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The mode of action of warfarin. An intracellular precursor for factor VII in rat liver
BIOCHIMICA ET BIOPHYSICAACTA
495
BBA R e p o r t BBA 21312 T h e m o d e o f a c t i o n o f warfarin. A n intracellular p r e c u r s o r f o r F a c t o r VII in rat liver
GABOR REZ~and HANS PRYDZ Department of Microbiology, Dental Faculty, University of Oslo, Blindern, Oslo 3 (Norway}
(Received July 21st, 1971)
SUMMARY Direct evidence for an intracellular precursor of the coagulation Factor VII in liver from warfarin-treated rats was obtained by giving cycloheximide and subsequently vitamin K1. Even in complete absence of cytoplasmic protein synthesis, vitamin KI in vivo caused an increase of Factor VII present in the isolated submicrosomal fraction to about 60% of the controls.
There is now ample evidence for the presence of circulating precursors for the vitamin K-dependent coagulation Factors II (refs. 1-3), IX (ref. 4) and X (refs. 5, 6) in the plasma of patients under anticoagulant treatment. These precursors have been demonstrated by immunological methods. They all crossreact with antisera against the respective complete factors. Earlier attempts to demonstrate a similar circulating precursor for Factor VII by means of an anti-Factor VII antiserum were, however, unsuccessful 7 . Since Factor VII among the vitamin K-dependent factors is the predominant activity synthesized or detected in various in vitro systems (isolated liver cell suspensions s , hepatoma cell cultures 9, liver slices 1° and submicrosomal fractions from rat liver 1~,12), possible differences between Factor VII and the three other factors would be of interest. This paper reports experiments carried out with a subcellular fraction from rat liver 11'~2 demonstrating the presence of a warfarin-induced precursor also for Factor VII. Factor VII deficiency plasma was kindly provided by Dr. O. Egeberg, Institute for Thrombosis Research, Oslo. Cycloheximide (Sigma) and warfarin (Marevan, Evans) were dissolved in distilled water. These solutions and vitamin K1 (Konakion, Roche) were given by intraperitoneal injection. *Permanent address: Department of General Zoology, E.L.T.E~University,Budapest, Hungary. Biochint Biophys. Acta, 244 (1971) 495-499
496
BBA REPORT
A microsomal fraction was prepared from perfused livers of fasting male rats (CD*F inbred rats, Charles River, Wilmington, Mass.), and incubated, sonicated and ultracentrifuged in a solution of 0.25 M sucrose, 1 mM disodium EDTA and 35 mM Tris-HC1 (pH 7.8) 11,12. In normal rats this fraction contains 1 - 2 units of Factor VII activity per mg protein. Factor VII activity and protein were estimated as described ~3. The level of Factor VII in normal rat plasma was 100 units/ml. In rats killed 12 to 36 h after a single injection of warfarin (0.1 mg/100 g rat weight) the Factor VII-carrying microsomal subfraction H,~2 contained no measurable Factor VII activity, even after incubation for 60 min at 37 ° (refs. 11, 12). However, 4 8 - 7 2 h after the administration of warfarin the activity had reached 40-50% of the normal level. A single injection of vitamin K1 24 h after warfarin resulted, as expected, in an increase in Factor VII in the microsomal fraction. This increase was essentially linearly dependent upon the dose of vitamin K~ up to about 0,1 mg/lO0 g rat weight (Table I) and was detectable in rats killed 30 min after vitamin K~ administration (Fig. 1). The same level of Factor VII as in untreated controls was reached after 9 0 - 1 5 0 min, followed by an overshoot of variable size and duration. The absence of any increase in Factor VII activity when vitamin K 1 is not given excludes the possibility of an unspecific activation. TABLE I FACTOR;VII ACTIVITY IN MICROSOMALFRACTION 90 rain AFTER VITAMIN K1 ADMINISTRATION Dose o f vitamin K l [lag/1O0 g)
Factor VII (% o f COntrois)
1
0
10 50 100 200 250 500 1000
13 75 100 100 150 110 113
In plasma from similarly treated rats the increase in Factor VII appeared not until 6 0 - 9 0 min after vitamin K~ injection confirming that this intracellular pool of Factor VII kinetically is a precursor for plasma Factor VII (ref. 14). Warfarin and vitamin K1 probably exert a post-translational control on the biosynthesis of Factor VII (ref. 14). One would expect then that warfarin-treated rats synthesized a precursor for Factor VII analogous to that for Factor X in man s , monkey s and cow 6. To demonstrate such a precursor, cycloheximide (1 mg/100 g), reducing protein synthesis by 95% within 5 min ~4, was given to rats at various times between 22.5 and 24 h after warfarin treatment. The precursor present at the onset of the cycloheximide block might for a certain time period be available to "finalization" by the vitamin K-dependent process. Vitamin K1 was therefore given to the warfarin and cycloheximide-treated rats 24 h after warfarin and 0 - 9 0 min after cycloheximide. The rats were killed 90 min later (25.5 h after warfarin). The increase in Factor VII activity in the submicrosomal fraction after vitamin Biochim. 8iophys. Acta, 244 (1971) 495-499
BBA REPORT
497
250
20C
5
~ 150. c
~, ioo.
50
o
f
~ Time(h)
Fig.1. Activity of Factor VII in the incubated (60 min, 37~) mlcrosomal subfraction l:J2 from warfarinized rats (0.1 mg/100 g rat) killed at various intervals after vitamin K1 injection (0.25 mg/100 g). Warfarin given 24 h before vitamin K1. The activity is given as % of the activity in the same subfraction prepared from normal, untreated rats. K~ administration to these rats with complete block of cytoplasmic protein synthesis never reached the level seen in control rats receiving warfarin and vitamin K~ only. But even when given 3 0 - 4 0 rain after cycloheximide vitamin K1 caused an increase to about 60% of the control activity. When cycloheximide was given 6 0 - 9 0 min before vitamin K1, no increase in Factor VII activity was found (Fig.2). These data indicated that, when treated with warfarin, the rats synthesized a precursor for Factor VII which in a vitamin K/warfarin (K-w)-sensitive step might undergo the necessary changes to become an active procoagulant independent of simultaneous polypeptide synthesis. Probably due to flow o f this precursor through an intracellular compartment it is only available for modification by the K - w process for about 30 min. After 6 0 - 9 0 min all precursor molecules have passed this stage. This interval of 30 min, in which the precursor is available, coincides with the time lag between warfarin administration and its effect upon the intracellular subBiochim. Biophys. Acta, 244 (1971) 495-499
498
BBA REPORT
E "6 40
= >
20'
30
Interval be[ween ¢ycloheximide and vitamin K 1 (rn*n)
Fig.2. Effect of vitamin KI (0.25 mg/100 g) upon intracellular Factor VII activity in rats treated with warfarin (0.1 rag/100 g) and cycloheximide (1 rag/100 g). The activity is given as % of the activity in the same subfraction prepared from rats treated with warfarin and vitamin K1 only. microsomal Factor VII pool 14, which is beyond the K-w-sensitive step 14. It is tempting to conclude that warfarin and vitamin K act immediately after the polypeptide synthesis is finished ~4 and that the precursor remains available to vitamin K-dependent modification in the time interval before it reaches the pool in the smooth microsomal fraction. Studies by Hill et al. ~s of the prothrombin content of the total microsomal fraction were interpreted to mean that vitamin K functioned at a late stage in translation of the prothrombin messenger RNA, possibly in establishing disulphide bridges. They were unable, however, to demonstrate any vitamin K-activatable precursor after puromycin administration. This may be due to the 1-h interval between puromycin and vitamin K administration. As demonstrated here for Factor VII, the prothrombin precursor might not be available for modification by the vitamin K-dependent process 1 h after blocking protein synthesis. The results of Babior and Kipnes ~6 may to a large extent be explained by the existence of an intracellular precursor. The requirement for calcium might be due to a possible effect on the release of Factor VII when sonication is not used. It is noteworthy that the Factor VII activity observed by Babior and Kipnes ~6 is 5-10-fold smaller than that seen in our experiments n,~2,~4. The nature of these precursors and the K - w process is unknown. Kinetic ~4, immunologic s,6 and peptide map studies ~7 indicate that the differences from the complete factors are due to structural and/or conformational changes occurring after the main part of the polypeptide chain has been synthesized. The failure of earlier attempts to demonstrate a Factor VII precursor in plasma from normal persons on warfarin treatment might be due to the specificity of the antiserum being directed against antigenic determinants present only on the complete Factor VII molecule. The structural differences between the precursor and the complete molecule may be more pronounced for Factor VII than for the other factors. Alternatively, the heavy doses of warfarin used to suppress the Factor VII synthesis may have suppressed the synthesis of the precursor as well. Biochim. Biophys. Acta, 244 (1971) 495-499
BBA REPORT
499
The apparent overshoot of Factor VII activity in the microsomal fraction after vitamin K~ administration might be due to a suboptimal amount of vitamin K in the control rats, so that some precursor normally passes the K - w process without being modified. Alternatively, vitamin K~ may lead to a temporary inhibition of the flow rate of Factor VII and its precursor, thus increasing the pool size. Finally, vitamin K~ may have a stimulatory effect on transcription and/or translation of mRNA for Factor VII in addition to its post-translational effect. G.R. is a Norwegian Government Scholarship holder. REFERENCES I F. Josso, J.M. Lavergne, M. Gonault, O. Prou-Wartelle and J.P. Soulier, Thromb. Diath. Haemorrhag., 20 (1968) 88. 2 J.E. Nil6hn and P.O.Ganrot, Scand. J. Clin. Lab. lnvest., 22 (1968) 17. 3 P.O. Ganrot and J.E. Nil6hn, Scand. J. Clin. Lab. Invest., 22 (1968) 23. 4 M.J. Larrieu and D. Meyer, Lancet, 2 (1970) 1085. 5 H. Prydz and ~ . Gladhaug, Thromb. Diath. Haemorrhag., 25 (1971) 157. 6 P. Reekers, ~ Berre, H.C. Hemker and H. Prydz, in preparation. 7 H. Prydz, Scand. J. Clin. Lab. Invest., 17 (1965) 66. 8 H. Prydz, Scand. J. Clin. Lab. lnvest., 16 (1964) 540. 9 H.E. Rugstad, H. Prydz and B. Johansson, in preparation. 10 J. Pool and J. Robinson, Am. J. Physiol., 196 (1959) 423. 11 A. Gaarder and H. Prydz, Biochim. Biophys. Acta, 140 (1967) 545. 12 A. Gaarder and H. Prydz, Biochim. Biophys. Acta, 184 (1969) 220. 13 ~ . Gladhaug and H. Prydz, Biochim. Biophys. Acta, 215 (1970) 105. 14 H. Prydz and G. Gaudernack, Biochim. Biophys. Acta, 230 (1971) 373. 15 R.B. Hill, S. Gaetani, A.M. Paolucci, P.B. RamaRao, R. Alden, G.S. Ranhotra, D.V. Shah, V.K. Shah and B. Connor Johnson, J. Biol. Chem., 243 (1968) 3930. 16 B.M. Babior and R.S. Kipnes, Biochemistry, 9 (1970) 2564. 17 J. Stenflo, Acta Chem. Scand., 24 (1970) 3762. Biochim. Biophys. Acta, 244 (1971) 495-499
495
BBA R e p o r t BBA 21312 T h e m o d e o f a c t i o n o f warfarin. A n intracellular p r e c u r s o r f o r F a c t o r VII in rat liver
GABOR REZ~and HANS PRYDZ Department of Microbiology, Dental Faculty, University of Oslo, Blindern, Oslo 3 (Norway}
(Received July 21st, 1971)
SUMMARY Direct evidence for an intracellular precursor of the coagulation Factor VII in liver from warfarin-treated rats was obtained by giving cycloheximide and subsequently vitamin K1. Even in complete absence of cytoplasmic protein synthesis, vitamin KI in vivo caused an increase of Factor VII present in the isolated submicrosomal fraction to about 60% of the controls.
There is now ample evidence for the presence of circulating precursors for the vitamin K-dependent coagulation Factors II (refs. 1-3), IX (ref. 4) and X (refs. 5, 6) in the plasma of patients under anticoagulant treatment. These precursors have been demonstrated by immunological methods. They all crossreact with antisera against the respective complete factors. Earlier attempts to demonstrate a similar circulating precursor for Factor VII by means of an anti-Factor VII antiserum were, however, unsuccessful 7 . Since Factor VII among the vitamin K-dependent factors is the predominant activity synthesized or detected in various in vitro systems (isolated liver cell suspensions s , hepatoma cell cultures 9, liver slices 1° and submicrosomal fractions from rat liver 1~,12), possible differences between Factor VII and the three other factors would be of interest. This paper reports experiments carried out with a subcellular fraction from rat liver 11'~2 demonstrating the presence of a warfarin-induced precursor also for Factor VII. Factor VII deficiency plasma was kindly provided by Dr. O. Egeberg, Institute for Thrombosis Research, Oslo. Cycloheximide (Sigma) and warfarin (Marevan, Evans) were dissolved in distilled water. These solutions and vitamin K1 (Konakion, Roche) were given by intraperitoneal injection. *Permanent address: Department of General Zoology, E.L.T.E~University,Budapest, Hungary. Biochint Biophys. Acta, 244 (1971) 495-499
496
BBA REPORT
A microsomal fraction was prepared from perfused livers of fasting male rats (CD*F inbred rats, Charles River, Wilmington, Mass.), and incubated, sonicated and ultracentrifuged in a solution of 0.25 M sucrose, 1 mM disodium EDTA and 35 mM Tris-HC1 (pH 7.8) 11,12. In normal rats this fraction contains 1 - 2 units of Factor VII activity per mg protein. Factor VII activity and protein were estimated as described ~3. The level of Factor VII in normal rat plasma was 100 units/ml. In rats killed 12 to 36 h after a single injection of warfarin (0.1 mg/100 g rat weight) the Factor VII-carrying microsomal subfraction H,~2 contained no measurable Factor VII activity, even after incubation for 60 min at 37 ° (refs. 11, 12). However, 4 8 - 7 2 h after the administration of warfarin the activity had reached 40-50% of the normal level. A single injection of vitamin K1 24 h after warfarin resulted, as expected, in an increase in Factor VII in the microsomal fraction. This increase was essentially linearly dependent upon the dose of vitamin K~ up to about 0,1 mg/lO0 g rat weight (Table I) and was detectable in rats killed 30 min after vitamin K~ administration (Fig. 1). The same level of Factor VII as in untreated controls was reached after 9 0 - 1 5 0 min, followed by an overshoot of variable size and duration. The absence of any increase in Factor VII activity when vitamin K 1 is not given excludes the possibility of an unspecific activation. TABLE I FACTOR;VII ACTIVITY IN MICROSOMALFRACTION 90 rain AFTER VITAMIN K1 ADMINISTRATION Dose o f vitamin K l [lag/1O0 g)
Factor VII (% o f COntrois)
1
0
10 50 100 200 250 500 1000
13 75 100 100 150 110 113
In plasma from similarly treated rats the increase in Factor VII appeared not until 6 0 - 9 0 min after vitamin K~ injection confirming that this intracellular pool of Factor VII kinetically is a precursor for plasma Factor VII (ref. 14). Warfarin and vitamin K1 probably exert a post-translational control on the biosynthesis of Factor VII (ref. 14). One would expect then that warfarin-treated rats synthesized a precursor for Factor VII analogous to that for Factor X in man s , monkey s and cow 6. To demonstrate such a precursor, cycloheximide (1 mg/100 g), reducing protein synthesis by 95% within 5 min ~4, was given to rats at various times between 22.5 and 24 h after warfarin treatment. The precursor present at the onset of the cycloheximide block might for a certain time period be available to "finalization" by the vitamin K-dependent process. Vitamin K1 was therefore given to the warfarin and cycloheximide-treated rats 24 h after warfarin and 0 - 9 0 min after cycloheximide. The rats were killed 90 min later (25.5 h after warfarin). The increase in Factor VII activity in the submicrosomal fraction after vitamin Biochim. 8iophys. Acta, 244 (1971) 495-499
BBA REPORT
497
250
20C
5
~ 150. c
~, ioo.
50
o
f
~ Time(h)
Fig.1. Activity of Factor VII in the incubated (60 min, 37~) mlcrosomal subfraction l:J2 from warfarinized rats (0.1 mg/100 g rat) killed at various intervals after vitamin K1 injection (0.25 mg/100 g). Warfarin given 24 h before vitamin K1. The activity is given as % of the activity in the same subfraction prepared from normal, untreated rats. K~ administration to these rats with complete block of cytoplasmic protein synthesis never reached the level seen in control rats receiving warfarin and vitamin K~ only. But even when given 3 0 - 4 0 rain after cycloheximide vitamin K1 caused an increase to about 60% of the control activity. When cycloheximide was given 6 0 - 9 0 min before vitamin K1, no increase in Factor VII activity was found (Fig.2). These data indicated that, when treated with warfarin, the rats synthesized a precursor for Factor VII which in a vitamin K/warfarin (K-w)-sensitive step might undergo the necessary changes to become an active procoagulant independent of simultaneous polypeptide synthesis. Probably due to flow o f this precursor through an intracellular compartment it is only available for modification by the K - w process for about 30 min. After 6 0 - 9 0 min all precursor molecules have passed this stage. This interval of 30 min, in which the precursor is available, coincides with the time lag between warfarin administration and its effect upon the intracellular subBiochim. Biophys. Acta, 244 (1971) 495-499
498
BBA REPORT
E "6 40
= >
20'
30
Interval be[ween ¢ycloheximide and vitamin K 1 (rn*n)
Fig.2. Effect of vitamin KI (0.25 mg/100 g) upon intracellular Factor VII activity in rats treated with warfarin (0.1 rag/100 g) and cycloheximide (1 rag/100 g). The activity is given as % of the activity in the same subfraction prepared from rats treated with warfarin and vitamin K1 only. microsomal Factor VII pool 14, which is beyond the K-w-sensitive step 14. It is tempting to conclude that warfarin and vitamin K act immediately after the polypeptide synthesis is finished ~4 and that the precursor remains available to vitamin K-dependent modification in the time interval before it reaches the pool in the smooth microsomal fraction. Studies by Hill et al. ~s of the prothrombin content of the total microsomal fraction were interpreted to mean that vitamin K functioned at a late stage in translation of the prothrombin messenger RNA, possibly in establishing disulphide bridges. They were unable, however, to demonstrate any vitamin K-activatable precursor after puromycin administration. This may be due to the 1-h interval between puromycin and vitamin K administration. As demonstrated here for Factor VII, the prothrombin precursor might not be available for modification by the vitamin K-dependent process 1 h after blocking protein synthesis. The results of Babior and Kipnes ~6 may to a large extent be explained by the existence of an intracellular precursor. The requirement for calcium might be due to a possible effect on the release of Factor VII when sonication is not used. It is noteworthy that the Factor VII activity observed by Babior and Kipnes ~6 is 5-10-fold smaller than that seen in our experiments n,~2,~4. The nature of these precursors and the K - w process is unknown. Kinetic ~4, immunologic s,6 and peptide map studies ~7 indicate that the differences from the complete factors are due to structural and/or conformational changes occurring after the main part of the polypeptide chain has been synthesized. The failure of earlier attempts to demonstrate a Factor VII precursor in plasma from normal persons on warfarin treatment might be due to the specificity of the antiserum being directed against antigenic determinants present only on the complete Factor VII molecule. The structural differences between the precursor and the complete molecule may be more pronounced for Factor VII than for the other factors. Alternatively, the heavy doses of warfarin used to suppress the Factor VII synthesis may have suppressed the synthesis of the precursor as well. Biochim. Biophys. Acta, 244 (1971) 495-499
BBA REPORT
499
The apparent overshoot of Factor VII activity in the microsomal fraction after vitamin K~ administration might be due to a suboptimal amount of vitamin K in the control rats, so that some precursor normally passes the K - w process without being modified. Alternatively, vitamin K~ may lead to a temporary inhibition of the flow rate of Factor VII and its precursor, thus increasing the pool size. Finally, vitamin K~ may have a stimulatory effect on transcription and/or translation of mRNA for Factor VII in addition to its post-translational effect. G.R. is a Norwegian Government Scholarship holder. REFERENCES I F. Josso, J.M. Lavergne, M. Gonault, O. Prou-Wartelle and J.P. Soulier, Thromb. Diath. Haemorrhag., 20 (1968) 88. 2 J.E. Nil6hn and P.O.Ganrot, Scand. J. Clin. Lab. lnvest., 22 (1968) 17. 3 P.O. Ganrot and J.E. Nil6hn, Scand. J. Clin. Lab. Invest., 22 (1968) 23. 4 M.J. Larrieu and D. Meyer, Lancet, 2 (1970) 1085. 5 H. Prydz and ~ . Gladhaug, Thromb. Diath. Haemorrhag., 25 (1971) 157. 6 P. Reekers, ~ Berre, H.C. Hemker and H. Prydz, in preparation. 7 H. Prydz, Scand. J. Clin. Lab. Invest., 17 (1965) 66. 8 H. Prydz, Scand. J. Clin. Lab. lnvest., 16 (1964) 540. 9 H.E. Rugstad, H. Prydz and B. Johansson, in preparation. 10 J. Pool and J. Robinson, Am. J. Physiol., 196 (1959) 423. 11 A. Gaarder and H. Prydz, Biochim. Biophys. Acta, 140 (1967) 545. 12 A. Gaarder and H. Prydz, Biochim. Biophys. Acta, 184 (1969) 220. 13 ~ . Gladhaug and H. Prydz, Biochim. Biophys. Acta, 215 (1970) 105. 14 H. Prydz and G. Gaudernack, Biochim. Biophys. Acta, 230 (1971) 373. 15 R.B. Hill, S. Gaetani, A.M. Paolucci, P.B. RamaRao, R. Alden, G.S. Ranhotra, D.V. Shah, V.K. Shah and B. Connor Johnson, J. Biol. Chem., 243 (1968) 3930. 16 B.M. Babior and R.S. Kipnes, Biochemistry, 9 (1970) 2564. 17 J. Stenflo, Acta Chem. Scand., 24 (1970) 3762. Biochim. Biophys. Acta, 244 (1971) 495-499