Biochimica et Biophysica Acta, 386 (1975) 556-566

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 37013 PARTIAL P U R I F I C A T I O N A N D C H A R A C T E R I Z A T I O N OF T H E E N Z Y M E W H I C H CONVERTS P R E C U R S O R LIVER P R O T E I N TO F A C T O R X

G. C. H. CHUNG*, R. DELANEY, D. MACK and B. CONNOR JOHNSON** Oklahoma Medical Research Foundation and Department of Biochemistry and Molecular Biology, Oklahoma University Health Sciences Center, Oklahoma City, Okla. 73104 (U.S.A.)

(Received September 9th, 1974)

SUMMARY A rat liver post-microsomal supernatant enzyme, which carries out an epigenetic conversion of a protein contained in liver microsomes to Factor X, has been partially purified 250-fold in 50 % yield by a combination of salt fractionation and gel filtration. The crude enzyme is stable to freezing and thawing but unstable at 4 °C. However, the partially purified enzyme is more stable at 4 °C. It requires Ca z+ and HCOa- for optimum formation of Factor X activity. The supernatant enzyme is vitamin K dependent and exhibits its maximum rate of formation of Factor X between pH 8 and 8.5.

INTRODUCTION Goswami and Munro [1] reported that the in vitro formation of prothrombin occurs in the absence of the components necessary for protein synthesis, and thus such formation must be due to conversion of a protein precursor rather than to protein synthesis. Following vitamin K administration to deficient animals, Hill et al. [2] showed prothrombin formation to be a post-translational event. Babior [3] reported in vitro formation of Factor VII using liver slices and demonstrated the difference between vitamin K normal liver and liver from warfarintreated animals. This was extended to isolated, intact functional liver cells by Ranhotra and Johnson [4]. The in vitro formation of the clotting factor was not affected by the addition of protein synthesis blocking agents such as cycloheximide or puromycin. The formation of clotting factor zymogens during incubation appears to be due to modification of some precursor. Administration of vitamin K to vitamin Kdeficient animals restored this ability of the in vitro system within 25 min. A cell-free system was developed by Babior and Kipnes [5] in which formation of clotting factor assayed as Factor VII could be obtained by incubation of liver

* Present Address: University of Washington, School of Medicine, Division of Biology and Biomedical Sciences, St. Louis, Mo. 63110, U.S.A. ** To whom all communications for reprints should be addressed.

557 homogenate mitochondrial supernatants from normal but not from warfarin-treated rats. Although they concluded from their data that Factor VII formation occurred with microsomal supernatant alone, we will present evidence that microsomes are also required. In all cases in vitro addition of vitamin K did not restore the system derived from warfarin-treated rats. We now report the partial purification and characterization of the vitamin Kdependent enzyme which completes Factor X formation. A preliminary report of this work has appeared [6]. MATERIALS AND METHODS

Materials. Gibco Medium 199 was obtained from General Biochemicals, sodium warfarin from Endo Laboratories and rabbit brain thromboplastin from Dade. Folin and Ciocalteu phenol reagent was obtained from Harleco; crystalline bovine serum albumin from Pentex; and Factor VII-X-deficient bovine plasma, cephalin, and Russell's viper venom from Sigma. Factor VII-deficient human plasma was obtained from Medical Sciences International. Phenylmethylsulfonyl fluoride (PhMeSO~F) was obtained from the Eastman Kodak Co. Estimation of protein. Quantitative estimation of protein concentration was by the method of Lowry et al. [7] using bovine serum albumin as standard. Absorbance at 280 nm was used only to monitor effluent from gel filtration columns and not for quantitative estimation of protein concentration. Preparation of animals. Livers for homogenization were obtained from 150200 g male Sprague-Dawley rats. Vitamin K deficiency was produced by the administration of warfarin at a dose of 2.5 mg/100 g body weight 18 h prior to killing [5]. Preparation of liver sub-cellular fractions. After 18 h fasting, the rats were anesthetized and the livers freed of blood by perfusion with 40 ml of 0.154 M NaCI at room temperature followed by 40 ml of the same solution at 0 °C. The liver was homogenized at 0 °C in five volumes of 0.25 M sucrose, using a Potter-Elvehjem homogenizer with a Teflon pestle. Whole cells, nuclei and mitochondria were removed by 10 min centrifugation at 24 000 X g. Microsomal supernatants were prepared by centrifugation at 105 000 x g for 1 or 2 h and 200 000 × g for 1 h. The microsomal pellet was washed and resuspended in a quantity of 0.25 M sucrose equal to 0.2 times the volume of the 24 000 x g supernatant from which the pellet was obtained. Incubation system. Clotting Factor X was formed by incubation of 0.25 ml of post-microsomal supernatant, 0.25ml of cell culture Medium 199 or of 0.1 M Tris. HC1 in 20 mM ammonium bicarbonate, pH 8.0 (unless another pH is stated), 0.10 ml of 10 mM CaCI2 and 0.05 ml of microsomal suspension (approx. 0.8-1.0 mg of protein) at 37 ° C for 20 min. The reaction was terminated by chilling in melting ice and clotting factor activity was immediately determined. Assay of incubation mixture for product clotting factor activity using Factor VII-X-deficient plasma. The material to be assayed was diluted, if necessary, with an equal volume of 0.25 M imidazole. HC1 buffer, pH 7.3; 0.10 ml of this solution was placed in a small polyethylene vessel together with 0.1 ml of Factors VII- and Xdeficient bovine plasma. This mixture was brought to 37 °C and coagulation initiated by the addition of 0.2 ml of a 1:1 mixture of tissue thromboplastin and 20 mM CaC12. The time from the addition of thromboplastin to the formation of a clot was measured

558 automatically with a fibrometer (Baltimore Biological Laboratory). Standard curves were used to calculate units, and were prepared by measuring clotting factor levels in dilutions of normal rat plasma in the presence of Factors VII-X-deficient bovine plasma. The clotting factor content of normal rat plasma was arbitrarily set at 100 units/ml and the clotting factor levels formed during incubation are reported in these units after subtracting the number of units found in the identical unincubated control. Thus, all results reported are units formed during incubation. Assay for Factor VII or Factor VIIa activity. Factor VII was assayed similarly to the above by substituting Factor VII-deficient human plasma for the Factor VII-Xdeficient plasma. Factor VIIa was determined by direct assay using Factor VIIdeficient human plasma, but without the thromboplastin preparation. Clotting time was taken from the addition of calcium. Assay for Factor X or Factor Xa activity. Assay for Factor X was carried out by the Bachmann procedure [8], as modified by Woolf et al. [9] as follows: 0.1 ml Factor VII-X-deficient plasma q- 0.1 ml Russell's viper venom and cephalin were incubated together for 5 rain at 37 °C; then 0.1 ml sample and 0.1 ml of 25 m M CaCl2 were added. Clotting time was determined in a fibrometer from the addition of calcium as before. Factor Xa was assayed similarly with the omission of Russell's viper venom and the 5-min incubation. Factor Xa activity was estimated from the Factor X standard curve prepared with normal rat plasma. RESULTS

Location of the component(s) dependent on the vitamin K status of the animal responsible for clotting factor formation Microsomes and post-microsomal supernatants were prepared from livers from normal or warfarin-treated rats. The ability of various combinations of these microsomal and supernatant preparations to support clotting factor formation is given in Table I. Microsomes from either animal can function in any combination with normal supernatant. In contrast, supernatant from warfarin-treated animals could not funcTABLE I EFFECT OF WARFARIN TREATMENT ON FACTOR X FORMATION IN VITRO Microsomal pellets and supernatants were prepared at 200 000 g for 1 h from mitochondrial supernatants prepared from liver homogenates of normal or warfarin-treated rats. The incubation medium contained 0.1 M Tris.HC1 buffer (pH 8.0) with 0.04 M NH(HCO3 and 1.5 mM CaC12. The other conditions for incubation are described in the text. The assays for Factor X and Factor Xa are described in the text. Units/ml of incubation mixture

Source

Supernatant

Microsomes

Factor X

Factor Xa

Normal Warfarin Normal Warfarin Warfarin None

Normal Warfarin Warfarin Normal None Warfarin

3.05 0 3.4 0.25 0 0

4.5 0 4.1 0.5 0 0

559 TABLE II EFFECT OF CENTRIFUGATION ON CLOTTING FACTOR FORMATION Cell fractions were prepared by centrifuging at 24 000 x g for 10 min to remove mitochondria and at either 105 000 g for 1 h or 200 000 g for 1 h to recover microsomes. The final microsomal pellet was resuspended in a quantity of 0.25 M sucrose equal to 0.2 times the volume of the 24 000 x g supernatant from which the pellet was obtained. The reaction mixtures contained 0.25 ml of 105 000 x g supernatant or 200 000 x g supernatant, 0.05 ml of 105 000 x g microsomes or 200 000 x g microsomes, 0.25 ml of Gibco Medium 199 (pH 7.3) and 0.1 ml of 0.01 M CaC12 in a total volume of 0.65 ml. As necessary, 0.25 M sucrose was used in place of either cell fraction. Reaction mixtures were incubated and assayed for Factor VII-X activity using Factors VII and X-deficient bovine plasma. Liver fractions incubated

105 000 x g microsomes plus supernatant 105 000 x g supernatant alone 105 000 x g microsomes alone 200 000 x g microsomes plus supernatant 200 000 x g supernatant alone 200 000 x g microsomes alone

Clotting time (s)

Clotting factor activity (units/ml incubation mixture)

Unincubated

Incubated

> 200 >200 >200

45 60 >200.0

7.50 4.20 0.00

> 200 > 200 >200

43 >200.0 >200.0

8.25 0.00 0.00

t i o n in c o m b i n a t i o n with m i c r o s o m e s o f n o r m a l o r w a r f a r i n - t r e a t e d animals. The p o s s i b i l i t y still r e m a i n e d t h a t n o r m a l s u p e r n a t a n t s alone c o u l d p r o d u c e clotting factor f o r m a t i o n . S u p e r n a t a n t s f r o m centrifugations o f 105 000 x g for 2 h r e t a i n e d a small b u t variable ability to f o r m clotting factor w i t h o u t a d d i t i o n o f m i c r o s o m e s alt h o u g h B a b i o r a n d K i p n e s [5] h a d r e p o r t e d t h a t a m i c r o s o m a l s u p e r n a t a n t p r e p a r e d at 105 000 x g for 1 h was as active as a p o s t - m i t o c h o n d r i a l s u p e r n a t a n t . T h e r e f o r e s u p e r n a t a n t s p r e p a r e d by centrifugations o f 1 h at 105 000 x g a n d 200 000 x g were tested for clotting factor f o r m a t i o n w i t h o u t m i c r o s o m e s a d d e d b a c k (Table II). F o r m a t i o n o f clotting factor c o u l d n o t be detected in either m i c r o s o m a l p r e p a r a t i o n alone n o r in the 200 000 x g s u p e r n a t a n t . T h e 105 000 x g s u p e r n a t a n t was a l m o s t h a l f as active as the r e c o n s t i t u t e d m i c r o s o m e s plus supernatant. T h u s the s u p e r n a t a n t c o n t a i n e d the v i t a m i n K - d e p e n d e n t c o m p o n e n t .

Partial purification of the supernatant component I n i t i a l a t t e m p t s to f r a c t i o n a t e the s u p e r n a t a n t were unsuccessful due to the loss o f u p to 70 ~ o f the activity in 24 h at 4 °C. F r a c t i o n a t i o n o f the v i t a m i n K d e p e n d e n t c o m p o n e n t p r o v e d p r a c t i c a l when the activity was d e m o n s t r a t e d stable to freeze-thawing a n d was relatively stable frozen ( - - 2 0 °C) for u p to 1 m o n t h . T h e a m o u n t o f time t h a t a p r e p a r a t i o n was in the u n f r o z e n state was k e p t to a m i n i m u m d i c t a t e d b y the r e q u i r e m e n t s for f r a c t i o n a t i o n . A l l f r a c t i o n a t i o n steps were at 4 °C. P o s t - m i c r o s o m a l s u p e r n a t a n t in 0.25 M sucrose was d i l u t e d with an equal v o l u m e o f 52 m M NH4HCO3, 0.13 M T r i s - H C 1 ( p H 8.0) to buffer the mixture. Solid (NH4)2SO4 (5.7 g/25 m l diluted s u p e r n a t a n t ) was dissolved to 40~o s a t u r a t i o n with c o n s t a n t stirring. T h e p r e c i p i t a t e was r e m o v e d b y c e n t r i f u g a t i o n a t 34 000 x g for 20 m i n and 2.57 g o f solid (NH4)2SO4 was dissolved to achieve a 55 ~o saturation. This

560 last precipitate was collected after centrifugation at 34 000 × g for 20 min and dissolved in 5 ml of 0.25 M sucrose. This redissolved precipitate was placed on a 2.5 × 76 cm column of Sephadex G-200 equilibrated with 0.1 M NH4HCOa/0.25 M Tris. HCI (pH 8.0). The column was developed with this buffer at 12 ml/h. The fractions from (NH4)zSO4 precipitation and from the column were assayed by incubation with normal microsomes followed by the Factor X assay (which measures any Factor X and Factor Xa formed during the incubation). The void volume of this column was 118 ml. The fractions, between 150 and 185 ml of effluent, containing the vitamin K-dependent activity were pooled (approx. 30 ml) and concentrated to 10 ml with dry Sephadex G-25 by the method of Flodin et al. [10]. Further purification of this fraction was achieved by elution from a column of Bio-gel P-300 (2.5 × 55 cm) with the same buffer at a flow rate of 10 ml/h. While Dextran Blue 2000 appeared at 43 ml, activity to form Factor X eluted between 90 and 115 ml of effluent in a skewed peak with a maximum activity at l l 0 ml. This material was concentrated and frozen. A summary of these steps is contained in Table III. TABLE III PURIFICATION OF SUPERNATANT VITAMIN K-DEPENDENT ENZYME The details of fractionation, incubation and assay are described in the text with the incubation medium containing 0.1 M Tris.HC1 buffer (pH 8.0) with 0.04 M NH4HCO3 and 1.5 mM CaC12. One unit of enzyme will catalyze the formation of one unit of Factor X in 20 min under these conditions. Purification step

Total activity (units)

Total protein (mg)

Spec. act. (units/mg)

Recovery (~)

Purification

Post-microsomal supernatant (NH4)2SO4 fractionation Sephadex G-200 Bio-gel P-300

304 200 174 165

520.0 33.6 4.1 1.2

0.59 6.0 42.4 135

100 65.8 57.2 54.2

1.0 10.2 72.4 230.8

The degree of heterogeneity of the preparation after a 250-fold purification was estimated by polyacrylamide gel electrophoresis at p H 8.8. One major staining region and many minor staining regions indicated that the sample was still impure.

Identity of the product(s) of incubation of normal microsomes and post-microsomal supernatant Assay of incubation mixtures before and after incubation for 20 min for clotting factors VII-X, VII, VIIa, X and Xa produced the data in Table IV. Before incubation no Factor Xa activity was detected but low levels of activity were measured in the other assays. After incubation for 20 min Factor Xa activity 'was high and all other activities had increased 20-30-fold. Because Factor Xa acts directly on prothrombin to produce thrombin, any Factor Xa present in an incubation mixture will exert a major effect on activity measurements by assays for any of the other factors; VII, VIIa or X.

561 TABLE IV CLOTTING FACTOR FORMATION DURING INCUBATION ASSAYED AS FACTOR VIIX, FACTOR VII, FACTOR VIIa, FACTOR X and FACTOR Xa Incubations used the conditions in Table I. Assays for each clotting factor are described in the text. Assay

Factor VII-X Factor VII Factor VIIa Factor X Factor Xa

Unincubated

Incubated

Clotting time (s)

Incubation mixture (units/ml)

Clotting time (s)

Incubation mixture (units/ml)

131 35 71 82 > 200

0.09 0.23 -0.18 0

38 23 29 36 36

2.3 6.5 -3.7 4.2

Factor X formation and activation to Factor Xa T h e a p p e a r a n c e o f F a c t o r X a activity d u r i n g i n c u b a t i o n c o u l d be due to a c t i v a t i o n o f preexisting F a c t o r X to F a c t o r X a b y newly c o m b i n e d F a c t o r V I I a n d Tissue F a c t o r ( F a c t o r VIIa). T o d i s t i n g u i s h between this a n d f o r m a t i o n o f F a c t o r X, which is subsequently c o n v e r t e d to F a c t o r X a d u r i n g i n c u b a t i o n , a d v a n t a g e was t a k e n o f the irreversible "serine p r o t e a s e " i n h i b i t o r , P h M e S O 2 F [11], to inactivate F a c t o r s VII, V I I a a n d X a as they a p p e a r e d in the i n c u b a t i o n mixture. T a b l e V c o m p a r e s the F a c t o r s VII, X a n d X a c l o t t i n g times before i n c u b a t i o n , after i n c u b a t i o n a n d after i n c u b a t i o n with P h M e S O 2 F present. B o t h F a c t o r V I I a n d F a c t o r X a clotting times r e m a i n e d at the u n i n c u b a t e d levels when the P h M e S O 2 F was r e m o v e d b y dialysis a n d the mixtures assayed. H o w e v e r , F a c t o r X showed a s m a l l b u t significant decrease in c l o t t i n g time over the u n i n c u b a t e d mixture. F o r m a t i o n o f F a c t o r X was a p p a r e n t in the absence o f the a c t i v a t i o n system. W h e n the subsequent a c t i v a t i o n system was n o t i n h i b i t e d b y P h M e S O 2 F , a m u c h higher level o f F a c t o r X a activity was p r o d u c e d during incubation.

Requirements of the incubation system for Factor X formation T h e c o m p l e x c o m p o n e n t s o f tissue culture m e d i u m , G i b c o M e d i u m 199 were studied to d e t e r m i n e the r e q u i r e m e n t s o f the i n c u b a t i o n system. M o s t o f these nuTABLE V FORMATION OF FACTOR X IN THE PRESENCE OF PhMeSO2F The incubation mixture containing 20 mM PhMeSO2F was incubated for 30 rain at 37 °C, dialysed at 4 °C and then assayed. The other incubations were for the standard 20 rain. All incubations were otherwise as described in Table I. Assays are described in the text. Treatment

Factor VII clotting time (s)

Factor X clotting time (s)

Factor Xa clotting time (s)

Unincubated Incubated Incubated with PhMeSO2F

40 29 40

105 43 58

> 200 40 > 200

562 TABLE VI EFFECTS OF MEDIUM COMPONENTS ON CLOTTING FACTOR FORMATION Incubation mixture contained 0.25 ml of post-microsomal supernatant, 0.05 ml of microsomes and 0.1 ml of 0.01 M CaCl2. Various concentrations of various buffers (pH 7.3) were added to make a final volume of 0.65 ml. Concentrations given are final concentrations. Incubations were conducted at 37 °C for 20 rain. Factor VII-X-deficient bovine plasma was used in the assay mixtures as described in the text. Incubation medium

Activity (%)

Gibco Medium 199 0.1 M phosphate buffer, pH 7.3 0.1 M NaHCO3.HC1, pH 7.3 0.1 M imidazole. HCI, pH 7.3 0.1 M imidazole.HCl, pH 7.3 + 0.06 M NaC1 0.01 M imidazole. HC1 + 0.06 M NaC1 0.01 M imidazole. HC1 + 0.06 M NH4C1 0.01 M imidazole.HC1 + 0.035 M NaHCOa 0.1 M Tris.HCl 0.1 M Tris.HC1 ÷ 0.02 M NH4HCO3 0.1 M Tris-HC1 ÷ 0.04 M NH4HCOa 0.1 M Tris.HC1 q- 0.06 M NH4HCO3

100 0 67 29 8 26 22 83 52 100 100 70

trients were f o u n d to have n o effect o n the i n c u b a t i o n and c o u l d be eliminated. While the b i c a r b o n a t e a p p e a r e d essential, v a r io u s buffers co u l d be used (Table VI). A buffer o f 0.1 M T r i s . H C 1 , p H 7.3, c o n t a i n i n g 20-40 m M H C O 3 - was o p t i m a l for clotting factor f o r m a t i o n a n d its subsequent assay. A l l w o r k r e p o r t e d here except for the d a t a in T a b l e II used 40 m M NH4HCOa/0.1 M Tris. HC1 m e d i u m . T h e step at which C a 2+ was required was d e m o n s t r a t e d by incubating in the presence or absence o f 1.5 m M CaC12 (Table VII). F a c t o r X - X a f o r m a t i o n was n o t d e m o n s t r a b l e w h e n Ca 2+ was o m i t t e d d u r i n g the incubation. M g 2+, Ba 2+, Cu 2+ or M n 2+ co u l d n o t replace C a 2+. In a similar m a n n e r the i n c u b a t i o n step was d e m o n strated to require the H C O 3- (Table VIII). W h e n H C O a - was o m i t t ed during the

TABLE VII EFFECT OF CaC12 ON CLOTTING FACTOR FORMATION Incubation mixture contained 0.25 ml of post-microsomal supernatant, 0.05 ml of microsomes and 0.25 ml of 0.1 M NH4HCO3 in 0.25 M Tris. HC1 buffer (pH 7.3), final NH4HCO3 concentration of 40 mM and final Tris.HCl concentration of 100raM. 0.1 ml of 0.01 M CaC12 or distilled water was added to incubation mixture to make final CaC12 concentration of 1.5 mM or 0 raM. Incubations were conducted at 37 °C for 20 min. The Factor VII-X assay was used to estimate clotting factor formation as described in the text. Clotting time (s) . . . . . . . . . . . Unincubated Incubated Incubation mixture with CaCI, >200 Assay mixture with CaCl2 Incubation mixture without CaCl2 > 200 Assay mixture with CaCl2

Clotting factor activity (unit/ml incubation mixture)

31

7.5

> 200

0.0

563 TABLE VIII EFFECT OF HCO3- ON THE YIELD OF CLOTTING FACTOR Incubation mixture contained 0.25 ml of post-microsomal supernatant (approx. 1.0 mg protein) and 0.05 ml microsomal suspension (approx. 0.8-1.0 mg protein) both from vitamin K normal animals, 0.1 ml of 0.01 M CaCI2, in a total volume of 0.65 ml of 0.1 M Tris.HCl buffer, pH 7.3. The Factor VII-X assay was used to estimate clotting factor formation as described in the text. Presence or absence of HCO3Incubation medium - -

-+ HCO3-, 0.025 M ÷ HCOa-, 0.025 M

Assay medium

Units clotting factor activity formed/ml incubation mixture

-+ HCO3-, 0.025 M ÷ HCOa-, 0.025 M --

0.58 0.66 5.1 4.4

incubation but was included during the assay for Factor V I I - X formation, only 0.7 unit/ml of incubation mixture were produced. The presence of HCO3- in the incubation medium resulted in seven times more Factor VII-X formation than its omission from the incubation medium. Once the salts requirements were established the p H for optimal formation of Factor X-Xa was investigated over a p H range of 5.6 to 9.0. The m a x i m u m activity was found between p H 8 and 8.5. The p H activity profile was not symmetric about the optimum but was skewed to more acid values. No activity was detected below p H 6.7 and little above p H 9.0. I f the Tris.HC1 buffer was replaced by imidazole. HC1, or Tris/acetate, a similar activity curve was produced but the values were lower in imidazole. HC1 buffer. The experiments presented in Table II demonstrated the absolute requirements for microsomes and for the post-microsomal supernatant in order for formation of Factor X-Xa to occur during incubation. The Factor X-Xa activity formed was shown to be mainly associated with the microsomes after incubation by reisolation of the microsomes and the post-microsomal supernatant after the 20-min incubation at 37 °C. Factor X-Xa activity was three times greater in the microsomes than in the supernatant. This suggests that the microsomes serve as a source of Factor X precursor and not just as a source of Tissue Factor. DISCUSSION Incubation of liver homogenates at 37 °C produced increased clotting factor activity which Babior and Kipnes [5] ascribed to components almost totally present in a microsomal supernatant for the following reasons: the supernatant alone produced almost as much Factor VII activity as the whole liver homogenate and addition of the microsomal fraction resulted in little increase in Factor VII activity formed. It is evident from examination of the supernatant and microsomes prepared at 200 000 × g, a centrifugal force which depletes the supernatant of all microsomes and sub-microsomal particles, that neither microsomes nor supernatant alone result in clotting factor formation during incubation. Similar findings have been reported by Lowenthal and Wang [12]. The recombined supernatant and microsomal fractions

564 completely restore the clotting factor formation activity of the liver homogenate. Although we have not demonstrated that the microsomes contain partially completed molecules of Factor X, others have shown that prothrombin [13, 14] and Factor VII [15] are associated with the microsomes of normal livers. We find that most of the Factors X and Xa formed are associated also with the microsomal fraction after the incubation at 37 °C. The component contributed by the microsomal supernatant was inhibited by warfarin treatment of the rat while the microsomal component, partially completed Factor X, was not affected. The supernatant component was considered to be an enzyme because it increased Factor X activity with time, increasing concentrations of supernatant protein increased the rate and quantity of Factor X formed up to a limit, it was heat unstable, non-dialyzable, salt precipitable, and its activity varied with pH [6]. The actual specificity of this enzyme is somewhat uncertain due to the nature of the clotting factor assays which are used to estimate the product. The presence of clotting factors in their activated states show dominance in an assay with Factor IIa ~ Factor Xa > Factor VIIa. Because direct assays for Factor IIa activity indicate very little apparent activity and the presence of anti-thrombin in the liver microsomes reduces the contribution of Factor IIa formed [16], the activity measured in the assays reported here are not ascribed to Factor IIa. The similarity of results when Factor VII-deficient plasma or Factor VII-X-deficient plasma is used to assay the incubation products suggests that Factor VII and VIIa activities are not great enough to substantially increase the activity measured in the Factor X and Xa assays. Factor Xa activity, present after incubation at 37 °C is of the same order of magnitude in activity as is measured in the Factor X assay, and Factor VII and VIIa activities if present do not add appreciably to the Factor X and Xa activities which predominates. The supernatant enzyme completes Factor X formation during the incubation rather than converting preexisting Factor X to Factor Xa. The absence of Factor X activity in microsomal or supernatant fractions, pre- or post-incubation, indicates that there is insufficient Factor X preexisting in this system to account for the quantity of Factor Xa found after incubation of the whole system. Thus we believe that Factor X formation occurs as the result of the action of the supernatant enzyme. This was confirmed by use of PhMeSOzF to inhibit Factor VII, Factor VIIa, and Factor Xa during the incubation. An increase in Factor X was demonstrable after incubation and removal of the PhMeSO2F. It is very likely that a small quantity of Factor VII is present in the microsomal fraction and is present as Factor VIIa because of the presence of Tissue Factor [17] and calcium. This complex would convert any Factor X formed during the incubation to Factor Xa and account for our findings of high Factor Xa activity after the incubation. It is of extreme interest to locate the exact position in the microsomes and state of completion of the Factor X precursor protein when this enzyme acts upon it. Most of the published evidence [2, 4, 18] indicate that the precursor proteins polypeptide has been formed. It would normally be on the ribosome, being inserted into the endoplasmic reticulum, traversing the endoplasmic reticulum to the Golgi apparatus, or being pinocytosed out of the cell. The only step where it is easily accessible to the supernatant enzyme would be on the ribosome or before insertion into the endoplasmic reticulum. Although a 20 min incubation would allow time for the enzyme to

565 penetrate portions of the endoplasmic reticulum and modify the precursor protein, there is little likelihood that the completed protein could act on prothrombin in the Factor Xa assay within seconds if it was contained inside the endoplasmic reticulum. Thus the assay appears to measure only completed Factor X or Xa molecules on the ribosomes, attached (bound) to the outer membrane of the endoplasmic reticulum, or free in solution. The nature of the reaction catalyzed by the enzyme is still uncertain. Recent findings by Howard and Nelsestuen [19] and by Magnusson et al. [20] indicate that the vitamin K-dependent step in Factor X and Factor II formation involves carboxylation of the protein near the amino terminus. The bicarbonate requirement for the enzyme shown here and the report by Girardot et al. [21] that H~4CO3- is incorporated more rapidly than normal into Factor II in vivo when vitamin K is administered to a warfarin-treated rat suggest that this enzyme might be involved in carboxylation of the precursor proteins to form Factor X and possibly the other vitamin K-dependent clotting factors. The requirement for calcium in the incubation medium is clear as it is for bicarbonate. How the calcium functions is not clear. By analogy to its reaction with most of the clotting factors, calcium should aid in binding the enzyme or a cofactor to the substrate and/or phospholipid. Further purification of the enzyme and the identification of a simple substrate to replace the microsomes appear to be necessary before the nature of the reaction catalyzed by this enzyme can be identified and the mechanism by which it is accomplished determined. ACKNOWLEDGEMENT This work was supported by a research grant H L 16515 from the National Institutes of Health.

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