid content is normal in vitamin K-dependent plasma proteins isolated from scor-

*These investigations were supported by Swedish Medical Re- search Council Project No B85-03X-04487-llC and by Albert Pihls- sons Stiftelse and Bergvalls Stiftelse. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$. Supported by a visiting scientist fellowship from the University of Lund during the course of this work and by the Yoshida Foundation for Science and Technology. Present address: Institute of Hematol- ogy, Jichi Medical School, Minamikawachi, Tochigi, 329-04 Japan.

J To whom correspondence should be addressed.

butic guinea pigs (14). It thus seems that the required hydrox- ylase, unlike proline hydroxylase (15,16), is not ascorbic acid- dependent. It should be determined whether vitamin K is involved in the hydroxylation of the aspartic acid residues. To this end, we have purified and partially characterized protein C from the plasma of a dicoumarol-treated cow. This protein C was only partially carboxylated (-10% of normal) and biologically inactive but was found to have a P-hydrox- yaspartic acid content that was not significantly lowered. Thus, we have obtained no evidence for the coupling of vitamin K-dependent carboxylation and the hydroxylation of certain aspartic acid residues in the vitamin K-dependent protein.

MATERIALS AND METHODS AND RESULTS’

Characterization of Purified Abnormal Protein C-The ab- normal protein C obtained after gel filtration was apparently homogeneous, and its mobility on sodium dodecyl sulfate slab gel electrophoresis was identical with that of normal protein C before and after reduction of disulfide bonds (Fig. 3). The molecular weight of either form of protein C was estimated to be 58,000 and that of the heavy chain 41,500. The light chains were not stained by the silver staining procedure. Fig. 4 shows the crossed immunoelectrophoretic patterns obtained when the first dimension was run with Ca2+- or EDTA-containing buffers. The antiserum was a monospecific antiserum against normal protein C. The immunoprecipitation arcs of abnormal protein C in both buffers were weaker than those of normal protein C. In the presence of Ca”, the abnormal protein C had a more anodal position than that of normal protein C, but in the presence of EDTA the precipitation arc was in approximately the same position as that found for normal protein C. This indicates that the abnormal protein C in contrast to normal protein C does not bind Ca2+. Similar results have previously been obtained with uncarboxylated forms of prothrombin and factor X (25, 35, 36).

The amino acid composition of the abnormal protein C (not shown) was in good agreement with the composition calcu- lated from the amino acid sequence. The content of y-carbox- yglutamic acid (Table 111) showed that all preparations of abnormal protein C contained 1-2 residues of y-carboxyglu- tamic acid/mol of protein. The purified protein C was presum- ably partially carboxylated since a contamination with ap-

Portions of this paper (including “Materials and Methods,” part of “Results,” Table I and 11, and Figs. 1 and 2) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, MD 20814. Request Document No. 85M-461, cite the authors, and include a check or money order for $3.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

10453

10454 Protein C in Bovine Plasma after Warfarin Treatment

1 2 3 4 5 FIG. 3. Silver-stained sodium dodecyl sulfate slab gel elec-

trophoresis of purified abnormal and normal protein C. Track I shows molecular weight markers (phosphorylase, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor). Track 2 shows normal protein C after reduction. Track 3 shows abnormal protein C after reduction. Track 4 shows unreduced normal protein C. Track 5 shows unreduced abnormal protein C.

A B

Ca2+ +

EDTA + .- + k , .

FIG. 4. Crossed immunoelectrophoresis of purified abnor- mal and normal protein C. The first dimension electrophoresis was performed in EDTA- or Ca2+-containing buffer. A, abnormal protein C; B, normal protein C.

proximately 10% of normal, fully carboxylated protein C would have been detected in the crossed immunoelectropho- resis. The P-hydroxyaspartic acid content of five preparations of abnormal protein C was only slightly lower than that of normal protein C (Table 111). However, the amounts of puri- fied abnormal protein C were small, and so the homogeneity of the final product could therefore not be as rigorously established as for normal protein C. The slight reduction of P-hydroxyaspartic acid content may therefore not be signifi- cant. Furthermore, the abnormal protein C was obtained from one animal, whereas the normal protein C was obtained from pooled bovine plasma. The results of the P-hydroxyaspartic acid measurements argue against a coupling between vitamin

TABLE I11 y-Carboxyglutamic acid and p-hydroxyaspartic acid in purified

dicoumnrol-induced protein C Hydrolysis time was 24 h. The protein content was calculated from

the content of ASD. Glu. Ala. and Leu.

Protein C

Dicoumarol-induced protein C

Preparation 1 Preparation 2 Preparation 3 Preparation 4 Preparation 5

Normal protein C

y-Carboxyglutamic 8-Hydroxyaspartic acid acid

mollmol protein

0.83 0.39 0.89 0.48 1.07 0.34 1.71 0.59 1.73 0.57

9.6.11.4" 0.62. O.7gb 'Two experiments (Ref. 28). From Ref. 6.

I I

0 30 60 90 120

Incubation time (min) FIG. 5. Activation of normal and abnormal protein C by a-

thrombin in Ca2+-free buffer. Protein C at a concentration of 0.48 mg/ml was incubated with a-thrombin at an enzyme to substrate ratio 1:50 (w/v). A t intervals, 5-pl aliquots were withdrawn and mixed with 5-pl aliquots of 600 p~ dansylarginine-N-(3-ethyl-l,5-pentane- diyl)amide* to stop further activation. The amidolytic activity of each aliquot was measured with a synthetic substrate, and the degree of activation was calculated from a standard curve made with completely activated purified protein .C. 0, normal protein C as substrate; A, abnormal protein C as substrate.

K-dependent carboxylation and the hydroxylation of the as- partic acid residue in position 71.

Inactivation of Bovine Factor V, by Activated Abnormal Protein C-Incubation of abnormal protein C with a-throm- bin resulted in the generation of amidolytic activity. As shown in Fig. 5, there was no difference between normal and abnor- mal protein C in the rate of activation of a-thrombin in the absence of Ca2+ ions, even at the low protein concentration used. The activated proteins were also identical, as adjudged by sodium dodecyl sulfate slab gel electrophoresis, both before and after reduction (not shown). However, the rate of acti- vation of protein C by the thrombin-thrombomodulin complex in the presence of CaC12 was different for normal and abnor- mal protein C . Two preparations of abnormal protein C were activated at 25 and 29% of the rate obtained for normal protein C. It thus appears that abnormal protein C is as

The abbreviations used are: dansyl, 5-dimethylaminonaphthal- ene-1-sulfonyb APT", activated partial thromboplastin time; SDS, sodium dodecyl sulfate, ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; DAPA, dansylarginine-N-(3-ethyl-1,5- pentanediy1)amide.

Protein C in Bovine Plasma after Warfarin Treatment 10455

300

2 200

Y u)

+ I-

4 n

100

0 0 0.1 0.2 0.3 0.4

Concentration of activated protein C (pg/ml plasma)

FIG, 6. Anticoagulant activity of thrombin-activated ab- normal and normal protein C. Activated partial thromboplastin time ( A P T T ) was measured in duplicate as described under "Mate- rials and Methods." U, abnormal activated protein C; -, normal activated protein C.

susceptible to activation by thrombin as normal protein C in the absence of cofactors but not in their presence.

Activated protein C is a potent anticoagulantly, rapidly inactivating factor V. and VIII. by limited proteolysis in the presence of Caz+ ions and negatively charged phospholipid. The experiments shown in Fig. 6 were performed to learn whether the Gla residues in protein C influence its anticoag- ulant activity. Addition of activated normal protein C to plasma prolonged the activated partial thromboplastin time, but in contrast, activated abnormal protein C failed to prolong the activated partial thromboplastin time, even in the pres- ence of a large amount (300 ng) of the enzyme. However, in experiments with purified bovine factor V., activated normal and abnormal protein C degraded the factor V. equally effec- tively in the absence of phospholipid. In the presence of phospholipid, the inactivation of factor V. by the normal enzyme proceeded faster than in the absence of phospholipid, whereas the inactivation by the abnormal enzyme was not influenced (not shown). The results show that the Gla resi- dues are a prerequisite for the anticoagulant activity of acti- vated protein C.

DISCUSSION

Erythro-P-hydroxyaspartic acid is formed by postribosomal hydroxylation of certain aspartic acid residues (4). Up to now, this modified amino acid has been found in all the vitamin K-dependent plasma proteins except prothrombin but not in other proteins (6, 9). We have reported previously that the enzyme hydroxylating aspartic acid residues, in contrast to prolylhydroxylase, does not seem to be ascorbate-dependent in uiuo (14). Another possibility is that vitamin K is involved in the hydroxylation reaction. Vitamin K might be involved either directly in the hydroxylation reaction or indirectly, i.e. the hydroxylase might require an already carboxylated protein as substrate. To investigate whether vitamin K may be in- volved in the hydroxylation, we have purified protein C from the plasma of a cow treated with a vitamin K antagonist. The purified abnormal protein C contained 1-2 mol of y-carbox- yglutamic acid/mol of protein, in contrast to 9-11 mol/mol for normal protein C. The abnormal protein C had virtually no anticoagulant activity. Both barium citrate adsorption

experiments performed on plasma and crossed immunoelec- trophoresis experiments indicated that the purified material was partially carboxylated rather than contaminated with 10% of fully carboxylated protein C. Furthermore, the lack of biological activity of the abnormal activated protein C indi- cated that the level of contamination with normal protein C was clearly below 10%. Our data indicate that there is no coupling of vitamin K-dependent carboxylation and the hy- droxylation of the aspartic acid residue in position 71 in protein C. However, it can be argued that the partially car- boxylated protein, even with only 1 residue of y-carboxyglu- tamic acid, may be an adequate substrate for the hydroxylase, whereas when completely devoid of y-carboxyglutamic acid protein C would not be hydroxylated. Our data do not preclude this possibility nor do they preclude an involvement of vita- min K by way of an enzyme system less susceptible to inhi- bition by warfarin than the vitamin K-metabolizing enzymes involved in glutamate carboxylation.

Acknowledgments-We would like to thank Marianne Holmberg for expert technical assistance and Dr. Timothy Carlson for critically reading the manuscript.

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Supplementary material to

Prote in C i n Bovine Plasma af ter Warfar in Treatment . pur i f icat ion, P a r t i a l C h d r a c t e ~ i z a t i o n and E-Hydroxyaspartic Acid Content

TERUKO SUGO, ULLA PERSSON and JOHAN STENFLO

MATERIALS AND METHODS

Chemicals - Benzamidine hydmchlor ide, phenylmethanesul fonyl f luor ide (FUSF), 2,Z"azino-di- m e n z e t h i a m l i n su l fon i c ac id , (ABTS), L-phosphatidyl-L-rerine. L-=-phosphatidyl cho-

a c i d were fmm Fluka. Dimethyl p imel imidate hydrochlor ide was obtained from Pierce and dicou- l i n e w e ~ e obtained frm Sigma. DiisDpropylfluOrOphosphate (DFP) and threa-8-hydmxyarpar t ic

mar01 f rom Fermran, Malmd, Sweden. r-Carboxyglutamic acid Was from Calbiochem. Boc-Leu-Ser- Thr-Arg-4 methylcauaaryl-7-anide (MCA) was f rom the Prote in Research Foundation and Simplastin'

DEAE-Sephadex wwe f r om Phamc ia and U l t m g e l AcA-44 fmm LKB. Dansyl -arg in ine N-(3-ethy l - and automated APTT reagent were purchased fms General Diagnostics. Pmtein A-Sephamre and

1,5-pentamediyl)-amide was synthesized as described by Nesheim e t a l . (17) and erythro-E-hyd- m x y a r p a r t i c a c i d was synthesized as we have described previously (4).

Prote ins - Ovalbumin was obtained from Sigma, and marker proteins far SDS-gel e lect rophores is l r o m r m a c i a . Horseradish pemxidase conjugated rabbit antimouse IgG antiserum was t h e pro- duct o f DAKO. Rabbit ant imouse ant isera far isatype determinat ion were obtained fmm Mi les Laborator ies Inc. Bovine p m t e i n C, f a c t o r V and s-thrombin were p u r i f i e d as descr ibed pw- v i o u s l y (19.21). Rabbi t thmmbomdul in was a k i n d g i f t f m m Drs Charles and Naomi Esmon.

line ( S p 210 Ag 14 ce l l s of BALBIc o r i g i n ) d e f i c i e n t i n h y p o x a n t h i n e ph0spho~ibosyltr.nsferare Product ion of nxlnoclonal ant ibodies against bov ine prote in C - An 8-amguanine resistant cel l

was used. The mice were i n j e c t e d i n t r a d e m l l y w i t h I O ug o f bov ine pmte in C e m u l s i f i e d i n Freund.s cmple te ad juvant . The imnunizatians were repeated a f t e r 1 and 3 weeks w i th t he p ro - t e i n e m u l s i f i e d i n Freund.s incomplete adJuvant. Three days p r i o r t o t h e f u s i o n 200 ug o f p m -

farmed as described by Eorrebaeck and Eylar (22). t e i n C was given in t raper i toneal ly . Spleens were removed and c e l l fusion and c l o n i n g were per-

s tandard so l id DhaSe ELISA orocedure (23) . The w e l l s o f t h e m i c m t i t e r D l a t e s were Coated w i t h Ant ibouies against bov ine prote in C produced by the hyb r ide cu l twes were detected by a

p r o t e i n C ( I O uk per ml i n 0.05 M sodium carbonate buffer pH 9.6) a t 4'C ovenright. Subsequent

Horseradish peroxidase canjuqated rabbit antimouse imunoulobulins were used a s a second a n t i - washes were made w i t h I O d phosphate buffer pH 8.0 conta in ing 0.5 M NaCl and 0.1 % TWEEN 20.

body. ABTS was used as rubit ;ate. For pmduction o f mnoc iona l an t ibod ies mice were i n j e c t e d

5.101 c e l l s each. A f t e r 9-11 days t h e a s c i t i c f l u i d was c o l l e c t e d and the imnunoglobulin frac- t i o n p r e c i p i t a t e d by adding sol id amnonium s u l f a t e t o 50 % (w /v ) saturation (ambient tempera- t u r e ) . The p r e c i p i t a t e was d i l S o l v e d i n w a t e r and d ia lyzed against 50 d trir-HCl pH 7.4 and

dures. The i s o t y p e s o f t h e p u r i f i e d n'anoclanal ant ibodies were determined by the dolble ant i - then subjected to chromatography on a column o f DEAE-Sepharose column us ing standard proce-

ox idase conjugated goat ant i rabbi t IgG. body sandwich ELISA using isotype Speci f ic rabbi t ant inxluse ant isera i n c o n j u n c t i o n w i t h per-

i n t r a p e r i t o n e a l l y w i t h 500 u l of 2 , 6 , 1 0 , 1 4 - t e t r a m e t h y l p ~ ~ t ~ d ~ ~ ~ ~ ~ and four days l a t e r w i t h

Pre a ra t ion o f imnunoaf f in i t mat r i x - Monoclonal antibodies were i m b i l i z e d on p m t e i n A-

cubated wi th 3.0 rnl O f p m t e i n A-Sepharose and subsequently crossl inked with dimethyl pimeli- SephPamre 31 described by S c k i d e r e t a l . ( 2 4 ) . B r i e f l y 34 mg o f monoclonal antibody was i n -

middte. The r e s u l t i n g a f f i n i t y r e s i n was poured i n t o I column and washed once w i t h 3 M amno- n ium th iocyanate in 0.1 M phosphate buffer pH 6.8 p l i o r t o use.

~/ P r o t e i n C assa ~ Abnormal p m t e i n C was assayed by solid-phase ELISA. Po lys ty rene mic ro t i te r

S i t e s wem blocked by incubating 15 m in w i th bu f fe r con ta in ing 2 % ovalbumin. After washing p r o t e i n C an t i se rum, d i l u ted t o 1 t o 1000 w i t h c o a t i n g b u f f e r (see above). The f ree b ind ing

as deswibed above 40 V I o f I 1: l m ix tu re O f p r o t e i n C and a so lu t ion con ta in ing monaclonal ant ibody were added t o t h e w e l l s . The l a t t e r s o l u t i o n c o n t a i n e d 5 pglrnl o f t h e a n t i b o d y i n

4'C f o r 24 h a 1 t o IO00 d i l u t i o n O f horseradish peroxidase conjugated goat antimouse imnuno- 0.25 % ovalbumin i n 10 d phosphate buffer pH 8.0 conta in ing 0.5 M NaCI. A f t e r i n c u b a t i o n a t

g l o b u l i n was added, and i n c u b a t l o n a t mom temperature was continued fov 30 mi". Fo l low ing the add i t i on o f ABTS and H 0 i n 0.05 M c i t r a t e b u f f e r pH 5.0 and I 20 min incubat ion the absor- bance at 405 nrn war rnoh?wed on a Titerteck autoreader (Flow). The amount o f p m t e i n C i n t h e sample s o l u t i o n was calculated from a Standard C U N ~ made w i t h normal p m t e i n C, and assuming t h a t t h e r e was no d i f f e r e n c e i n t h e i m u n o c h e m i c a l r e a c t i v i t y between normal and abnormal p m - t e i n C.

ynateck) were coated for 16 h a t ambient temperature with 50 y l o f rabb i t an t i -bov ine

E l e c t m p h o r e t i c and imnunochemical methods - Crossed i n u n o e l e c t r a p h o r e s i r was perfomed using standard procedures (25). SIIS-polyacrylamide s l a b gel electmphores is was performed on I O t o 15 P urddlent qels (20) which were s i l v e r s t a i n e d as described by Marrisrey (26). UeStern blot- t i n g war p e r f o h e d as described by Burnet te (27) .

Pro te in concent ra t ion - The concentrat ion o f normal p r a t e i n C used to ppepare the ELlSA r tan- dard curve was determined from the amino a c i d c o n t e n t o f a n acid hydro lysate andl&e composi- t i o n o f b o v i n e p m t e i n C repopted earl ier (28.29). The e x t i n c t i o n c o e f f i c i e n t , El% a t 280 nm, used for both normal and abnormal p r o t e i n was 13.7 (1s) and the molecular weight Was 56 000

weight 37 000 (30). (1 '3) . For bov ine thmmbin the ext inct ion coef f ic ient used war 21.0 (30) and the molecular

Amino a c i d ana1 s i 5 - The amino ac id composi t ions o f noma1 and abnormal p r o t e i n C were deter- mmned a f t e r hyd:olysis ( 2 4 h i n 6 M HCI a t llO°C i n vacuo) using a Beckman Model 6300 amino ac id ana lyzer and t h e ma rams and b u f f e r s v o v i d e d bv the manufartucer vCarbaxv4Iutamic acid . _ was ,,,arured as deSCPlOed prev~ously I J i ) . 8-Hydroxyaspart ic acid war analyzed a f t e r a c i d hydmlySiS an t h e Beckman 6300 automatic analyser equipped with I n inhyd r in de tec t i on system. The erythro-E-hydmxyaspartic acid Content i n t he hyd ro l ysa tes was re la ted to the Conten t Of glutamic acid. Th~eo-8-hydTOXy.Splrt ic acid was not measured, because we have shown prev ious ly that appmXimatelY 20 % o f t h e t o t a l E - h y d r o x y a s p a r t i c a c i d c o n t e n t i n a 24 h pmte in hydroxy- l a t e I S i n t h e t h r e o form (6).

A c t i v a t i o n o f abnormal r o t e i n C b a-thrombin - Normal and abnormal p r o t e i n C (0.48 mg/ml i n 30 mM Tris-HCI, 6;1 M NaPCI, pH 7.5: were a c t i v a t e d by bovine a-thrornbin a t an enzyme t o sub- s t r a t e r a t i o o f 1 /50 ( v l w ) a t 37°C. A t i n t e r v a l s , 5 y1 o f a l i q u o t s were withdrawn and mixed w i t h 5 u l o f 600 uM DAPA. The a c t i v a t e d p m t e i n C was assayed by measuring the amidolytic ac- t i v i t y u s i n g t h e s y n t h e t i c f l u o m g e n i c S u b s t r a t e , Boc-Leu-Ser-Thr-Arg-MCA i n 50 d Trir-HCI,

by comparison w t h t h a a m d o l y t i c a c t i v i t y O f pur i f ied , comple te ly ac t i va ted p ro te in C. A c t i - 0.1 M NaCl, 1 mM CaCl pH 8.0 (32). The anaunt o f ac t i va ted pmte in C oenerated was ca lcu lated

method of Esmon e t a l . (33) va t ion o f abnormal p r o t e i n C by t h e o-thrombin-thrombom0dulin cmplex was performed by the

. ~" . ~ "

Anticoagulant assay O f ac t i va ted bov ine p ro te in C -Ac t i va ted pa r t i a l t h rombop las t i n t ime 7APTTl was determined by incubating 100 u l o f normal bovine plasma and 100 "1 of phospholipid and ac t iva tor so lu t ion lau tomated APTT reaoent) a t 37OC f a r 4.75 mi". Thereaf ter I O u l o f ac-

A f t e r 15 rec 100 u l O f 25 d Cat1 was added and the c lo t t ing t ime recorded. The average APTT t i v a t e d b o v i n e p m t e i n C d i l u t e d w i t h 1 % k A i n 50 mM Tris-HCI 0.1 M NaCl pH 7.5 wa; added;

Of normal bo;ine plasma was 30 lec?

To s tudy t he i nac t i va t i on O f f a c t o r V , a- thrombin-act ivated bov ine factor V was incubated w i t h a c t i v a t e d n o m 1 O P abnormal proteinaC. Thus factor . V a t 8 U/ml i n 50 d T?ir-HCl, 0.1 M NaCl, 5 mM CaCl 0 1 % BSA and 0.2 d OAPA was incubated wfth each p r o t e i n C a t a f i n a l con- c e n t r a t i o n o f 32yg;ml i n t h e presence or absence of phosphol ip id ( I 0 0 ug lml ) . A t in te rva ls , 1 "1 a l i q u o t s were withdrawn and arrayed f o r f a c t o r V a c t i v i t y e s s e n t i a l l y as described pre- v ious ly (34) .

v i o u s l y ( 2 5 ) and t h e amount o f abnormal p m t h r m b i n i n b l o o d samples measured by Crossed im- I n d u c t i o n o f s y n t h e s i s o f abnormal p r o t e i n C - Oicoumarol was given to I cow as described pre-

mal pmthmbmin was about 25 % of the pretreatment value, 900 m l of b lood was drawn i n t o poJy- munoelectmphoresir using rabbi t ant iserum against bovine pmthrombin. When the Pemaining nor-

e thy lene bo t t l es con ta in ing 100 rnl Of 0.1 M t r i s o d i u m c i t r a t e . The plasma was s t o r e d a t -20 C u n t i l processed.

Pu r i f i ca t i on o f abnormal p m t e i n C - TO the plasma obtained f m m the d icoumam-t rea ted cow, an Were a e t o a ina concentrat ion of 1 mM each. A f t e r 20 m i " a t 22OC the

mal Prote in C was removed by a d s o v t i o n t o bar ium c i t ra te, i .e . e ighty ml 1 M BaCl was 110wly Esmadw:?kde 3 midi: benzai id i j le hydrochlor ide. Subsequent steps were performed i t 4OC. Nor-

added per 1 of Plasma w i t h s t i r r i n g . A f t e r one hour t he p rec ip i t a te was mnxlved by'centrifuga- t i o n a t 5000 x 9 f o r 20 min. The supernatant so lu t ion was f ract ionated by a d d i t i o n o f s o l i d amnonium Sul fa te. The p r e c i p i t a t e f o m d between 25 and 65 % ( w l v ) Saturat ion was col lected, d i s r o l v e d i n a minimum amount O f water and dialyzed for 16 h against 8 1 of water containing 1 mM bemamidine hydmchlor ide, and then for 24 h against 2 changer o f 8 1 of 0.1 M phosphate

med which was r ~ n o v e d by c e n t r i f u g a t i o n a t 5000 x g f o r 20 min. The clear supernatant was app- buf fer pH 6.0 conta in ing 3 d bemamidine hydrachlor ide. A small amount o f p r e c i p i t a t e was f o r -

l i e d t o a DUE-Sephadex column (6 x 50 cm) equ i l i b ra ted W i th t he same b u f f e r a t f l o w r a t e

bance a t 280 MI had decreased below 2.0. The p m t e i n s were e lu ted w i th a 4 1 l i n e a r g r a d i e n t 33 nl per h (F ig . 1 ) . and t h e column was washed w i t h t h e e q u i l i b r a t i o n b u f f e r u n t i l t h e absor-

of NaCl (0 t o 0.35 M) i n t h e phosphate b u f f e r . Abnormal p m t e i n C i n t h e Column e f f l u e n t a s measured by using Solid-phase ELISA e l u t e d j u s t a f t e r t h e p m t h r o n b i n peak. The l a t t e r was mo- ni tored by rocket imnunoelectmphores is . Fmct ions conta in ing the abnormal p m t e i n C were p ~ - led, OFP ra . added ta 1 d f ina l concent ra t ion and the pool war concen t ra ted by u l t ra f i l t r a - t i o n . It was t hen app l i ed t o t he i nmunoa f f i n i t y colmn (0.9 x 4.5 cm), which had been e q u i l i - b r r t e d w i t h 0.1 M phosphate pH 6.0 (F ig . 2 ) . The f lw Pate was 10 ml per h and t h e column was washed exhaus t i ve l y w i th 0.1 M phosphate buf fer conta in ing 2 M NaCl pH 6.0. The adsorbed pra- t e i n s were e l u t e d w i t h 3 M a m n i u m t h i o c y a n a t e i n 0.1 M phosphate buf fer pH 6.8 and t h e i n d i - v idua l f rac t ions d ia lyzed aga ins t 50 d i r i s - H C I 0.1 M NaCl pH 7.5. Fract ions conta in ing ab- normal p r o t e i n C (monitored by ELISA assay) were pooled, concentrated ta 2.0 rnl by u l t r a f i l t r a - t i o n . The concentmte was subjected to gel f i l t r a t i o n t o remove I high molecular weight conta- minant. Thus a f t e r a p p l i c a t i o n t o a column o f U l t r o g e l AcA-44 ( 1 . 6 x 100 cm) e q u i l i b r a t e d w i t h 50 mM Tr i r -HCI , pH 7.5 0.1 M NaCl t h e column was developed a t a f l a w r a t e of 18 rnl per h. The r e s u l t i n g abnormal p r o t e i n C was t h e n c o n c e n t r a t e d b y u l t r a f i l t r a t i o n and s t o r e d a t -7OOC.

RESULTS

Charac teF imt ion o f monoclonal ant ibodies against bov ine prote in C - The concentrat ion of pm-

or D a r t i a l l v c a r b o x v l a t e d f o r m o f t h e o m t e i n i s oresumablv l w e r . Monoclonal antibodies were t e l n C i n bovine plasma i s on l y abou t 5 ng per 1 and the concentrat ion of the uncarboxylated

t h e k e f a r e b o d u c e d t o ~ f i i i l i t a t e t h e p u r i f i c a t i o n . I n a d d i t i o n t o p m t e i n C the c lones w e ~ e also t e s t e d w i t h a c t i v a t e d p m t e i n C and prote in C fmm which the I-carboxyglutamic acid (Gla) conta in ing domain had been removed by l im i ted pmteo lys i s (G la -doma in less pmte ins l . As expec- ted, an an t ibody recogn iz ing t he G la -dma in le r r pmte in also recognized uncarboxylated protein C (Table I ) . It i s r m r k a b l e tha t th ree ou t o f the f i ve nx lnoc lona l an t ibod ies d id no t Pecog- n i r e a c t i v a t e d p r o t e i n C by t h e ELISA assay. None o f t h e f i v e monoclonal a n t i b o d i e s i n h l b i t e d t h e a m i d o l y t i c a c t i v i t y o f a c t i v a t e d p m t e i n C. and fu r thermore the ac t i va t ion o f pmte in C by o-thmmbin O P t h e thr0mbin-thro.bom.dulin complex was no t i nh ib i t ed . The antibody, Bpc-5

o f reduved p m t e i n C, whereas Bpc-4 o n l y reacted very weakly w i t h reduced p m t e i n C. Bpc 1-3 recognized t h e heavy chain o f p m t e i n C i n Western b l o t t i n g experiments a f t e r e l e c t r o p h a r e r i r

however recogorred unreduced intact and act ivated pmtein C on Western b l o t t i n g b u t d i d n o t re- cognize the reduced p m t e i n . BPC-4 was used i n t h e p u r i f i c a t i o n of uncarboxylated bovine pro- t e i n C.

P u r i f i c a t i o n o f a b n o m l m t e i n C - '"Rocket i n m u n o e l e c t m p h ~ r e r i s " pmved u n r e l i a b l e f o r t h e quan t i t a t i on o f pmte in CP in plasma fmm w a r f a r i n i d cols and there fore the ELISA w t h o d was develpped. Plasma obta ined dur ing war far in admin is t ra t ion was found to con ta in pmte in C i n the range o f 1.6 t o 4.4 uglml. which i s 30 t o BO % o f t h e p m t e i n C c o n c e n t r a t i o n i n normal

mal D r o t h m r b i n as judqed bv crossed imnunoelectmphoresis (25). Af te r adsorp t ion of normal plasma. The plasma obtained f m m t h e war fa r i n t rea ted c o l contained appmximately 30 % of no?-

supernatant, but when an i d e n t i f i c a l e x p e r i m e n t w i t h plasma f m m an untreated cow was perfor- prothrombin to b a r i h ; i t r a t e mre than 60 S o f t h e p m t e i n C i m n u n o r e a c t i v i t y r m i n e d i n t h e

med no p r o t e i n C was detected in the supernatan t . There fore bar ium c i t ra te adsorp t ion was used I S t h e f i r s t p u r i f i c a t i o n s t e p t o remove r m i n i n g normal p m t e i n C fmm t h e plasma.

matography step employing the insolubi l ized monoclonal ant ibody (Bpc-4) r e s u l t e d i n a 320-fold The p u r i f i c a t i o n o f a b n o m 1 p m t e i n C i s summarized i n Table 11. The inmunoaff ini ty chm-

p u r i f i c a t i o n w i t h o u t s i g n i f i c a n t loss o f p m t e i n C. Holevev. i n s p i t e o f e x h a u s t i v e w a s h i n g o f the colmn w i t h b u f f e r c o n t a i n i n g 2 M NaCl p r i o r t o e l u t i o n an un iden t i f i ed h igh molecular weight , pmte in contaminated the pmte in C preparation. This was r m v e d by the gel f i l t r a t i u n Step. The overall y i e l d o f a b n a m l p r o t e i n C was low and only 0.35 t o 0.55 ng of.abnormal p m - t e i n C was obtained from 1.8 t o 2.5 1 Of war far in ized plasma.

Protein C in Bovine Plasma after Warfarin Treatment 10457

30 E 3 K c

m 20

e 9 51

10

0 A Protein c

300 400 500 600 Fraction numbel

%&ography o f ammonium sul fate f ract ionated bar ium supernatant on a column Df OEAE-Sepha- dex. A f t e r application of sample t o t h e column (6 x 50 un) which had been e q u i l i b r a t e d w i t h

4 1 l i n e a r NaCl gradient of NaCl (0-0.35 MI i n t h e e q u i l i b r a t i o n b u f f e r . 11 m l f r a c t i o n s were 0.1 M phosphate buffer, 3 nM benzamidine hydmchlor ide pH 6.0, e l u t i o n was performed wi th a

co l lected. Absorbance It 280 M1 was detennined (-*-) and a b n o m l p m t e i n C arrayed by ELISA (bo-) . Fract ions were pooled a s i nd ica ted by the hor izon ta l bar .

I I I I

15 2 M NaCl I

3 M NH,SCN

1 300

E a

w Prater C

Effluent volume (ml)

400

c E

200 3 0 C ._ m P c

100

0

TRBLE I

S p e c i f i c i t y o f a n t i - b w i n e p m t e i n C monoclonal antibodies

ELISA assay, absorbance &t 405 m b

Clone Typea P r o t e i n C Gla-domainless Oicoumarol-Induced Activated p r o t e i n c p r o t e i n C PPDteln c

Bpc 1 LgGlk 0.77 0.89 0.76 0

Bpc 2 IgGIk 0.73 0.67 0.73 0

BPC 3 1 q e a k 0.67 0.67 0.63 0

8pc 4 IgG2ak 0.69 0.69 0.65 0.10

Bpc 5 IgG2bk 0.70 0.69 0.63 0.45 ~

a . I ro type de terminat ion was pevfanned by u s i n g s o l i d phase ELISA as descr'ibed under MaterlalE and Methods.

b. Refevr t o t h e amount O f ABTS hydro lyzed a f te r 20 m i n , a t mom temperature Wlth standard ELISA d ~ l a y .

F r a c t i o n

Plasma

BlCl rupe?natant

A m n i u m s u l f a t e

Sephadex DEAE-

McAb-protein -8epharose

AcA-44 ~-

a. A r b i t r a r y u n i t s

2600 171000 11400 100 1

2720 163000 708 62 0.64

1480 107000 2760 24 0.37

216 2020 1010 8.8 7.4

6.6 6.9 940 8.2 2370

11 .9 0.77 451 3.9 a730

W a f f i n i t y c h m m t o g m p h y of the OEAE-Sephadex pool . Af ter concentrat ion by u l t r a f i l t r a t i o n the Mter i .31 f rom the DEAE-Sephadex column was a p p l i e d t o a column (0.9 I 4 cml packed w i t h

w i t h 0.1 M phosphate buffer pH 6.0 Containing 2 M NaCl, t h e column was e l u t e d w i t h 3 M a m - protein A-Sephamre to which the monoclonal antibody Bpc-4 had been crorrlinked. After washing

nium th iocyanate i n 0.1 H hosphate buf fer pH 6.8 a t a f l o w r a t e o f 10 m1 per h. Absorbance a t 280 nm was determined (-e-? and abnonnal p m t e i n C detennined by ELISA ( -0 -1 . Fract ions were pooled as i n d i c a t e d by the bar .