the effect of cycloheximide administration on vitamin k-stimulated prothrombin formation

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 141, 571-578 (1970)

The Effect of Cycloheximide Administration on Vitamin

K-Stimulated Prothrombin Formation’

J. W. SUTTIE

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 63706

Received August 5, 1970; accepted September 8, 1970

The administration of vitamin K to severely hypoprothrombinemic, vitamin K- deficient rats results in the appearance of about 50% of the normal steady state levels of plasma prothrombin within 1 hr, followed by a slower rate of repletion. This rapid initial burst of prothrombin is inhibited only about 25% by a dose of cycloheximide which almost completely blocks total plasma protein synthesis. The same level of cycloheximide treatment will block the synthesis of prothrombin which is seen between 1 and 2 hr after vitamin K administration, and will also block the hydrocortisone-induced synthesis of tyrosine amino transferase in the same animals. It has been reported that in a rat liver perfusion system there is a specific antagonism between the action of vitamin K and cycloheximide. No evidence for such a relationship could be seen in intact animals. The data strongly suggest that the action of vitamin K may involve the conversion of some precursor protein to prothrombin in a step that does not involve ribosomal protein synthesis.

The mechanism by which vitamin K regu- lates the synthesis of the plasma protein, prothrombin, and the other vitamin K-dependent clotting factors (VII, IX and X)z is unknown. Th.e vitamin has no generalized influence on protein synthesis in intact animals (1, 2) or in an isolated perfused liver (3, 4), nor does it release active, preformed prothrombin from storage sites in the liver (5). The original postulations (6) that the vitamin exerted its action by controlling the synthesis of the specific messenger RNA for prothrombin synthesis have not been con- firmed (1, 4, 7), and although Bell and Matschiner (8) have shown that it is the rate of synthesis, not the rate of destruction of the protein which is controlled, there is disagreement as to whether the vitamin is needed to initiate de novo synthesis of the protein, or if it may act by aiding the conversion of some presently undefined pre-

1 This investigation was supported in part by Grant AM 09305 from the National Institutes of Health, U. S. Public Health Service.

2 For a list of commonly used synonyms for the various clotting factors, see Ref. 29.

cursor protein to the physiologically active form of prothrombin.

Much of the confusion concerns the effects of inhibitors of protein biosynthesis on the response of tissue preparations or intact animals to the vitamin, and the appropriate control of these experiments. Puromycin has been reported by Suttie to completely block the factor VII response to vitamin K which is seen in an isolated perfused liver from a deficient rat. (4), while Olson et al. (9) have reported a lack of effect of puromycin on clotting factor release from a perfused liver, and Babior has reported factor VII synthesis in liver slices which is insensitive to puromycin (10). More recently it has been claimed by Kipfer et al. (3) that both puromycin and cycloheximide would block prothrombin production in a liver perfusion system, but that the effect of cycloheximide, which blocks protein synthesis at the ribosomal level could be overcome if high levels of vitamin K were added to the perfusate. On the basis of these data, they have postulated that vitamin 1-C and cycloheximide are competing for the same site on those ribo-

571

572 SUTTIE

somes that are active in synthesizing prothrombin.

Hill et al. (1) have indicated that in intact vitamin K-deficient animals, puromycin will block the vitamin K-induced response to prothrombin, but that cycloheximide is ineffective. A similar lack of effect of cyclohexi- n-ride was seen by Bell and Matschiner (8). This lack of effect of cycloheximide has been taken to indicate that vitamin K is involved in some step subsequent to peptide bond formation, and that it may somehow func- tion in the conversion of a precursor molecule to prothrombin. Li et al. (11) have, however, obtained data from rat liver perfusions which are interpreted as indicating that the vitamin initiates de novo synthesis, and preliminary data on the incorporation of labeled amino acids into prothrombin following the administration of vitamin K to a warfarin- treated, hypoprothrombinemic dog (12) are also consistent with an initiation of de novo synthesis.

This report presents the results of a series of investigations into the nature of the response of prothrombin to vitamin K administration and the sensitivity of this response to cycloheximide.

METHODS

Animds. Male 140-g rats of the Holtzman strain were housed in coprophagy-preventing cages (13) and fed a diet low in vitamin K (14) for 6-8 days. At this time their plasma prothrombin concentration was usually less than 20 units/ml plasma (normal values are 210-240 units).

Prothrombin assay. Animals were placed under light ether anesthesia, and blood was obtained from the exposed jugular vein or by cardiac puncture, mixed 9:l with 0.1 M potassium oxalate, re- frigerated, and centrifuged for 20 min at 20008. Prothrombin concentrations were measured by the two-stage method of Ware and Seegers as modified by Shapiro and Waugh (15), and expressed as Iowa units per milliliter of plasma.

Injections. Freshly prepared aqueous solutions of cycloheximide (Sigma) were administered intraperitoneally in a total volume of less than 0.5 ml. Vitamin K1 was administered intramuscularly as Aqua Mephyton (Merck) or this emulsion was diluted with saline for intrajugular injection.

Radioactive compounds. Effectiveness of the cycloheximide dose in blocking total protein synthesis was determined following an intraperitoneal injection of 4 pCi of reconstituted protein hydrol-

ysate-‘4C (Schwarz BioResearch). At the indicated times, blood was drawn by heart puncture, the animals were killed, and the liver removed. The liver was homogenized in 2.3 vol of a 0.35 M buf- fered sucrose medium (16) and centrifuged for 15 min at 15,000g. This postmitochondrial supernate was diluted and centrifuged for 1 hr at 105,OOOg to obtain a microsomal pellet which was resuspended in the sucrose medium and a supernatant fraction. Carrier amino acids (yeast hydrol- yzate) were added to these fractions as well as the plasma samples, and the proteins were pre- cipitated with an equal volume of 10% trichloro- acetic acid. The precipitate was dissolved in alkali, reprecipitated, and dissolved in NCS reagent (Nuclear-Chicago). An organic scintillator was added and the samples were counted in a Packard Tri-Carb liquid scintillation spectrometer with external standardization.

To follow the localization and distribution of vitamin K in intact animals, 2-‘4C-methyl-1,4- naphthoquinone (55 pCi/mg) which had been pre- viously prepared in our laboratory (17) was mixed with, phylloquinone (Aqua Mephyton), diluted with saline, and administered as indicated. At the indicated times, the animals were killed, the liver was removed and homogenized in saline, and an aliquot was dissolved in NCS, and the radioactivity determined as described above.

Tyrosine amino transferase. The enzyme was induced by an injection of hydrocortisone sodium succinate (Solu-Cortef, Upjohn) and the animals killed at the times indicated. The liver was homogenized in 4 vol of 0.14 M KCl, centrifuged 30 min at 31,000g and the tyrosine amino transferase activity was assayed in the supernatant by a modification (18) of the method of Diamond- stone (19). Protein was assayed by a modification of the Lowry method (29).

RESULTS

The data in Fig. 1 illustrate the nature of the response of intact vitamin K-deficient, hypoprothrombinemic rats to an intramuscular injection of phylloquinone. The response is characterized by a large increase in the units of prothrombin which appears in the blood during the first hour following injection, and a slow increase at subsequent times. The solid line is the theoretical induction curve for the initiation of de novo prothrombin synthesis based on the half-life of 6 hr which has been determined for this protein by Bell and Matchiner (8). The response during the first hour is much greater than this, but the rate of synthesis following

VITAMIN K-STIMULATED PROTHROMBIN FORMATION 573

‘O” RESPONSE TO - VITAMIN K,

0 I 2 3 4 5 6 HOURS AFTER K,

FIG. 1. Response of hypoprothrombinemic- deficient rats to vitamin K,. Male, 150-g rats were fed a vitamin K-deficient diet in coprophagy- preventing cages for 6-8 days, and were then given 1 mg of vitamin K1 im at 0 time. Values are expressed as Iowa Units as determined by a two- stage assay on blood drawn from the jugular vein or obtained by cardiac puncture. Data from 81 animals are included, and the vertical lines at the top of the shaded bars represent the standard error for 5-26 anima,ls per group. The solid curve repre- sents the theoretical curve for the repletion of prothrombin assuming a biological half-life of 6 hr and a steady state prothrombin level of 206 units/ ml.

the first hour is very similar to the theoretical rate.

The rapid appearance of prothrombin which is seen during the first hour is relatively insensitive to the action of cycloheximide (Fig. 2). About 70 % as much prothrombin was produced during the first hour in those animals given vitamin K and cycloheximide as was seen in those rats given only the vitamin. The level of cycloheximide used, 5.0 mg/kg body wt, is considerably in excess of the 1.5 mg/kg which has been shown (8) to be sufficient to block prothrombin synthesis in a normal rat, and the data in Fig. 3 indicate that it was sufficient in these experiments to block prothrombin formation after the first hour. The same dose of cycloheximide which was relatively ineffective during the first hour was able to completely block the small increase in this protein which was seen during the second hour after vitamin K administration. The timing of the cycloheximide administration was the same in both

/pJ Control

Cycloheximide

0 I Hr 0 IHr

FIG. 2. Effect of cycloheximide treatment on the vitamin K-dependent synthesis of prothrombin in deficient rats. Prothrombin response was measured in 150-g male vitamin K-deficient rats given vitamin K1 only, or given an ip injection of 0.5 mg cycloheximide/lOO g body wt at 0 time, and 1 mg vitamin K1 im at 30 min. Blood was drawn for two- stage assay at 30 min from the jugular vein and at 90 min by cardiac puncture. The data are expressed as unit,s of prothrombin at 0 time and 1 hr after vitamin K1 administration. The vertical lines at the top of the bars are standard errors for seven animals per group.

cases, 30 min prior to the 1-hr time period during which prothrombin synthesis was being studied. The rate of release of vitamin K from the intramuscular injection site to the liver was followed by the use of radioactive phylloquinone, and it can be seen (Table I) that the level of vitamin K or its metabolites in the liver was as high or higher between 1 and 2 hr than it was between 0 time and 1 hr. The lack of an effect of cycloheximide on the prothrombin response during the first hour would indicate that the initial burst of prothrombin production and/ or release is mediated by an action of the vitamin at some step beyond the point of cycloheximide inhibition and strongly sug- gests some conversion of a precursor to an active form.

To insure that the timing of the cycloheximide dose and its concentration were sufficient to block protein synthesis in the

574 SUTTIE

E I20 - ;:

ii: too - -i \ g 80 -

E 0 L 60 - f 2 a 40 - u) .‘- s 20 -

w Control

Cycloheximide

IHr 2Hr IHr 2Hr

FIG. 3. Effect of cycloheximide treatment on the synthesis of prothrombin between 1 and 2 hr following vitamin K administration. Vitamin K-deficient, 150-g male rats were given 1 mg of vitamin K1 im at 0 time and blood was drawn by cardiac puncture at 60 and 120 min. Cyclohexi- mide-treated rats were given an injection of 0.5 mg/lOO g body wt ip at 30 min. Vertical lines are standard errors for 10 rats in the control group and 14 in the cycloheximide group. All animals in the control group showed an increase in prothrombin over the 1-hr period, while half of the cycloheximide-treated animals showed a decrease.

TABLE I DISTRIBUTION OF PHYLLOQUINONE AND ITS METABOLITES FOLLOWING INTRAMUSCULAR

INGESTION”

Plasma (dpm/ml) Liver (dpm/organ)

30 195 f 48 1770 f 460 60 236 f 70 1940 f 260

120 287 f 83 3040 f 650 180 525 f 101 3430 f 260

a Male rats (150 g) were given 1 mg (3 X 105 dpm) phylloquinone intramuscularly. Values are mean f SE for six rats per group.

intact rat, the effect of varying doses of cycloheximide and varying times of its administration on amino acid incorporation into plasma and liver proteins was investi- gated. The data in Tables II and III indicate that all of the doses of cycloheximide used

TABLE II

EFFECT OF AMOUNT OF CYCLOHEXIMIDE ON THE INCORPORATION OF RADIOACTIVE AMINO

ACIDS INTO PROTEINS

Cyclohexi- mide @/kg) Plasma

dpm/mg protein

Microsome Microsomal supernate

0 269 f 16 387 f 17 172 f 9 2.5 17 f 2 75 f 2 20 f 3 5.0 B&4 77 f 22 20 f 4 7.5 12 f 2 90 f 9 19 f 2

10.0 10 f 3 78 f 12 17 * 2

(L Male rats (150 g) were given the indicated dose of cycloheximide intraperitoneally at 0 time and 4 pCi of 14C amino acids intraperitoneally at 30 min. The animals were killed and samples prepared at 90 min. Values are mean f SE for four rats per group.

were able to decrease the amount of radioactive amino acid incorporation into plasma proteins during a 1-hr period by about 95 %, and that the administration of cycloheximide 30 min prior to the labeling period was as effective as any other time tested. The cycloheximide treatment which was routinely used, 5 “g/kg, was effective in blocking almost all incorporation of radioactive amino acids into plasma proteins but was, however, able to block only about 25% of the prothrombin response (Figs. 2 and 4).

Olson has suggested (11) that at least in the perfused liver, there is a specific interac- tion between vitamin K and cycloheximide at the physiologically important receptor site for the vitamin, and that high concentrations of vitamin K can overcome the inhibitory effect of cycloheximide on prothrombin synthesis. This dose dependency does not appear to be present in the case of the intact rat. The data in Fig. 4 clearly indicate that if the dose of cycloheximide is held constant at 5 mg/kg and the dose of vitamin K varied from 1.0 pg/lOO g, which is not quite sufficient to give a maximum prothrombin response, to 100 pg/rat, the effectiveness of the cycloheximide treatment is unchanged. At all levels of vitamin K, the amount of the control prothrombin response which was sensitive to cycloheximide was 20% or less. If the vitamin can reverse the effect of cycloheximide in viva some difference in the ef-


TABLE III

EFFEW OF TIMING OF CYCLOHEXIMIDE DOSE ON THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO PROTEIN’

Cycloheximide treatment dpm/mg of protein

Microsomes Microsomal supernate

No cycloheximide 269 f 16 387 f 17 172 f 9 15 min before a.a. 12 zk 2 91 f 16 28 zt 3 30 min before a.a. 8Ik4 77 f 22 20 f 3 60 min before a.a. 21 * 3 95 f 11 28 f 3

120 min before a.a. 21 f 2 93 l 15 26 f 6

(1 Male rats (150 g) were given 5 mg cycloheximide/kg body wt intraperitoneally at the indicated times prior to 4 &i of 1% amino acids intraperitoneally. The animals were killed and samples prepared 60 min after the amino acid injection. Values are mean &SE for four rats per group.

’ ‘Jg 5 lJ4 50 lJ4 ‘00 ‘Jg

phylloquinone / rot

FIG. 4. Effect of varying levels of vitamin K and a constant level of cycloheximide on prothrombin synthesis. Vitamin K-deficient, 150-g male rats were given an intravascular dose of phylloquinone and a sample of blood was drawn at 0 time. At 60 min a second sample of blood was drawn. The cycloheximide group received 0.5 mg/ 100 g body wt 30 min prior to the vitamin administration. The data are expressed as the increase in prothrombin between 0 time and 1 hr, and the vertical lines are standard errors for from six to nine rats per group.

fectiveness of the antibiotic should have been seen when the vitamin concentration was varied over a lOO-fold range. The data in Fig. 5 indicate that under these conditions, the amount of the vitamin or its metabolites actually reaching the target organ was roughly proportional to that which was administered. The results of the converse experiment, where the dose of vitamin K was held constant at 5 pg/rat, and the amount

5 50 100

pg Phylloqumne 1 rat

FIG. 5. Retention of varying amounts of in- jected phylloquinone in rat liver. Vitamin K-deficient, 150-g male rats were given varying amounts of phylloquinone containing 2-W-methyl-phylloquinone by intrajugular injection and killed after 1 hr. The data plotted are the average of five rats per group, and the total radioactivity found in the liver has been expressed as phylloquinone or its metabolites.

of cycloheximide varied from 2.5 to 20 mg/kg are shown in Fig. 6. The highest dose of cycloheximide had no more effect on prothrombin synthesis than the lowest amount tested.

The large response in prothrombin production following vitamin K administration is to some extent dependent, on the degree of deficiency of the animals. If the animals are left on the vitamin K-deficient diet for 6-S days, at least 90 % of them will have plasma prothrombin concentrations of less than 10 % of normal, and many show essentially no prothrombin by the two-stage assay. After 3 days on the deficient diet there is a consider- able variation in degree of prothrombin de- pletion of individual rats, but many animals

576 SUTTIE

0 2.5 5.0 10.0 20.0

Cycloheximide (mg/kd

FIG. 6. Effect of varying levels of cycloheximide on the vitamin K-induced prothrombin response. Vitamin K-deficient, 150-g male rats were given varying amounts of cycloheximide by intraperitoneal injection at 0 time, and 5 pg phylloquinone iv at 30 min. Blood was taken at 30 and 90 min. The data are expressed as a percentage of the mean increase seen in controls given only vitamin K. The 5 mg/kg data are taken from Fig. 4, and for the other levels the control increase in plasma prothrombin during the 1-hr period was 106 units/ ml. Vertical lines are standard errors for from five to seven rats per group.

have plasma concentrations in the range of from 30-60 units of prothrombin/ml. The first hour response in prothrombin production following vitamin K administration in these animals was found to be in the range of from 60-80 units/ml of plasma, rather than the loo-120 normally seen, but the degree to which it was inhibited by cycloheximide was unchanged. Likewise, if the hypoprothrombinemia was produced by warfarin injection 24 hr prior to the experiment, a normal response could be seen if sufficient amounts of vitamin K were used, and the response was again inhibited only about 20 % by cycloheximide.

It is clear from the data presented that the level of cycloheximide used in these experiments should have been sufficient to block a vitamin K initiation of de novo prothrombin production. There remained, however, the possibility that the induced synthesis of a specific protein with a short half-life might not be as sensitive to cycloheximide administration as was indicated by its effect on radioactive amino acid incorporation into total liver and plasma proteins. The liver enzyme, tyrosine amino transferase (TAT), can be induced by a number of agents including hydrocortisone (21). Preliminary experiments indicated that

)-

, .

I-

)-

)-

m Control

m Cycloheximide I

Prothrombin TAT

FIG. 7. Effect of cycloheximide on induction of tyrosine amino transferase and prothrombin synthesis. Hypoprothrombinemic rats were given 30 mg/kg hydrocortisone ip at 0 time, 0.5 mg/lMl g body wt, cycloheximide (if given) at 30 min, and vitamin K, at 60 min. Blood was drawn from the jugular vein at 60 min, and by cardiac puncture at 120 min. The animals were killed and livers taken for enzyme assay at 126 min. The data are expressed as the increase in units of prothrombin during the 1 hr, and as milliunits of tyrosine amino transferase per mg of liver supernate protein. The data include both vitamin K-deficient rats which were given 20 pg of Kr and warfarin-treated animals given 100 rg K1. The tyrosine amino transferase activity in noninduced controls was 14.5 f 1.6 milliunits/mg protein. Vertical lines are standard errors for nine rats/group.

a substantial increase in the enzyme could be seen 2 hr after a hydrocortisone injection and that this increase could be completely blocked by cycloheximide. Vitamin K-deficient or warfarin-treated hypoprothrombinemic rats were therefore given both hydrocortisone and vitamin K, and the effect of cycloheximide on the induction of prothrombin and of TAT was established. The data in Fig. 7 indicate that although the cycloheximide treatment was able to completely prevent induction of TAT, it had only the usual limited effect on the production of prothrombin.

DISCUSSION

These data confirm the early observations of Hill et al. (1) that cycloheximide was ineffective in blocking the vitamin K-induced production of clotting factor activity in the


hypoprothrombinemic rat. As their data were obtained using a one-stage prothrombin assay, they are difficult to interpret in terms of the production of one of the specific clotting factors and the quantitative significance of the response was questionable. The data presented here clearly indicate that there is a portion of the early production of prothrombin which is cycloheximide sensitive, but that 70.-80 % of it is not.

These observations suggest that the vitamin may be involved in transforming some type of precursor protein to active prothrombin in a step which does not require protein synthesis, and that there may be a hepatic build-up of this precursor in the deficient state. Cycloheximide has been shown to effectively block protein synthesis at the ribosomal level (22), and there seems little doubt that the dose routinely used (5 mg/ kg) should have effectively blocked the synthesis of prothrombin. It was very effective in blocking total plasma protein synthesis; it did block any increase in plasma prothrombin which occurred more than 1 hr after vitamin K administration; less than this amount of cycloheximide has been shown (8) to block. synthesis of prothrombin in a normal rat; and it was sufficient to block the induction of another hepatic protein, tyrosine amino transferase, which has a rapid turnover rate.

Based on observations in an isolated perfused rat liver system, Li et al. (11) have postulated that vitamin K can act to reverse the inhibitory effect of cycloheximide, but only for those ribosomes that are active in synthesizing prothrombin. The attempts reported here to demonstrate some type of dose-response relationship between vitamin K and cycloheximide were not successful. The relative ratios of concentrations tested fell within t:hose which did show a response in the perfusion system, but whether that observation has some relevance to the physiological effect of the vitamin in the intact animal or whether it is some type of artifact of the perfusion system cannot yet be determined.

The possibility that the vitamin may be functioning to activate a precursor has been suggested by a number of investigators (1, 8, 10, 23) and there have been claims of

a protein in the plasma of hypoprothrombinemic animals which might be related to such a precursor protein (24-28). Li et al. (11) have, however, pointed to the incorporation, in an isolated perfused liver, of labeled amino acids into newly synthesized prothrombin as proof of de novo synt,hesis of prothrombin. However, most of the data presented fail to show ayy. significant increase in specific radioactivity of the prothrombin produced in response to the vitamin compared to the specific activity of the small amount of prothrombin formed in control perfusions. Rigorous proof of de nova synthesis under these experimental conditions would seem to require the demonstra- tion of a large increase in both total and specific radioactivity. Experiments in our laboratory (12) would also indicate that the majority of the newly synthesized prothrombin in the dog is the result of new protein formation. The isolated prothrombin in these experiments was undoubtedly impure, and a significant amount of a precursor could have gone undetected. It should also be pointed out that if there is a hepatic protein which is a precursor to active prothrombin, it might well be expected to undergo turnover and degradation to amino acids as well as conversion to prothrombin. If its turnover rate were faster than that of prothrombin by an appreciable factor, experiments designed to study the incorporation of labeled amino acids into newly synthesized prothrombin would not be expected to detect its existence unless they were directed only to the initial phase of the synthesis.

These experiments strongly suggest, on the basis of the very rapid initial response of prothrombin to vitamin K and lack of sensitivity of this initial response to cycloheximide, that ribosomal protein synthesis is not involved in the physiological action of vitamin K. However, experiments where anti- biotics which block protein synthesis are used in intact animals have always been difficult to interpret, and the response seen may have an alternate explanation. In any event, any hypothesis of the mechanism of action of vitamin K must explain the very rapid response which is seen immediately after vitamin administration, and the in- ability of levels of cycloheximide which

578 SUTTIE

block subsequent formation of prothrombin to significantly affect this initial response.

ACKNOWLEDGMENTS

The author acknowledges the technical assist- ance of Mrs. Kathleen Nelson in these experiments and thanks Dr. M. Hermodson for the synthesis of the radioactive phylloquinone used.

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the effect of cycloheximide administration on vitamin k-stimulated prothrombin formation

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