Vitamin K and prothrombin formation

Life S c i e n c e s Vol. 9, P a r t II, pp. 79-86, 1970. P r i n t e d in G r e a t Britain

Pergamon Press

VITAMIN K AND PROTHROMBIN FORMATION G. S. Ranhotra and B. Connor Johnson

University of Oklahoma, School of Medicine, Department of Biochemistry and Molecular Biology and Oklahoma Medical Research Foundation, Biochemistry Sect ion Oklahoma City, Oklahoma 73104 (Received 24 July 1969; in final form 20 October 1969) IT HAS BEEN WELL ESTABLISHED (1-4) that vitamin K does not effect the synthesis of the vitamin K dependent proteins (prothrombin and factors VII, IX and X) at the site of transcription of DNA for the production of specific messenger-RNA. However, rigorous evidence as to whether vitamin K brings about prothrombin synthesis at the translation level of peptide has not been available. Puromycin has been variously reported to block (3,4) or not to block (5-7) the induction of vitamin K dependent clotting proteins in liver preparations, thus another approach to the problem appeared necessary. We have, therefore, examined the incorporation of labeled amino acids into prothrombin in the normal animal, in the vitamin K deficient animal and in the vitamin K deficient animal given vitamin K I. The data obtained appear to demonstrate that vitamin K does not function in the de novo synthesis of prothrombin but at a step in prothrombin formation following synthesis of the polypeptide chain. E xpe rime nt a l Male, 200 gm Sprague-Dawley rats, were made hypoprothrombinemic by housing them in coprophagy-prevention cages (8) and feeding a vitamin K deficient diet (9). All rats (Normal and vitamin K deficient) were given 14C(14.3 mc/mg) (U) or 3H(28.9 mc/mg)(G) labeled L-amino acid mixture intraperitoneally and where indicated vitamin K I (i rag) one hour prior to obtaining blood by heart puncture. Vitamin KI, Supported

in part

by NIH

grant 79

AM

10282.

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I00 rag, was solubilized with 1 ml of Tween 80 and the solution brought to 50 ml with 0.9% NaCI. The clotting proteins of oxalated blood plasma were adsorbed on powdered BaSO4 (125 mg per ml) by slowly stirring (all steps henceforth at 4 o ) for one hour followed by centrifuging and washing the resulting BaS04 sediment once with 0.i M potassium oxalate in 0.9% NaCI and twice with 0.9% NaCI. The adsorbed proteins were eluted then from the BaSO4 by gently agitating the sediment on a Vortex mixer with 1/100th plasma volume of cold 0.17 M sodium citrate, pH 7.8 for 30 rain. After centrifugation, the BaSO 4 eluate was used (a) for the determination of protein concentration by the method of Lowry et al. (i0), (b) for the measurement of radioactivity in a Packard Tri-Carb Liquid Scintillation Spectrometer and (c) for the purification and identification of prothrombin by polyacrylamide gel electrophoresis. Gels were stained with a 1.0% solution of Amido Black. Stained prothrombin in the gel was quantitated by scanning with a Gelman Gel Scanner. Results and Discussion TABLE 1 The Uptake of Labeled Amino Acids¢ by Plasma Proteins Treatment

Normal r a t s vitamin K

Rat No.

+

Normal rats Deficient rat + vitamin K

DPM/mg p r o t e i n BaSO 4 e l u a t e

BaS04 t r e a t e d plasma

1 2

420 337

258 172

3 4

314 302

200 220

5

416

283

L-Amino Acids-14C(U) mixture (14.3 mc/mg), New England Nuclear, Boston, Mass. All rats were injected labeled amino acids (5 ~c/100 ~l body weight) intraperitoneally and where indicated l mg vitamin K I one hour prior to blood withdrawal. In one hour incorporation

studies,

it was found that

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81

radioactivity was i n c o r p o r a t e d into both the vitamin K d e p e n d e n t p l a s m a p r o t e i n s a d s o r b a b l e on BaS04 a n d t h o s e n o n - v i t a m i n K d e p e n d e n t p r o t e i n s n o t a d s o r b e d on BaS04 (Table i). T h e s e d a t a i n d i c a t e d de novo s y n t h e s i s o f t h e BaSO 4 a d s o r b a b l e c l o t t i n g p r o t e i n s t o an e x t e n t s i m i l a r t o t h a t o f o t h e r p l a s m a p r o t e i n s d u r i n g t h e one h o u r p e r i o d . From T a b l e 2, i t c a n be s e e n t h a t t h e c l o t t i n g proteins a d s o r b e d on, a n d e l u t e d f r o m BaS04 showed no s i g n i f i c a n t differences in radioactivity between the normal, vitamin K-deficient, and vitamin K deficient r a t s g i v e n v i t a m i n K1. TABLE 2 The U p t a k e o f L a b e l e d Amino Acids¢ by P l a s m a P r o t e i n s A d s o r b e d on BaS04 T r e a t m e n t ¢¢

No. Rat s

Plasma Prothrombin % of normalttt

DPM/mg P r o t e i n i n BaSO4 e l u a t e

1 Normal r a t s Deficient rats + v i t a m i n K1 Deficient rats

Rat No. 2 3

4

4 4

100 + 4 100 + 5

260 120

64 191

64 154

237 31

4

5 + 5

120

69

26

75

L Amino Acids-3H(G) mixture (28.9 mc/mg), New England Nuclear, Boston, Mass. All rats were administered, intraperitoneally, labeled amino acids (5 ~Lc/100 gm body weight) and where indicated 1 mg vitamin KI, one hour prior to withdrawal of blood by heart puncture. At the time of blood withdrawal. Measured by the method of Quick et al.(ll). If vitamin K administration b r o u g h t a b o u t de novo s y n t h e s i s o f t h e v i t a m i n K d e p e n d e n t p r o t e i n s one w o u l d e x p e c t c o m p a r a t i v e l y much more i n c o r p o r a t i o n of label in the case of deficient a n i m a l s in which p r o t h r o m b i n s y n t h e s i s had j u s t b e e n " i n d u c e d " by v i t a m i n K1 a n d w o u l d e x p e c t l i t t l e o r no radioactivity in the vitamin K dependent clotting proteins

82

PROTHROMBIN FORMATION

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Vol. 9, No. 2

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PROTHROMBIN FORMATION

83

Since certain non-vitamin K dependent clotting proteins, (factor V and fibrinogen) have been shown to also be somewhat adsorbed on BaS04 in addition to the vitamin K dependnet clotting proteins, it was essential to further purify one of the dependent proteins and prothrombin was selected. The prothrombin in the BaS04 eluate was purified by means of poly acrylamide disc gel electrophoresis and identified on the gel by simultaneously running a control gel with added carrier bovine prothrombin isolated by the method of Moore et al. (12). In Figure 1 are given scans of the gels from vitamin K normal, vitamin K deficient and vitamin K deficient animals treated with vitamin K I. From the results in Figure 1 and Table 3, it is immediately observed that the amount of prothrombin is essentially the same proportion of the total protein in each of the BaSO 4 eluates and if expressed as a fraction of the total blood taken is essentially the same proportion in the case of normal, deficient and deficient animals treated with vitamin K I. TABLE 3 The Amount and Radioactivity of Prothrombin in BaSO 4 Eluate Rat P l a s m a T o t a l p r o t e i n Prothrombin no. v o l u m e i n 10 1 B a S O 4 p r o t e i n i n t eluate ~ 10 ~ 1 B a S 0 4 eluate?~t ml ~g ~ g 1 2 3 4 Deficient 5 rats + 6 vitamin 7 KI 8 9 Deficient i0 rats ll 12 Normal rats

t tt t~t

4.0 3.5 4.0 5.0 5.0 5.0 4.5 6.0 5.0 4.0 3.0 4.5

37 25 25 35 42 80 45 95 50 95 35 45

19 12 11 15 23 35 21 45 25 40 17 22

1 / 1 0 0 t h plasma volume o f sodium c i t r a t e e l u t e p r o t e i n s a d s o r b e d on BaS04. By m e t h o d o f Lowry e t a l . ( 1 0 ) . By s c a n n i n g s t a i n e d g e l s .

DPM/mg Prothrombin

630 500 1363 200 783 171 429 131 360 150 529 273 was u s e d t o

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T h e r e a p p e a r s t o be no d e c r e a s e i n " p r o t h r o m b i n p r o t e i n " l e v e l w i t h v i t a m i n K d e f i c i e n c y a n d no i n c r e a s e i n p r o t h r o m bin protein level with vitamin K treatment. When t h e p r o t h r o m b i n b a n d was c u t f r o m t h e g e l and m e a s u r e d f o r r a d i o activity, similar amounts of radioactivity were d e t e c t e d w h e t h e r t h e b a n d was o b t a i n e d f r o m n o r m a l r a t s , d e f i c i e n t rats or deficient r a t s g i v e n v i t a m i n K~. Similar experiments with similar results have been c a r r i e d out u s i n g warfarin treated rats. The d a t a p r e s e n t e d d e m o n s t r a t e t h a t t h e e f f e c t o f vitamin K is not at the translation level of prothrombin synthesis but i s s u b s e q u e n t t o p e p t i d e bond f o r m a t i o n . L o w e n t h a l a n d B i r n b a u m (14) and S u t t i e (15) h a v e p r e s e n t e d data indicating i n v i t r o f a c t o r VII s y n t h e s i s following vitamin K administration, w h i c h i s l e s s b l o c k e d d u r i n g an initial s h o r t p e r i o d by p r o t e i n s y n t h e s i s blocking agents a l t h o u g h w i t h l o n g e r t i m e s b l o c k i n g becomes r e l a t i v e l y complete. T h i s a p p e a r s t o be i n c o n f i r m a t i o n o f o u r d a t a a n d s u g g e s t s t h e f o r m a t i o n o f a c t i v e p r o t h r o m b i n f r o m an "unfinished" precursor present in liver cells, which becomes r a p i d l y c o n s u m e d when c y c l o h e x i m i d e b l o c k s i t s f u r t h e r synthesis. Using rat-prothrombin a n t i b o d y , L i e t a l . (13) h a v e shown, in 6 h o u r l i v e r p e r f u s i o n e x p e r i m e n t s w i t h normal rats, a proportional incorporation of 14C-leucine into p r o t h r o m b i n i n r e s p o n s e t o v i t a m i n K. In c o n t r a s t t o t h e i r 6 h o u r e x p e r i m e n t s i n w h i c h de novo s y n t h e s i s o c c u r r e d , o u r d a t a w e r e o b t a i n e d d u r i n g one h o u r c u r e o f v i t a m i n K d e f i c i e n c y h y p o p r o t h r o m b i n e m i a by v i t a m i n K 1. D u r i n g t h i s one hour a very marked response in plasma prothrombin activity o c c u r r e d w h i c h was n o t a c c o m p a n i e d by an i n c r e a s e d i n c o r poration of labeled amino acids into the prothrombin protein. On t h e b a s i s o f o u r d a t a , t h e s i t e o f a c t i o n o f v i t a m i n K a p p e a r s t o be t h e c o n v e r s i o n o f a p r o t h r o m b i n p r e c u r s o r into prothrombin in a vitamin K dependent step subsequent to de novo s y n t h e s i s o f t h e p r e c u r s o r p r o t e i n . Possibly, the "inactive-prothrombin" protein found in blood of the vitamin K deficient animals is the prothrombin precursor released f r o m t h e l i v e r c e l l s non a c t i v a t e d due t o l a c k o f t i m vitamin K dependent "activation" step.

i

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Summary The s i t e o f a c t i o n o f v i t a m i n K i n t h e p a t h w a y o f t h e formation of active prothrombin has been re-examined. The controversy as to whether the vitamin functions at a transcription, a translation or a later level has been resolved by f i n d i n g t h a t l a b e l i n g o f p r o t h r o m b i n by l a b e l e d a m i n o acids given to vitamin K deficient rats along with a curat i v e d o s e o f v i t a m i n K i s (1) no d i f f e r e n t than the labeli n g o f p r o t h r o m b i n i n v i t a m i n K n o r m a l u n t r e a t e d r a t s and (2) no d i f f e r e n t t h a n t h e l a b e l i n g o f a p r o t e i n which a p p e a r s t o be i n a c t i v e p r o t h r o m b i n p r e s e n t i n t h e p l a s m a o f vitamin K deficient rats. These data indicate that the site action of vitamin K is in the formation of active prot h r o m b i n f r o m some " u n f i n i s h e d " i n a c t i v e p r e c u r s o r and not a t a l e v e l o f de n o v o s y n t h e s i s . Acknowledgment s We w i s h t o a c k n o w l e d g e t h e t e c h n i c a l a s s i s t a n c e o f Mrs. C a t h e r i n e Boyd a n d t o t h a n k Mr. G e o r g e V a l k o v i c h f o r t h e preparation of bovine prothrombin. Re f e r e nce s 1. 2.

3. 4. 5. 6, 7. 8. 9.

B.C. JOHNSON, R. B. HILL, R. ALDEN and G. S. RANHOTRA, L i f e S c i e n c e s 5, 3 8 5 ( 1 9 6 6 ) . R . B . HILL, S. GAETANI, A. M. PAOLUCCI, P. B. RAMA RAO, R. ALDEN, G. S. RANHOTRA, D. V. SHAH, V. K. SHAH a n d B. C. JOHNSON, J . B i o l . Chem. 243, 3 9 3 0 ( 1 9 6 8 ) . J . W . SUITIE, A r c h . B i o c h e m . B i o p h y s . 118, 1 6 6 ( 1 9 6 7 ) . R.E. OLSON, G. PHILLIPS a n d N. WANG, Adv. i n Enzyme Reg. v 6 , 2 1 3 ( 1 9 6 8 ) . J.P. OLSON, L. L. MILLER and S. B. TROUP, J. C l i n . I n v e s . 45, 6 9 0 ( 1 9 6 6 ) . BERNARDM. BABIOR, B i o c h i m . B i o p h y s . A c t a 1 2 3 , 6 0 6 ( 1 9 6 6 ) . G.S. HANHOTRA a n d B. C. JOHNSON, F e d . P r o c . 28, 385 (1969). V . C . METTA, L. NASH and B. C. JOHNSON, J . Nut r . 74, 473(1961). ~I. S. MA~W.ESH and B. C. JOHNSON, Proc. S o c . Exptl. Biol. Med. 101, 467(1959).

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12. 13. 14. 15.

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O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR a n d R. J. RANDALL, J . B i o l . Chem. 193, 2 6 5 ( 1 9 5 1 ) . A. J. QUICK, M. STANLEY-BROWN a n d F. W. BANCROFT, Amer. J . Med. S c i . 190, 5 0 1 ( 1 9 3 5 ) . H. C. MOORE, S. E. LUX, O. P. MALHOTRA, S. BAKERMAN and J. R. CARTER, Biochim. Biophys. Acta iii, 174(1965). L. F. LI, R. K. KIPFER and R. E. OLSON, Fed. Proc. 28, 385(1969). J. LOWENTHAL and H. BIRNBAUM, Fed. Proc. 28, 385(1969). J. W. suTrIE, presented in Symposium paper - Control of Clotting Factor Biosynthesis by Vitamin K. Fed. Proc. 28, 248(1969).