THE JOURNAL UIOLOGIC~L CHEMKWRY
Vol. 243,No. 14,Issueof July 25,pp. 3939-3939,
P&&d in U.S.A.
Vitamin I. and Biosynthesis of Protein and Prothrombin”
(Received for publication, August l&1967)
ROBERTA B. HILL,~ SANCIA GAETANI,~ ANNA MARIA PAOLUCCI,$ P. B. RAMARAO,~
ROSEMARIE ALDEN, AND G. S. RANHOTRA
From the Division of Nutritional Biocherristry, University of Illinois, Urbana, Illinois 61803
D. V. SHAH,~( V. Ii. SHAH,~[ AND B. CONNOR JOHNSON
From the Department of Biochemistry, University of Oklahoma School of Medicine and Oklahoma Medical
ResearchFoundation, Oklahoma City, Oklahoma 73104
SUMMARY gave an essentially normal prothrombin response. At higher
The role of vitamin K in the synthesis of prothrombin has cycloheximide levels the response to vitamin KI, while much
been examined with the following results. less complete, was clear and definite.
1. Vitamin K deficiency has no effect on general protein 6. The response of vitamin K-deficient rats to vitamin K1,
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synthesis as studied by amino acid incorporation into protein administered following treatment with blocking doses of
in vivo and in vitro and by tryptophan pyrrolase production puromycin, appears significant.
following tryptophan feeding. 7. These data appear to indicate that the site of function
2. Prothrombin activity was found in normal rat liver of vitamin K is not at the genetic level, as has been reported,
microsomes with increased release following ultrasonic but at a late stage in translation of prothrombin messenger
treatment, but could not be found in microsomes from the RNA to form a functional prothrombin molecule.
livers of vitamin K-deficient rats or dicumarol-treated rats.
Administration of suboptimal levels of vitamin K to vitamin
K-deficient rats resulted in detectable microsomal prothrom-
bin in 2 hours and essentially normal values within 3 hours.
3. Adequate vitamin KI, given by injection, completely Despite the numerous possible functions which have been de-
restored the blood prothrombin levels of vitamin K-deficient scribed for vitamin K in the living cell, e.g., in oxidative phos-
rats in 1 hour, and of dicumarol- or warfarin-treated rats in phorylation (l-4), in electron transport (5-8), and steroid de-
5 to 7 hours depending on the amounts of warfarin and hydrogenation (9), the only recognized symptom of vitamin
vitamin K1 given. K deficiency in man and experimental animals is lengthened
4. Following administration of actinomycin D or ethionine clotting time of the blood, with subsequent internal hemorrhag-
to vitamin K-deficient rats, treatment with vitamin K1 ing. The increased clotting time in blood or plasma is due to a
markedly stimulated prothrombin production, indicating decreased concentration of prothrombin and of certain other
that the site of action of vitamin K is beyond the level of clotting factors, e.g. VII (extrinsic), IX, and X. There is no
transcription of DNA to prothrombin messenger RNA. evidence that vitamin K1 occurs in the prothrombin molecule
5. Treatment with vitamin K1, following administration
to vitamin K-deficient rats of cycloheximide at a level just Prothrombin is synthesized in liver parenchymal cells (ll),
sufficient to block synthesis of prothrombin for 6 to 8 hours, and upon incubation of a cell-free system from rat liver, its con-
* These investigations were supported in part by Grants AM tent in microsomes has been shown to increase (12, 13). Our
06005 and AM 10282 from the National Institutes of Health and by laboratory has been particularly interested in the problem of
Contract DA-49-193 MD-2830 between the Office of the Surgeon vitamin K function, and these studies are a part of a continuing
General, Department of the Army, and the University of Illinois.
The opinions expressed in this publication are those of the authors program on the role of vitamin K in prothrombin formation.
and not necessarily those of the Army. Since these clotting factors are proteins, the possibility was first
$ Present address, Department of Nutrition and Food Science, considered that vitamin K was involved generaliy in all protein
University of Kentucky, Lexingt,on, Kentucky 4050G.
0 Present address, Department of Nutrition, University of synthesis, even though the postulation of Martius and Nitz-
Rome, Rome, Italy. Litzow (l), that this was at the level of ATP synthesis, seemed to
1 Present address, Central Food Technological Research In- be untenable (4, 5). We have investigated the possible involve-
stitute, Mysore-2, India.
)I Present address, Faculty of Science, Maharaja Sayajirao, ment of vitamin K in general protein synthesis by studying the
University of Baroda, Baroda, India. incorporation in &JO and in vitro of radioactive L-(1J4C)-leucine
Issue of July 25, 1968 Hill et al. 3931
into protein and the induction in vitro of tryptophan pyrrolase were washed twice with 10% trichloracetic acid, twice with hot
(14). 95% ethanol, once with ethanol-ether (3 : I), and twice with
In view of the specificity of response of vitamin K of only cer- ether. Weighed amounts (2 to 3 mg) of dried sample were
tain blood-clotting proteins, it was logical to consider the possi- dissolved in 1 ml of 1 M hyamine hydroxide in methanol at 50”,
bility that the vitamin might function at the genetic level in the and 14 ml of scintillation grade toluene containing 6 g of 2,5-
specific biosynthesis of the clotting factors (15-19). Studies on diphenyloxazole per liter were added.
prothrombin activity in normal and vitamin K-deficient rats
after treatment with agents which block protein synthesis at Incorporation in Vitro of L-(1 J4C)-Leucine into Liver
different levels indicate, however, that vitamin K functions at a
site later than transcription from DNA (20, 21). Microsomes and pH 5 enzymes, prepared from the livers of
control and vitamin K-deficient rats, were incubated with L-
EXPERIMENTAL PROCEDURE (I-14C)-leucine, following the procedure of Keller and Zamecnik
Male Sprague-Dawley rats were fed a vitamin K-deficient (31). The proteins precipitated wit,h 10% trichloracetic acid
diet (22). Controls were given, in addition, 40 pg of the diphos- were washed twice with hot 10% trichloracetic acid, hot 95%
phosodium ester of menadione orally, weekly. The deficient ethanol, and acetone-ether (1:l) and then dried. The samples
animals were housed in tubular coprophagy-preventing cages were subsequently handled like those from the studies of in-
(23). Plasma was obtained from oxalated blood samples for the corporation in viva.
determination of prothrombin time (24-27) and for the studies
of the incorporation in vivo of radioactive leucine into blood Experiments Involving Treatment with Protein
proteins. Animals were killed by decapitation and all organs Synthesis-blocking Agents
were rinsed twice in ice-cold isotonic solutions of sodium chloride Vitamin K Treatment Experiments-nn-Ethionine and puro-
or sucrose, followed by immediate homogenization with approxi- mycin dihydrochloride were Nutritional Biochemicals products.
mately 3 volumes of cold 0.25 M sucrose in a Potter-Elvehjem
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Warfarin was obtained from K and K Laboratories, Inc., New
type homogenizer. Protein concentrations were measured by York, and vitamin K1 from General Biochemicals. Actidione
the method of Lowry et’al. (a), except in the incorporation (cycloheximide) was supplied by Upjohn Company, Kalamazoo,
studies in viuo, in which total nitrogen was determined by the Michigan. Simplastin was obt’ained from Warner-Chilcott
boric acid modification of the micro-Kjeldahl procedure (29). Institute Division, Morris Plains, New Jersey.
Radioactivity measurements were made in a Packard Tri-Carb Control, vitamin K-deficient, and warfarin-treated rats were
liquid scintillation spectrometer. used to investigate prothrombin time after treatment with actino-
mycin D, ethionine, cycloheximide (Actidione), or puromycin.
Prothrombin Activity in Rat Liver Microsomes
Warfarin-treated animals were controls receiving intraperitoneal
Microsomal fractions were obtained by centrifugation of injections of sodium warfarin (0.1 mg or 1 mg/lOO g of body
homogenates prepared in 0.25 M sucrose from livers of control, weight) 20 to 24 hours before injection of the protein synthesis-
vitamin K-deficient, and dicumarol-treated rats, after removal blocking agent. The doses of actinomycin D used were 500,
of cell debris, nuclei, and mitochondrial fractions. Heavy 1000, and 2000 pg/lOO g of body weight, and those of cyclo-
microsomes were obtained by centrifugation at 17,500 x g for heximide were 50, 250, 750, and 1500 pg/lOO g of body weight.
60 min; light microsomes were obtained by centrifugation at They were suspended in wat.er, such that the intraperitoneal
105,000 x g for 60 min; “total microsomes” indicates heavy plus injection never exceeded 0.5 ml.
light. Heavy microsomes were resuspended and recentrifuged Normal and vitamin K-deficient rats received injections of
twice in 0.25 M sucrose, while the surfaces of the light and total ethionine intraperitoneally in two doses (100 mg/lOO g of body
microsomal pellets were rinsed twice with a sucrose solution. weight each), one at zero time, and the other 3 hours later. The
Finally, the fractions were suspended in the citrate buffer of amount administered at each injection was dissolved in warm
Helgeland and Laland (30). After ultrasonic treatment in a 0.9% NaCl immediately before injection and was injected at
lo-kc ultrasonic oscillator, the suspensions were recentrifuged at body temperature. For comparative purposes, in each group
105,000 x g for 60 min before the prothrombin activities were two or three rat.s received inject,ions of the same volume of 0.9%
determined in the supernatant. Results are reported in units NaCl.
per g of protein in the microsomal fraction used, on the basis that Puromycin was given to normal and vitamin K-deficient rats
1 ml of standard human plasma contains 100 units, the minimum at the rate of 20 mg, 30 mg, and 40 mg/lOO g of body weight.
for our procedure being 2.5 units per g of protein. Of a freshly prepared solution containing 20 mg of puromycin
dihydrochloride in 0.9% NaCl and brought to pH 5.5 with
Incorporation in Vivo of L-(i-%J-Leucine into NaOH, 1 ml was injected intraperitoneally.
Proteins of Organs and Plasma Vitamin K1 solutions were prepared by mixing well 50 mg of
Male rats, 12 vitamin K-deficient and 12 control, were paired vitamin K1 into 0.6 ml of Tween 80 and bringing the solution to
according to time of birth and weight. After the intraperitoneal 10 ml wit.h 0.90/, NaCl. This solution could then be diluted
injection of L-(1-I%)-leucine (5 C/l00 g of body weight), three with 0.9% NaCl for lesser concentrations of vitamin K1. The
animals from the control group and three from the vitamin K- usual curative dosages used were 40 pg for vitamin K-deficient
deficient group were killed 1, 2, 4, and 6 hours following injection rats and 100 pg and 1 mg for warfarin-treated rats, given sodium
of the labeled amino acid. The livers, spleens, kidneys, and warfarin in doses of 0.1 and 1 mg/lOO g of body weight, respec-
hearts were homogenized in demineralized water. The proteins tively. The vitamin was given at various times (0 to 24 hours)
of the organs and the plasma were precipitated overnight at 2” after injection of the protein synthesis-blocking agent, as in-
with equal volumes of 20% trichloracetic acid. The proteins dicated in the figures. In some experiments the Quick (24, 27)
3932 Vitamin K and Biosynthesis of Protein and Prothrombin Vol. 243, No. 14
prothrombin time assay was used to follow prothrombin activity TABLE II
over a period of time in the same animal. The tips of the tails Plasma and liver microsomal prothrombin activity in
of the rats were cut off, blood was withdrawn in a capillary tube, dicumarol-treated rats
the volume was marked and silicone-treated for microprocedures,
Plasma pro- Tot~aJmm$ro-
and the tails were cauterized. One volume of blood was trans- No. of rats Dicumarol treatment” TiTFvEp
thrombin time prothrombin
ferred quickly into 2 volumes of a thromboplastin-calcium-
sodium chloride mixture (Simplastin) in a partially hollowed -I hr set mits/gfiroteinb
glass plate, heated continuously to 37.5”. Time required for the 10 None 15 16
clotting in these experiments is expressed as percentage of normal 6 Fed for 3 days 80->500 0
(Figs. 1, 3a, 3b, and 5) based on the Quick prothrombin time 2 Injected 2 14-14 16.9”
from control animals (15 f 1 set). 1 Injected 6 14 9.8
In those experiments in which the Allington (25) method for 3 Injected 12 25-25-75 0
prothrombin time was used, the data are expressed as deter- 1 Injected 24 61 0
mined in set (Figs. 2, 4, 6, and 7). Both methods of expressing a The rats were fed 0.25yo dicumarol in vitamin K-free ration
prothrombin time have their merits. The partial cures ob- or they received intraperitoneal injections of a Tween 80 suspen-
tainable following high levels of blocking agents cannot be clearly sion containing 25 mg/lOO g of body weight.
seen in the “percentage of normal” plots, while they can be seen b After sonic treatment.
on a direct plot. On the other hand, the complete response of c Pooled livers.
the vitamin K-deficient animal to vitamin K is most dramatically
shown on the percentage of normal plot. TABLE III
Radioactive iZmino Acid Incorporation Experiments-Two or Prothrombin activity in plasma and in total microsomes from livers
four hours after treatment with actinomycin D or cycloheximide, of vitamin K-dejicient rats (two rats in each group) at
normal rats received intraperitoneal injections of uniformly various times after intraperitoneal injection of
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labeled mixed W-amino acids (5 C/100 g of body weight) and vitamin K,
were killed 1 hour later. Control animals received the labeled
amino acids but no antibiotic. The proteins of plasma and of Vitamin KI Plasma after ultrasonic
injected prothrombin time treatment
heavy microsomes, prepared in 0.25 M sucrose as previously
described, were precipitated with equal volumes of cold 20% Pg set units/g protein
trichloracetic acid, containing 25 mg/lOO ml of cold mixed amino 10 92 0
acids (Beckman standard calibration mixture, type 1). After 20 52 0
15 min the samples were centrifuged. The protein precipitates 10 20 2.0,3.0
were then each washed once with 10% trichloracetic acid con- 10 16 4.6, 3.4
taining 25 mg/lOO ml of cold mixed amino acids, 10% trichlor- 20 14 15.7,9.3
acetic acid, water, and absolute ethanol; then twice with ethanol-
ether (3:1), and twice with anhydrous ether. Of the dried phenyloxazolyl)]benzene (dimethylPOPOP)) were added, and
samples, 5 to 10 mg, dissolved in 0.2 ml of 2.5 N NaOH, were the vials were quickly shaken.
pipetted into a scintillation vial filled with a thixotropic gel
powder (Cab-0-Sil, Packard). Following this, 15 ml of scintillator
(800 ml of scintillation grade toluene, 200 ml of ethanol, 5 g of Microsomal Prothromlrin
2,5-diphenyloxazole (PPO), and 0.3 g of 1,4-bis-[2-(4-methyl-5-
It has been shown that liver microsomal prothrombin can be
“completed” or activated or released by incubation (12) or ultra-
sonic treatment (30), and that ultrasonic treatment is the more
Prothrombin activity in liver microsomes and cell sap before and efficient (13). This apparent “release” of prothrombin from
after sonic treatment from control and vitamin
microsomes led us to study the effect of vitamin K deficiency on
prothrombin activity at the microsomal level. These data are
Average values are given, and ranges are shown in parentheses. given in Tables I through III, in which the designation “0”
<a of animalr Control K-deficient signifies that the prothrombin activity was unmeasurable in our
Cell fraction Before After
Before sonic After sonic sonic sonic
Heavy microsomes contained more prothrombin activity than
+K -K treatment treatment treat- treat- light microsomes (Table I) and no activity was found in mito-
chondria, in agreement with the results of Goswami and Munro
(12). No prothrombin activity could be detected in liver micro-
Total micro- 15 5 (4.7813.4) (11.:!23.9) 0 somes from vitamin K-deficient rats.
somes Prothrombin activity was not found in microsomes from rats
Heavy 6 5 (7.2!%2) (17.z7.3) 0 fed a dicumarol-containing diet for 3 days (Table II). Twelve
hours after an injection of dicumarol (25 mg/lOO g of body
Light 2 3 6 (l&2.2) 0 weight) to control rats, microsomal prothrombin activity was
unmeasurable, while that of plasma had decreased to 30% of
Cell sap 5 0
normal. Plasma prothrombin levels thus decreased as micro
-I somal prothrombin decreased.
Issue of July 25, 1968 Hill et al. 3933
The data in Table III show the opposite process. Vitamin TABLE IV
K-deficient rats with plasma prothrombin times greater than E$ect of actinemycin D and cycloheximide on incorporation in vivo
100 set were given suboptimal quantities of vitamin K1. A dose of mixed 14C-amino acids (5 C/l00 g of body weight,
of 40 wg will restore plasma prothrombin activity of vitamin K- injected 1 hour prior to sacrijice) into microsomal
deficient rats to normal in 1 hour; doses in 10 and 20 pg require a and plasma proteins of vitamin K normal rats
longer time interval. Simultaneously with the decreases in Figures in parentheses indicate the number of rats.
plasma prothrombin times, increases in microsomal prothrombin Incorporation into Incorporation into
were observed (Table III). Dose of microsomes pl~SIllil
The fact that vitamin K has not been found in the prothrombin agentQ
3 hrs 5 hrs 3 hrs 5 hrs
molecule (10) does not eliminate the possiblity that it might be
specifically responsible for “activating” or for joining together
subunits of the protein, or that it might be needed for the syn-
thesis of the peptide chains or for assembling the molecule in its Actinomycin 500 114.2 (2) 80.7 (3) 116.8 (2) 80.9 (3)
entirety. It is also possible that the vitamin may be required D 1000 55.4 (1) 66.2 (3) 103.8 (2) 70.1 (3)
2000 44.1 (1) 61.8 (3) 85.3 (2) 64.2 (3)
in all protein syntheses (e.g. for ATP synthesis, as suggested by
Martius and Nitz-Litzow (1)) but that this effect is observed Cyclohexi- 750 36 (1) 12.5 (1) 5 (1) 6 (1)
in the synthesis of the clotting proteins because of their very mide 1500 20 (1) 11 (1) 4 (1) 9 (1)
rapid rate of turnover. To study the effect of vitamin K defi-
ciency on general protein synthesis, the induction of tryptophan 5 Protein synthesis-blocking agents given 3 and 5 hours prior
pyrrolase activity by tryptophan (14) and the incorporation to sacrifice.
of L-(l-i4C)-leucine into proteins, both in tivo and in z&o, were b Controls (3 rats) did not receive any antibiotic. Mean numer-
examined. ical values for l-hour incorporations were 377 cpm per mg of pro-
tein for microsomes and 300 cpm per mg of protein for plasma.
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Amino Acid Incorporation
When rats were injected with L-(1-14C)-leucine, the radioac- normal rats at zero time and 5 PC of mixed 14C-amino acids were
tivity found per mg of protein in heart, spleen, kidney, liver, and injected intraperitoneally 1 hour prior to decapitation at 3 or 5
plasma did not differ between vitamin K-deficient animals and hours, the incorporation of radioactivity into microsomal protein
controls at any time from 1 to 6 hours after injection of the was essentially unaltered compared to that in controls not in-
labeled amino acid. Liver microsomes plus pH 5 enzymes (31) jected with actinomycin D. At a level of 2000 pg of actinomycin
from vitamin K-deficient rats incorporated L-(l-14C)-leucine as D per 100 g of body weight, incorporation of radioactivity into
well as did those from control animals, even though the deficiency both microsomal and plasma proteins was somewhat decreased.
was severe, as shown by plasma prothrombin times (70 to over In contrast, injections of cycloheximide at levels of 750 pg and
120 set). 1500 pg/lOO g of body weight decreased incorporation of radio-
The induction of tryptophan pyrrolase by tryptophan feeding activity from the mixed 14C-amino acid into plasma proteins to
was also found to be unaffected by vitamin K deficiency, both less than 10% of the controls and into microsomal proteins to
in the case of intact and adrenalectomized rats (14). low values. These differences are consistent with the site of
Thus the effects of vitamin K deficiency on protein synthesis action of actinomycin D in blocking synthesis of RNA (32) and
appear to be confined to prothrombin and related clotting pro- of cycloheximide in preventing the growth of nascent polypeptide
teins. Vitamin K-deficient rats respond completely in 1 hour, chains (33) and peptide bond formation (34).
and warfarin- or dicumarol-treated rats in 5 to 7 hours, to ade-
quate injections of vitamin K1 (Fig. lc). Only when there has Response of Hypoprothrombinemic Animals to Vitamin K1 Given
been extensive internal hemorrhaging, resulting in a marked de- after Administration of Protein Synthesis-blocking Agents
crease in blood cell volume, will the vitamin K-deficient animal Control Experiments with Normal Animals to Establish Levels
sometimes fail to respond immediately t.o treatment with vitamin of Protein Synthesis-blocking Agents Required to Block Prothrombin
6. Synthesis-Fig. 1 gives the data on the effects of actinomycin D
and cycloheximide on blood prothrombin levels. After injection
Experiments with Agents Which Block Protein Synthesis of actinomycin D into control rats, decreased prothrombin ac-
Possible hereditary relationships between the vitamin K-de- tivity in blood can be observed. This is shown in Fig. la for
pendent blood-clotting factors led Olson (15, 19) to postulate that doses of 500 and 2000 pg/lOO g of body weight of actinomycin D
vitamin K acted as an effector molecule at the genetic level in the (the curve for doses of 1000 fig lies between the two given). At
synthesis of these blood-clotting factors. It was reported that all these levels of actinomycin D, all animals died within 8 to 24
menadione given to vitamin K-deficient chicks (15, 17) and hours.
vitamin K1 given to dicumarol-treated rats (16, 17) were unable Cycloheximide, since it blocks after mRNA, caused an immedi-
to restore plasma prothrombin activity after protein synthesis ate decrease in prothrombin activity, which reflects the rapid
had been blocked by actinomycin D. The results of our experi- turnover rate of prothrombin (21). Blood prothrombin activity
ments with control, vitamin K-deficient, and warfarin-treated decreased, essentially linearly, to low levels in about 6 hours
rats, in which protein synthesis has been blocked by actinomycin following all dose levels of cycloheximide (Fig. lb). The rate of
D or by cycloheximide (Actidione), are shown in Table IV and decrease was only slightly slower following 50 pg of cycloheximide
Fig. 1 through 3. per 100 g of body weight than that following 750 or 1500 g. All
From the data given in Table IV, it can be seen that when animals that received 50 pg of cycloheximide per 100 g of body
500 pg of actinomycin D were injected intraperitoneally into weight recovered and had normal prothrombin levels 24 hours
3934 Vitamin K and Biosynthesis of Protein and Prothrombin Vol. 243, No. 14
after treatment; at 250 pg all animals lived and prothrombin re-
coveries, ranging from 30 to 100% of normal, were found 30
hours after treatment, although prothrombin levels had remained
low for all these rats from the 6th to the 15th hour after treat-
ment. Following the administration of 750 and 1500 fig of
cycloheximide per 100 g of body weight, all animals were dead
within 6 to 16 hours after treatment. The toxicity of these pro-
tein synthesis-blocking agents is apparently not related to their
effect on prothrombin activity, but rather to their general block-
ing of protein synthesis, since the dead animals showed no
Similar control data on the blocking of prothrombin synthesis
in normal rats by ethionine and puromycin are given in Fig. 4,
Curve A and Fig. 7A, respectively.
Control Experiments to Establish Recovery Times of Dejkient
and Warfarin-treated Animals following Vitamin K1 Administra-
tion-It was found that 50 mg of sodium warfarin injected per
100 g of body weight would kill a rat in 20 min without decreasing
prothrombin activity, yet a rat given 20 mg/lOO g of body weight
may live 30 hours or longer, while prothrombin activity de-
creases to less than 1% of normal (prothrombin time being
greater than 180 set). In Fig. lc, prothrombin curves are shown
for 13 rats given injections of 1 mg of sodium warfarin per 100 g of
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body weight, 24 hours before zero time. At zero time, five of the
rats received injections of 1 mg of vitamin K, and the pro- I I 1 I
thrombin times returned to 100% of normal within 8 hours 2 4 6 8 I
(1~5) ; eight rats remained untreated (1~8). When 0.1 mg of HOURS
sodium warfarin per 100 g of body weight was given to four rats FIG. 2. Effect of vitamin K1 treatment on vitamin K-deficient
24 hours before zero time, prothrombin activity did not decrease and warfarin-treated rats. Curve A, four vitamin K-deficient
below 20% of normal, and 0.1 mg of vitamin K1 restored pro- rats given 40 rg of vitamin Kt per rat at zero time; Curve B, five
normal rats given sodium warfarin (0.1 mg/lOO g of body weight)
thrombin activity to normal levels within 6 hours (1~4). In 24 hours before zero time and given vitamin K, (0.1 mg/lOO g of
body weight) at zero time; Curve C, seven normal rats given sodium
warfarin (1 mg/lOO g of body weight) 24 hours before zero time
a b C and given vitamin KI (1 mg/lOO g of body weight) at zero time.
addition, 40 pg of vitamin K1 restored prothrombin activity
of vitamin K-deficient rats in 1 to 1s hours (leg), and 500 pg
of vitamin Ki will act even more rapidly.
Similar data obtained by the Allington (25) method of pro-
thrombin determination are plotted in Fig. 2 and show again an
almost complete response to vitamin K1 injection in 1 hour in the
case of the vitamin K-deficient animals (Curve A), and a much
more delayed response in the case of animals treated with
warfarin (Curves B and C).
Experiments with Hypoprothrombinemic Animals-With these
IO0 controls available, it was then possible to study the response of
0 5 5 IO 0 5 lo
vitamin K-deficient or warfarin-treated rats to vitamin Ki given
simultaneously or at various time intervals following administra-
FIG. 1. Prothrombin activity, expressed as percentage of
normal and also expressed in set, plotted against treatment time tion of the different protein synthesis-blocking agents.
in hours. a, actinomycin D was given intraperitoneally to con-
trol rats, of which five were given 500 pg/lOO g of body weight and Response to Vitamin K1 in Presence of Actinomycin D
five were given 2000 pg/lOO g of body weight. b, cycloheximide
was given intraperitoneally to control rats, of which five were In Fig. 3a (Curves 1 and 9) it is shown that vitamin K-deficient
given 50 pg/lOO g of body weight, 10 were given 250 pg/lOO g of rats given vitamin Ki, simultaneously with or 3 hours after an
body weight and 11 were given 750 fig/100 g of body weight. c, 10, injection of 500 pg of actinomycin D per 100 g of body weight,
vitamin K-deficient rats, not treated with vitamin K. 3, vitamin
K-deficient rats treated at zero time with 40 rg of vitamin K1; recover their prothrombin activity. Warfarin-treated rats
8, rats given 1 mg of warfarin per 100 g of body weight 24 hours given actinomycin D also responded to vitamin Ki (Curves 4-W
before zero time, but not treated with vitamin K; 6, rats given 1 and I-W), but less rapidly, corresponding to the time curve for
mg of warfarin per 100 g of body weight 24 hours before zero time recovery after warfarin injections shown in Fig. 1~5. Obviously,
and given 1 mg of vitamin K, at zero time; 4, rats given 0.1 mg of
100% of normal cannot be reattained since prothrombin activity
warfarin per 100 g of body weight 24 hours before zero time and
given 0.1 mg of vitamin KI at zero time. Numbers on the graph is slowly lost in control rats given the same quantity of actino-
indicate the number of rats. mycin D. Recovery of prothrombin activity after injection of
Issue of July 25, 1968 Hill et al.
2000 pg of actinomycin D was also studied because of the results
of incorporation of mixed 14C-amino acids into microsomal and
plasma protein (Table IV). From the data on control rats
given 2000 pg/lOO g of body weight of actinomycin D (Fig. 3b,
Curve C-5), it appeared that prothrombin synthesis was continu-
ing at a rate sufficient to keep blood prothrombin above 50% of
normal for 6 hours, but that after 6 hours, a sharp break in the
curve occurs presumably due to exhaustion of mRNA for pro-
thrombin synthesis; and thereafter a rapid fall in blood pro-
thrombin, similar to that seen after cycloheximide, occurs.
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IO’ I I I I
0 6 12 18 24 : 1
FIG. 4. Effect of vitamin K1 treatment on rats treated with two
equal doses of 100 mg of ethionine per 100 g of body weight each.
Curve A, seven normal rats given ethionine at 0 and 3 hours;
Curve B, five vitamin K-deficient rats given ethionine at 0 and 3
HOURS hours and vitamin K1 (40 pg per rat) at 5 hours; Curve C, three
vitamin K-deficient rats given ethionine at 0 and 3 hours and
vitamin K1 (40 pg per rat) at 24 hours.
Nonetheless, as can be seen from Fig. 3b (Curves 5, 2 and S)
b vitamin K-deficient rats (5% or less of normal prothrombin levels,
prothrombin time being greater than 80 set) responded to vitamin
Kr by increased blood prothrombin levels up to the control values,
even when (Curve S) the vitamin was given 5 hours after the high
actinomycin D dose and no further mRNA for prothrombin ap-
peared available. These data indicate that vitamin K functions
after prothrombin mRNA formation in the regulation of the syn-
thesis of active prothrombin.
It has been stated that ethionine blocks protein synthesis by
blocking conversion of nucleolar RNA to cytoplasmic ribosomal
RNA rather than by blocking mRNA synthesis (35, 36). While
ethionine thus blocks at a step later than does actinomycin D, it
can be seen from Fig. 4 that vitamin Kr treatment at 5 hours and
even at 24 hours after starting ethionine injection in vitamin K-
deficient rats still gave a definite prothrombin response.
8 ‘O Response to Vitamin K1 in Presence of Cycloheximide
FIG. 3. Prothrombin activity of control, vitamin K-deficient, Since the above data indicated that the site of vitamin K action
and warfarin-treated rats given 500 (Fig. 3~) and 2000 (Fig. 36) was definitely beyond the transcription level, further experiments
fig of actinomycin D per 160 g of body weight at zero time, fol-
lowed by vitamin K1 at times as indicated. C, control rats; W, were carried out with cycloheximide, which blocks protein syn-
warfarin-treated rats (1 mg/lOO g of body weight, 24 hours before thesis at the translation level (33, 34). Restoration of pro-
zero time). Numbers on graphs represent the number of rats. thrombin levels upon vitamin Kr administration to vitamin K-
Vitamin K1 (40 pg) was given to vitamin K-deficient rats, except deficient animals treated with cycloheximide would indicate that
for the group in Fig. 3b, Curve 3, which was given 500 pg (5 hours
after actinomycin D). Vitamin Kl (1 mg) was given to the the vitamin acts at a site later than the site of action of cyclo-
warfarin-treated rats. heximide.
3936 Vitamin K and Biosynthesis of Protein and Prothrombin Vol. 243, No. 14
From Fig. 5, it is clearly seen that vitamin K1 (100 pg), given
1 hour after cycloheximide (50 fig/100 g of body weight) ad-
ministration, resulted in an essentially complete restoration of
prothrombin levels in the case of vitamin K-deficient rats
(Curve 4) and warfarin-treated rats (Curve 9). At the same time,
control rats, given the same level of cycloheximide, showed an
almost 80% reduction in prothrombin level in about 7 hours
(Curve 5), while warfarin-treated and vitamin K-deficient rats,
given cycloheximide and no vitamin K, showed no increase in
prothrombin (curves not shown).
Data on warfarin-treated animals given higher levels of cyclo-
heximide are presented in Fig. 6. These animals received in-
jections of 0.1 mg/lOO g of body weight (Fig. 6A) and 1 mg/lOO
g of body weight (Fig. 6@ of sodium warfarin 24 hours before the
cycloheximide injections. Groups received 250 pg (Fig. 6A)
or 750 pg (Fig. 6B) of cycloheximide and 0.1 mg (Fig. 6A, Curve
A) or 1 mg (Fig. 6B, Curve A) of vitamin K1 per 100 g of body
weight. Control groups were given 250 pg (Fig. 6A, Curve B)
or 750 pg (Fig. 6B, Curve B) of cycloheximide per 100 g of body
weight, but no vitamin K1. It can be seen from Fig. 6, A and B,
that the prothrombin time of the warfarin-treated animals given
201 I I 1 I
only cycloheximide increased steadily, while, in contrast, the 0 2 4 6 8
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prothrombin time of warfarin-treated animals given cyclo- t HOURS
heximide and vitamin Ii1 showed marked decreases over several
Because of the rapid fall in prothrombin after the administra-
tion of cycloheximide to normal rats and because of the possible
01 I I I I
2 4 6 8 1
FIG. 6. Effect of vitamin K1 administration on warfarin-treated
rats given cycloheximide. A, Curve A, five rats given 0.1 mg of
sodium warfarin per 100 g of body weight 24 hours before zero time,
250 fig of cycloheximide per 100 g of body weight at zero time, and
0.1 mg of vitamin K, per 100 g of body weight at 1 hour; Curve B,
three rats given the same warfarin and cycloheximide treatments
but no vitamin K1. B, Curve A, five rats given 1 mg of sodium
warfarin per 100 g of body weight 24 hours before zero time, 750
pg of cycloheximide per 100 g of body weight at zero time, a.nd 1
mg of vitamin K1 per 100 g of body weight at zero time; Curve B,
HOURS three rats given the same warfarin and cycloheximide treatment
FIG. Effect of cycloheximide
5. treatment on prothrombin but no vitamin KI.
response to vitamin K,. Curve 5, five normal rats given 50 rg of
cycloheximide at zero time; Curve 4, four vitamin K-deficient rats structural antagonism between cycloheximide and vitamin K,
given 50 pg of cycloheximide at zero time and 100 Mg of vitamin K, large doses of vitamin K1 were given to cycloheximide-treated
at 1 hour; Curve 9, nine rats given 0.1 mg of sodium warfarin per
100 g of body weight 24 hours before zero time, 50 pg of cyclo- hypoprothrombinemic, but not vitamin K-deficient, rats; how-
heximide at zero time, and 100 pg of vitamin KI at 1 hour. ever, no response was obtained.
Issue of July 25, 1968 Hill et al. 3937
tion by the two-stage procedure of Ware and Seegers (26) was
attempted in recovery experiments following administration of
cycloheximide and ethionine to vitamin K-deficient rats. In
these cases, blood for plasma preparation was obtained by heart
puncture from control and experimental animals at different time
intervals. The recovery patterns for the animals studied con-
firm entirely the foregoing data; however, since blood could not
be repeatedly drawn by heart puncture, the two-stage prothrom-
bin assay procedure was used only for confirmation of isolated
The results of amino acid incorporation and tryptophan pyrro-
lase induction experiments (14) show that general protein syn-
thesis is not interfered with in vitamin K deficiency, even though
the protein prothrombin (and other vitamin K-dependent pro-
teins) almost disappear from the blood and can no longer be
found in the microsomes.
The data show that vitamin K1 treatment of the deficient ani-
mal shortly after administration of high doses of actinomycin D
will stimulate prothrombin production. Reversal of vitamin K
deficiency was also obtained within 1 hour, when vitamin K1 was
given 1 hour after an injection of cycloheximide at a level which
had been shown to block prothrombin synthesis in control ani-
mals for more than 6 hours. Following much higher levels of
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I I I I
2 4 6 8 cycloheximide, a less complete but still clear response to vitamin
HOURS K was obtained in vitamin K-deficient rats. Vitamin K-de-
FIG. 7. Effect of puromycin treatment on prothrombin response ficient animals also responded to vitamin K1 administered 5 or
to vitamin K1. A, Curve a, three normal rats given 20 mg of 24 hours after ethionine injection. Vitamin Ki given to normal
puromycin per 100 g of body weight. Curve b, two normal rats rats 24 hours after ethionine injection has no effect on prothrom-
given 30 mg of puromycin per 100 g of body weight. Curve c, two
normal rats given 40 mg of puromycin per 100 g of body weight; bin level.
Curve d. five vitamin K-deficient rats given 20 mg of puromycin In the case of puromycin, vitamin K1 given simultaneously
per 1OO’g of body weight at zero time and vitamin KI-(40 pg per with the antibiotic provoked a slight response in vitamin K-
rat) at zero time. B, Curve a, one vitamin K-deficient rat given deficient rats in the presence of 40 mg of puromycin per 100 g of
40 mg of puromycin per 100 g of body weight at zero time and
body weight and more response in the presence of 20 mg. There
vitamin K1 (40 pg per rat) at zero time; Curve b, two vitamin K-
deficient rats given 40 mg of puromycin per 100 g of body weight was little response to vitamin Ki administered 1 or 3 hours after a
at zero time and vitamin Kr (4Opg per rat) at 1 hour; Curve c, two proven prothrombin synthesis-blocking level (40 mg/lOO g of
vitamin K-deficient rats given 40 mg of puromycin per 100 g of body weight) of puromycin had been given to vitamin K-de-
body weight at zero time and vitamin Kr (40 .ng per rat) at 3 ficient rats. These latter data are in agreement with the results
obtained by Suttie (37) in liver perfusion experiments. Prydz
(38) has found that puromycin and warfarin both inhibit Factor
Response to Vitamin K1 in Presence of Puromycin VII biosynthesis in suspensions of rat liver cells; however, he has
The determination of the amount of puromycin required to not examined the effect of puromycin on vitamin K-initiated
block prothrombin synthesis in normal rats is presented in Fig. formation of Factor VII.
7A which shows that with a puromycin dose of 40 mg/lOO g of In interpreting these results, several factors need to be taken
body weight the prothrombin time increases rapidly and the into consideration. We determined the amount of protein syn-
blood prothrombin level drops to about 15% of normal (Fig. 7A, thesis-blocking agent to be used in recovery experiments on the
Curve c). A puromycin dose of 50 mg/lOO g of body weight to basis of the amount required in the vitamin K-adequate animal
normal rats was found to be lethal, the rats dying in 5 to 5+ to reduce active circulating blood prothrombin to a low level.
hours. The data on puromycin treatment of vitamin K-deficient The use of such control animals appears essential, since tech-
rats are presented in Fig. 7A, Curve d and Fig. 7B, Curves a, b, niques are not available to measure the incorporation of labeled
and c. While it is evident from Fig. 7A, Curve d that animals nucleic acid into specific prothrombin messenger RNA and since
do show some response to a dose of 40 pg of vitamin K1 given the rat has insufficient isolatable blood prothrombin to readily
simultaneously with a suboptimal level of puromycin (20 mg/ permit the measurement of the incorporation of labeled amino
100 g of body weight), when the puromycin dose is increased to acids into prothrombin. In any case, since it is the formation of
the complete prothrombin synthesis-blocking level of 40 mg/ active prothrombin following vitamin K administration that was
100 g of body weight (Fig. 7B, Curves a, b, and c), the response being studied, a level of protein synthesis-blocking agent proven
to subsequent vitamin Kr administration is very small (Fig. 7B, to block synthesis of this particular protein (or group of proteins)
Curves b and c) (negligible if plotted as percentage of normal). was established and was used. Such controls have been lacking
in other studies (15, 37). If low doses of blocking agent are
given to vitamin K-deficient rats over a 24-hour period, the
Since the prothrombin data reported here were obtained by the animals frequently die of hemorrhage within that period due to
Quick (24, 27) or Allington (25) single-stage methods, confuma- the vitamin K deficiency even though the antibiotic dose given
3938 Vitamin K and Biosynthesis of Protein and Prothrombin Vol. 243, No. 14
may be too low to completely block prothrombin synthesis. On or puromycin should not block their conversion to prothrombin
the other hand, the fact that a vitamin K-deficient rat responds by vitamin K, given either simultaneously or 1 hour later.
within 1 or 2 hours to vitamin Ki treatment makes possible the Losito (48) was unable to confirm the presence of this prothrom-
use of essentially totally blocking doses of these agents, instead bin inhibitor using vitamin K-deficient and dicumarol-treated
of the low levels used in many longer term animal experiments. chicks.
The blocking of prothrombin synthesis, primarily at one site in Our data are in agreement with the report of Anderson and
protein synthesis, by a protein synthesis-blocking agent still Barnhart (ll), who found that no fluorescence due to prothrom-
leaves all sites between the block and the final “activation” open bin is seen in the liver of the dicumarol-treated animal upon
for vitamin K action, if it functions beyond the blocking site. examination with specific prothrombin fluorescent antibody, but
However, while the site of action of vitamin K and all subsequent that fluorescence appears within 2 hours after treatment of such
sites are still open, lack of prothrombin regeneration upon treat- animals with vitamin K, indicating a rapid “turnon” of pro-
ment with vitamin K could also mean that the blocking agent thrombin formation in the liver by vitamin K.
acting at an earlier site had blocked the availability of required It is assumed that vitamin K deficiency blocks only one site of
precursors or enzymes. On the other hand, good responses ob- prothrombin synthesis and that prothrombin synthesis proceeds
tained with vitamin K do indicate that the level of action of the normally up to that step in the vitamin K-deficient animal.
vitamin is beyond the site blocked. Puromycin has been reported at various times to completely
Warfarin- or dicumarol-treated rats are less likely to die from block vitamin K-induced Factor VII formation (Suttie (37).\, not
hemorrhage during antibiotic treatment, but are considered less to block vitamin K-induced prothrombin formation (Olson et
useful experimental animals because (a) these vitamin K antago- al. (43)) in liver perfusions, and not to block vitamin K-induced
nists have let’hal effects unrelated to blood clotting, as demon- Factor VII formation in liver slices (Babior (49)). Our data
strated by 2- to 4-hour nonhemorrhagic death following high indicate an effect similar to that of high levels of cycloheximide,
doses (20 mg or more) of warfarin, and (b) because the presense i.e. inhibition but not a complete block. Since puromycin blocks
of the antagonist makes the cure with vitamin K more difficult, protein synthesis by terminating peptide synthesis, becoming
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requiring higher vitamin K levels and longer times, due pre- itself incorporated into the growing peptide chain (50-52), it is
sumably to competition for binding sites occupied by the drug. thus inactivated and large amounts must be used. This action
The data from the actinomycin D and ethionine experiments of puromycin is accompanied by a rapid breakdown of rat liver
indicate clearly that the site of vitamin K function is beyond the polysomes to a new steady state level, characterized by a shift to
the transcription level, and the data from the cycloheximide ex- smaller aggregates (53). These data and those obtained at high
periments indicate that the vitamin functions beyond the site at levels of cycloheximide indicate that polysomal breakdown
which cycloheximide blocks protein synthesis. interferes with the prothrombin response to vitamin K adminis-
Cycloheximide acts at a site beyond the attachment of the tration.
amino acid-charged tRNA to the ribosome and appears to block On the basis of the data presented in this paper, it appears that
transfer of amino acids from aminoacyl-tRNA to form poly- a possible model for the role of vitamin K in the synthesis of
peptide (34). Its activity appears to depend on the character of prothrombin (and by inference, of the other vitamin K-dependent
the microsomes (39-41). Cycloheximide has been found to not clotting proteins, Factors VII, IX, and X) is as a precursor of a
inhibit polyribosome formation, but to prevent conversion of cofactor of a protein (the binding site of which is competitive
polyribosomes to single ribosomes under the usual stimuli (33, with warfarin) which is involved in the removal of the precursor
42). This may be further evidence that it blocks at the level of peptide from the polysome into its proper folded tertiary struc-
peptide synthesis (33). Godchaux, Adamson, and Herbert (42) ture. It may be that formation of this folded structure (8 S-S
have shown the ribosomal changes to be very much greater at bonds have been found in canine prothrombin (54) and in bovine
higher cycloheximide concentrations, and this may well explain prothrombin (55)) is an integral part of the complete synthesis
the relatively poor cures obtained at the higher dose levels of of the molecule at the level of peptide bond formation and that
cycloheximide. this is the site of translation regulation of the synthesis of pro-
Subsequent to the first repor& (20,21) on the ability of vitamin thrombin. This model bears some resemblance to that proposed
K to bring about restoration of blood prothrombin levels in the by Cline and Bock (56) for translation regulation of specific pro-
vitamin K-deficient or warfarin-treated rat in the presence of tein synthesis.
actinomycin D, confirmation with the use of intact animals, Fieser (57) showed that the vitamin K quinones are active
liver perfusion, and liver slice techniques has been reported by -SH reagents and vitamin K-dependent clotting proteins are
J. P. Olson, Miller, and Troup (43), Suttie (37), and Lowenthal inactivated by sulhydryl compounds; thus it is possible that
and Simmons (44). Recently R. E. Olson et al. (45, 46) have vitamin K functions by virtue of its ability to bind -SH com-
withdrawn their earlier conclusion of transcription control by pounds.
vitamin K (15-19) and are now in agreement with the findings From the duration of the plateau level for blood prothrombin
that vitamin K exerts its control at a later level. at the highest level of actinomycin D (Fig. la), the turnover time
The proposal of Hemker et al. (47) that a preprothrombin syn- of prothrombin messenger RNA can be est.imated as approxi-
thesized in the liver is converted to prothrombin in a vitamin mately 6 hours. From the cycloheximide data (Fig. lb), one can
K-dependent step appears unlikely in view of the cycloheximide estimate the turnover time of final active prothrombin as also
data. If, as Hemker has proposed, this preprothrombin piles up about 6 hours. These data, of course, give no evidence as to
to the extent that it spills over into the blood, where it acts as an whether the decay of blood prothrombin activity is due to degra-
inhibitor of prothrombin conversion, these high levels of prepro- dation or deactivation of the protein.
thrombin would have been present in our vitamin K-deficient The data on the blocking of estrogen-induced reactions by
rats and the administration of even high levels of cycloheximide cycloheximide (58) is similar to the data on vitamin K-induced
Issue of July 25, 1968 Hill et al. 3939
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