[ l l l BOVINE COAGULATION FACTOR X 95

During the activation of factor X by the intrinsic and extrinsic path- ways, a specific peptide bond is hydrolyzed in the amino-terminal region of the heavy chain, as illustrated in Fig. 1.15-1~ This cleavage occurs between Argo1 and Ile~.,, giving rise to factor Xa~ (MW 45,300) and an activation peptide (MW 9500).

Factor Xa~ is then converted to factor Xa~ (MW 42,600) by hydroly- sis of a second specific peptide bond in the carboxyl-terminal region of the heavy chain. This cleavage occurs between Arg29o and Gly~91, giving rise to a degradation glycopeptide (MW 2700). Factor X ~ and factor X ~ have equivalent coagulant activity. Thus, the critical event in the activation reaction is the liberation of a new amino-terminal isoleucine in the first step, and this residue probably forms an internal ion pair with Asps32, which is adjacent to the active center Ser~33. This probably leads to the charge-relay network analogous to that found in the pan- creatic proteases? s-~l

The amino acid and carbohydrate compositions for factor X~, factor X~,, and factor X ~ are shown in Table II. 17

15 K. Fujikawa, M. E. Legaz, and E. W. Davie, Biochemistry 11, 4892 (1972). ~ K. Fujikawa, M. E. Legaz, and E. W. Davie, Biochemistry 13, 4508 (1974). 1~ K. Fujikawa, K. Titani, and E. W. Davie, Proc. Natl. Acad. Sci. U.S.A. 72, 3359

(1975). '~ B. W. Matthews, P. B. Sigler, R. Henderson, and D. M. Blow, Nature (London)

214, 652 (1967). '~P. B. Sigler, D. M. Blow, B. W. Matthews, and R. Henderson, J. Mol. Biol. 35,

143 (1968). :o D. M. Blow, J. J. Birktoft, and B. S. Hartley, Nature (London) 221, 337 (1969). 2~ D. M. Shotton and H. C. Watson, Nature (London) 225, 811 (1970).

[11] The Activation of Bovine Coagulation Factor X 1

By JOLYON JESTY and YALE NEMERSON

Factor X (Stuart-Prower factor) is one of the four vitamin K-depen- dent clotting factors; the others are prothrombin, factor VII, and factor IX. The activation of factor X is the point at which the intrinsic and extrinsic pathways of coagulation converge, the activators being, respec- tively, a complex of factor IXa + factor VIII + phospholipid + cal- cium ions,2, 3 and a complex of factor VII + tissue factor (a lipoprotein)

'Supported in part by Grant HL 16126 from National Institutes of Health, U.S. Public Health Service.

2 C. Hougie, K. W. E. Denson, and R. Biggs, Thromb. Diath. Haemorrh. 18, 211 (1967). P. G. Barton, Nature (London) 215, 1508 (1967).

96 SLOOD CLOTTING ENZYMES [11]

calcium ions. ~,~ Factor X can also be specifically act ivated in the presence of Ca 2÷ by a fraction from Russell 's viper venom (RVV), ~-9 and it is largely owing to this tha t the present knowledge of factor X and its act ivat ion is so comprehensive.

Activated factor X part icipates in the coagulation sequence by acti- vat ing (in the presence of a cofactor, factor V, phospholipid, and Ca 2÷) prothrombin to thrombin, which then cleaves fibrinogen and factor X I I I , leading to clot formation and covalent cross-linking.

The act ivat ion of factor X is known to be proteolytic, an NH~-ter- minal glycopeptide being released from the heavy chain of factor X to give a new NH2-terminal sequence in factor Xa, I l e -Va l -Gly-Gly2 ,1° This cleavage is probably identical in all mechanisms of act ivat ion; Radcliffe and Bar ton used RVV, the intrinsic and extrinsic act ivat ing complexes, and trypsin, and found isoleucine as one of the NH2-terminal residues in all the isolated forms of factor Xa (the other terminal residue, alanine, is on the light chain).11

In act ivations of factor X in the presence of l ip id- - for instance, in act ivat ions by the tissue fac tor - fac tor V I I complex ( T F - V I I ) or by RVV in the presence of l ip id- -another proteolytic cleavage occurs to release a COOH-termina l glycopeptide from the heavy chain of either factor X or X~; this is a result of the action of factor X~. 1°,~2

The act ivat ion of factor X results in the appearance of esterase act ivi ty and the abil i ty to incorporate, and be inhibited by, diisopropyl phosphorofluoridate (DFP) . This inhibitor reacts with an active serine residue in the now-famil iar sequence Gly-Asp-Ser -Gly-Gly-Pro2 T M How- ever, the rate of the inactivation of factor Xa by D F P is considerably lower than tha t observed with many other serine esterases.

Factor X, the zymogen, is a glycoprotein of about 55,000 monomeric molecular weight tha t tends to aggregate, especially in the presence of

4 y. Nemerson, Biochemistry 5, 601 (1966). 5 W. J. Williams and D. G. Norris, J. Biol. Chem. 241, 1847 (1966). 6 Abbreviations used are: RVV, Russell's viper venom; TF-VII, the tissue factor- factor VII complex; DFP, diisopropylphosphorofluoridate; TBS, Trisobuffered saline (0.1 M NaC1-50 mM TrisoC1, pH 7.5) ; SDS, sodium dodecyl sulfate.

7 R. G. Macfarlane, Br. J. Haematol. 7, 496 (1961). s M. P. Esnouf and W. J. Williams, Biochem. J. 84, 62 (1962). K. Fujikawa, M. E. Legaz, and E. W. Davie, Biochemistry l l , 4892 (1972).

1o j. Jesty, A. K. Spencer, Y. Nakashima, Y. Nemerson, and W. Konigsberg J. Biol. Chem. 250, 4497 (1975).

1~ R. D. Radcliffe and P. G. Barton, J. Biol. Chem. 248, 6788 (1973). ~2 j. Jesty, A. K. Spencer, and Y. Nemerson, J. Biol. Chem. 249, 5614 (1974). laj. E. Leveson and M. P. Esnouf, Br. J. Haematol . 17, 173 (1969). 14 K. Titani, M. A. Hermodson, K. Fujikawa, L. H. Ericsson, K. Walsh, H. Neurath,

and E. W. Davie, Biochemistry l l , 4899 (1972).

[11] BOVINE COAGULATION FACTOR X 97

X a + Lipid Intermediate I x

T.F- ® / ar ,IC) Xo / RVV / +

Lipid/~. F.- ~ZE ~ Z o o_, o ® +

I / ~ RVV Lipid

* P 7 B - ~ ' a ~" -- Intermediate 2

FIG. 1. Pathways of activation of factor X. Reproduced from J. 3esty, A. K. Spencer, Y. Nakashima, Y. Nemerson, and W. Konigsberg, J. Biol. Chem. 250, 4497 (1975).

Ca-°÷. 15-~7 It contains about 11% carbohydrate and consists of two poly- peptide chains linked by disulfide bonds; the heavy chain contains all the carbohydrate) 6,~8 About 70% of the carbohydrate is contained in the NH2-terminal peptide lost on activation, 9 and the remaining carbohy- drate is apparently all contained in the COOH-terminal peptide lost through the action of factor X~; the final product, fl-Xa, contains no carbohydrate. 11

The two cleavages, of the NH._,- and COOH-termini of the heavy chain of factor X, can occur in either order, resulting in alternative pathways of activation in mixtures that contain lipid22 These pathways are summarized in Fig. 1. As the pathway via I1 is initiated by the action of the product, factor Xa, it occurs to a greater extent in slower activations. 12 In acti- vations with high concentrations of TF-VII, the major pathway is via ~-Xa, but the rate of conversion of ~-X.~ to fl-X~ (by the loss of the COOH-terminal peptide) is sufficient to prohibit the isolation of a-Xa from such mixtures free of f l - X , C ~ However, it has been shown that the NH..,-terminal sequence of fl-Xa produced in this way is identical with that of ~-Xa produced by RVV, and that the same NHo.-terminal pep- tide is produced in both cases? ° The COOH-terminal peptide produced is identical with that we obtain by incubation of factor X with factor X,~ to produce I1. As shown in Fig. 1, I~ can be activated directly to fl-X~ by the release of the same NH.,-terminal peptide as is released in the

1~ C. M. Jackson and D. J. Hanahan, Biochemistry 7, 4506 (1968). 1~'K. Fujikawa, M. E. Legaz, and E. W. Davie, Biochemistry 11, 4882 (1972). ~7 M. P. Esnouf, P. H. Lloyd, and J. Jesty, Biochem. J. 131,781 (1973). 1~ C. M. Jackson, Biochemistry l l , 4873 (1972).

98 BLOOD CLOTTING ENZYMES [11]

AMINO ACID AND CARBOHYDRATE COMPOSITIONS OF DERIVATIVES OF FACTOR X

Chain or peptide a

N-ter- C-ter- minal minal

X X I1 a-X~ ~-Xa pep- pep- Compo- L.C. H.C. H.C. H.C. H.C. tide t ide nent MWb: 17,000 37,000 34,000 30,000 27,000 7600 2700

Amino acid ~ Lys 7.4 15.7 15.8 15.3 14.9 0.2 0 ~ His 3.~3 8.8 7.8 6.9 5.8 1.9 1 Arg 8.2 17.8 17.1 15.5 14.6 2.0 0 Asx 15.2 25.7 25.6 19.2 20.6 7.2 0 Thr 16.5 24.1 22.6 20.2 18.0 2.9 2 Ser 11.1 18.9 19.4 13.3 13.3 6.8 1 Glx 28.1 33.4 32.5 26.3 25.1 7.2 1 Pro 2.4 15.4 10.9 12.6 8.4 3.0 5 Gly 15.3 27.3 26.8 23.7 22.9 3.6 1 Ala 6.5 24.6 22.8 19.5 17.1 5.0 2 Cys/2, 15.9 9.4 7.7 9.3 8.6 0 0 Val 5.4 20.0 19.7 18.4 16.9 1.9 1 Met 0 4.7 5.3 4.9 4.9 0 0 Ile 2.1 9.0 9.2 8.0 8.2 0.9 0 Leu 7.7 23.1 20.9 16.3 14.4 6.8 2 Tyr 2.9 6.9 6.4 5.9 6.3 1.0 0 Phe 8.1 12.4 13.1 12.3 11.4 0.2 0 TrpS 1.5 7.5 6.5 5.2 4.2 1.4 1

Carbohydrate c Hexose 0 8. lg ND h 3.2 0 4.1~ 1.6 Hexosamine 0 6.3 - - 0.7 0 3.0 0 .9 Sialic acid 0 5.3 - - 1.4 0 2.4 1.3

a Abbreviations used: L.C., light chain; H.C., heavy chain; b The molecular weights of factor X L.C. and H.C. are from C. M. Jackson [Bio-

chemistry 11, 4873 (1972)]. That of the NH~-terminal peptide is the sum of the polypeptide weight (calculated by adjustment to nearest-integer values for each residue) and the carbohydrate weight. The polypeptide molecular weight of the COOH-terminal peptide is from the amino acid sequence; the carbohydrate weight was added to this.

c Amino acid and carbohydrate contents are expressed as moles of residue per mole of chain or peptide. Calculated from the amino acid sequence.

e Determined as the S-carboxymethyl derivative. / The t ryp tophan contents of factor X L.C. and H.C., a-Xa H.C., and the NH.o-

terminal peptide are from K. Fujikawa, M. E. Legaz, and E. W. Davie [Bio- chemistry 11, 4882 and 4892 (1972)]. The t ryp tophan contents of I~ H.C. and f~-X~ H.C. are by subtract ion of the single Trp residue lost in the COOH-terminal peptide from factor H.C. and a-Xa H.C., respectively.

g From Fujikawa et a l / The figures for the NH2-terminal peptide are corrected for the lower molecular weight tha t we estimate, b

h ND, not determined.

[11] B O V I N E C O A G U L A T I O N F A C T O R X 99

I,I

aXa

Corboxy- terminal Activation Pept ide /gTa Peptide

TF- ~2~ RVV (Xa) "r a (xa)

1 HzN'TRP-ALA- ILE THR VAL- ARG °ILE-VAL" GLY" GLY - T - - ARG- GLY- HIS - - PRO- LEU-COOH

© ® 1 ®

[;In HOOC-ARG ] SER-ASX-ALA- NH 2

FIG. 2. Sites of cleavage during activation of factor X. Cleavage at A releases the activation peptide [reactions (1) and (4), Fig. 1] and results in the appearance of factor Xa activity. Cleavage at B releases the COOH-terminal peptide [reactions (2) and (3), Fig. 1]. Cleavage at C, in addition to B, occurs very slowly in the presence of factor X~ and results in the appearance of I~ [reaction (5), Fig. 1]. Reproduced from J. Jesty, A. K. Spencer, Y. Nakashima, Y. Nemerson, and W. Konigsberg, J. Biol. Chem. 250, 4497 (1975).

conversion of a-X~ to f i - X a ; and if RVV is used in this activation, lipid is not necessary. 1°,~2 f i -Xa and the two peptides produced in its formation by either pathway account entirely for the amino acid and carbohydrate contents, and the NH2- and COOH-termini, of factor X (table, Fig. 2).1°

Fujikawa et al. ~9 showed considerable homology in the amino acid sequences of the NH.,-terminal regions of prothrombin, factor IX, and the light chain of factor X; less comprehensive results on single-chain factor VI I indicate that this too has a similar NH~-terminal sequence. 2° Thus it is very likely that factor X is derived from a single-chain pre- cursor; Mat tock and Esnouf 2~ isolated a possible candidate from bovine plasma, but could not confirm that factor Xa was generated by RVV.

The heavy chains of thrombin, factor Xa, two-chain factor VII, and factor IXa are also clearly homologous with the pancreatic serine estera- ses in the NH2-terminal region. 1~,2°,22 And third, there is clear homology in the active-site regions of thrombin, factor Xa, the pancreatic enzymes, and what is probably the active-site region of factor IXa, although in this last case the fragment was obtained from factor IX. 14,22

1. K. Fujikawa, M. H. Coan, D. L. Enfield, K. Titani, L. H. Ericsson, and E. W. Davie, Proc. Natl. Acad. Sci. U.S.A. 71,427 (1974).

50 R. D. Radcliffe and Y. Nemerson, this volume [6]. 51 p. Mattock and M. P. Esnouf, Nature (London), New Biol. 242, 90 (1973). 55 D. L. Enfield, L. H. Ericsson, K. Fujikawa, K. Titani, K. A. Walsh, and H. Neu-

rath, FEBS Lett. 47, 132 (1974).

1 0 0 :BLOOD CLOTTING, ENZYMES [11]

The pancreatic enzymes and the B chain thrombin also show con- siderable homology in the immediate region of their COOH termini, which apparently remain intact during activation of the respective zymogens. ~-~ In contrast, the COOH terminus of factor X, which is cleaved by the action of factor X~, shows no homology with these other C00H- t e r mina l sequences. Its amino acid sequence is Gly-His-Ser-Glu- Ala -Pro-Ala -Thr -Trp-Thr (CHO) -Val-Pro-Pro-Pro-Leu-Pro-Leu, CHO indicating the carbohydrate moiety. 1° Not only is the composition of the terminal hexapeptide somewhat unusual, but it also seems that the carbohydrate moiety is attached to the peptide through a linkage that is rare in plasma glycoproteins, N-acetylgalactosaminyl-O-threonine2 °,26

Assay of Factor X

Principle. Factor X is activated specifically by a component of Russell's viper venom. ~ In single-stage assays for factor X, the sample is added to bovine plasma deficient in factor X; the venom is then added, and the assay is started by the addition of Ca 2+. The fact tha t factor X is always present at less than 10% of its normal value makes it the limiting factor, and the clotting time is a function of its concentration. This single-stage assay is a function not only of the concentration, but also the "act ivi ty ," of factor X, i.e., the rate at which it is activated by RVV. However, this distinction is not usually a problem in practice.

Procedure. Factor X-deficient plasma can be prepared by filtration of plasma through a Seitz filter, 27 or through charcoal, ~s but both these methods are prone to problems. So unless, there is a need for very large amounts of deficient plasma, it is simpler to buy it (Thame Diagnostics, Thame, Oxon, England; Sigma Chemical Co., St. Louis, Missouri). Before use, the plasma is reconstituted with lipid by adding 1/100 volume of "cephalin" to give a final phospholipid concentration of about 0.1 mg/ml. 29 A stock solution of Russell's viper venom is prepared in the following way: 2 mg of dried venom (Burroughs Wellcome, Tuckahoe, New York; Sigma Chemical Co.) is dissolved in 5 ml of 0.1 M NaCI-50 mM Tris-Cl pH 7.5 (TBS). Glycerol, 5 ml, is then added, and the solu-

23S. Magnusson, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 3, p. 277. Academic Press, New York, 1971. B. S. Hartley and D. M. Shotton, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 3, p. 323. Academic Press, New York, 1971.

25 j. Stenflo, J. Biol. Chem. 247, 8167 (1972). 2~ R. G. Spiro, Annu. Rev. Biochem. 39, 599 (1970). 27 F. Bachmann, F. Duckert, and F. Koller, Thromb. Diath. Haemorrh. 2, 24 (1958). 2s K. W. E. Denson, Acta Haematol. 25, 105 (1961). 29 W. N. Bell and I-t. G. Alton, Nature (London) 174, 880 (1954).

[11] BOVINE COAGULATION FACTOR X 101

tion is mixed well. This stock solution is stable for at least 4 months at --20 °. For use in the assay, 0.2 ml of this stock is diluted with 1.8 ml of TBS; the dilute solution is referred to as RVV/10.

Normal citrated bovine plasma (blood is collected into a ~o volume 0.12 M trisodium citrate, and the red cells are centrifuged down) is de- fined as containing 100 units of factor X per milliliter, and is used to calibrate the assay. Dilutions of normal plasma (e.g., ~o , ½o, ~o , Moo, and ½oo) are made in TBS and assayed in duplicate at 37°: to a glass tube add 0.1-ml sample, then 0.1 ml of factor X-deficient plasma, and then 0.1 ml RVV/10. Mix, and add 0.1 ml of 25 mM CaC12, and record the clotting time from this addition. A plot of log (clotting time) against log [factor X] should be linear over this range. To assay an un- known, the sample is diluted sufficiently for the clotting time to fall on the standard line and then assayed in the same way.

The Assay of Factor Xa

Principle. Factor Xa forms the prothrombin-converting complex with factor V, phospholipid, and Ca 2+, the activity of this complex on pro- thrombin being a function of the factor Xa concentration. The very low levels of factor Xa that are assayed mean that it is the limiting factor in the generation of thrombin in the assay.

Procedure. There is no standard preparation of factor Xa available, so the assay is calibrated with dilutions of purified factor Xa, from 1 to 20 ng/ml. The extremely good stability of factor Xa in 50% glycerol- TBS at --20 ° permits the use of a stock solution of known purity for years. The concentration of pure factor X~ can be estimated from its extinction; A I ~ _ 9.4.3o A suitable dilution of the sample in 0.02%

--280

ovalbumin-TBS (0.I ml) is added to 0.I m] of 25 mM CaCI2 in a tube at 37 ° . Factor X-deficient plasma is then added immediately, and the clotting time is recorded from this addition. A plot of log (clotting time) against log [factor Xa] should be linear over the range indicated.

The addition of plasma last to the assay mixture gives more repro- ducible results than the more usual methods where CaC12 is added last. This may be a result of the inactivation of factor X~ by plasma inhibi- tors before the addition of CaCl~ in these methods.

The Isolat ion of Factor X

Factor X, like the other vitamin K-dependent clotting factors, pro- thrombin, factor VII, and factor IX, can be adsorbed on a variety of salts of divalent metals. The majority of methods for factor X purifica-

so j. Jesty and M. P. Esnouf, Biochem. Y. 131, 791 (1973).

102 BLOOD CLOTTING ENZYMES [11]

tion use adsorption of factor X from bovine plasma on BaS04, and sub- sequent elution with citrate, as the first step in purification2 ,16,17,31-3~ It is, however, necessary to use small amounts of BaS04 in order to avoid contamination with high-molecular-weight material, and this has the dis- advantage that the associated adsorption of prothrombin, factor VII, and factor IX is low. Furthermore, BaSO~ with protein adsorbed is very difficult to handle in the subsequent washes and elutions. Barium citrate is more easily handled and results in higher yields of all four factors.

The procedure we prefer involves adsorption on barium citrate, fol- lowed by elution and precipitation with ammonium sulfate24 This part of the process is carried out for us on 50 liters of plasma by the New England Enzyme Center, and is described by Radcliffe and Nemerson. 2°

The subsequent chromatography of the barium salt eluate on an anion-exchanger is common to all published methods of purification of factor X, and is usually followed by rechromatography on DEAE- Sephadex A50 to obtain pure factor X. 16,~m~-33 In most cases factor X is eluted in the last chromatography as a double peak of almost constant specific activity. So far there are no very significant differences reported between the two forms of factor X; Jackson TM and Fujikawa et al. ~6 show only small differences in amino acid and carbohydrate compositions and molecular weights within the limits of error. Therefore in practice the two forms are generally pooled together.

The first chromatography of the barium citrate eluate is described by Radcliffe and Nemerson. 2° The presence of benzamidine-HC1 during this procedure is necessary for the prevention of activation of single- chain factor VII by contaminating factor X~. Factor X~ also degrades factor X to a species we call I~, and it is probably this that was observed by Jackson and Hanahan in their preparations of factor X~5; the pres- ence of the inhibitor completely prevents this. Factor X can be detected only by assay in the fractions of the first chromatography, owing to the high absorbance of the benzamidine-HC1. The rechromatography of factor X is carried out as follows.

Procedure. The fractions containing factor X are pooled (about 3 X 10 e units are obtained from 50 liters of starting plasma). Such pools, in 25 mM benzamidine-HC1, can be stored at - 2 0 ° for some weeks before rechromatography. The pool is concentrated by ultrafiltration (Amicon Corporation, PM-10 membrane) to about 100 ml and then dialyzed against 0.08 M sodium citrate pH 6.5. The solution is then applied at 35

31D. Papahadjopoulos, E. T. Yin, and D. J. Hanahan, Biochemistry 3, 1931 (1964). 32 C. M. Jackson, T. F. Johnson, and D. J. Hanahan, Biochemistry 7, 4492 (1968). 33 S. P. Bajaj and I/:. G. Mann, J. Biol. Chem. 248, 7729 (1973). 2, D. L. Aronson and D. M~nach~, Biochemistry 5, 2635 (1966).

[lll BOVINE COAGULATION FACTOR X 103

ml/hr to a column of DEAE-Sephadex A50 (2.5 × 30 cm) previously equilibrated in the same buffer. The chromatography is done at 4 ° . After application of the sample, the protein is eluted with a l-liter gradient, from 0.13 to 0.26 M sodium citrate pH 6.5, at the same flow rate. The fractions are assayed and their extinctions measured at 280 nm. Fractions of specific activity more than 8000 units/mg (A~sV~ = 9.617) are pooled, concentrated by ultrafiltration to about 20 ml, and finally dialyzed against 50% glycerol-TBS before storage at - 2 0 °. Such solutions are stable for years.

By disc electrophoresis and SDS-gel electrophoresis factor X pro- duced by this method is homogeneous and free of prothrombin, factor IX, and factor VII by bioassay. Such preparations do, however, contain trace amounts of factor Xa--typically about 0.01-0.1%. This level can be reduced by treatment with 20 mM DFP at pH 7.5 for 2 hr at room temperature before storage, but factor Xa activity can never be com- pletely removed by this method. It is contamination with factor Xa that is probably responsible for the generation of factor Xa activity during storage, but such generation can be detected only by assay.

Preparation of ~-Xa

The routine preparation of factor Xa that we use involves activation with the coagulant fraction of RVV in the absence of lipid, followed by chromatography of the activated mixture on DEAE-Sephadex to separate the RVV fraction from factor X~. ~° Although a-Xa is formed initially by RVV in the absence of lipid, over the course of purification this is converted autocatalytically to fl-Xa.

Procedure. Factor X, 60 mg in 120 ml of 0.15 M NaC1-50 mM Tris-C1, pH 7.5, is incubated with 0.1 absorbance unit of the coagulant fraction of RVV ~2 in the presence of 5 mM CaCl~ for 30 min at 37 °. The reaction is stopped by the addition of trisodium citrate to a concentra- tion of 6 mM, and the mixture is applied directly at 20 ml/hr tb a column (1.5 X 16 cm) of DEAE-Sephadex A50 previously equilibrated in 0.15 M NaC1-50 mM Tris-C1, pH 7.5. The protein is eluted at the same flow rate with a linear 800-ml gradient, 0.2-0.55 M NaC1, in 50 mM Tris-C1 pH 7.5. A breakthrough peak consists largely of the COOH- terminal peptide, which may be isolated by gel filtration on Sephadex G-50, as described for the preparation of I1. The venom coagulant frac- tion is eluted at about 0.25 M NaCl, close to a small peak that may represent a small proportion of the activation peptide. The factor X is eluted last. The pool of active material is concentrated by ultrafiltration (Amicon Corp., PM-10) and dialyzed against 50% glycerol-TBS before

1 0 4 BLOOD CLOTTING ENZYMES [11]

storage at --20 °. The amount of fl-X, obtained is about 70% of the theoretical yield.

Preparation of ~-X a by Activation with TF-VII

In order to obtain activation of factor X by the tissue factor-factor VII complex, sufficient of the complex must be used to attain activation in less than 15 min. This is a result of the inactivation of factor VII by factor Xa. Under these conditions the major pathway of activation is via a-Xa. 1~ Continued incubation of the mixture ensures the complete conversion of ~-Xa to fl-Xa.

Preparation of the TF-VII Complex. Tissue factor apoprotein is re- constituted with lipid as described in this volume, 35 and dialyzed against 0.1 M NaC1-50 mM Tris-C1, pH 7.5. The lipoprotein is then centrifuged down at 100,000 g for 1 hr at 4 °, and resuspended in the same buffer to give a final protein concentration of 1 mg/ml. Two-chain factor VII is added to the suspension to a final concentration of 1000 units/ml, and the solution is made 5 mM in CaC12. After incubation for 10 min at 37 °, the mixture is kept on ice before use; it is stable for 1-2 hr in this form, but slowly loses activity owing to inactivation by endogeneous factor Xa in the factor VII.

Factor X (15 mg in 30 ml of 0.1 M NaC1-50 mM Tris-C1, pH 7.5) is incubated with factor VII (as the TF-VII complex) at a final concen- tration of 150 units/ml in the presence of 5 mM CaC12 at 37 ° for 30 min. The reaction is then stopped by the addition of trisodium citrate to a concentration of 12 mM, and the mixture is then centrifuged for 1 hr at 4 ° at 100,000 g to remove the lipid. The resulting solution containing the reaction products is then chromatographed on a column of DEAE- Sephadex A50 exactly as described for the preparation of fl-Xa by activa- tion of factor X with RVV.

It should be noted that fl-Xa produced by the method described is identical in amino acid composition and NH2-terminal and C00H- terminal sequences as fl-X~ produced by the simpler method involving activation by RVV in the absence of lipid.

Preparation of 11 and the COOH-Terminal Peptide

Procedure. Factor X (30 mg in 60 ml of 25 mM Tris-C1, pH 7.5) is incubated for 110 min at 22 ° with 1.5 mg of fl-X~ in the presence of an equimolar dispersion of phosphatidylcholine (Sigma Chemical Co.) and

8~ F. A. Pitlick and Y. Nemerson, this volume [5].

[11] BOVINE COAGULATION FACTOR X 105

phosphatidylserine (ICN Pharmaceuticals) to a final phospholipid con- centration of 0.1 mg/ml, and 5 mM CaCl~. The reaction is stopped by the addition of trisodium citrate to a concentration of 12 mM. The mix- ture is then centrifuged at 100,000 g for 1 hr at 4 ° to remove the phos- pholipid. Factor X~ is removed from the mixture by stirring with 2 ml of Sepharose 4B to which soybean trypsin inhibitor has been coupled ~6 (substitution is about 5 mg of inhibitor per milliliter of gel). After 20 min at room temperature, the inhibitor-Sepharose is removed by filtration and the mixture is lyophilized. The dried material is taken up in 5 ml of 1 M guanidine-HC1 and chromatographed on a column of Sephadex G-50 (2 X 90 cm) equilibrated in 0.2 M NH4HC03 or other suitable buffer. The gel filtration is done at 4 ° at a flow rate of 10 ml/hr, and clearly separates I~ from the C00H-terminal peptide. The I~ pool is con- centrated by ultrafiltration to about 5 ml, dialyzed against 50% glycerol- TBS, and stored at --20 °. The COOH-terminal peptide is stable in water at --20 ° .

Proteolytic Action of Factor Xa

Until recently it was generally a tacit assumption that the only major role of factor X~ is the activation of prothrombin. Recent results, how- ever, show that this is not the case; factor Xa can also cleave at sub- stantial rates two bonds each in factor VII and factor X as well as the two bonds it cleaves in prothrombin. All the cleavages by factor Xa so far studied are accelerated at least 100-fold by phospholipid and calcium ions, and in the case of prothrombin activation a cofactor, factor V, is also involved.

In the activation of prothrombin, factor Xa cleaves fi~st an Arg-Thr bond to form P3 (I2), the inactive immediate precursor of thrombin2 ms P~ is then cleaved at an Arg-Ile bond to form the two-chain enzyme. 23,8~ The cleavage of prothrombin to form an alternative intermediate, P2 (I1), is not an action of factor Xa, and is solely a result of thrombin action29 Both cleavages of prothrombin by factor X~ are lipid-dependent and require the lipid-binding sites of prothrombin either intact, in the case of P3 formation, or, in the case of P3 conversion to thrombin, in the form of a complex between P3 and its activation peptide, F1-2. 4°

~° P. Cuatrecasas, d. Biol. Chem. 245, 3059 (1970). 37 C. M. Heldebrant , R. J. Butkowski, S. P. Bajaj, and K. G. Mann, J. Biol. Chem.

248, 7149 (1973). 3~j. Reuterby, D. A. Walz, L. E. McCoy, and W. H. Seegers, Thrombosis Res. 4, 885

(1974). 39 W. Kisiel and D. J. Hanahan, Biochem. Biophys. Res. Commun. 59, 570 (1974). ~° C. T. Esmon, W. G. Owen, and C. M. Jackson, J. Biol. Chem. 249, 8045 (1974).

106 BLOOD CLOTTING ENZYMES [11]

Factor X~ cleaves factor VII at two sites in lipid- and Ca2÷-dependent reactions. The first is the rapid cleavage of an Arg-Ile bond, which cor- responds with activation and the formation of 2-chain factor VII. s° The second is the slower inactivation, by the cleavage of a Y-Gly bond, where the nature of Y is not yet known, but is presumed to be Arg. This cleavage occurs in the C00H-terminal region of the heavy chain of the activated material, releasing a 12,000-dalton peptide that contains the active serine residue. 2°

Factor Xa cleaves factor X at three sites in the heavy chain, but the third cleavage [the formation of Is; reaction (5), Fig. 1] is relatively insignificant even in very slow activations of factor X. The most rapid cleavage is that of an Arg-Gly bond in the COOH-terminal region of the heavy chain of either factor X or a - X a as discussed in this chapter. A slower cleavage is that of the Arg-Ile bond that is cleaved by other acti- vators to release the normal NHs-terminal activation peptide, and re- sults in the appearance of factor Xa activity.

So far all the protein substrates that are known for factor X~ are vitamin K-dependent clotting factors, except for one very poor substrate, chymotrypsinogen. ~1 And it appears that the lipid-binding sites of the substrate must be either intact or in the form of a complex with the sub- strate. Both Stenflo et al. 42 and Nelsestuen et al. 4~ have demonstrated the existence of ,/-carboxylglutamic acid residues in CaS÷-binding peptides from the NHs-terminal region of prothrombin, and the latter group has preliminary evidence for the existence of "extra" negative charges in a homologous peptide from factor X, 44 whose amino acid composition cor- responds closely with residues 5 to 43 of the light chain? s In support of the idea that the light chain of factors X and Xa contains the Ca 2÷- binding sites of the molecules is the identical sequence of a tetrapeptide in the NH~-terminal regions of prothrombin and the light chain of factor X, Leu-Glu-Glu-Val (residues 6 to 9 in prothrombin, and 5 to 8 in the light chain of factor X).19 Stenflo et al. ~ showed that both the glutamic residues of this peptide in normal prothrombin and ~-carboxylated, whereas in dicoumarol-induced prothrombin they are not. Thus it seems very likely that factor X contains such residues in the NHs-terminal region of the light chain, and that these are the Ca2÷-binding sites of factors X and X~. ~1 j. I-I. Milstone and V. K. Milstone, Proc. Soc. Exp. Biol. Med. 117, 290 (1964).

J. Stenflo, P. Fernlund, W. Egan, and P. Roepstorff, Proc. Natl . Acad. Sci. U.S.A. 71, 2730 (1974).

a G. L. Nelsestuen, T. H. Zytkovicz, and J. B. Howard, J. Biol. Chem. 249, 6347 (1974).

4~j. B. Howard and G. L. Nelsestuen, Fed. Proc., Fed. Am. Soc. Exp. Biol. 33, 1473 (1974).

[12] FACTOR V 107

Of the cleavages by factor Xa described (including that of chymo- trypsinogen), four are at Arg-Ile bonds and correspond with the activa- tion of zymogens, while two are at Arg-Gly bonds, and two are at Arg-Thr bonds. Thus although factor Xa is apparently completely specific for arginyl bonds, the distal residue does not in itself appear to specify the site of cleavage.

Acknowledgment We thank Dr. C. M. Jackson for providing copies of papers before publication.

[12] Factor V By ROBERT W. COLMAN and ROBERT M. WEINBERG

Bovine Factor V

Assay Method

Principle. Factor V 1 is a plasma protein that is necessary for the optimal rate of conversion of prothrombin to thrombin in both the in- trinsic and extrinsic system of blood coagulation. The enzyme responsi- ble for the hydrolytic cleavage of prothrombin to thrombin is activated factor X (Xa),~ which, in addition to factor V, requires phospholipid and ionic calcium.

Prothrombin

Factor V +

Phospholipid +

Factor X a +

C a 2+

Fibrinogen

Thrombin l '

Fibrin

Thus, to assay factor V, one must measure the rate of activation of prothrombin to thrombin. Thrombin activity can be measured by its

1 A. G. Ware, R. C. Murphy, and W. H. Seegers, Science 106, 618 (1947). 2j. H. Milstone, Fed. Proc., Fed. Am. Soc. Exp. Biol. 23, 742 (1964).