56
BLOOD CLOTTING ENZYMES
[7]
tion of 1-2% of the single-chain factor VII during the assay, rather than inherent single-chain activity. A trace level of double-chain factor VII could generate enough factor Xa to feed back and activate single-chain material, since the activation of factor VII by factor Xa in the presence of phospholipids and calcium ions is rapid2 Possible activation by other enzymes generated during the assay, such as intrinsic pathway enzymes, must also be considered.
[7] H u m a n
Factor XII (Hageman Factor)
By JOHN H. GRIFFIN and CHARLES G. COCHRANE Factor X I I is an enzyme that circulates in zymogen form in blood and is capable, upon activation, of initiating the clotting, fibrinolytic, and kinin-generating systems. 1-6 Initially described by Ratnoff, 7 factor XII-deficient persons are asymptomatic. In addition to factor XII, the activation in plasma of clotting, fibrinolysis, and kinin generation by negatively charged foreign surfaces also appears to require prekallikrein 4 and a high-molecular-weight form of kininogen, s The precise description of the mechanism(s) of activation of the Hageman factor pathways has been limited by the difficulty of isolating satisfactory amounts of the factor X I I zymogen and of stabilizing preparations of factor X I I in zymogen form. Factor X I I in zymogen form has been isolated from human and rabbit blood, 9-11 and structural changes accompanying the i j . Margolis, Ann. N.Y. Acad. Sci. 104, 133 (1963). 20. D. Ratnoff, Prog. Hematol. 5, 204 (1965). *C. G. Cochrane, S. Revak, B. S. Aikin, and K. D. Wuepper, in "Inflammation: Mechanisms and Control" (I. H. Lepow and P. A. Ward, eds.), p. 119. Academic Press, New York, 1972. 4K. D. Wuepper, in "Inflammation: Mechanisms and Control" (I. H. Lepow and P. A. Ward, eds.), p. 93. Academic Press, New York, 1972. 5E. H. Magoon, J. Spragg, and K. F. Austen, Adv. Biosci. 12, 225 (1974). C. G. Cochrane, S. D. Revak, K. D. Wuepper, A. Johnston, and D. C. Morrison, Adv. Biosci. 12, 237 (1974). ' O. D. Ra~noff and J. E. Colopy, J. Clin. Invest. 34, 602 (1955). 8I~. D. Wuepper, D. R. Miller, and M. Lacombe, Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 859 (1975). 9 C. G. Cochrane and K. D. Wuepper, J. Exp. Med. 134, 986 (1971). 1oS. D. Revak, C. G. Cochrane, A. R. Johnston, and T. E. Hugli, J. Clin. Invest. 54, 619 (1974). 11H. Saito, O. D. Ratnoff, and V. H. Donaldson, Circ. Res. 34, 641 (i974).
[7]
HUMAN FACTOR XII
57
activation of human and rabbit factor X I I have been reported2 °,12-14 Nonetheless, variability in reproducing the isolation 9,1° of factor X I I and in stabilizing the isolated zymogen ~5 led us to develop more reliable methods 1~ of isolating and stabilizing factor XII which are described here. In all manipulations involving factor XII, exposure to glass must be avoided by using plasticware or freshly siliconized glassware (Siliclad, Clay-Adams, Inc.) since factor XII sticks very readily to glass.
Assay Methods Factor X I I activity can be conveniently assayed for its ability to activate isolated plasma prekallikrein or to clot factor XII-deficient plasma, as described below. Less conveniently, it can be assayed for its ability to activate fibrinolysis. 17 Highly purified factor XII can hydrolyze, albeit inefficiently, N-Ac-Gly-Lys-0Et. is
Prekallikrein Activation Assay After activation of factor XII by kaolin or by trypsin, factor XIIa is allowed to convert the zymogen prekallikrein to its active form, kallikrein, which is then conveniently assayed spectrophotometrically for its ability to hydrolyze Bz-Arg-OEt.
Reagents Tris-buffered saline (TBS): 0.01 M Tris-C1, 0.15 M NaC1, pH 7.4 Kaolin (Fisher Scientific Co.) : acid-washed, 5 mg/ml in TBS Bz-Arg-OEt (BAEE): (Mann Research): 1 mM in 0.1 M Tris-C1, 0.05 M NaCI, pH 8.0 (prepared daily and kept at 4 ° until needed) Trypsin ("Trypsin-TPCK", Worthington Biochemical Corp.): Stock solution, 1 mg/ml, in 1 mM acetic acid kept at 4 ° and diluted 1:100 into trypsin assay buffer immediately before each assay. Trypsin assay buffer: 0.1 M sodium phosphate, 0.05 M NaC1, pH 7.6 12C. G. Cochrane, S. D. Revak, and K. D. Wuepper, J. Exp. Med. 138, 1564 (1973). 1~A. P. Kaplan and K. F. Austen, J. Exp. Med. 133, 696 (1971). 14C. R. McMillin, H. Saito, O. D. Ratnoff, and A. C. Walton, J. Clin. Invest. 54, 1312 (1974). 15H. Z. M o v a t and A. H. Ozge-Anwar, J. Lab. Clin. Med. 84, 861 (1974). 1~j . H. Griffin and C. G. Coehrane, manuscript in preparation. i~ A. P. Kaplan and K. F. Austen, J. Exp. Med. 136, 1378 (1972). 18R. J. Ulevitch, D. Letchford, and C. G. Cochrane. Thromb. Diath. Haemorrh. 31, 30 (1974).
58
BLOOD CLOTTING ENZYMES
[7]
Ovomucoid trypsin inhibitor (Worthington Biochemical Corp.): 2 mg/ml in TBS (solution stable at 4 ° at least 1 month) Prekallikrein: Substrate rabbit prekallikrein is partially purified from citrated rabbit plasma as described elsewhere. 19 Briefly, the ~/-globulin fraction obtained from a DEAE-Sephadex column is further fractionated on a CM-Sephadex column with elution effected by a NaC1 gradient. The pool of prekallikrein activity obtained from the CM-Sephadex column is dialyzed against PBS (0.01 M Na phosphate, 0.15 M NaC1, pH 7.0). The concentration of the pool of prekallikrein is adjusted so that, after total conversion of prekallikrein to kallikrein, a 100-~l aliquot will hydrolyze 0.5 ~mole of BAEE per minute in the assay described below. Aliquots of the substrate prekallikrein are stored at --20 ° in plastic tubes. Aliquot tubes are thawed only once and, thereafter, stored at 4 ° . Disposable plastic pipettes are used to handle the prekallikrein solution. Procedure. Factor XII is activated (1) on the negatively charged surface of kaolin or (2) proteolytically by trypsin. (1) Kaolin activation can be useful during early stages of purification because factor XII stays bound to kaolin and plasma protease inhibitors can be washed away so that they do not interfere with prekallikrein activation or with BAEE hydrolysis. Kaolin activation is performed by incubating at 37 ° for 15 min a 5- to 100-~1 aliquot of the test solution containing 0.03-0.5 ~g of factor XII with 5.0-500/~g of kaolin in a final volume of 100-200 ~l in a 12 X 75 mm plastic test tube. The optimal amount of kaolin must be empirically established for each test solution, and it depends on the amount of interfering protein that can bind to kaolin and thereby compete for the factor XII activation sites. Excess kaolin must be avoided since it binds and inhibits kallikrein at a later stage in this coupled assay. After this incubation, the plastic test tube containing the activation mixture is centrifuged 3 min at 2000 rpm at ambient temperature to form a kaolin pellet, which is washed two times with 200 ~l of TBS at 20 °. The supernatants are discartted and factor XIIa is bound to the kaolin pellet. (2) Trypsin activation is accomplished by adding a 5-50-~1 aliquot of the test solution containing 0.03-0.5 ~g of factor XII to a 12 X 75 mm plastic test tube containing 100 ~1 of trypsin assay buffer and 20 ~l of trypsin (0.2 ~g). This activation mixture is incubated at 37 ° for 15 min, then 100 ~l of ovomucoid trypsin inhibitor (200 ~g) is added to the tube for a further 10 min at 37 °. Ovomucoid trypsin inhibitor inhibits trypsin, but not factor XIIa or kallikrein. 19K. D. Wuepper and C. G. Cochrane, J. Exp. Med. 135, 1 (1972).
[7]
HUMAN FACTOR XII
59
The conversion of the substrate prekallikrein to kallikrein by factor XIIa is accomplished by adding 100 ~l of the rabbit prekallikrein reagent solution to the test tube containing activated factor X I I and incubating the reaction mixture at 37 ° for 10-30 min, depending on the amount of factor XIIa present. Then the hydrolysis of B A E E by kallikrein is determined by adding 1.5 ml of 1 m M B A E E to the test tube containing the reaction mixture. After an incubation at 37 ° if 5-30 min depending on the amount of kallikrein generated, the absorbance increase at 253 nm due to the hydrolysis of B A E E 2° is measured in cuvettes with a 0.5-cm pathlength. It is necessary to have control reaction mixtures lacking factor X I I or factor XIIa or prekallikrein and also to use in the reference cuvette an appropriately diluted B A E E solution since 1 m M B A E E has A~53 of ,-~ 1.2 per 0.5 cm. The hydrolysis of B A E E by kallikrein is approximately linear for an absorbance change between 0 and 0.2. Total hydrolysis of 1 mM B A E E gives AA,.,53of 0.44 for a 0.5-cm cuvette. Kallikrein can also be assayed spectrophotometrically based on its hydrolytic activity against the tripeptide substrate, Bz-Pro-Phe-Argparanitroanilide (Pentapharm, A. G., Basel). 2°a
Clotting Assay After activation of factor X I I by kaolin, factor XIIa activity is assayed for its ability to clot factor XII-deficient human plasma.
Reagents TBS Kaolin (Fisher Scientific) : acid washed, 10 mg/ml in TBS CaC12, 0.050 M, 37 ° Rabbit brain cephalin (Sigma) Citrated factor XII-deficient and normal human plasmas Bovine serum albumin (Sigma), 1.0 mg/ml in TBS All reagents are at 4 ° except the CaC12.
Procedure. The following are added in order to a disposable 10 X 75 mm glass tube: 0.1 ml of kaolin, 0.1 ml of cephalin, 0.1 ml of test sample, and 0.1 ml of factor XII-deficient plasma. The tube is mixed gently and placed in a 37 ° H20 bath for 8.0 min. Then 75 t~l of CaCl~ is added, at which point a stop watch is started. The tube contents are rapidly mixed, and the tube is left standing in the bath until the watch reads 30 sec. The tube is then held in the bath and tilted continually 50D. C. Morrison and C. G. Cochrane, J. Exp. Med. 140, 797 (1974). :~"B. Svenson, in "Chemistry and Biology of the Kallikrein-Kinin System in Health and Disease," U.S. Govt. Printing Office, Washington, D.C., in press.
60
BLOOD CLOTTING ENZYMES
[7]
until clotting occurs. A firm clot should appear suddenly, at which point the stop watch is stopped. The reference normal human plasma should be diluted with bovine serum albumin and assayed at dilutions varying from ~ o to ~oo, giving clotting times between 50 and 140 sec. Various dilutions of the sample factor XII solution with bovine serum albumin are made and assayed. To determine the clotting activity of the unknown factor X I I solution, the data are plotted on log-log paper showing clotting time versus dilution. By interpolation the clotting activity of the factor XII sample is determined. Unit o] Activity. One unit of factor XII clotting activity is arbitrarily defined as that amount found in 1 ml of a standard pool of normal citrated human plasma. Highly purified human factor XII exhibits specific clotting activities of 59 units/mg 14 to 80 units/mg. 16 Purification Procedure Freshly prepared citrated plasma is the starting material for factor XII isolation from human blood. Blood is collected from the antecubital vein of normal healthy volunteers into 50-ml plastic centrifuge tubes containing one-sixth volume acid-citrate-dextrose anticoagulant (ACD: 1 liter contains 13.6 g of citric acid, 25 g of sodium citrate, 20 g of dextrose). The blood is centrifuged at 3000 rpm for 20 min at 20 °, and the plasma obtained is then centrifuged again at 5000 rpm for 40 min at 20 °. The following is a convenient scheme for isolating factor XII from 500 ml of starting ACD-plasma which contains ca. 12 mg of factor XII. 16 All steps are carried out in plasticware or siliconized glassware and at 4 ° (except steps 1 and 2). All buffers contain 1 mM Na4EDTA, 50 ~g of hexadimethrine bromide (Polybrene, Aldrich) per milliliter, 1 mM benzamidine-HCl (Aldrich), and 0.02% NAN3. Buffers are prepared fresh for each step to minimize bacterial contamination, since bacterial endotoxin is a potent activator of factor XII. 21 Dialysis tubing and all containers are prerinsed with a 2 g/liter Polybrene solution and then rinsed with H~O before contacting the solution containing factor XII. Step 1. Heat Treatment. ACD plasma, 500 ml, in a plastic centrifuge bottle is rapidly brought to 56 ° in a 75 ° H~O bath with stirring of the plasma by a siliconized thermometer and then placed in a 56 ° H20 bath for 30 min. The visible precipitate which includes denatured fibrinogen and prekallikrein is removed by centrifugation at 2000 g for 30 min. This defibrination step can be omitted without affecting the final yield, purity, or stability of factor XII. 21K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969).
[7]
HUMAN FACTOR XlI
61
Step 2. DEAE-Sephadex Chromatography. The supernatant is dialyzed against the starting buffer, 40 mM Tris, 10 mM succinic acid, pH 8.4, for chromatography on DEAE-Sephadex A-50 (Pharmacia). One hundred grams of resin is allowed to settle in 3.8 liters of starting buffer in a 4-liter beaker and allowed to swell overnight at 56 °. The resin is poured into a 10 X 40 cm siliconized glass column and washed with 4 liters of starting buffer. The 500-ml sample is applied at 700-900 ml/hr, and 4 liters of starting buffer washes the column at 800 ml/hr. Factor XII is then eluted from the column using a linear gradient formed by 6 liters of starting buffer in the proximal chamber and 6 liters of 0.3 M Tris, 0.12 M succinic acid, 0.3 M NaC1, pH 7.8, in the distal chamber. A flow rate of 600-900 ml/hr is observed for a hydrostatic pressure of 50-80 cm; 45-ml fractions can be collected in 18 X 250 mm siliconized glass tubes. The fractions are assayed for factor XII activity and for protein by the Lowry method. Factor XII elutes at pH 8.0-8.1 at a buffer conductivity of 8-13 millimho. Fractions containing factor XII activity are found in the ascending portion of the albumin peak, and they are pooled to give 2 liters. This entire step can be performed at 20 ° and it preferably should be if high molecular weight kininogen is to be purified from the same column. Pools of high molecular weight kininogen, plasminogen, factor XI, prekallikrein, and plasminogen proactivator have been obtained from this column for further purification. The latter three proteins elute in the ,/-fraction which does not bind to the column. Plasminogen elutes just prior to factor XII while high molecular weight kininogen elutes much later at a conductivity of 20-23 mmho. Step 3. Lysine-Sepharose Affinity Chromatography. The 2-liter pool of factor XII is passed through a lysine-Sepharose affinity column in order to remove plasminogen since plasmin is a potent proteolytic activator of factor XII. Lysine-Sepharose is prepared as described elsewhere, TM and a 200-ml volume of resin is poured into a 6.5 X 30 cm plastic column and equilibrated with TBS buffer. The 2-liter pool is passed through the lysine-Sepharose column at 300 ml/hr. The 2-liter pool is then brought to 60% saturation with (NH4),.,S04 in order to precipitate and to concentrate factor XII. Three liters of saturated (NH4) 2SO4 (767 g/liter) at 4 ° are added dropwise over 5 hr to the stirred 2-liter pool of factor XII in a 10-1iter plastic bucket. Stirring continues for 45 rain before centrifugation at 2500 rpm for 1 hr at 4 ° in l-liter plastic bottles to collect the precipitate, which is redissolved in 250 ml of the starting buffer for the next DEAE-Sephadex column. Step 4. Second DEAE-Sephadex Chromatography. A descending pH gradient is used to elute factor XII from DEAE-Sephadex. Seven grams
62
BLOOD CLOTTING ENZYMES
[7]
of DEAE-Sephadex A-50 are allowed to swell at 56 ° overnight in the starting buffer, 0.03 M imidazole, 0.001 M succinic acid, pH 8.1, and poured into a 2.6 X 35 cm siliconized glass column. The resin is washed with 450 ml of starting buffer before the 250-ml sample which has been dialyzed against starting buffer is applied. The column is again washed with 450 ml of starting buffer. Gradient elution is effected with 600 ml of starting buffer in the stirred proximal chamber and 600 ml of 0.12 M imidazole, 0.045 M succinic acid, pH 6.6, in the distal chamber. The flow rate is controlled at 56 ml/hr by a peristaltic pump, and 8-ml fractions are collected. Factor XII is eluted before the bulk of the protein, and it is found in fractions between pH 7.2 and 6.9. The pool of factor XII activity (about 250 ml) is brought to 60% saturation with (NH4)~S04 at 4 °, as outlined above, and the precipitate containing factor XII is dissolved in 30 ml of the starting buffer for the following SP-Sephadex column. Step 5. SP-Sephadex Chromatograph. An increasing salt gradient at constant pH is employed to elute factor XII from SP-Sephadex. Two grams of SP-Sephadex C-50 are allowed to swell at 56 ° overnight in the starting buffer, 0.10 M acetate, 0.15 M NaC1, pH 5.3, and sufficient resin to give a bed height of 9 cm is poured into a siliconized glass column, 2.6 cm in diameter. The resin is washed with 100 ml of starting buffer, and the 30-ml sample which has been dialyzed against starting buffer is applied, at which point collection of 6-ml fractions at a flow rate of 56 ml/hr is begun. A 100-ml wash with starting buffer is made. Gradient elution is effected with 150 ml of starting buffer in the stirred pi'oximal chamber and 150 ml of 0.1 M acetate 0.5 M in NaC1, pH 5.3, in the distal chamber. As seen in Fig. 1, the bulk of the protein does not bind to the column, whereas factor XII is bound and elutes between 0.24 M and 0.28 M NaC1. The pool of factor XII obtained from this column contains 2.4-3.0 mg of protein which is greater than 95% factor XII, corresponding to a yield of 20-25% of the factor XII present in the initial 500 ml of citrated plasma. The clotting activity of factor XII purified with this procedure is 80 ± 11 units/mg, and less than 2% of factor XII is present as factor XIIa. The procedure described above for isolation of human factor XII has been used to isolate rabbit factor XII in similar yield and purity.
Properties
Stability. Maintenance of factor XII in zymogen form poses a difficult problem, and it has often been observed that factor XII can be
[7]
HUMAN FACTOR XII
63
1.8
..../•0"5
r,,,.
x
0.s o
0.3
\ .. _ ~
~ 0 . I - - ~ 0 . 4 ~
z.,
,"-,~ . . . . . . . . .
l 0
~.
15
30 45 60 75 FractionNumber[6ml) FIG. 1. Elution of human factor XII (Hageman factor) from a sulfopropylSephadex column. converted to factor XIIa over a period of several weeks even when stored frozen at --70% Nonetheless, factor X I I can be successfully and totally preserved in zymogen form for longer than 9 months when kept at 4 ° in solution in 6 m M acetate, pH 5.0, 0.5 m M EDTA, 0.15 M NaC1. P u r i t y . The chromatographed preparations of human factor X I I described above are found to be greater than 95% homogeneous on SDS polyacrylamide gels 21 and on alkaline polyacrylamide gels. 2-~ P h y s i c a l and Chemical Properties. The electrophoretic mobility of human factor X I I is that of a fl-globulin and an isoelectric point of 6.3 has been reported? 3 The sedimentation coefficient determined in sucrose density gradients is 4.5 S at 20% 1° A molecular weight of 76,000 is observed in sodium dodecyl sulfate (SDS) polyacrylamide gel experiments, 1° but less reliable values determined by exclusion chromatography on Sephadex G-200 have ranged from 100,000 to 120,0002 ,23 Enzymic activation of factor X I I in solution results in the appearance of polypeptide fragments of 38,000, 28,000, and about 10,000 MW, 1°- and the 28,000 M W fragment contains the enzymic active site while the 38,000 and 10,000 M W fragments contain high affinity binding sites for negatively charged surfaces. 24 Enzymic activation by kallikrein of factor X I I bound to negative surfaces either in plasma or in purified systems results in a single cleavage yielding 48,000 and 28,000 M W fragments. 25 The amino acid content of human factor X I I has been determined in B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964). :~J. Spragg, A. P. Kaplan, and K. F. Austen, Ann. N.Y. Acad. Sci. 209, 372 (1973). :4 S. D. Revak and C. G. Cochrane, J. Clin. Invest. in press. :~J. It. Griffin and C. G. Cochrane, Proc. Nat. Acad. Sci. U.S.A. in press. 2:
64
BLOOD CLOTTING ENZYMES
[7]
AMINO ACID COMPOSITION OF FACTOR X I I Amino acid a
(1) Human ~
(2) Human e
(3) Human s
(4) Rabbit ~
Lysine Histidine Arginine Aspartic acid and asparagine Threonine Serine Glutamic acid and glutamine Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leueine Tyrosine Phenylalanine
6.45 3.57 5.52 8.22 5.58 9.73 12.02 5.81 9.71 7.10 3.56 5.11 0.283 2.77 8.34 2.71 3.54
4.2 4.5 5.9 6.3 5.8 8.5 11.3 8.9 10.5 9.2 4.2 5~ 4 0.1 _+ 0.1 1.4 7.6 2.8 2.9
3.73 4.73 6.91 6.56 5.98 6.43 12.31 9.94 9.02 9.33 (3.07) 5.97 0.210 1.48 9.46 2.43 2.60
3.55 5.33 6.54 7.09 5.45 7.16 11.31 10.00 8.91 9.30 (2.54) 5.60 0.223 1.80 9.85 2.31 3.02
a Tryptophan was not reported. Cysteine values in columns (3) and (4) are minimal values; those in column (1) were determined as CM-Cys. b S. D. Revak, C. G. Cochrane, A. R. Johnston, and T. E. Hugli, J. Clin. Invest. 54, 619 (1974). c C. R. McMillin, H. Saito, O. D. Ratnoff, and A. C. Walton, J. Clin. Invest. 54, 1312 (1974). d j. H. Griffin and C. G. Cochrane, manuscript in preparation. s e v e r a l l a b o r a t o r i e s on different p r e p a r a t i o n s , a n d t h e d a t a a r e shown in t h e t a b l e . T h e a m i n o a c i d c o n t e n t of r a b b i t f a c t o r X I I is also r e p o r t e d in t h e t a b l e a n d i t shows a r e m a r k a b l e s i m i l a r i t y to our m o r e r e c e n t d a t a for h u m a n f a c t o r X I I . 16 T h e c i r c u l a r d i c h r o i s m s p e c t r u m of h u m a n f a c t o r X I I h a s been p u b lished, 14 a n d t h e d a t a suggest t h a t t h e s e c o n d a r y s t r u c t u r e of m o s t of f a c t o r X I I is n o n p e r i o d i c . R e c e n t s t u d i e s 2~ h a v e led to t h e suggestion t h a t c o n t a c t a c t i v a t i o n of f a c t o r X I I can be e n t i r e l y e x p l a i n e d in t e r m s of i n t e r a c t i o n s b e t w e e n f a c t o r X I I , p r e k a l l i k r e i n , high m o l e c u l a r w e i g h t k i n i n o g e n , a n d an a c t i v a t ing surface. T h e h y p o t h e s i s has been a d v a n c e d 25 t h a t f a c t o r X I I a n d high m o l e c u l a r w e i g h t k i n i n o g e n f o r m a c o m p l e x on t h e s u r f a c e w h i c h a l t e r s t h e s t r u c t u r e of f a c t o r X I I such t h a t i t can be r a p i d l y c o n v e r t e d to f a c t o r X I I a b y a single p r o t e o l y t i c c l e a v a g e b y k a l l i k r e i n . T h e n f a c t o r X I I a can r e c i p r o c a l l y a c t i v a t e m o r e p r e k a l l i k r e i n to k a l l i k r e i n as well as a c t i v a t e f a c t o r X I a n d p l a s m i n o g e n p r o a c t i v a t o r l e a d i n g to a c t i v a t i o n of t h e
[Ta]
BOVINE FACTOR XI (PLASMA THROMBOPLASTIN ANTECEDENT)
65
various factor X I I - d e p e n d e n t pathways, i.e., kinin-generation, intrinsic coagulation, and fibrinolysis. I m m u n o c h e m i c a l Properties. Specific goat precipitating antibodies have been prepared against h u m a n factor X I I . 1° The absorption of the antiserum with h u m a n factor XII-deficient plasma was notably useful in eliminating antibodies directed against trace contaminants of factor X I I preparations. The specific antiserum against factor X I I is routinely used to determine concentrations of factor X I I in plasma samples and in other solutions according to the radial immunodiffusion method of Mancini. ~6 The average concentration of factor X I I in human plasma has been determined by this method to be 29 ~g/ml, i.e., 24 ~g/ml in citrated normal human plasma. 1° Inhibitors. H u m a n factor X I I is inhibited by C1 esterase inhibitor 2~,27 and by lima bean trypsin inhibitor, but not by soybean or ovomucoid trypsin inhibitor.
Acknowledgments The studies reported from the authors' laboratory were supported by U.S. Public Health Service Grants NIH AI 07007 and NHLI Grant 16411-01. The skilled technical assistance of Greg Beretta, Alice Kleiss, and Susan Revak is gratefully acknowledged. ~G. A. Mancini, A. O. Carbonaro, and J. F. Heremans, Immunochemistry 2, 235 (1965). 5, A. B. Schreiber, A. P. Kaplan, and K. F. Austen. J. Clin. Invest. 52, 1402 (1973).
[7a] Bovine Factor XI (Plasma Thromboplastin Antecedent) B y TAKEHIKO KOIOE, HISAO KATO, and EARL W. DAVIE
Factor X I is a plasma glycoprotein t h a t participates in the early stage of intrinsic blood coagulation. 1 I t is absent, or present as an inert protein, in plasma of individuals with a congenital bleeding disorder called plasma thromboplastin antecedent deficiency. ~,3 In normal plasma, factor X I is present in a precursor form and is converted to an active form (factor XIa) by another coagulation factor(s). However, the physiological 1E. W. Davie and K. Fujikawa, A~nu. Rev. Biochem. 44, 799 (1975). 2 R. L. Rosenthal, O. H. Dreskin, and N. Rosenthal, Proc. Soc. Exp. Biol. Med. 8g, 171 (1953). C. D. Forbes and O. D. Ratnoff, J. Lab. Cli~. Meal. 79, 113 (1972).
BLOOD CLOTTING ENZYMES
[7]
tion of 1-2% of the single-chain factor VII during the assay, rather than inherent single-chain activity. A trace level of double-chain factor VII could generate enough factor Xa to feed back and activate single-chain material, since the activation of factor VII by factor Xa in the presence of phospholipids and calcium ions is rapid2 Possible activation by other enzymes generated during the assay, such as intrinsic pathway enzymes, must also be considered.
[7] H u m a n
Factor XII (Hageman Factor)
By JOHN H. GRIFFIN and CHARLES G. COCHRANE Factor X I I is an enzyme that circulates in zymogen form in blood and is capable, upon activation, of initiating the clotting, fibrinolytic, and kinin-generating systems. 1-6 Initially described by Ratnoff, 7 factor XII-deficient persons are asymptomatic. In addition to factor XII, the activation in plasma of clotting, fibrinolysis, and kinin generation by negatively charged foreign surfaces also appears to require prekallikrein 4 and a high-molecular-weight form of kininogen, s The precise description of the mechanism(s) of activation of the Hageman factor pathways has been limited by the difficulty of isolating satisfactory amounts of the factor X I I zymogen and of stabilizing preparations of factor X I I in zymogen form. Factor X I I in zymogen form has been isolated from human and rabbit blood, 9-11 and structural changes accompanying the i j . Margolis, Ann. N.Y. Acad. Sci. 104, 133 (1963). 20. D. Ratnoff, Prog. Hematol. 5, 204 (1965). *C. G. Cochrane, S. Revak, B. S. Aikin, and K. D. Wuepper, in "Inflammation: Mechanisms and Control" (I. H. Lepow and P. A. Ward, eds.), p. 119. Academic Press, New York, 1972. 4K. D. Wuepper, in "Inflammation: Mechanisms and Control" (I. H. Lepow and P. A. Ward, eds.), p. 93. Academic Press, New York, 1972. 5E. H. Magoon, J. Spragg, and K. F. Austen, Adv. Biosci. 12, 225 (1974). C. G. Cochrane, S. D. Revak, K. D. Wuepper, A. Johnston, and D. C. Morrison, Adv. Biosci. 12, 237 (1974). ' O. D. Ra~noff and J. E. Colopy, J. Clin. Invest. 34, 602 (1955). 8I~. D. Wuepper, D. R. Miller, and M. Lacombe, Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 859 (1975). 9 C. G. Cochrane and K. D. Wuepper, J. Exp. Med. 134, 986 (1971). 1oS. D. Revak, C. G. Cochrane, A. R. Johnston, and T. E. Hugli, J. Clin. Invest. 54, 619 (1974). 11H. Saito, O. D. Ratnoff, and V. H. Donaldson, Circ. Res. 34, 641 (i974).
[7]
HUMAN FACTOR XII
57
activation of human and rabbit factor X I I have been reported2 °,12-14 Nonetheless, variability in reproducing the isolation 9,1° of factor X I I and in stabilizing the isolated zymogen ~5 led us to develop more reliable methods 1~ of isolating and stabilizing factor XII which are described here. In all manipulations involving factor XII, exposure to glass must be avoided by using plasticware or freshly siliconized glassware (Siliclad, Clay-Adams, Inc.) since factor XII sticks very readily to glass.
Assay Methods Factor X I I activity can be conveniently assayed for its ability to activate isolated plasma prekallikrein or to clot factor XII-deficient plasma, as described below. Less conveniently, it can be assayed for its ability to activate fibrinolysis. 17 Highly purified factor XII can hydrolyze, albeit inefficiently, N-Ac-Gly-Lys-0Et. is
Prekallikrein Activation Assay After activation of factor XII by kaolin or by trypsin, factor XIIa is allowed to convert the zymogen prekallikrein to its active form, kallikrein, which is then conveniently assayed spectrophotometrically for its ability to hydrolyze Bz-Arg-OEt.
Reagents Tris-buffered saline (TBS): 0.01 M Tris-C1, 0.15 M NaC1, pH 7.4 Kaolin (Fisher Scientific Co.) : acid-washed, 5 mg/ml in TBS Bz-Arg-OEt (BAEE): (Mann Research): 1 mM in 0.1 M Tris-C1, 0.05 M NaCI, pH 8.0 (prepared daily and kept at 4 ° until needed) Trypsin ("Trypsin-TPCK", Worthington Biochemical Corp.): Stock solution, 1 mg/ml, in 1 mM acetic acid kept at 4 ° and diluted 1:100 into trypsin assay buffer immediately before each assay. Trypsin assay buffer: 0.1 M sodium phosphate, 0.05 M NaC1, pH 7.6 12C. G. Cochrane, S. D. Revak, and K. D. Wuepper, J. Exp. Med. 138, 1564 (1973). 1~A. P. Kaplan and K. F. Austen, J. Exp. Med. 133, 696 (1971). 14C. R. McMillin, H. Saito, O. D. Ratnoff, and A. C. Walton, J. Clin. Invest. 54, 1312 (1974). 15H. Z. M o v a t and A. H. Ozge-Anwar, J. Lab. Clin. Med. 84, 861 (1974). 1~j . H. Griffin and C. G. Coehrane, manuscript in preparation. i~ A. P. Kaplan and K. F. Austen, J. Exp. Med. 136, 1378 (1972). 18R. J. Ulevitch, D. Letchford, and C. G. Cochrane. Thromb. Diath. Haemorrh. 31, 30 (1974).
58
BLOOD CLOTTING ENZYMES
[7]
Ovomucoid trypsin inhibitor (Worthington Biochemical Corp.): 2 mg/ml in TBS (solution stable at 4 ° at least 1 month) Prekallikrein: Substrate rabbit prekallikrein is partially purified from citrated rabbit plasma as described elsewhere. 19 Briefly, the ~/-globulin fraction obtained from a DEAE-Sephadex column is further fractionated on a CM-Sephadex column with elution effected by a NaC1 gradient. The pool of prekallikrein activity obtained from the CM-Sephadex column is dialyzed against PBS (0.01 M Na phosphate, 0.15 M NaC1, pH 7.0). The concentration of the pool of prekallikrein is adjusted so that, after total conversion of prekallikrein to kallikrein, a 100-~l aliquot will hydrolyze 0.5 ~mole of BAEE per minute in the assay described below. Aliquots of the substrate prekallikrein are stored at --20 ° in plastic tubes. Aliquot tubes are thawed only once and, thereafter, stored at 4 ° . Disposable plastic pipettes are used to handle the prekallikrein solution. Procedure. Factor XII is activated (1) on the negatively charged surface of kaolin or (2) proteolytically by trypsin. (1) Kaolin activation can be useful during early stages of purification because factor XII stays bound to kaolin and plasma protease inhibitors can be washed away so that they do not interfere with prekallikrein activation or with BAEE hydrolysis. Kaolin activation is performed by incubating at 37 ° for 15 min a 5- to 100-~1 aliquot of the test solution containing 0.03-0.5 ~g of factor XII with 5.0-500/~g of kaolin in a final volume of 100-200 ~l in a 12 X 75 mm plastic test tube. The optimal amount of kaolin must be empirically established for each test solution, and it depends on the amount of interfering protein that can bind to kaolin and thereby compete for the factor XII activation sites. Excess kaolin must be avoided since it binds and inhibits kallikrein at a later stage in this coupled assay. After this incubation, the plastic test tube containing the activation mixture is centrifuged 3 min at 2000 rpm at ambient temperature to form a kaolin pellet, which is washed two times with 200 ~l of TBS at 20 °. The supernatants are discartted and factor XIIa is bound to the kaolin pellet. (2) Trypsin activation is accomplished by adding a 5-50-~1 aliquot of the test solution containing 0.03-0.5 ~g of factor XII to a 12 X 75 mm plastic test tube containing 100 ~1 of trypsin assay buffer and 20 ~l of trypsin (0.2 ~g). This activation mixture is incubated at 37 ° for 15 min, then 100 ~l of ovomucoid trypsin inhibitor (200 ~g) is added to the tube for a further 10 min at 37 °. Ovomucoid trypsin inhibitor inhibits trypsin, but not factor XIIa or kallikrein. 19K. D. Wuepper and C. G. Cochrane, J. Exp. Med. 135, 1 (1972).
[7]
HUMAN FACTOR XII
59
The conversion of the substrate prekallikrein to kallikrein by factor XIIa is accomplished by adding 100 ~l of the rabbit prekallikrein reagent solution to the test tube containing activated factor X I I and incubating the reaction mixture at 37 ° for 10-30 min, depending on the amount of factor XIIa present. Then the hydrolysis of B A E E by kallikrein is determined by adding 1.5 ml of 1 m M B A E E to the test tube containing the reaction mixture. After an incubation at 37 ° if 5-30 min depending on the amount of kallikrein generated, the absorbance increase at 253 nm due to the hydrolysis of B A E E 2° is measured in cuvettes with a 0.5-cm pathlength. It is necessary to have control reaction mixtures lacking factor X I I or factor XIIa or prekallikrein and also to use in the reference cuvette an appropriately diluted B A E E solution since 1 m M B A E E has A~53 of ,-~ 1.2 per 0.5 cm. The hydrolysis of B A E E by kallikrein is approximately linear for an absorbance change between 0 and 0.2. Total hydrolysis of 1 mM B A E E gives AA,.,53of 0.44 for a 0.5-cm cuvette. Kallikrein can also be assayed spectrophotometrically based on its hydrolytic activity against the tripeptide substrate, Bz-Pro-Phe-Argparanitroanilide (Pentapharm, A. G., Basel). 2°a
Clotting Assay After activation of factor X I I by kaolin, factor XIIa activity is assayed for its ability to clot factor XII-deficient human plasma.
Reagents TBS Kaolin (Fisher Scientific) : acid washed, 10 mg/ml in TBS CaC12, 0.050 M, 37 ° Rabbit brain cephalin (Sigma) Citrated factor XII-deficient and normal human plasmas Bovine serum albumin (Sigma), 1.0 mg/ml in TBS All reagents are at 4 ° except the CaC12.
Procedure. The following are added in order to a disposable 10 X 75 mm glass tube: 0.1 ml of kaolin, 0.1 ml of cephalin, 0.1 ml of test sample, and 0.1 ml of factor XII-deficient plasma. The tube is mixed gently and placed in a 37 ° H20 bath for 8.0 min. Then 75 t~l of CaCl~ is added, at which point a stop watch is started. The tube contents are rapidly mixed, and the tube is left standing in the bath until the watch reads 30 sec. The tube is then held in the bath and tilted continually 50D. C. Morrison and C. G. Cochrane, J. Exp. Med. 140, 797 (1974). :~"B. Svenson, in "Chemistry and Biology of the Kallikrein-Kinin System in Health and Disease," U.S. Govt. Printing Office, Washington, D.C., in press.
60
BLOOD CLOTTING ENZYMES
[7]
until clotting occurs. A firm clot should appear suddenly, at which point the stop watch is stopped. The reference normal human plasma should be diluted with bovine serum albumin and assayed at dilutions varying from ~ o to ~oo, giving clotting times between 50 and 140 sec. Various dilutions of the sample factor XII solution with bovine serum albumin are made and assayed. To determine the clotting activity of the unknown factor X I I solution, the data are plotted on log-log paper showing clotting time versus dilution. By interpolation the clotting activity of the factor XII sample is determined. Unit o] Activity. One unit of factor XII clotting activity is arbitrarily defined as that amount found in 1 ml of a standard pool of normal citrated human plasma. Highly purified human factor XII exhibits specific clotting activities of 59 units/mg 14 to 80 units/mg. 16 Purification Procedure Freshly prepared citrated plasma is the starting material for factor XII isolation from human blood. Blood is collected from the antecubital vein of normal healthy volunteers into 50-ml plastic centrifuge tubes containing one-sixth volume acid-citrate-dextrose anticoagulant (ACD: 1 liter contains 13.6 g of citric acid, 25 g of sodium citrate, 20 g of dextrose). The blood is centrifuged at 3000 rpm for 20 min at 20 °, and the plasma obtained is then centrifuged again at 5000 rpm for 40 min at 20 °. The following is a convenient scheme for isolating factor XII from 500 ml of starting ACD-plasma which contains ca. 12 mg of factor XII. 16 All steps are carried out in plasticware or siliconized glassware and at 4 ° (except steps 1 and 2). All buffers contain 1 mM Na4EDTA, 50 ~g of hexadimethrine bromide (Polybrene, Aldrich) per milliliter, 1 mM benzamidine-HCl (Aldrich), and 0.02% NAN3. Buffers are prepared fresh for each step to minimize bacterial contamination, since bacterial endotoxin is a potent activator of factor XII. 21 Dialysis tubing and all containers are prerinsed with a 2 g/liter Polybrene solution and then rinsed with H~O before contacting the solution containing factor XII. Step 1. Heat Treatment. ACD plasma, 500 ml, in a plastic centrifuge bottle is rapidly brought to 56 ° in a 75 ° H~O bath with stirring of the plasma by a siliconized thermometer and then placed in a 56 ° H20 bath for 30 min. The visible precipitate which includes denatured fibrinogen and prekallikrein is removed by centrifugation at 2000 g for 30 min. This defibrination step can be omitted without affecting the final yield, purity, or stability of factor XII. 21K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969).
[7]
HUMAN FACTOR XlI
61
Step 2. DEAE-Sephadex Chromatography. The supernatant is dialyzed against the starting buffer, 40 mM Tris, 10 mM succinic acid, pH 8.4, for chromatography on DEAE-Sephadex A-50 (Pharmacia). One hundred grams of resin is allowed to settle in 3.8 liters of starting buffer in a 4-liter beaker and allowed to swell overnight at 56 °. The resin is poured into a 10 X 40 cm siliconized glass column and washed with 4 liters of starting buffer. The 500-ml sample is applied at 700-900 ml/hr, and 4 liters of starting buffer washes the column at 800 ml/hr. Factor XII is then eluted from the column using a linear gradient formed by 6 liters of starting buffer in the proximal chamber and 6 liters of 0.3 M Tris, 0.12 M succinic acid, 0.3 M NaC1, pH 7.8, in the distal chamber. A flow rate of 600-900 ml/hr is observed for a hydrostatic pressure of 50-80 cm; 45-ml fractions can be collected in 18 X 250 mm siliconized glass tubes. The fractions are assayed for factor XII activity and for protein by the Lowry method. Factor XII elutes at pH 8.0-8.1 at a buffer conductivity of 8-13 millimho. Fractions containing factor XII activity are found in the ascending portion of the albumin peak, and they are pooled to give 2 liters. This entire step can be performed at 20 ° and it preferably should be if high molecular weight kininogen is to be purified from the same column. Pools of high molecular weight kininogen, plasminogen, factor XI, prekallikrein, and plasminogen proactivator have been obtained from this column for further purification. The latter three proteins elute in the ,/-fraction which does not bind to the column. Plasminogen elutes just prior to factor XII while high molecular weight kininogen elutes much later at a conductivity of 20-23 mmho. Step 3. Lysine-Sepharose Affinity Chromatography. The 2-liter pool of factor XII is passed through a lysine-Sepharose affinity column in order to remove plasminogen since plasmin is a potent proteolytic activator of factor XII. Lysine-Sepharose is prepared as described elsewhere, TM and a 200-ml volume of resin is poured into a 6.5 X 30 cm plastic column and equilibrated with TBS buffer. The 2-liter pool is passed through the lysine-Sepharose column at 300 ml/hr. The 2-liter pool is then brought to 60% saturation with (NH4),.,S04 in order to precipitate and to concentrate factor XII. Three liters of saturated (NH4) 2SO4 (767 g/liter) at 4 ° are added dropwise over 5 hr to the stirred 2-liter pool of factor XII in a 10-1iter plastic bucket. Stirring continues for 45 rain before centrifugation at 2500 rpm for 1 hr at 4 ° in l-liter plastic bottles to collect the precipitate, which is redissolved in 250 ml of the starting buffer for the next DEAE-Sephadex column. Step 4. Second DEAE-Sephadex Chromatography. A descending pH gradient is used to elute factor XII from DEAE-Sephadex. Seven grams
62
BLOOD CLOTTING ENZYMES
[7]
of DEAE-Sephadex A-50 are allowed to swell at 56 ° overnight in the starting buffer, 0.03 M imidazole, 0.001 M succinic acid, pH 8.1, and poured into a 2.6 X 35 cm siliconized glass column. The resin is washed with 450 ml of starting buffer before the 250-ml sample which has been dialyzed against starting buffer is applied. The column is again washed with 450 ml of starting buffer. Gradient elution is effected with 600 ml of starting buffer in the stirred proximal chamber and 600 ml of 0.12 M imidazole, 0.045 M succinic acid, pH 6.6, in the distal chamber. The flow rate is controlled at 56 ml/hr by a peristaltic pump, and 8-ml fractions are collected. Factor XII is eluted before the bulk of the protein, and it is found in fractions between pH 7.2 and 6.9. The pool of factor XII activity (about 250 ml) is brought to 60% saturation with (NH4)~S04 at 4 °, as outlined above, and the precipitate containing factor XII is dissolved in 30 ml of the starting buffer for the following SP-Sephadex column. Step 5. SP-Sephadex Chromatograph. An increasing salt gradient at constant pH is employed to elute factor XII from SP-Sephadex. Two grams of SP-Sephadex C-50 are allowed to swell at 56 ° overnight in the starting buffer, 0.10 M acetate, 0.15 M NaC1, pH 5.3, and sufficient resin to give a bed height of 9 cm is poured into a siliconized glass column, 2.6 cm in diameter. The resin is washed with 100 ml of starting buffer, and the 30-ml sample which has been dialyzed against starting buffer is applied, at which point collection of 6-ml fractions at a flow rate of 56 ml/hr is begun. A 100-ml wash with starting buffer is made. Gradient elution is effected with 150 ml of starting buffer in the stirred pi'oximal chamber and 150 ml of 0.1 M acetate 0.5 M in NaC1, pH 5.3, in the distal chamber. As seen in Fig. 1, the bulk of the protein does not bind to the column, whereas factor XII is bound and elutes between 0.24 M and 0.28 M NaC1. The pool of factor XII obtained from this column contains 2.4-3.0 mg of protein which is greater than 95% factor XII, corresponding to a yield of 20-25% of the factor XII present in the initial 500 ml of citrated plasma. The clotting activity of factor XII purified with this procedure is 80 ± 11 units/mg, and less than 2% of factor XII is present as factor XIIa. The procedure described above for isolation of human factor XII has been used to isolate rabbit factor XII in similar yield and purity.
Properties
Stability. Maintenance of factor XII in zymogen form poses a difficult problem, and it has often been observed that factor XII can be
[7]
HUMAN FACTOR XII
63
1.8
..../•0"5
r,,,.
x
0.s o
0.3
\ .. _ ~
~ 0 . I - - ~ 0 . 4 ~
z.,
,"-,~ . . . . . . . . .
l 0
~.
15
30 45 60 75 FractionNumber[6ml) FIG. 1. Elution of human factor XII (Hageman factor) from a sulfopropylSephadex column. converted to factor XIIa over a period of several weeks even when stored frozen at --70% Nonetheless, factor X I I can be successfully and totally preserved in zymogen form for longer than 9 months when kept at 4 ° in solution in 6 m M acetate, pH 5.0, 0.5 m M EDTA, 0.15 M NaC1. P u r i t y . The chromatographed preparations of human factor X I I described above are found to be greater than 95% homogeneous on SDS polyacrylamide gels 21 and on alkaline polyacrylamide gels. 2-~ P h y s i c a l and Chemical Properties. The electrophoretic mobility of human factor X I I is that of a fl-globulin and an isoelectric point of 6.3 has been reported? 3 The sedimentation coefficient determined in sucrose density gradients is 4.5 S at 20% 1° A molecular weight of 76,000 is observed in sodium dodecyl sulfate (SDS) polyacrylamide gel experiments, 1° but less reliable values determined by exclusion chromatography on Sephadex G-200 have ranged from 100,000 to 120,0002 ,23 Enzymic activation of factor X I I in solution results in the appearance of polypeptide fragments of 38,000, 28,000, and about 10,000 MW, 1°- and the 28,000 M W fragment contains the enzymic active site while the 38,000 and 10,000 M W fragments contain high affinity binding sites for negatively charged surfaces. 24 Enzymic activation by kallikrein of factor X I I bound to negative surfaces either in plasma or in purified systems results in a single cleavage yielding 48,000 and 28,000 M W fragments. 25 The amino acid content of human factor X I I has been determined in B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964). :~J. Spragg, A. P. Kaplan, and K. F. Austen, Ann. N.Y. Acad. Sci. 209, 372 (1973). :4 S. D. Revak and C. G. Cochrane, J. Clin. Invest. in press. :~J. It. Griffin and C. G. Cochrane, Proc. Nat. Acad. Sci. U.S.A. in press. 2:
64
BLOOD CLOTTING ENZYMES
[7]
AMINO ACID COMPOSITION OF FACTOR X I I Amino acid a
(1) Human ~
(2) Human e
(3) Human s
(4) Rabbit ~
Lysine Histidine Arginine Aspartic acid and asparagine Threonine Serine Glutamic acid and glutamine Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leueine Tyrosine Phenylalanine
6.45 3.57 5.52 8.22 5.58 9.73 12.02 5.81 9.71 7.10 3.56 5.11 0.283 2.77 8.34 2.71 3.54
4.2 4.5 5.9 6.3 5.8 8.5 11.3 8.9 10.5 9.2 4.2 5~ 4 0.1 _+ 0.1 1.4 7.6 2.8 2.9
3.73 4.73 6.91 6.56 5.98 6.43 12.31 9.94 9.02 9.33 (3.07) 5.97 0.210 1.48 9.46 2.43 2.60
3.55 5.33 6.54 7.09 5.45 7.16 11.31 10.00 8.91 9.30 (2.54) 5.60 0.223 1.80 9.85 2.31 3.02
a Tryptophan was not reported. Cysteine values in columns (3) and (4) are minimal values; those in column (1) were determined as CM-Cys. b S. D. Revak, C. G. Cochrane, A. R. Johnston, and T. E. Hugli, J. Clin. Invest. 54, 619 (1974). c C. R. McMillin, H. Saito, O. D. Ratnoff, and A. C. Walton, J. Clin. Invest. 54, 1312 (1974). d j. H. Griffin and C. G. Cochrane, manuscript in preparation. s e v e r a l l a b o r a t o r i e s on different p r e p a r a t i o n s , a n d t h e d a t a a r e shown in t h e t a b l e . T h e a m i n o a c i d c o n t e n t of r a b b i t f a c t o r X I I is also r e p o r t e d in t h e t a b l e a n d i t shows a r e m a r k a b l e s i m i l a r i t y to our m o r e r e c e n t d a t a for h u m a n f a c t o r X I I . 16 T h e c i r c u l a r d i c h r o i s m s p e c t r u m of h u m a n f a c t o r X I I h a s been p u b lished, 14 a n d t h e d a t a suggest t h a t t h e s e c o n d a r y s t r u c t u r e of m o s t of f a c t o r X I I is n o n p e r i o d i c . R e c e n t s t u d i e s 2~ h a v e led to t h e suggestion t h a t c o n t a c t a c t i v a t i o n of f a c t o r X I I can be e n t i r e l y e x p l a i n e d in t e r m s of i n t e r a c t i o n s b e t w e e n f a c t o r X I I , p r e k a l l i k r e i n , high m o l e c u l a r w e i g h t k i n i n o g e n , a n d an a c t i v a t ing surface. T h e h y p o t h e s i s has been a d v a n c e d 25 t h a t f a c t o r X I I a n d high m o l e c u l a r w e i g h t k i n i n o g e n f o r m a c o m p l e x on t h e s u r f a c e w h i c h a l t e r s t h e s t r u c t u r e of f a c t o r X I I such t h a t i t can be r a p i d l y c o n v e r t e d to f a c t o r X I I a b y a single p r o t e o l y t i c c l e a v a g e b y k a l l i k r e i n . T h e n f a c t o r X I I a can r e c i p r o c a l l y a c t i v a t e m o r e p r e k a l l i k r e i n to k a l l i k r e i n as well as a c t i v a t e f a c t o r X I a n d p l a s m i n o g e n p r o a c t i v a t o r l e a d i n g to a c t i v a t i o n of t h e
[Ta]
BOVINE FACTOR XI (PLASMA THROMBOPLASTIN ANTECEDENT)
65
various factor X I I - d e p e n d e n t pathways, i.e., kinin-generation, intrinsic coagulation, and fibrinolysis. I m m u n o c h e m i c a l Properties. Specific goat precipitating antibodies have been prepared against h u m a n factor X I I . 1° The absorption of the antiserum with h u m a n factor XII-deficient plasma was notably useful in eliminating antibodies directed against trace contaminants of factor X I I preparations. The specific antiserum against factor X I I is routinely used to determine concentrations of factor X I I in plasma samples and in other solutions according to the radial immunodiffusion method of Mancini. ~6 The average concentration of factor X I I in human plasma has been determined by this method to be 29 ~g/ml, i.e., 24 ~g/ml in citrated normal human plasma. 1° Inhibitors. H u m a n factor X I I is inhibited by C1 esterase inhibitor 2~,27 and by lima bean trypsin inhibitor, but not by soybean or ovomucoid trypsin inhibitor.
Acknowledgments The studies reported from the authors' laboratory were supported by U.S. Public Health Service Grants NIH AI 07007 and NHLI Grant 16411-01. The skilled technical assistance of Greg Beretta, Alice Kleiss, and Susan Revak is gratefully acknowledged. ~G. A. Mancini, A. O. Carbonaro, and J. F. Heremans, Immunochemistry 2, 235 (1965). 5, A. B. Schreiber, A. P. Kaplan, and K. F. Austen. J. Clin. Invest. 52, 1402 (1973).
[7a] Bovine Factor XI (Plasma Thromboplastin Antecedent) B y TAKEHIKO KOIOE, HISAO KATO, and EARL W. DAVIE
Factor X I is a plasma glycoprotein t h a t participates in the early stage of intrinsic blood coagulation. 1 I t is absent, or present as an inert protein, in plasma of individuals with a congenital bleeding disorder called plasma thromboplastin antecedent deficiency. ~,3 In normal plasma, factor X I is present in a precursor form and is converted to an active form (factor XIa) by another coagulation factor(s). However, the physiological 1E. W. Davie and K. Fujikawa, A~nu. Rev. Biochem. 44, 799 (1975). 2 R. L. Rosenthal, O. H. Dreskin, and N. Rosenthal, Proc. Soc. Exp. Biol. Med. 8g, 171 (1953). C. D. Forbes and O. D. Ratnoff, J. Lab. Cli~. Meal. 79, 113 (1972).
