Effects of Vitamin K Deficiency, Warfarin, and Inhibitors of Protein Synthesis upon the Plasma Levels of Vitamin K-dependent Clotting Factors in the Chick1 DANIEL A. WALZ,2 ROGER K. KIFFER3 AND ROBERT E. OLSON Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104 ABSTRACT Two-week-old chicks adequate in vitamin K showed a relative lack of vitamin K-dependent clotting factors when compared with the rat, cow, and man. Chick prothrombin was 50%, IX 8%, and X 6% of respective levels in the rat. Factor VII was not detectable in chick plasma. When 1-day-old chicks were fed a vitamin K-deficient diet, prothrombin levels fell to 5% in 5 days, whereas factors IX and X fell to only 60% of normal. After warfarin administration to normal chicks, prothrombin levels fell to 20% in 6 hours, whereas factors IX and X fell to 60%. When cycloheximide was given to normal chicks, all vitamin K-dependent factors fell at the same relative rate with a half time of 2 hours. Cycloheximide also completely blocked the effect of physiological doses (10 /tg) of phylloquinone upon prothrombin synthesis, but only partially blocked the effect of pharmacological doses (2.5 mg) of phyllo quinone, suggesting an antagonism between cycloheximide and vitamin K at the ribosomal level. Puromycin was effective in blocking the action of vitamin K at both physiological and pharmacological doses. In the chick, unlike the rat, it appears that ( 1 ) cycloheximide is fully effective in blocking the action of physiological doses of vitamin K, and (2) the regulatory systems for factors IX and X appear to have a higher affinity for vitamin K and a lower affinity for warfarin than the regulatory system for prothrombin. J. Nutr. 105: 972-981, 1975. INDEXING KEY WORDS vitamin warfarin •coagulation factors
K
Vitamin K was discovered in nutritional studies with chicks (1), and the hemorrhagic disease caused by vitamin K deficiency in chicks has been shown to be due to the absence of prothrombin (2). Relatively little, however, has been done to study the mechanism of the regulatory effect of vitamin K upon prothrombin synthesis in this species, Olson et al. (3) showed that the response Of prothrombin
levels
in the deficient •»•V. «--~. With time,
and
to
il . that
il the
~ response
• cycloheximide
•
of bovine and rat prothrombins ), forms a stable dimer, and contains no sialic acid (4). Others have observed that chick plasma has a reduced clotting potential when compared with that of mammals (5, 6). Particularly noteworthy were the low levels of factor XII ( Hageman factor ) and the vitamin K-dependent coagulation fac-
menaquinOne-O
chick was essentially I
• phylloquinone
Received for publication December 5, 1974.
linear
>supported in part by grant AM 09992 from the
National Institutes of Health, United Health Service, Bethesda, Md. 20014.
was
States
Public
blocked hilt actino'Predoctoral of the National of Uiix-Keuuybv niiromvoin puiumycm uui not iiui bv uy dcunuHealtn USPHS. fellow Supported by grant AM Institutes 446. Present mycin U. Chick prothrombin diners from address: Department of Physiology, Wayne State „.< i- *L. .1 „_-L- •4.1,„i;i • University School of Medicine, Detroit, Mich. 48201.
other mammalian prothrombins in mat it is not dQSoroea adsorbed on on arv>ys, SrCOo nds has aa lower lower ^pe snenor.
s postdoctoral fellow of the National Heart institute,^ USPHS. SupportedAs|0^atlon. by grant 5Present PO2 HE 30228 an(J MlssourJHeart address:
cine tnrombogenic activity ( ca. 40% or that
Pontiac,in. 972
Downloaded from https://academic.oup.com/jn/article-abstract/105/8/972/4763472 by Washington University in St. Louis user on 08 June 2018
VITAMIN K-DEPENDENT COAGULATION FACTORS IN THE CHICK
tors X (Stuart-Prower factor), IX (plasma thromboplastin component), and VII (proconvertin). The purpose of this investigation was to study the levels of the vitamin K-dependent coagulation proenzymes in chick plasma, as compared with those of the rat, cow, and man, and to describe the effects of vitamin K deficiency, cycloheximide, and warfarin treatment upon the concentration of these factors in the chick. The effect of inhibitors of protein synthesis upon the re sponse of prothrombin levels in the vita min K-deficient chick to phylloquinone was also investigated.
973
formed in duplicate. In the in vivo experi ments on chickens, the one-stage assays for the various vitamin K-dependent clotting factors were applied to individual samples. The one-state assay of Hjort et al. ( 10 ) em ploying Russell's viper venom7 was used
for estimation of prothrombin. If factor X was below 5%, the imidazole buffer citrated saline was replaced with 10% horse serum in the imidazole buffer as a source of factor X. Factor X was assayed by a combination of the methods of Densen (11) and of Bachmann et al. (12). In these assays, citrated bovine plasma was first mixed with charcoal, the resultant slurry was poured into a Seitz filter fitted with a standard number 7, 30% asbestos pad, and fractions MATERIALS AND METHODS were collected. Those fractions devoid of Animals. One-day-old White Rock chicks factors VII and X but rich in prothrombin in groups of 24 were placed in raised floor were pooled and frozen and referred to as wire-bottomed brooders with contact heat.4 VII-X-deficient plasma. Substitution of Room temperature was maintained at 22°, chicken plasma for the bovine plasma did and the overhead lights were on between not alter the results. Factor VII was as 6 AM and midnight and off between mid sayed by the method of Owren and Aas night and 6 AM.Fresh food and water were (13). Thromboplastin was prepared from supplied daily. The chicks were fed a nor chicken brain as an acetone dried powder mal stock diet5 for 1 day and then a puri (14). Factor IX was measured by the assay fied vitamin K-deficient diet (8) or a con of Stapp ( 15) as modified below. An ali trol diet containing 10 mg/kg of phyllo quot of celite ( 50 mg/ml ) was added to all quinone 6 ad libitum. The chicks grew nor assays to provide a surface-adhering agent mally and weighed 200 ±10 ( SEM) g at for better clot resolution. This had no ef 14 days. At intervals during this 2-week fect on clotting time or recovery. For all of period, 1.8 ml of blood was obtained by the aforementioned assays, barium sulfatecardiac puncture and drawn into 0.2 ml of absorbed bovine plasma was used as a 3.8% sodium citrate. The blood was mixed, source of factor V. centrifuged for 20 minutes at 2,000 X g, and Reagents and drugs. The sodium warfarin the plasma was recovered and stored at preparation used was Coumadin.8 Phyllo —20° until assayed. No changes in clotting quinone (vitamin K!) was administered in factors were noted during 6 months of a water-miscible form as AquaMEPHYTON.9 Uniformly labeled [I4C]Ieucine ad storage. Clotting assays. Plasma from 50 St. Louis ministered had a specific activity of mCi/ University albino rats (originally of Wistar mmole.10 Cycloheximide, puromycin, and strain) was pooled and used as the refer actinomycin D were highly purified prod ence sample to compare with plasma from ucts.11 The fibrinogen was bovine in other species. Pooled plasma from 3 cows, * Obtained from James Manufacturing Company, 25 chicks, and 4 healthy human subjects Ft. Atkinson, Wls. was analyzed for the vitamin K-dependent 6 Startena, obtained from Ralston Purina Com St. Louis, Mo. factors. The two-stage method of Ware and pany, • A number of the chicks fed the vitamin K-deflcient diet of Griminger and Donis (7) showed no differ Seegers (9) was used for the comparative in growth rate or response to phylloquinone. study of prothrombin levels. Rabbit Sim- ences 7 Obtained from Ross Allen Reptile Institute. 8 Obtained from Endo Laboratories, Garden City, plastin was used as a source of thrombo N.Y. 8 Obtained from Merck Sharp & Dohme, Quebec, plastin for all four species. The substitu Canada. tion of chick brain thromboplastin for rabbit 10Obtained from New England Nuclear, Boston, Mass. Simplastin did not alter the results ob 11Obtained from Upjohn Company, North Haven, tained in the chick. All assays were per Conn.
Downloaded from https://academic.oup.com/jn/article-abstract/105/8/972/4763472 by Washington University in St. Louis user on 08 June 2018
974
WALZ, KIFFER AND OLSON
origin.12 All gel filtration resins13 and mycin D was administered by intraperiWhatman DEAE-cellulose obtained as toneal injection. DE-1 floe14 were of the highest quality. RESULTS All remaining reagents were common chem icals of the highest purity.15-17 1. Comparative study of vitamin K-de Use of inhibitors of protein synthesis. pendent coagulation factors. The concen Puromycin is a well-established irreversible trations of vitamin K-dependent clotting inhibitor of protein synthesis that acts at factors in rat, chick, bovine, and human the ribosome by terminating mRNA trans plasma are shown in table 1. Rat plasma lation (16, 17). In preliminary studies, it was arbitrarily assigned 100%. There was was demonstrated that 2.5 mg of puro- good agreement (within 10%) between the one- and two-stage assays for pro mycin/100 g of chick body weight injected in 0.05 M phosphate buffer pH 7.0 intra- thrombin. By the two-stage method, the peritoneally 2 hours before L-[U-14C]leu- average values for prothrombin in Iowa cine, and repeated at 2-hour intervals for Units/ml (21) were: rat, 145; cow, 250; the duration of the experiment, inhibited human, 290; and chick, 75. The range for incorporation of labeled amino acid into the four human subjects tested individually liver and plasma proteins by 90%. Cyclo- as well as in a pooled sample was 270-312 heximide is a reversible inhibitor of ribo- Iowa Units/ml. This variance was typical somal translocation ( 18, 19 ) that has been of individual rat and chick samples taken extensively used to study protein synthesis from animals well nourished with respect in mammals. A dose-response study in to vitamin K. Factor VII varied from 137% chicks showed that 0.5 mg of cyclohex- in human plasma to less than 1% in the imide/100 g of chick body weight given chick. The cow had only 10% of the VII 0.5 to 2.0 hours before injection of L-[U- activity of the rat. Factor IX was lower in "C]leucine inhibited incorporation of la the rat than in all other plasmas tested. beled amino acid into plasma and liver Man had 45% the amount of factor IX in proteins by 90%. A single dose of cyclo- the rat, the cow had 27%, and the chicken heximide was effective for 8 hours. Actino- had only 8%. Factor X levels showed the mycin D, an inhibitor of DNA-dependent same pattern as IX, man having 55%, the RNA polymerase (20), was employed to cow 68%, and the chicken only 6% of the determine the rate of decay of prothrombin rat. Clearly, the levels of all of the vitamin mRNA in normal chicks. A stock actino- K-dependent factors in the chicken were mycin D solution containing 2.5 mg/ml depressed in comparison with levels of was made by dissolving 10 mg of actino- other mammals. 2. Effects of vitamin K deficiency, cyclomycin D in 1.5 ml 95% ethanol and dilut ing the solution to 5 ml in propylene glycol. heximide, actinomycin D, and warfarin For the biological experiments, this stock treatment on coagulation factors. The ef fects upon the concentration of vitamin Ksolution was further diluted to 1.5 mg/ml with physiological saline, and the actino- dependent clotting factors of feeding 1-dayold chicks a vitamin K-deficient diet for 7 days are shown in figure 1. The initial TABLE 1 values for these factors were in the normal Comparison of the vitamin K-dependent factor levels range. Prothrombin value was 75 ±6 in rat, human, bovine, and chicken plasmas (SEM) Iowa Units/ml in 12 chicks. Pro thrombin levels fell to less than 5% in 5 Levels of vitamin K-dependent factor days with a half time of 1.5 days. Con IX' VII' Species versely, factors IX and X fell much more RatHumanBovineChicken1002101675010013710
K
Vitamin K was discovered in nutritional studies with chicks (1), and the hemorrhagic disease caused by vitamin K deficiency in chicks has been shown to be due to the absence of prothrombin (2). Relatively little, however, has been done to study the mechanism of the regulatory effect of vitamin K upon prothrombin synthesis in this species, Olson et al. (3) showed that the response Of prothrombin
levels
in the deficient •»•V. «--~. With time,
and
to
il . that
il the
~ response
• cycloheximide
•
of bovine and rat prothrombins ), forms a stable dimer, and contains no sialic acid (4). Others have observed that chick plasma has a reduced clotting potential when compared with that of mammals (5, 6). Particularly noteworthy were the low levels of factor XII ( Hageman factor ) and the vitamin K-dependent coagulation fac-
menaquinOne-O
chick was essentially I
• phylloquinone
Received for publication December 5, 1974.
linear
>supported in part by grant AM 09992 from the
National Institutes of Health, United Health Service, Bethesda, Md. 20014.
was
States
Public
blocked hilt actino'Predoctoral of the National of Uiix-Keuuybv niiromvoin puiumycm uui not iiui bv uy dcunuHealtn USPHS. fellow Supported by grant AM Institutes 446. Present mycin U. Chick prothrombin diners from address: Department of Physiology, Wayne State „.< i- *L. .1 „_-L- •4.1,„i;i • University School of Medicine, Detroit, Mich. 48201.
other mammalian prothrombins in mat it is not dQSoroea adsorbed on on arv>ys, SrCOo nds has aa lower lower ^pe snenor.
s postdoctoral fellow of the National Heart institute,^ USPHS. SupportedAs|0^atlon. by grant 5Present PO2 HE 30228 an(J MlssourJHeart address:
cine tnrombogenic activity ( ca. 40% or that
Pontiac,in. 972
Downloaded from https://academic.oup.com/jn/article-abstract/105/8/972/4763472 by Washington University in St. Louis user on 08 June 2018
VITAMIN K-DEPENDENT COAGULATION FACTORS IN THE CHICK
tors X (Stuart-Prower factor), IX (plasma thromboplastin component), and VII (proconvertin). The purpose of this investigation was to study the levels of the vitamin K-dependent coagulation proenzymes in chick plasma, as compared with those of the rat, cow, and man, and to describe the effects of vitamin K deficiency, cycloheximide, and warfarin treatment upon the concentration of these factors in the chick. The effect of inhibitors of protein synthesis upon the re sponse of prothrombin levels in the vita min K-deficient chick to phylloquinone was also investigated.
973
formed in duplicate. In the in vivo experi ments on chickens, the one-stage assays for the various vitamin K-dependent clotting factors were applied to individual samples. The one-state assay of Hjort et al. ( 10 ) em ploying Russell's viper venom7 was used
for estimation of prothrombin. If factor X was below 5%, the imidazole buffer citrated saline was replaced with 10% horse serum in the imidazole buffer as a source of factor X. Factor X was assayed by a combination of the methods of Densen (11) and of Bachmann et al. (12). In these assays, citrated bovine plasma was first mixed with charcoal, the resultant slurry was poured into a Seitz filter fitted with a standard number 7, 30% asbestos pad, and fractions MATERIALS AND METHODS were collected. Those fractions devoid of Animals. One-day-old White Rock chicks factors VII and X but rich in prothrombin in groups of 24 were placed in raised floor were pooled and frozen and referred to as wire-bottomed brooders with contact heat.4 VII-X-deficient plasma. Substitution of Room temperature was maintained at 22°, chicken plasma for the bovine plasma did and the overhead lights were on between not alter the results. Factor VII was as 6 AM and midnight and off between mid sayed by the method of Owren and Aas night and 6 AM.Fresh food and water were (13). Thromboplastin was prepared from supplied daily. The chicks were fed a nor chicken brain as an acetone dried powder mal stock diet5 for 1 day and then a puri (14). Factor IX was measured by the assay fied vitamin K-deficient diet (8) or a con of Stapp ( 15) as modified below. An ali trol diet containing 10 mg/kg of phyllo quot of celite ( 50 mg/ml ) was added to all quinone 6 ad libitum. The chicks grew nor assays to provide a surface-adhering agent mally and weighed 200 ±10 ( SEM) g at for better clot resolution. This had no ef 14 days. At intervals during this 2-week fect on clotting time or recovery. For all of period, 1.8 ml of blood was obtained by the aforementioned assays, barium sulfatecardiac puncture and drawn into 0.2 ml of absorbed bovine plasma was used as a 3.8% sodium citrate. The blood was mixed, source of factor V. centrifuged for 20 minutes at 2,000 X g, and Reagents and drugs. The sodium warfarin the plasma was recovered and stored at preparation used was Coumadin.8 Phyllo —20° until assayed. No changes in clotting quinone (vitamin K!) was administered in factors were noted during 6 months of a water-miscible form as AquaMEPHYTON.9 Uniformly labeled [I4C]Ieucine ad storage. Clotting assays. Plasma from 50 St. Louis ministered had a specific activity of mCi/ University albino rats (originally of Wistar mmole.10 Cycloheximide, puromycin, and strain) was pooled and used as the refer actinomycin D were highly purified prod ence sample to compare with plasma from ucts.11 The fibrinogen was bovine in other species. Pooled plasma from 3 cows, * Obtained from James Manufacturing Company, 25 chicks, and 4 healthy human subjects Ft. Atkinson, Wls. was analyzed for the vitamin K-dependent 6 Startena, obtained from Ralston Purina Com St. Louis, Mo. factors. The two-stage method of Ware and pany, • A number of the chicks fed the vitamin K-deflcient diet of Griminger and Donis (7) showed no differ Seegers (9) was used for the comparative in growth rate or response to phylloquinone. study of prothrombin levels. Rabbit Sim- ences 7 Obtained from Ross Allen Reptile Institute. 8 Obtained from Endo Laboratories, Garden City, plastin was used as a source of thrombo N.Y. 8 Obtained from Merck Sharp & Dohme, Quebec, plastin for all four species. The substitu Canada. tion of chick brain thromboplastin for rabbit 10Obtained from New England Nuclear, Boston, Mass. Simplastin did not alter the results ob 11Obtained from Upjohn Company, North Haven, tained in the chick. All assays were per Conn.
Downloaded from https://academic.oup.com/jn/article-abstract/105/8/972/4763472 by Washington University in St. Louis user on 08 June 2018
974
WALZ, KIFFER AND OLSON
origin.12 All gel filtration resins13 and mycin D was administered by intraperiWhatman DEAE-cellulose obtained as toneal injection. DE-1 floe14 were of the highest quality. RESULTS All remaining reagents were common chem icals of the highest purity.15-17 1. Comparative study of vitamin K-de Use of inhibitors of protein synthesis. pendent coagulation factors. The concen Puromycin is a well-established irreversible trations of vitamin K-dependent clotting inhibitor of protein synthesis that acts at factors in rat, chick, bovine, and human the ribosome by terminating mRNA trans plasma are shown in table 1. Rat plasma lation (16, 17). In preliminary studies, it was arbitrarily assigned 100%. There was was demonstrated that 2.5 mg of puro- good agreement (within 10%) between the one- and two-stage assays for pro mycin/100 g of chick body weight injected in 0.05 M phosphate buffer pH 7.0 intra- thrombin. By the two-stage method, the peritoneally 2 hours before L-[U-14C]leu- average values for prothrombin in Iowa cine, and repeated at 2-hour intervals for Units/ml (21) were: rat, 145; cow, 250; the duration of the experiment, inhibited human, 290; and chick, 75. The range for incorporation of labeled amino acid into the four human subjects tested individually liver and plasma proteins by 90%. Cyclo- as well as in a pooled sample was 270-312 heximide is a reversible inhibitor of ribo- Iowa Units/ml. This variance was typical somal translocation ( 18, 19 ) that has been of individual rat and chick samples taken extensively used to study protein synthesis from animals well nourished with respect in mammals. A dose-response study in to vitamin K. Factor VII varied from 137% chicks showed that 0.5 mg of cyclohex- in human plasma to less than 1% in the imide/100 g of chick body weight given chick. The cow had only 10% of the VII 0.5 to 2.0 hours before injection of L-[U- activity of the rat. Factor IX was lower in "C]leucine inhibited incorporation of la the rat than in all other plasmas tested. beled amino acid into plasma and liver Man had 45% the amount of factor IX in proteins by 90%. A single dose of cyclo- the rat, the cow had 27%, and the chicken heximide was effective for 8 hours. Actino- had only 8%. Factor X levels showed the mycin D, an inhibitor of DNA-dependent same pattern as IX, man having 55%, the RNA polymerase (20), was employed to cow 68%, and the chicken only 6% of the determine the rate of decay of prothrombin rat. Clearly, the levels of all of the vitamin mRNA in normal chicks. A stock actino- K-dependent factors in the chicken were mycin D solution containing 2.5 mg/ml depressed in comparison with levels of was made by dissolving 10 mg of actino- other mammals. 2. Effects of vitamin K deficiency, cyclomycin D in 1.5 ml 95% ethanol and dilut ing the solution to 5 ml in propylene glycol. heximide, actinomycin D, and warfarin For the biological experiments, this stock treatment on coagulation factors. The ef fects upon the concentration of vitamin Ksolution was further diluted to 1.5 mg/ml with physiological saline, and the actino- dependent clotting factors of feeding 1-dayold chicks a vitamin K-deficient diet for 7 days are shown in figure 1. The initial TABLE 1 values for these factors were in the normal Comparison of the vitamin K-dependent factor levels range. Prothrombin value was 75 ±6 in rat, human, bovine, and chicken plasmas (SEM) Iowa Units/ml in 12 chicks. Pro thrombin levels fell to less than 5% in 5 Levels of vitamin K-dependent factor days with a half time of 1.5 days. Con IX' VII' Species versely, factors IX and X fell much more RatHumanBovineChicken1002101675010013710
