- Email: [email protected]
[10] Bovine factor X (Stuart factor)
[10]
BOVINE FACTOR X (STUART FACTOR)
89
Properties of Bovine Factor VIII Factor V I I I is a large glycoprotein with a molecular weight greater than one millionJ -~ Factor VIII isolated by the present procedure has both coagulant activity and platelet aggregating activity (yon Willebrand factor). The coagulant activity is increased 50- to 100-fold in the presence of proteases such as thrombin, factor Xa, and trypsin. Whether the coagulant activity and platelet aggregating activity reside in the same molecule has not been established. Thus, it is possible that the protein isolated from the gel filtration step is primarily yon Willebrand factor associated with a trace amount of factor VIII.
[10] Bovine Factor X (Stuart Factor) B y KAzuo FUJIKAWA and EARL W. DAVIE
Factor X is a glycoprotein which plays a central role in blood coagulation. 1 It is present in a precursor form in normal plasma, but is inactive in patients with Stuart disease. Factor X is activated by two physiological pathways called the intrinsic and extrinsic systems. In the intrinsic coagulation system, factor X is activated by factor IXa in the presence of factor VIII, phospholipid, and calcium ions. In the extrinsic system, it is activated by tissue factor and another plasma protein called factor VII. Factor X is also activated by other proteolytic enzymes, such as pancreatic trypsin and a protease from Russell's viper venom. Factor X~ is a serine protease, which in turn activates prothrombin in the presence of factor V, calcium ions, and phospholipid. Recently, other important functions of factor Xa have been reported. Factor X~ activates factor VII, and this reaction may play an important role in the extrinsic coagulation pathway. 2 Factor X.~ also modifies factor V I I I and potentiares its effect in the intrinsic pathway. 3 Bovine factor X has been isolated in two different forms, factor X1 and factor X~, which are separated by column chromatography on DEAE-Sephadex. 4,~ These two proteins have the same specific activity 1E. W. Davie and K. Fujikawa, Annu. Rev. Biochem. 44, 99 (1975). ~. R. ]%dcliffe and Y. Nemerson, J. Biol. Chem. 250, 388 (1975). 3E. W. Davie, K. Fujikawa, M. E. Legaz, and H. Kato, Cold Spring Harbor Con]. Cell Proli]eration, 2, 65 (1975). 4K. Fujikawa, M. W. Legaz, and E. W. Davie, Biochemistry II, 4892 (1972). 5C. M. Jackson, Biochemistry ll, 4873 (1972).
90
BLOOD CLOTTING ENZYMES
[10]
and amino acid content. They also have the same migration on sodium dodecyl sulfate (SDS) or disc gel electrophoresis. The two proteins may differ in their carbohydrate content, ~ although this has not been confirmed.4 Factor X was first isolated by Esnouf and Williams as the protein substrate for the Russell's viper venom protease2 Thereafter, more efficient methods have been developed by several different groups of investigators in which the yield and quality of this protein have been improved. 4,5,7-9 The use of protease inhibitors during the purification steps has been of considerable importance for the isolation of undegraded protein in the precursor form. Thus far, a homogeneous preparation of human factor X has not been described.
Reagents Factor X-deficient plasma: Normal bovine blood is collected in one-tenth volume of 0.1 M sodium oxalate and plasma is obtained by centrifugation at 7000 g for 15 min. The plasma is then filtered through a Seitz filter to remove factor X, 1° and the deficient plasma is stored in small aliquots at --60 °. If available, congenital factor X-deficient plasma can also be used. Russell's viper venom--phospholipid mixture: One-half milligram of crude Russell's viper venom (Ross Allen Reptile Institute, Silver Springs, Florida, or Sigma Chemical .Co., St. Louis, Missouri) is mixed with 10 ml of the stock phospholipid suspension described in the previous section under factor IX isolation. This mixture is stored in small aliquots (0.5 ml) at --20 ° and diluted 10-fold with 0.15 M NaC1 before use. This solution is stable for several hours at 4 ° . Michaelis buffer. Michaelis buffer is made as follows: 4.9 g of sodium acetate, 7.2 g of sodium barbital, 8.4 g of sodium chloride, and 100 mg of bovine serum albumin are dissolved in 1 liter of distilled water, and the pH is adjusted to 7.4 with 1 N HC1. This buffer is stored at 4 ° . Protein is determined by the biuret method H using crystalline bovine serum albumin as a standard or the spectrophotometric method is em6 M. P. Esnouf and W. J. Williams, Biochem. J. 84, 62 (1962). 7 C. M. Jackson and D. J. Hanahan, Biochemistry 7, 4506 (1968). 8 j . Jesty and M. P. Esnouf, Biochem. d. 131, 791 (1973). 9S. P. Bajaj and K. G. Mann, J. Biol. Chem. 248, 7729 (1973). lo F. Bachmann, F. Duckert, and F. Koller, Thromb. Diath. Haemorrh. 2, 24 (1958). 11A. G. Gornall, C. S. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
[10]
BOVINE FACTOR X (STUART FACTOR)
91
ployed for the homogeneous preparations using an extinction coefficient of 14.5.
Assay Procedure A test sample is diluted with Michaelis buffer to give a clotting time between 20 and 60 sec. A 0.1-ml aliquot of the diluted sample is incubated at 37 ° for 30 sec with 0.1 ml of the diluted Russell's viper venomphospholipid mixture and 0.1 ml of deficient plasma. A 0.1-ml solution of 0.025 M CaCI., is then added to the mixture and the clotting time is recorded. A standard activity curve is made by diluting normal plasma with Michaelis buffer at 1:10, 1:20, 1:40, 1:80, and 1:160. Duplicate assays are recommended for each sample. For the assay of factor Xa, Russell's viper venom is deleted from the Russell's viper venom-phospholipid mixture. Purification Procedure
Reagents. All stock solutions and the preparation of DEAE-Sephadex are the same as those previously described in this volume in Chapters on factor VIII [9] and factor IX [8]. Purification Steps The purification steps up to the batchwise DEAE-Sephadex fraction are the same as those described in this volume [8]. Fifteen hundred milliliters of 0.2 M sodium citrate eluate from the DEAE-Sephadex is diluted with an equal volume of cold water and diisopropyl phosphorofluoridate (DFP) is added to give a final concentration of 0.1 mM. This solution is then applied to a column of DEAE-Sephadex (4.0 X 20 cm) which was previously equilibrated with 0.05 M sodium citrate buffer, pH 7.0, containing 1 mM benzamidine-HC1. After application of the sample, the protein is eluted by a linear gradient composed of 3 liters of 0.1 M sodium citrate buffer and 3 liters of 0.2 M sodium citrate, both containing 1 mM benzamidine-H.C1. The first 1.5 liters of the eluate are discarded; the remaining eluate is collected in 25-ml fractions. When the fractions (dilution 1:100 with Michaelis buffer) are assayed, factor X activity is found in two peaks, which parallel two protein peaks eluted between 0.12 and 0.13 M sodium citrate buffer. The fractions of factor X1 and factor X~ are pooled, and DFP is added to a final concentration of 0.1 mM. The solution is then concentrated to about 50 ml by
92
[10]
:BLOOD CLOTTING ENZYMES
Diaflo ultrafiltration using a PM-10 filter membrane. An equal volume of glycerol is added to the concentrated sample, and the solution is stored at --20 ° . Factor X activity is stable for a number of months under these conditions. About 100 mg of factor X1 and factor X2 are obtained from 45 liters of plasma with a recovery of 45%. Before use, the stock sample is dialyzed against an appropriate buffer such as 0.02 M Tris-HC1, pH 7.2, containing 0.2 M NaC1. NH4HC03, 0.1 M, is used for the preparation of salt-free preparations. One unit of factor X is defined as that amount of activity present in 1.0 ml of normal human plasma, and specific activity is expressed as units per milligram of protein. A typical purification is shown in Table I. (One to two nanograms of factor Xa gives a clotting time of about 30 sec with bovine factor X-deficient plasma in the one-stage assay described above.)
Other General Properties of Factor X
Bovine factor X has a molecular weight of 55,100. It is composed of a heavy and a light chain, and these two chains are held together by a disulfide bond(s). A single-chain factor X has also been reported, but
TABLE I PURIFICATION OF BOVINE FACTOR X 4a
Purification step
Volume (ml)
Plasma 44,000 BaSO4 eluate 5,000 Batchwise 1,680 DEAE-Sephadex D E A E Sephadex 1,000 column
Total protein (mg)
Total activity (units)
Specific activity (units/ mg)
Recoyery (%)
Purification factor
3.32 X 1 0 6 b 1.69 X 1 0 4 b 722
32,600 c 27,400 d 23,400
0. 0088 1.6 32
100 84 71
1 190 3,700
15,900
98
49
10,100
163 ~
a From K. Fujikawa, M. W. Legaz, and E. W. Davie, Biochemistry 11, 4892 (1972). b Protein concentration was determined by the biuret method. c The original factor X activity was determined on a small sample collected in the absence of heparin, benzamidine, and soybean trypsin inhibitor. d This sample was passed through a D E A E - S e p h a d e x column (1.0 X 1.0 cm) which was equilibrated with 0.2 M sodium citrate to remove heparin. e Protein concentration was determined by absorbance at 280 nm.
[10]
BOVINE FACTOR X (STUART FACTOR)
Ala 1 S
I
93
COOH
IleArg ~
Trp CHO
COOH
Arg Gly ~ CHO Factor X
O COOH
Ala
(
t S !
@
Ile
ArgGly~
COOH CHO
Factor
Xaa
0 Ala
COOH
/ s S
t (
@
Ile Arg Factor Xa0
Fie. 1. Mechanism of activation of bovine factor X. The active center Ser-233 is circled. CI-IO refers to carbohydrate. Modified from K. Fujikawa, M. E. Legaz, and E. W. Davie, Biochemistry 13, 1508 (1974) and K. Fujikawa, K. Titani, and E. W. Davie, Proc. Natl. Acad. Sci. U.S.A. 72, 3359 (1975).
this preparation has not been well characterized. 12 Recently, the total amino acid sequences of the heavy and light chains of bovine factor X1 have been reported by Enfield e t al. 13 and Titani e t al. TM These studies have shown a great deal of homology between factor X, prothrombin, and factor IX. These three proteins, in addition to factor VII, require vitamin K for their biosynthesis. ,2 p. Mattock and M. P. Esnouf, Nature (London) N e w Biol. 242, 90 (1973). ,3 D. L. Enfield, L. I-I. Ericsson, K. A. Walsh, It. Neurath, and K. Titani, Proc. Natl. Acad. Sci. U.S.A. "/2, 16 (1975). 14K. Titani, K. Fujikawa, D. L. Enfield, L. H. Ericsson, K. A. Walsh, and I-I. Neurath, Proc. Natl. Acad. Sci. U.S.A. 72, 3082 (1975).
94
BLOOD CLOTTING ENZYMES
[10]
TABLE II AMINO ACID AND CARBOHYDRATE COMPOSITIONS OF FACTOR X~ AND FACTOR
Component A m i n o acid ~ Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Asparagine Glutamine Molecular weight (protein) c Carbohydrate Hexose Hexosamine Neuraminic acid C a r b o h y d r a t e (%) Protein (%) ~ Molecular weight (glycoprotcin) a
Xla a
Factor X,
Factor Xaa
Factor Xat~
Actiration peptide
Degradation peptide
23 12 25 27 31 31 45 18 41 31 24 26 5 12 30 10 21 7 14 14 49,801
23 10 23 21 28 24 39 15 38 26 24 24 5 11 23 9 21 6 13 13 44,368
23 9 23 21 26 23 38 10 37 24 24 23 5 11 21 9 21 5 13 13 42,625
0 2 2 6 3 7 6 3 3 5 0 2 0 1 7 1 0 1 1 1 5,452
0 1 0 0 2 1 1 5 1 2 0 1 0 0 2 0 0 1 0 0 1,761
10 10 7
2-3 1 1
9.6 90.4 55,070
2.1 97.9 45,310
0 0 0 0 100 42,630
9 7 5 42.6 57.4 9,490
2-3 1 1 34.8 65.2 2,700
a From K. Fujikawa, K. Titani, a n d E. W. Davie, Proc. Natl. Acad. Sci. U.S.A. 72, 3359 (1975). b Calculated from the amino acid sequence. Ten glutamic acid residues were assumed to be 3,-carboxyglutamic acid residues [D. L. Enfield, L. H. Ericsson, K. A. Walsh, H. N e u r a t h , a n d K. Titani, Proc. Natl. Acad. Sci. U.S.A. 72, 16 (1975)]. Calculated on the basis of three residues of hexose in factor X~a and the degradation peptide. Some revision will be necessary for these values, however, since the c a r b o h y d r a t e analyses have shown some variation. Consequently, a s u m m a t i o n of the hexose, hexosamine, a n d neuraminic acid in factor X ~ and the activation peptide does not equal factor X~.
[lll
BOVINE COAGULATION FACTOR X
95
During the activation of factor X by the intrinsic and extrinsic pathways, 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 hydrolysis 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 pancreatic proteases? s-~l The amino acid and carbohydrate compositions for factor X~, factor X~,, and factor X ~ are shown in Table II. 17 15K. 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 a n d YALE NEMERSON Factor X (Stuart-Prower factor) is one of the four vitamin K-dependent 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, respectively, a complex of factor IXa + factor VIII + phospholipid + calcium 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).
BOVINE FACTOR X (STUART FACTOR)
89
Properties of Bovine Factor VIII Factor V I I I is a large glycoprotein with a molecular weight greater than one millionJ -~ Factor VIII isolated by the present procedure has both coagulant activity and platelet aggregating activity (yon Willebrand factor). The coagulant activity is increased 50- to 100-fold in the presence of proteases such as thrombin, factor Xa, and trypsin. Whether the coagulant activity and platelet aggregating activity reside in the same molecule has not been established. Thus, it is possible that the protein isolated from the gel filtration step is primarily yon Willebrand factor associated with a trace amount of factor VIII.
[10] Bovine Factor X (Stuart Factor) B y KAzuo FUJIKAWA and EARL W. DAVIE
Factor X is a glycoprotein which plays a central role in blood coagulation. 1 It is present in a precursor form in normal plasma, but is inactive in patients with Stuart disease. Factor X is activated by two physiological pathways called the intrinsic and extrinsic systems. In the intrinsic coagulation system, factor X is activated by factor IXa in the presence of factor VIII, phospholipid, and calcium ions. In the extrinsic system, it is activated by tissue factor and another plasma protein called factor VII. Factor X is also activated by other proteolytic enzymes, such as pancreatic trypsin and a protease from Russell's viper venom. Factor X~ is a serine protease, which in turn activates prothrombin in the presence of factor V, calcium ions, and phospholipid. Recently, other important functions of factor Xa have been reported. Factor X~ activates factor VII, and this reaction may play an important role in the extrinsic coagulation pathway. 2 Factor X.~ also modifies factor V I I I and potentiares its effect in the intrinsic pathway. 3 Bovine factor X has been isolated in two different forms, factor X1 and factor X~, which are separated by column chromatography on DEAE-Sephadex. 4,~ These two proteins have the same specific activity 1E. W. Davie and K. Fujikawa, Annu. Rev. Biochem. 44, 99 (1975). ~. R. ]%dcliffe and Y. Nemerson, J. Biol. Chem. 250, 388 (1975). 3E. W. Davie, K. Fujikawa, M. E. Legaz, and H. Kato, Cold Spring Harbor Con]. Cell Proli]eration, 2, 65 (1975). 4K. Fujikawa, M. W. Legaz, and E. W. Davie, Biochemistry II, 4892 (1972). 5C. M. Jackson, Biochemistry ll, 4873 (1972).
90
BLOOD CLOTTING ENZYMES
[10]
and amino acid content. They also have the same migration on sodium dodecyl sulfate (SDS) or disc gel electrophoresis. The two proteins may differ in their carbohydrate content, ~ although this has not been confirmed.4 Factor X was first isolated by Esnouf and Williams as the protein substrate for the Russell's viper venom protease2 Thereafter, more efficient methods have been developed by several different groups of investigators in which the yield and quality of this protein have been improved. 4,5,7-9 The use of protease inhibitors during the purification steps has been of considerable importance for the isolation of undegraded protein in the precursor form. Thus far, a homogeneous preparation of human factor X has not been described.
Reagents Factor X-deficient plasma: Normal bovine blood is collected in one-tenth volume of 0.1 M sodium oxalate and plasma is obtained by centrifugation at 7000 g for 15 min. The plasma is then filtered through a Seitz filter to remove factor X, 1° and the deficient plasma is stored in small aliquots at --60 °. If available, congenital factor X-deficient plasma can also be used. Russell's viper venom--phospholipid mixture: One-half milligram of crude Russell's viper venom (Ross Allen Reptile Institute, Silver Springs, Florida, or Sigma Chemical .Co., St. Louis, Missouri) is mixed with 10 ml of the stock phospholipid suspension described in the previous section under factor IX isolation. This mixture is stored in small aliquots (0.5 ml) at --20 ° and diluted 10-fold with 0.15 M NaC1 before use. This solution is stable for several hours at 4 ° . Michaelis buffer. Michaelis buffer is made as follows: 4.9 g of sodium acetate, 7.2 g of sodium barbital, 8.4 g of sodium chloride, and 100 mg of bovine serum albumin are dissolved in 1 liter of distilled water, and the pH is adjusted to 7.4 with 1 N HC1. This buffer is stored at 4 ° . Protein is determined by the biuret method H using crystalline bovine serum albumin as a standard or the spectrophotometric method is em6 M. P. Esnouf and W. J. Williams, Biochem. J. 84, 62 (1962). 7 C. M. Jackson and D. J. Hanahan, Biochemistry 7, 4506 (1968). 8 j . Jesty and M. P. Esnouf, Biochem. d. 131, 791 (1973). 9S. P. Bajaj and K. G. Mann, J. Biol. Chem. 248, 7729 (1973). lo F. Bachmann, F. Duckert, and F. Koller, Thromb. Diath. Haemorrh. 2, 24 (1958). 11A. G. Gornall, C. S. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
[10]
BOVINE FACTOR X (STUART FACTOR)
91
ployed for the homogeneous preparations using an extinction coefficient of 14.5.
Assay Procedure A test sample is diluted with Michaelis buffer to give a clotting time between 20 and 60 sec. A 0.1-ml aliquot of the diluted sample is incubated at 37 ° for 30 sec with 0.1 ml of the diluted Russell's viper venomphospholipid mixture and 0.1 ml of deficient plasma. A 0.1-ml solution of 0.025 M CaCI., is then added to the mixture and the clotting time is recorded. A standard activity curve is made by diluting normal plasma with Michaelis buffer at 1:10, 1:20, 1:40, 1:80, and 1:160. Duplicate assays are recommended for each sample. For the assay of factor Xa, Russell's viper venom is deleted from the Russell's viper venom-phospholipid mixture. Purification Procedure
Reagents. All stock solutions and the preparation of DEAE-Sephadex are the same as those previously described in this volume in Chapters on factor VIII [9] and factor IX [8]. Purification Steps The purification steps up to the batchwise DEAE-Sephadex fraction are the same as those described in this volume [8]. Fifteen hundred milliliters of 0.2 M sodium citrate eluate from the DEAE-Sephadex is diluted with an equal volume of cold water and diisopropyl phosphorofluoridate (DFP) is added to give a final concentration of 0.1 mM. This solution is then applied to a column of DEAE-Sephadex (4.0 X 20 cm) which was previously equilibrated with 0.05 M sodium citrate buffer, pH 7.0, containing 1 mM benzamidine-HC1. After application of the sample, the protein is eluted by a linear gradient composed of 3 liters of 0.1 M sodium citrate buffer and 3 liters of 0.2 M sodium citrate, both containing 1 mM benzamidine-H.C1. The first 1.5 liters of the eluate are discarded; the remaining eluate is collected in 25-ml fractions. When the fractions (dilution 1:100 with Michaelis buffer) are assayed, factor X activity is found in two peaks, which parallel two protein peaks eluted between 0.12 and 0.13 M sodium citrate buffer. The fractions of factor X1 and factor X~ are pooled, and DFP is added to a final concentration of 0.1 mM. The solution is then concentrated to about 50 ml by
92
[10]
:BLOOD CLOTTING ENZYMES
Diaflo ultrafiltration using a PM-10 filter membrane. An equal volume of glycerol is added to the concentrated sample, and the solution is stored at --20 ° . Factor X activity is stable for a number of months under these conditions. About 100 mg of factor X1 and factor X2 are obtained from 45 liters of plasma with a recovery of 45%. Before use, the stock sample is dialyzed against an appropriate buffer such as 0.02 M Tris-HC1, pH 7.2, containing 0.2 M NaC1. NH4HC03, 0.1 M, is used for the preparation of salt-free preparations. One unit of factor X is defined as that amount of activity present in 1.0 ml of normal human plasma, and specific activity is expressed as units per milligram of protein. A typical purification is shown in Table I. (One to two nanograms of factor Xa gives a clotting time of about 30 sec with bovine factor X-deficient plasma in the one-stage assay described above.)
Other General Properties of Factor X
Bovine factor X has a molecular weight of 55,100. It is composed of a heavy and a light chain, and these two chains are held together by a disulfide bond(s). A single-chain factor X has also been reported, but
TABLE I PURIFICATION OF BOVINE FACTOR X 4a
Purification step
Volume (ml)
Plasma 44,000 BaSO4 eluate 5,000 Batchwise 1,680 DEAE-Sephadex D E A E Sephadex 1,000 column
Total protein (mg)
Total activity (units)
Specific activity (units/ mg)
Recoyery (%)
Purification factor
3.32 X 1 0 6 b 1.69 X 1 0 4 b 722
32,600 c 27,400 d 23,400
0. 0088 1.6 32
100 84 71
1 190 3,700
15,900
98
49
10,100
163 ~
a From K. Fujikawa, M. W. Legaz, and E. W. Davie, Biochemistry 11, 4892 (1972). b Protein concentration was determined by the biuret method. c The original factor X activity was determined on a small sample collected in the absence of heparin, benzamidine, and soybean trypsin inhibitor. d This sample was passed through a D E A E - S e p h a d e x column (1.0 X 1.0 cm) which was equilibrated with 0.2 M sodium citrate to remove heparin. e Protein concentration was determined by absorbance at 280 nm.
[10]
BOVINE FACTOR X (STUART FACTOR)
Ala 1 S
I
93
COOH
IleArg ~
Trp CHO
COOH
Arg Gly ~ CHO Factor X
O COOH
Ala
(
t S !
@
Ile
ArgGly~
COOH CHO
Factor
Xaa
0 Ala
COOH
/ s S
t (
@
Ile Arg Factor Xa0
Fie. 1. Mechanism of activation of bovine factor X. The active center Ser-233 is circled. CI-IO refers to carbohydrate. Modified from K. Fujikawa, M. E. Legaz, and E. W. Davie, Biochemistry 13, 1508 (1974) and K. Fujikawa, K. Titani, and E. W. Davie, Proc. Natl. Acad. Sci. U.S.A. 72, 3359 (1975).
this preparation has not been well characterized. 12 Recently, the total amino acid sequences of the heavy and light chains of bovine factor X1 have been reported by Enfield e t al. 13 and Titani e t al. TM These studies have shown a great deal of homology between factor X, prothrombin, and factor IX. These three proteins, in addition to factor VII, require vitamin K for their biosynthesis. ,2 p. Mattock and M. P. Esnouf, Nature (London) N e w Biol. 242, 90 (1973). ,3 D. L. Enfield, L. I-I. Ericsson, K. A. Walsh, It. Neurath, and K. Titani, Proc. Natl. Acad. Sci. U.S.A. "/2, 16 (1975). 14K. Titani, K. Fujikawa, D. L. Enfield, L. H. Ericsson, K. A. Walsh, and I-I. Neurath, Proc. Natl. Acad. Sci. U.S.A. 72, 3082 (1975).
94
BLOOD CLOTTING ENZYMES
[10]
TABLE II AMINO ACID AND CARBOHYDRATE COMPOSITIONS OF FACTOR X~ AND FACTOR
Component A m i n o acid ~ Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Asparagine Glutamine Molecular weight (protein) c Carbohydrate Hexose Hexosamine Neuraminic acid C a r b o h y d r a t e (%) Protein (%) ~ Molecular weight (glycoprotcin) a
Xla a
Factor X,
Factor Xaa
Factor Xat~
Actiration peptide
Degradation peptide
23 12 25 27 31 31 45 18 41 31 24 26 5 12 30 10 21 7 14 14 49,801
23 10 23 21 28 24 39 15 38 26 24 24 5 11 23 9 21 6 13 13 44,368
23 9 23 21 26 23 38 10 37 24 24 23 5 11 21 9 21 5 13 13 42,625
0 2 2 6 3 7 6 3 3 5 0 2 0 1 7 1 0 1 1 1 5,452
0 1 0 0 2 1 1 5 1 2 0 1 0 0 2 0 0 1 0 0 1,761
10 10 7
2-3 1 1
9.6 90.4 55,070
2.1 97.9 45,310
0 0 0 0 100 42,630
9 7 5 42.6 57.4 9,490
2-3 1 1 34.8 65.2 2,700
a From K. Fujikawa, K. Titani, a n d E. W. Davie, Proc. Natl. Acad. Sci. U.S.A. 72, 3359 (1975). b Calculated from the amino acid sequence. Ten glutamic acid residues were assumed to be 3,-carboxyglutamic acid residues [D. L. Enfield, L. H. Ericsson, K. A. Walsh, H. N e u r a t h , a n d K. Titani, Proc. Natl. Acad. Sci. U.S.A. 72, 16 (1975)]. Calculated on the basis of three residues of hexose in factor X~a and the degradation peptide. Some revision will be necessary for these values, however, since the c a r b o h y d r a t e analyses have shown some variation. Consequently, a s u m m a t i o n of the hexose, hexosamine, a n d neuraminic acid in factor X ~ and the activation peptide does not equal factor X~.
[lll
BOVINE COAGULATION FACTOR X
95
During the activation of factor X by the intrinsic and extrinsic pathways, 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 hydrolysis 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 pancreatic proteases? s-~l The amino acid and carbohydrate compositions for factor X~, factor X~,, and factor X ~ are shown in Table II. 17 15K. 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 a n d YALE NEMERSON Factor X (Stuart-Prower factor) is one of the four vitamin K-dependent 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, respectively, a complex of factor IXa + factor VIII + phospholipid + calcium 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).