- Email: [email protected]
The role of vitamin K in clotting factor synthesis
606
BIOCHIMICA ET BIOPHYSICAACTA
BBA 95430
T H E ROLE OF VITAMIN K IN CLOTTING FACTOR SYNTHESIS I. E V I D E N C E FOR T H E P A R T I C I P A T I O N OF VITAMIN K IN T H E CONV E R S I O N OF A P O L Y P E P T I D E P R E C U R S O R TO FACTOR VII
BERNARD M. BABIOR, M.D., Ph. D. Thorndike Memorial Laboratory, I I and I V (Harvard) Medical Services, Boston City Hospital, and Department o/ Medicine, Harvard Medical School, Boston, Mass. (U.S.A .)
(Received December i4th, 1965)
SUMMARY
Vitamin K-dependent Factor VII synthesis in rat-liver slices was not affected by puromycin although protein synthesis was inhibited by 9 8 %. The results indicate that vitamin K is required for the formation of Factor VII from a polypeptide precursor.
INTRODUCTION Vitamin K depletion or the administration of a vitamin K antagonist results in a Factor VII deficiency in the intact animal 1 and in vitro 2. Factor VII activity appears in homogenates of rat-liver slices after a 2-h incubation and is prevented by the addition of dicoumarol, a vitamin K antagonist a. Factor VII production is also inhibited if the animal from which the liver is obtained had been given a vitamin K deficient diet or treated with dicoumarol in vivo. Administration of vitamin K in vivo, but not in vitro, can overcome this inhibition. The precise means by which vitamin K acts in Factor V I I production (and that of other clotting factors) is unknown. The experiments to be reported were designed to determine whether vitamin K stimulates the synthesis de novo of Factor V I I from amino acids or whether vitamin K enhances the conversion to Factor V I I of a polypeptide already present in the liver. To decide between these possibilities, the relationship between vitamin K and Factor VII synthesis was examined in the presence and absence of puromycin 4. MATERIALS AND METHODS Sodium dicoumarol (Endo), 25 mg/kg, was administered intraperitoneally to male Sprague-Dawley rats (150-25o g), a dose which produced a consistent fall of Biochim. Biophys. Acta, 123 (1966) 606 61o
VITAMIN
607
K AND CLOTTING FACTOR SYNTHESIS
serum Factor V I I activity to less than 3 % of normal within 18 h. The animals were then fasted, but allowed water ad libitum. 24 h later the animals were anesthetized with intraperitoneal sodium pentobarbital (5 mg/Ioo g). Blood for Factor V I I determination (0.45 ml) was obtained b y cardiac puncture with a syringe containing 0.05 ml of 3.8 % (w/v) sodium citrate. The liver was then perfused in the intact animal through the portal vein with 30 ml of ice-cold Krebs-Ringer bicarbonate buffer (pH 7.4), then removed and slices prepared with a Stadie-Riggs microtome. Vitamin K 1, 2 mg (Aquamephyton, Merck) was administered b y intracardiac puncture IO min or less before perfusion was begun. Two liver slices were placed in 25-ml erlenmeyer flasks, 2 ml of incubation mixture* added, the flasks gassed for 15-3o sec with O2-CO2 (95:5), stoppered and incubated for 2 h at 37 ° in a Dubnoff shaker. The incubation was terminated by homogenizing the contents of the flask at o ° in a glass P o t t e r - E l v e h j e m homogenizer with a Teflon pestle. After dilution of the homogenate with an equal volume of imidazole buffer p H 7.3 (see ref. 5), Factor V I I was determined b y the method of Pechet using human Factor V I I deficient plasma e. The same method was used for the Factor V I I in rat plasma after I : I O dilution of the plasma with 0. 9 o/ /o saline. A standard curve for Factor V I I activity was obtained with normal rat plasma; b y definition, I ml of plasma contained IOO U of Factor VII. Factor V I I appearance in the homogenate was calculated as the difference between the Factor V I I activity at the end of the incubation and the Factor V I I activity of unincubated liver slices. This assay was not affected b y dicoumarol or puromycin**. Protein concentration was determined b y the biuret method 7. The incorporation of ['4C~leucine into protein as a measure of protein synthesis was assayed according to STEINBERG et al. s, preparing the protein for counting b y the method of MANCHESTER AND YOUNG9.
RESULTS
AND DISCUSSION
As shown in Table I, no production of Factor V I I occurred in the absence of vitamin K, whether or not puromycin was added to the incubation mixture. When vitamin K was given before sacrifice, however, Factor V I I was produced at nearly the control rate in spite of depression of protein synthesis to 2 % of control b y puromycin addition in vitro. This finding contrasts with that of other authors1°, n, who have emphasized the inhibition of hepatic Factor V I I synthesis b y puromycin. The results of these experiments suggest that a polypeptide precursor of Factor V I I accumulates in the liver under the experimental conditions, and is converted to Factor V I I in a vitamin K-dependent reaction: Amino acids >
Polypeptide precursor
Vitamin K >
Factor V I I (I}
A similar hypothesis was previously proposed b y HEMKER et al. TM. They found in the plasma of patients with vitamin K deficiency a protein which was a competitive inhibitor of prothrombin, one of the vitamin K-dependent clotting factors. T h e y * F o r c o m p o s i t i o n s e e T a b l e I. ** U n p u b l i s h e d d a t a .
Biochim. Biophys. Acta, 123 ( 1 9 6 6 ) 6 o 6 - 6 1 o
608 TABLE
e . M; BABIOR I
EFFECT OF PUROMYCIN ON THE FORMATION OF FACTOR
RAT-LIVER SLICES
VII IN
Incubation mixture contains liver slices, approx. 15o mg, Krebs-Ringer bicarbonate buffer (2 m l ) a n d w h e r e i n d i c a t e d , p u r o m y c i n , I m M ( S i g m a ) a n d [14C~leucine, 3 . 9 ' 1 °6 d i s i n t e g r a t i o n s / min (New England Nuclear). Each value represents one rat. The Factor VII activity of unincubat e d s l i c e s f r o m E x p t s . 4 - 7 , a n d o f s l i c e s f r o m t h e s a m e e x p e r i m e n t s i n c u b a t e d f o r 2 h a t o °, w a s i7~-6 units/g protein.
Expt.
I 2 3 4 5 6 7
Vitamin K
---+ + + +
Factor VII production (units/g protein)
[14C]Leucine incorporation (disintegrations~rain per mg protein)
No puromycin
Puromycin
No puromycin
Puromycin
< 2 <2 <2 16o 200 31o 270
< 2 <2 <2 21o 15 ° 33 ° 15o
io 400 9900 16 9 0 0 8400 ii 200 13 6 0 0 16 9 0 0
260 200 270 230 i8o 420 320
postulated t h a t this protein was a precursor of prothrombin, and that vitamin K was necessary for its conversion to prothrombin. In interpreting these data, two alternative hypotheses should be considered. They will be presented seriatim. I. The first of these alternative hypotheses states that there is a rapid, vitamin K-dependent, protein synthesizing (i.e., puromycin sensitive) step which takes place during the brief interval between administration of vitamin K to the rat and addition of puromycin to the incubation mixture. As a result of this rapid step, Factor V I I precursor is synthesized in large amounts. During the course of the subsequent incubation, the accumulated precursor is converted to Factor V I I in a vitamin K-independent reaction. If this were the case, dicoumarol added to the incubation mixture should have no effect on the reaction. As is shown in Table n , however, as well as b y POOL AND ROBINSON2, dicoumarol, a vitamin K antagonist, is a good inhibitor of Factor V I I synthesis in vitro. Thus, vitamin K is involved in a reaction which continues to take place throughout the course of the incubation. At the concentrations employed, dicoumarol depressed the incorporation of [14C~leucine into protein 13 (see Table I I I ) , raising the question of whether the in-
TABLE
II
EFFECT OF DICOUMAROL ON THE SYNTHESIS OF FACTOR VII
in vitro
I n c u b a t i o n s w e r e c a r r i e d o u t a s d e s c r i b e d i n T a b l e I. V i t a m i n before sacrifice.
Expt.
5 6 7
I( was given to each animal io rain
Factor VII production (units/g protein) No dicoumarol
I m M dicoumarol
5 m M dicoumarol
200 31o 270
69 5° 52
3 <2 9
Biochim. Biophys. Aeta, 123 ( 1 9 6 6 ) 6 o 6 - 6 1 o
VITAMIN
TABLE
K AND
609
CLOTTING FACTOR SYNTHESIS
III
EFFECT OF P U R O M Y C I N O N F A C T O R VII P R O D U C T I O N IN T H E P R E S E N C E OF D I C O U M A R O L Incubations were carried out as described in Table I. Vitamin I~ w a s given to each animal. T h e concentrations of both p u r o m y c i n a n d dicoumarol were I lumole/ml. [14C]Leucine, 3.9" IO~ disintegrations/rain, w a s added to the incubation mixtures.
Expt.
5 6 7
Dicoumarol
-+ -+ -+
Factor VII production (Units/g protein)
?aC]Leucine incorporation (disintegrations~rain per mg protein)
No puromycin
Puromycin
No puromycin
Puromycin
2oo 69 31o 5° 270 52
15o 49 33 ° 35 15o 29
i i 2oo 46oo 13 6 0 0 41oo 16 9 0 0 4500
18o 12o 420 12o 320 200
hibition of Factor V I I synthesis b y dicoumarol was due to its effect on protein synthesis. This does not seem to be the case, however, because puromycin had no effect on Factor V I I synthesis even when protein synthesis was almost completely inhibited. 2. The second hypothetical mechanism is shown in (II), in which the rate of synthesis of a Factor V I I precursor in a vitamin K-dependent reaction is very rapid compared with the rate of the conversion of the precursor to Factor V I I in a vitamin K-independent reaction.
Amino acids
Vitamin K Protein synthesis Precursor > Factor V I I > Slow Rapid
(II)
Under these circumstances, even with protein synthesis slowed as it was to 2 ~o of its original rate (Table I), it might be argued that the precursor could still be formed rapidly enough for no change in the rate of appearance of Factor V I I to be observed. Since here vitamin K would be participating in a reaction which continues throughout the incubation, Factor V I I synthesis could, in principle, be inhibited by adequate concentrations of dicoumarol (vide supra). Such an inhibition was actually observed (see Table II). According to this mechanism, the inhibition would result from an effect of dicoumarol on the first of the two reactions sufficient to curtail the production of precursor so that it would no longer accumulate but would at once be converted to Factor VII. Under these conditions, Factor V I I synthesis would reflect the rate of synthesis of the precursor, which by postulate is the immediate product of a protein-forming reaction. Therefore, the rate of appearance of Factor V I I would be proportional to the rate of protein synthesis. That this is not the case, however, is shown b y the results in Table I I I . When Factor V I I formation is partly inhibited b y dicoumarol, the presence of puromycin results only in a slight further inhibition of the synthesis of the clotting factor, despite the fact that protein synthesis is almost completely inhibited. Biochim. Biophys. Acta,
123
(I966)
6o6-61o
6Io
B.M. BABIOR
Failure of puromycin to inhibit the vitamin K-dependent synthesis of Factor VII implies that the vitamin probably does not act as an inducer of Factor VII synthesis at the level of messenger R N A 14. The small extent to which puromycin inhibits the reaction (approx. IO %) m a y represent the extent to which Factor VII arises from protein synthesis de novo.
ACKNOWLEDGEMENTS
I am indebted to Dr. H. S. STRAUSS for a generous gift of human Factor VIIdeficient plasma. I would like to thank Dr. L. PECHET for his assistance in setting up the Factor VII assay and Dr. C. S. DAVlDSON for his valuable encouragement and advice. This work was supported in part by Grants No. 5 TI-AM-5413 and AM-o9II5 from the National Institutes of Health, Bethesda, Md. (U.S.A.).
REFERENCES i M. M. WINTROBE, Clinical Hematology, 5th ed., Lea and Febiger, 1961, p. 882. 2 J. G. POOL AND J. ROBINSON, Am. J. Physiol., 196 (1959) 423 . 3 L. S. GOODMAN AND A. GILMAN, The Pharmacological Basis o[ Therapeutics, 2nd ed., Macmillan, N e w York, 1955, p. 15o9. 4 A. MORRIS, R. ARLINGHAUS, S. FAVELUKES AND R. SCHVCEET, Biochemistry, 2 (I963) lO84. 5 E. T. MERTZ AND C. A. OWEN, Proc. Soc. Exptl. Biol. Med., 43 (194 o) 204. 6 L. PECHET, ill L. M. TOCANTINS AND L. A. KAZAL, Blood Coagulation, Hemorrhage and Thrombosis, G r u n e alld S t r a t t o n , N e w York, 1964, p. 213. 7 E. LAYNE, ill S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, N e w York, 1957, p. 45 ° . 8 D. STEINBERG, M. VAUGHAN, C. B. ANFINSEN, j. D. GORRY AND J. LOGAN, in C. G. BELL AND F. N. HAVES, Liquid Scintillation Counting, P e r g a m o n , N e w York, 1958, p. 23 o. 9 I£. L. MANCHESTER AND F. G. YOUNG, Biochem. J., 7 ° (1958) 297. IO J. P. OLSON, L. L. MILLER AND S. B. TROUP, Blood, 22 (1963) 828. I I H. PRYDZ, Scand. J. Clin. Lab. Invest., 17 (1965) I43. 12 ]7I. C. HEMKER, J. J. VELTKAMP, A. HENSEN AND E. A. LOELIGER, Nature, 200 (1963) 589. 13 J. G. POOL AND C. F. BORCHGREVINK, Am. J. Physiol., 206 (1964) 229. 14 R. E. OLSON, Science, 145 (1964) 729 .
Biochim. Biophys. Acta, 123 (1966) 6o6-61o
BIOCHIMICA ET BIOPHYSICAACTA
BBA 95430
T H E ROLE OF VITAMIN K IN CLOTTING FACTOR SYNTHESIS I. E V I D E N C E FOR T H E P A R T I C I P A T I O N OF VITAMIN K IN T H E CONV E R S I O N OF A P O L Y P E P T I D E P R E C U R S O R TO FACTOR VII
BERNARD M. BABIOR, M.D., Ph. D. Thorndike Memorial Laboratory, I I and I V (Harvard) Medical Services, Boston City Hospital, and Department o/ Medicine, Harvard Medical School, Boston, Mass. (U.S.A .)
(Received December i4th, 1965)
SUMMARY
Vitamin K-dependent Factor VII synthesis in rat-liver slices was not affected by puromycin although protein synthesis was inhibited by 9 8 %. The results indicate that vitamin K is required for the formation of Factor VII from a polypeptide precursor.
INTRODUCTION Vitamin K depletion or the administration of a vitamin K antagonist results in a Factor VII deficiency in the intact animal 1 and in vitro 2. Factor VII activity appears in homogenates of rat-liver slices after a 2-h incubation and is prevented by the addition of dicoumarol, a vitamin K antagonist a. Factor VII production is also inhibited if the animal from which the liver is obtained had been given a vitamin K deficient diet or treated with dicoumarol in vivo. Administration of vitamin K in vivo, but not in vitro, can overcome this inhibition. The precise means by which vitamin K acts in Factor V I I production (and that of other clotting factors) is unknown. The experiments to be reported were designed to determine whether vitamin K stimulates the synthesis de novo of Factor V I I from amino acids or whether vitamin K enhances the conversion to Factor V I I of a polypeptide already present in the liver. To decide between these possibilities, the relationship between vitamin K and Factor VII synthesis was examined in the presence and absence of puromycin 4. MATERIALS AND METHODS Sodium dicoumarol (Endo), 25 mg/kg, was administered intraperitoneally to male Sprague-Dawley rats (150-25o g), a dose which produced a consistent fall of Biochim. Biophys. Acta, 123 (1966) 606 61o
VITAMIN
607
K AND CLOTTING FACTOR SYNTHESIS
serum Factor V I I activity to less than 3 % of normal within 18 h. The animals were then fasted, but allowed water ad libitum. 24 h later the animals were anesthetized with intraperitoneal sodium pentobarbital (5 mg/Ioo g). Blood for Factor V I I determination (0.45 ml) was obtained b y cardiac puncture with a syringe containing 0.05 ml of 3.8 % (w/v) sodium citrate. The liver was then perfused in the intact animal through the portal vein with 30 ml of ice-cold Krebs-Ringer bicarbonate buffer (pH 7.4), then removed and slices prepared with a Stadie-Riggs microtome. Vitamin K 1, 2 mg (Aquamephyton, Merck) was administered b y intracardiac puncture IO min or less before perfusion was begun. Two liver slices were placed in 25-ml erlenmeyer flasks, 2 ml of incubation mixture* added, the flasks gassed for 15-3o sec with O2-CO2 (95:5), stoppered and incubated for 2 h at 37 ° in a Dubnoff shaker. The incubation was terminated by homogenizing the contents of the flask at o ° in a glass P o t t e r - E l v e h j e m homogenizer with a Teflon pestle. After dilution of the homogenate with an equal volume of imidazole buffer p H 7.3 (see ref. 5), Factor V I I was determined b y the method of Pechet using human Factor V I I deficient plasma e. The same method was used for the Factor V I I in rat plasma after I : I O dilution of the plasma with 0. 9 o/ /o saline. A standard curve for Factor V I I activity was obtained with normal rat plasma; b y definition, I ml of plasma contained IOO U of Factor VII. Factor V I I appearance in the homogenate was calculated as the difference between the Factor V I I activity at the end of the incubation and the Factor V I I activity of unincubated liver slices. This assay was not affected b y dicoumarol or puromycin**. Protein concentration was determined b y the biuret method 7. The incorporation of ['4C~leucine into protein as a measure of protein synthesis was assayed according to STEINBERG et al. s, preparing the protein for counting b y the method of MANCHESTER AND YOUNG9.
RESULTS
AND DISCUSSION
As shown in Table I, no production of Factor V I I occurred in the absence of vitamin K, whether or not puromycin was added to the incubation mixture. When vitamin K was given before sacrifice, however, Factor V I I was produced at nearly the control rate in spite of depression of protein synthesis to 2 % of control b y puromycin addition in vitro. This finding contrasts with that of other authors1°, n, who have emphasized the inhibition of hepatic Factor V I I synthesis b y puromycin. The results of these experiments suggest that a polypeptide precursor of Factor V I I accumulates in the liver under the experimental conditions, and is converted to Factor V I I in a vitamin K-dependent reaction: Amino acids >
Polypeptide precursor
Vitamin K >
Factor V I I (I}
A similar hypothesis was previously proposed b y HEMKER et al. TM. They found in the plasma of patients with vitamin K deficiency a protein which was a competitive inhibitor of prothrombin, one of the vitamin K-dependent clotting factors. T h e y * F o r c o m p o s i t i o n s e e T a b l e I. ** U n p u b l i s h e d d a t a .
Biochim. Biophys. Acta, 123 ( 1 9 6 6 ) 6 o 6 - 6 1 o
608 TABLE
e . M; BABIOR I
EFFECT OF PUROMYCIN ON THE FORMATION OF FACTOR
RAT-LIVER SLICES
VII IN
Incubation mixture contains liver slices, approx. 15o mg, Krebs-Ringer bicarbonate buffer (2 m l ) a n d w h e r e i n d i c a t e d , p u r o m y c i n , I m M ( S i g m a ) a n d [14C~leucine, 3 . 9 ' 1 °6 d i s i n t e g r a t i o n s / min (New England Nuclear). Each value represents one rat. The Factor VII activity of unincubat e d s l i c e s f r o m E x p t s . 4 - 7 , a n d o f s l i c e s f r o m t h e s a m e e x p e r i m e n t s i n c u b a t e d f o r 2 h a t o °, w a s i7~-6 units/g protein.
Expt.
I 2 3 4 5 6 7
Vitamin K
---+ + + +
Factor VII production (units/g protein)
[14C]Leucine incorporation (disintegrations~rain per mg protein)
No puromycin
Puromycin
No puromycin
Puromycin
< 2 <2 <2 16o 200 31o 270
< 2 <2 <2 21o 15 ° 33 ° 15o
io 400 9900 16 9 0 0 8400 ii 200 13 6 0 0 16 9 0 0
260 200 270 230 i8o 420 320
postulated t h a t this protein was a precursor of prothrombin, and that vitamin K was necessary for its conversion to prothrombin. In interpreting these data, two alternative hypotheses should be considered. They will be presented seriatim. I. The first of these alternative hypotheses states that there is a rapid, vitamin K-dependent, protein synthesizing (i.e., puromycin sensitive) step which takes place during the brief interval between administration of vitamin K to the rat and addition of puromycin to the incubation mixture. As a result of this rapid step, Factor V I I precursor is synthesized in large amounts. During the course of the subsequent incubation, the accumulated precursor is converted to Factor V I I in a vitamin K-independent reaction. If this were the case, dicoumarol added to the incubation mixture should have no effect on the reaction. As is shown in Table n , however, as well as b y POOL AND ROBINSON2, dicoumarol, a vitamin K antagonist, is a good inhibitor of Factor V I I synthesis in vitro. Thus, vitamin K is involved in a reaction which continues to take place throughout the course of the incubation. At the concentrations employed, dicoumarol depressed the incorporation of [14C~leucine into protein 13 (see Table I I I ) , raising the question of whether the in-
TABLE
II
EFFECT OF DICOUMAROL ON THE SYNTHESIS OF FACTOR VII
in vitro
I n c u b a t i o n s w e r e c a r r i e d o u t a s d e s c r i b e d i n T a b l e I. V i t a m i n before sacrifice.
Expt.
5 6 7
I( was given to each animal io rain
Factor VII production (units/g protein) No dicoumarol
I m M dicoumarol
5 m M dicoumarol
200 31o 270
69 5° 52
3 <2 9
Biochim. Biophys. Aeta, 123 ( 1 9 6 6 ) 6 o 6 - 6 1 o
VITAMIN
TABLE
K AND
609
CLOTTING FACTOR SYNTHESIS
III
EFFECT OF P U R O M Y C I N O N F A C T O R VII P R O D U C T I O N IN T H E P R E S E N C E OF D I C O U M A R O L Incubations were carried out as described in Table I. Vitamin I~ w a s given to each animal. T h e concentrations of both p u r o m y c i n a n d dicoumarol were I lumole/ml. [14C]Leucine, 3.9" IO~ disintegrations/rain, w a s added to the incubation mixtures.
Expt.
5 6 7
Dicoumarol
-+ -+ -+
Factor VII production (Units/g protein)
?aC]Leucine incorporation (disintegrations~rain per mg protein)
No puromycin
Puromycin
No puromycin
Puromycin
2oo 69 31o 5° 270 52
15o 49 33 ° 35 15o 29
i i 2oo 46oo 13 6 0 0 41oo 16 9 0 0 4500
18o 12o 420 12o 320 200
hibition of Factor V I I synthesis b y dicoumarol was due to its effect on protein synthesis. This does not seem to be the case, however, because puromycin had no effect on Factor V I I synthesis even when protein synthesis was almost completely inhibited. 2. The second hypothetical mechanism is shown in (II), in which the rate of synthesis of a Factor V I I precursor in a vitamin K-dependent reaction is very rapid compared with the rate of the conversion of the precursor to Factor V I I in a vitamin K-independent reaction.
Amino acids
Vitamin K Protein synthesis Precursor > Factor V I I > Slow Rapid
(II)
Under these circumstances, even with protein synthesis slowed as it was to 2 ~o of its original rate (Table I), it might be argued that the precursor could still be formed rapidly enough for no change in the rate of appearance of Factor V I I to be observed. Since here vitamin K would be participating in a reaction which continues throughout the incubation, Factor V I I synthesis could, in principle, be inhibited by adequate concentrations of dicoumarol (vide supra). Such an inhibition was actually observed (see Table II). According to this mechanism, the inhibition would result from an effect of dicoumarol on the first of the two reactions sufficient to curtail the production of precursor so that it would no longer accumulate but would at once be converted to Factor VII. Under these conditions, Factor V I I synthesis would reflect the rate of synthesis of the precursor, which by postulate is the immediate product of a protein-forming reaction. Therefore, the rate of appearance of Factor V I I would be proportional to the rate of protein synthesis. That this is not the case, however, is shown b y the results in Table I I I . When Factor V I I formation is partly inhibited b y dicoumarol, the presence of puromycin results only in a slight further inhibition of the synthesis of the clotting factor, despite the fact that protein synthesis is almost completely inhibited. Biochim. Biophys. Acta,
123
(I966)
6o6-61o
6Io
B.M. BABIOR
Failure of puromycin to inhibit the vitamin K-dependent synthesis of Factor VII implies that the vitamin probably does not act as an inducer of Factor VII synthesis at the level of messenger R N A 14. The small extent to which puromycin inhibits the reaction (approx. IO %) m a y represent the extent to which Factor VII arises from protein synthesis de novo.
ACKNOWLEDGEMENTS
I am indebted to Dr. H. S. STRAUSS for a generous gift of human Factor VIIdeficient plasma. I would like to thank Dr. L. PECHET for his assistance in setting up the Factor VII assay and Dr. C. S. DAVlDSON for his valuable encouragement and advice. This work was supported in part by Grants No. 5 TI-AM-5413 and AM-o9II5 from the National Institutes of Health, Bethesda, Md. (U.S.A.).
REFERENCES i M. M. WINTROBE, Clinical Hematology, 5th ed., Lea and Febiger, 1961, p. 882. 2 J. G. POOL AND J. ROBINSON, Am. J. Physiol., 196 (1959) 423 . 3 L. S. GOODMAN AND A. GILMAN, The Pharmacological Basis o[ Therapeutics, 2nd ed., Macmillan, N e w York, 1955, p. 15o9. 4 A. MORRIS, R. ARLINGHAUS, S. FAVELUKES AND R. SCHVCEET, Biochemistry, 2 (I963) lO84. 5 E. T. MERTZ AND C. A. OWEN, Proc. Soc. Exptl. Biol. Med., 43 (194 o) 204. 6 L. PECHET, ill L. M. TOCANTINS AND L. A. KAZAL, Blood Coagulation, Hemorrhage and Thrombosis, G r u n e alld S t r a t t o n , N e w York, 1964, p. 213. 7 E. LAYNE, ill S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, N e w York, 1957, p. 45 ° . 8 D. STEINBERG, M. VAUGHAN, C. B. ANFINSEN, j. D. GORRY AND J. LOGAN, in C. G. BELL AND F. N. HAVES, Liquid Scintillation Counting, P e r g a m o n , N e w York, 1958, p. 23 o. 9 I£. L. MANCHESTER AND F. G. YOUNG, Biochem. J., 7 ° (1958) 297. IO J. P. OLSON, L. L. MILLER AND S. B. TROUP, Blood, 22 (1963) 828. I I H. PRYDZ, Scand. J. Clin. Lab. Invest., 17 (1965) I43. 12 ]7I. C. HEMKER, J. J. VELTKAMP, A. HENSEN AND E. A. LOELIGER, Nature, 200 (1963) 589. 13 J. G. POOL AND C. F. BORCHGREVINK, Am. J. Physiol., 206 (1964) 229. 14 R. E. OLSON, Science, 145 (1964) 729 .
Biochim. Biophys. Acta, 123 (1966) 6o6-61o