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[68] Preparation and assay of plasma antithrombin
[68]
ASSAY OF PLASMA ANTITHROMBIN
[68]
Preparation
and
Assay
of Plasma
915
Antithrombin
B y FRANX C. MONXHOUSZ
Introduction While antithrombin activity can be neutralized by a number of nonspecific agents and surfaces, the main progressive antithrombin of plasma is associated with the alpha globulins.1-a This protein fraction brings about the irreversible destruction of thrombin in a progressive manner typical of enzymatic degradation. ~,~ Assay Method Any method designed to estimate antithrombin activity should involve measurements over a time period. Since thrombin is known to be adsorbed on both dry glass and fibrinogen/,7 measurements should be carried out in silicone-treated glassware or plastic, and if the measurements are done on plasma, this should firstbe defibrinated. Since heating to 54 ° for 3-4 minutes has no measurable effect on antithrombin, this is : the preferable way to remove fibrinogen. In general, with small concentrations of thrombin relative to antithrombin, practically all thrombin is destroyed and the reaction is of the first order. With high concentrations of thrombin in relation to antithrombin, equilibrium is reached more slowly, with some thrombin always remaining in the active state. T w o basic types of assay are recommended therefore, one (Method A) based on the use of small concentrations of thrombin and the other (Method B) on large concentrations relative to antithrombin concentration. Method A
This is a first-order reaction and since clotting times are inversely proportional to the thrombin concentration, a straight line results when log clotting times are plotted against t h r o m b i n - a n t i t h r o m b i n incubation 1F. C. Monkhouse and S. Milojevic, Can. J. Physiol. Pharmaeol. 46, 347 (1968). P. Porter, M. C. Porter, and J. N. Shanberge, Clin. Chim. Aeta 17, 189 (1967). s V. Abilgaard, 8cand. J.Clin. Invest. 21, 89 (1968). ' E. Mihalyi, J. Gen. Physiol. 37, 139 (1953). ° F. C. Monkhouse, "Blood Clotting Enzymology," p. 323. Academic Press, New York, 1967. e W. H. Seegers, M. Nieft, and E. C. Loomis, Science 1Ol, 520 (1945). 'W. H. Seegers, K. D. Miller, E. B. Andrews, and R. C. Murphy, Ann. J. Physiol. 169, 700 (1952).
916
NATURALLY OCCURRINGACTIVATORSAND INHIBITORS (a)
[58]
(b)
2OOO
I ooo ,-,
600
400 2OO "6
I00
/
6O 40 2O 0% tO Incubation time (rain)
Fie. 1. The straight-line relation between the logarithm of the clotting time and the thrombin-antithrombin incubation time. (a) With varying dilutions of defibrinated plasma as the source of antithrombin, and (b) dilution of purified antithrombin. times. The slope of this line is the rate constant K, and varies directly with the antithrombin concentration. Validity of this statement is illustrated by Fig. 1. The simplest and best method was introduced by Gerendas. 8 Porcelain spot plates are used in place of test tubes and a fine glass hook is used to detect the first signs of coagulation. To 0.3 ml of plasma (or antithrombin fraction), 0.2 ml of 0.05 M phosphate buffer pH 7.8 and 0.3 ml of thrombin solution (10 / m l ) are added. At 30second and minute intervals thereafter, 0.1 ml of this incubation mixture is added to 0.2 ml of a fibrinogen solution and the clotting time noted. Clotting times over a period of 6 minutes are plotted and the best-fitting straight line is drawn. Antithrombin activity is expressed in terms of frozen pooled defibrinated beef plasma stored in small aliquots at --20 °, and prepared fresh at 6-month intervals. Specific activity refers to the activity per milligram of protein as compared to this standard beef plasma. When it is desirable to use units to give a quantitative comparison rather than percent of standard, 1 ml of standard beef plasma is considered to contain 100 units of antithrombin. In our hands this has proven to be much more satisfactory than attempting to express antis M. Gerendas, Nature 157, 837 (1946).
[68]
ASSAY OF PLASMA ANTITHROMBIN
917
thrombin in terms of units of thrombin destroyed, since a thrombin unit is itself a somewhat arbitrary figure.
Method B If one wishes to use larger quantities of thrombin and to avoid clotting time as a measure, then the following method is recommended. Two hundred units of thrombin are placed in each of a number of siliconized test tubes. Varying amounts of the antithrombin solution to be assayed are then added. At least one tube has no antithrombin added, and serves as a control. The volumes are then adjusted to 1 ml by the addition of Tris buffer and the tubes are allowed to incubate at 28 ° for 1 hour. The amount of residual thrombin is then estimated by adding 0.2-ml aliquots of the incubated thrombin-antithrombin mixture to 0.2 ml of a 0.4 M TAME (p-toluenesulfonyl arginine methyl ester) solution. The volumes are adjusted to 2 ml with Tris buffer, pH 8.5, the tubes are incubated at 37 ° for 30 minutes and the extent of TAME hydrolysis determined. A curve is then made by plotting volume of antithrombin solution against units of thrombin neutralized. For further details of the method, consult the publication by Monkhouse et al? Purification The first stage in the purification of antithrombin is adsorption on aluminum hydroxide, and since the preparation of the aluminum hydroxide is the key to successful purification, details are given.
Preparation o] Aluminum Hydroxide Gel ]or Adsorption o] Plasma Antithrombin Note: This procedure should be carried out as rapidly as possible, never over more than 21/~ hours. 1. Weigh out 22 g of ammonium sulfate. Dissolve in approximately 750 ml of distilled water. Bring to 63 °. 2. Weigh out 76.5 g of aluminum ammonium sulfate. Dissolve in approximately 2¼ liters of distilled water. Bring to 58 °. 3. Prepare a 100-ml quantity of a 50% solution of absolutely fresh ammonium hydroxide (ammonia analysis--minimum 28%, maximum 30%) from a previously unopened bottle. This is important. 4. Quickly pour 50% ammonium hydroxide in one lot into ammonium sulfate solution. The temperature will drop from 63 ° to approximately 60 ° . Immediately pour this solution in one lot into the aluminum ammonium sulfate solution. A precipitate will form immediately. Begin F. C. Monkhouse, E. S. France, and W. H. Seegers, Circulation Res. 3, 397 (1955).
918
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[58]
stirring the gel very vigorously, maintaining the temperature between 58 ° and 60 °, and continue for 10 minutes. 5. Centrifuge the gel rapidly (2000 rpm for 5 minutes sufficient). Use the centrifuge brake to speed up the process, and pour off the supernatant. 6. Suspend the precipitated gel by stirring or shaking in 1 liter of distilled water to which 0.44 ml of concentrated ammonium hydroxide has been added. 7. Centrifuge as before, suspending the precipitated gel in 1 liter of distilled water to which has been added 0.88 ml of ammonium hydroxide. 8. Centrifuge three times more, suspending the precipitated gel each time in 1 liter of distilled water (no ammonium hydroxide added). 9. Centrifuge and suspend gel in the least amount of distilled water which still allows the gel to be pipetted. Store at 4 °.
Adsorption o] Antithrombin Defibrinate the plasma by heating to 53 °, maintaining that temperature for 3 minutes and cooling quickly to room temperature. Remove the denatured fibrinogen 'by filtering the plasma through several layers of gauze. To remove the prothrombin, add 50 mg of BaCOs powder for each milliliter of plasma, stir gently for 10 minutes, and centrifuge. To each milliliter of supernatant add 0.2 ml of aluminum hydroxide, stir gently for 10 minutes, centrifuge, and discard the supernatant. Elute the antithrombin from the precipitate with 0.3 ml of 0.05 M sodium phosphate buffer (pH 7.8) per milliliter or original plasma.
Preparation ]or Chromatography Each liter of original plasma yields approximately 300 ml of eluate. For further purification, this eluate is reduced in volume to approximately 40 ml. Lyophilization frequently results in loss of specific activity. In our hands, pervaporation in cellophane bags has proven to give the most consistent results. Following the reduction in volume, dialyze the material ai~ainst a constantly stirred 0.05 M phosphate buffer for 5 hours, with a change of buffer every hour. The material is now referred to as crude antithrombin and usually contains a concentration of antithrombin activity 9-12 times that of normal defibrinated plasma. It can be maintained without loss of activity at this stage for an indefinite period of time if stored at --20 °. Before applying to a chromatographic column for further purification, dialyze the crude antithrombin for a further 2 hours against 0.065 M Tris buffer, pH 8.6.
Preparation o] Chromatographic Columns Suspend 120 g of N,N-diethylaminoethyl ether (DEAE) cellulose in 3.5 liters of distilled water to which is added approximately 5 g of so-
[68]
ASSAY OF PLASMA ANTITHROMBIN
919
dium hydroxide pellets. Stir thoroughly with a magnetic stirrer (pH will rise to 11 or higher) and filter. Add concentrated HC1 to the cellulose suspended in water until the p H is reduced to 2. Following this, add sufficient 5 N sodium hydroxide to raise the p H to 3. Filter and wash with distilled water and reconstitute in Tris buffer at pH 8.6. When the cellulose has been equilibrated to the buffer (this takes three to four changes of 2-liter quantities of buffer with 1 hour of equilibration per change), run the column for 3 hours with 0.065 M tris buffer, p H 8.6. The above amount of cellulose will normally prepare a column 40 mm in diameter and 120 cm in length.
Separation by a DEAE-Cellulose Column With the stopcock at the bottom of the column closed, pipette 25 ml of crude antithrombin (concentrated eluate) carefully onto the surface of the column. When the eluate has covered the column with an even layer, partially open the stopcock and allow the eluate to flow into the cellulose. Allow a head of 30 to 40 mm of the first eluting solution, Tris buffer containing 0.075 M sodium chloride, to build up, and seal the attached head. Adjust the flow rate to 3 ml per minute. Apply a one-bed volume of each of the following solutions in the order given: (1) 0.075 M, (2) 0.125 M, and (3) 0.200 M sodium chloride, all in Tris buffer at p H 3.0 A
_0 0.
0
2.5
2.C 1.5 1.0
p
05 0 1
2
3 Liters of eluate
4
FIG. 2. Chromatography on DEAE-cellulose of crude antithrombin from beef plasma. The column was equilibrated with Tris buffer, 0.065M, pH 8.6. The proteins were eluted from the column with gradients of 0.075M, 0.125M, 0200 M, and 2.0 M sodium chloride in Tris buffer. Arrows indicate where changes of concentration at the top of the column began. The shaded area indicates the area where antithrombin activity was recovered. The small numbers over the bars indicate specific fractions which were subjected to eleetrophoresis on cellulose acetate strips shown in Fig. 3.
920
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[68]
Antithrombin % specific activity
38
17
35
4
457
2885
6
3421 1-79
FI(~. 3. Electrophoretic patterns on cellulose acetate strips run 75 minutes at 200 V in 0.03 M phosphate buffer, pH 7~. Numbers 1-7 refer to the samples from the DEAE column in the regions indicated by the numbers over the bars in Fig. 2. 8.6. Figure 2 illustrates the pattern of fractionation and the shaded area indicates where the antithrombin activity is eluted. In Fig. 3, electrophoretic patterns of aliquots from this area are shown along with the antithrombin activity. '
Further Purification with Starch Gel Electrophoresis Small quantities of antithrombin with high specific activity can be obtained by subjecting the best material from the chromatographic column ~ starch gel electrophoresis according to the method of Smithies.1° By making the gel double the standard thickness, and using a slot double the thickness used by Smithies, up to 2 ml of elua~e can be processed a~ one time. The buffer used for both starch suspension and the bridge soluO. Smithies, Biochem. J. 61, 629 (1955).
[58]
ASSAY OF PLASMA ANTITHROMBIN
921
tion should be 0.006M in respect to phosphate and contain 1.85 g of NaH2PO, per liter. Best results have been obtained at pH 8.5 with runs for 17 hours at 280 V. During this time the current gradually increases from approximately 20 to 50 mA. Following the electrophoresis, a strip of gel cut longitudinally is stained with amido black and the remainder cut in sections perpendicular to the direction of flow, as indicated by bands on the stained portion. The sections are frozen and thawed and the fluid expressed by pressure. They are washed once with 0.05 M phosphate buffer. The extracts from a number of runs can be pooled and reduced in % specific activity Antithrombin
Cofactor
6625
175
2
5484
539
5
2217
0
4
4003
802
FXG.4. Electrophoretic patterns of material recovered from starch gel after electrophoresis. (1) Trailing edge of protein band after elution. (2) Center of band. (3) Leading edge of band. (4) Material applied to starch gel. (5) Standard plasma. Cofactor refers to heparin cofactor activity. Percent specific activity refers to the activity per milligramof protein compared to a standard plasma. volume. In Fig. 4, electrophoretic patterns of material prepared by the starch gel method are illustrated as they appear on cellulose acetate strips. Properties of Antithrombin Optimal activity of antithrombin occurs between pH 8 and 8.5.11 At pH's below 5.7, antithrombin loses its activity and if maintained at this pH for 1 hour or more, this loss of activity is irreversible. Antithrombin 11F. C. Monkhouse, Thromb. Diath. Hemorrhag. 9, 387 (1963).
922
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[68]
I'00i 80" 60" 4020-
0
20
4o
60
8'0
Incubationat 60° (rain) (0
FIa. 5. The decrease in antithrombin activity of plasma when incubated at 60 °. O ) Dog plasma; ( O O ) human plasma.
loses activity at low ionic concentration. 11 Full activity can be restored/ by additions of di- or trivalent anions, with phosphate ion the most active. Loss of activity occurs if antithrombin is kept in a low ionic solution for any length of time, thus it should be stored in a 0.05 M phosphate solution. Antithrombin is relatively stable below 60 ° but loses activity rapidly above 60 °. There is some species difference, with human antithrombin being most labile (Fig. 5)~ Human antithrombin is more labile than that of dog, rabbit, beef, or rat. Many organic solvents destroy antithrombin activity. Ethyl alcohol, anesthetic ~ether, and chloroform are particularly destructive, but petroleum ether has little effect. If anything, it tends to increase activity. At least three washes with 3 volumes of anesthetic ether are necessary to remove all antithrombin activity from plasma. When this is accomplished, the thrombin formed from the prothrombin in the ether-extracted plasma remains stable for several hours. 12 F. C. Monkhouse, Can. J. Physiol. Pharmacol. 43, 819 (1965).
[68]
ASSAY OF PLASMA ANTITHROMBIN
923
One of the oddities of the antithron~bin-thrombin relationship is that they mutually destroy each other. 13 This accounts for the somewhat lower antithrombin activity in serum than in plasma, and the greater the amount of thrombin generated during coagulation, the lower the antithrombin activity in the resulting serum.13 Similarly, the addition of thrombin to plasma lowers the antithrombin level in proportion to the amount of thrombin added. Highly purified fractions of antithrombin have been found by Seegers et al. to quantitatively inactivate their purified autoprothrombin C 14 and purified autoprothrombin I (probably equivalent to factor VII).1~ More recently, work in our laboratory has shown that highly purified antithrombin inactivates plasmin (Fig. 6), However, while the antithrombin fractions lose their ability to inactivate plasmin on freezing and storage at --20 °, they retain full antithrombin activity. No change in
95OI
1450
850 L
•~
25o
#
.c
.
c:
E
g
~ 150 .=:
E 150
5O
50 1
4
5 Fraction
6
7
number
FIG. 6. The antithrombin and antiplamnin activity of fractions of crude antithrombin subjected to purification by DEAE-cellulose chromatography. The antiplasmin is expressed in terms of micromoles of e-amino-N-caproic acid which would give an equivalent degree of inhibition. Solid bars represent antiplasmin activity, open bars antithrombin activity. F. C. Monkhouse, Am. J. Physiol. 197, 984 (1959). ~4W. H. Seegers, E. R. Cole, C. R. Harmison, and F. C. Monkhouse, Can. J. Biochem. 42, 359 (1964). W. H. Seeger~, H. Schroer, and K. Mitsyasu, Can. J. Biochem. 42, 1425 (1964).
924
NATURALLY OCCURRING ACTIVATORS AND INI-IIBITORS
[69]
protein pattern following the freezing has been demonstrable so far. It may be, therefore, that the two activities are functions of different parts of the same molecule. An association between antithrombin and antiplasrain has been reported by other workers. 16,1~ While the main antithrombin activity is associated with the alpha globulins, it has 'been shown that some antithrombin activity is associated with preparations of alpha macroglobulins, is This antithrombin apparently does not inhibit the esterase activity of antithrombin, la The main antithrombin, that associated with the ~.pha globulins, inactivates both the clotting activity and the esterase activity of thrombin. The exact relationship of these closely associated proteolytic activities cannot be settled until more chemically pure fractions are available. To obtain this purity without loss of activity is proving extremely difficult. N. Heimburger, First International Symposium on Tissue Factors in the Homeostasis o] the Coagulation,-Fibrinolysis System. Florence, Italy, May, 1967 p. 353. ~*H. Gans and B. It. Tan, Clin. Chim. Acta 17, 111 (1967). M. Steinbuch, C. Blatrix, and F. Josso, Rev. Franc. Etudes Clin. Biol. 13, 179 (1968). "M. Steinbuch, C. Blatrix, and F. Josso, Nature 2!6, 500 (1967).
[ 6 9 ] H i r u d i n a s a n I n h i b i t o r of T h r o m b i n B y :FRITZ MARKWhRDT
Introduction The salivary glands (also called neck glands or pharyngeal glands) of the leech Hirudo medicinalis contain a substance with anticoagulant properties which has been named hirudin. During leeching, the blood sucker secretes this anticoagulant into the wound in order to keep the blood from clotting. Hirudin was first isolated in 1955 and identified as a polypeptide. 1'2 The following amino acid composition was found (number of residues per mole): 10 Asp, 13 Glu, 6 Cys, 4 Ser, 9 Gly, 4 Thr, 1 Ala, 3 Val, 4 Leu, 2 Ile, 3 Pro, 2 Phe, 2 Tyr, 1 His, and 4 Lys. The amino acid sequence from the C-terminal end of the molecule is -Ala-Gly-Ser-Glu-Leu? The molecular weight based on the amino acid composition was 10,800. This is in agreement with a value of 9060 obtained by measurements with the analytical ultracentrifuge. The sedi1F. Markwardt, Natururissenscha]ten 42, 587 (1955). F. Markwardt, Hoppe-Seylers Z. Physiol. Chem. 308, 147 (1957). P. de la Llosa, C. Tertrin, and M. Jutisz, Biochim. Biophys. Acta 93, 40 (1964).
ASSAY OF PLASMA ANTITHROMBIN
[68]
Preparation
and
Assay
of Plasma
915
Antithrombin
B y FRANX C. MONXHOUSZ
Introduction While antithrombin activity can be neutralized by a number of nonspecific agents and surfaces, the main progressive antithrombin of plasma is associated with the alpha globulins.1-a This protein fraction brings about the irreversible destruction of thrombin in a progressive manner typical of enzymatic degradation. ~,~ Assay Method Any method designed to estimate antithrombin activity should involve measurements over a time period. Since thrombin is known to be adsorbed on both dry glass and fibrinogen/,7 measurements should be carried out in silicone-treated glassware or plastic, and if the measurements are done on plasma, this should firstbe defibrinated. Since heating to 54 ° for 3-4 minutes has no measurable effect on antithrombin, this is : the preferable way to remove fibrinogen. In general, with small concentrations of thrombin relative to antithrombin, practically all thrombin is destroyed and the reaction is of the first order. With high concentrations of thrombin in relation to antithrombin, equilibrium is reached more slowly, with some thrombin always remaining in the active state. T w o basic types of assay are recommended therefore, one (Method A) based on the use of small concentrations of thrombin and the other (Method B) on large concentrations relative to antithrombin concentration. Method A
This is a first-order reaction and since clotting times are inversely proportional to the thrombin concentration, a straight line results when log clotting times are plotted against t h r o m b i n - a n t i t h r o m b i n incubation 1F. C. Monkhouse and S. Milojevic, Can. J. Physiol. Pharmaeol. 46, 347 (1968). P. Porter, M. C. Porter, and J. N. Shanberge, Clin. Chim. Aeta 17, 189 (1967). s V. Abilgaard, 8cand. J.Clin. Invest. 21, 89 (1968). ' E. Mihalyi, J. Gen. Physiol. 37, 139 (1953). ° F. C. Monkhouse, "Blood Clotting Enzymology," p. 323. Academic Press, New York, 1967. e W. H. Seegers, M. Nieft, and E. C. Loomis, Science 1Ol, 520 (1945). 'W. H. Seegers, K. D. Miller, E. B. Andrews, and R. C. Murphy, Ann. J. Physiol. 169, 700 (1952).
916
NATURALLY OCCURRINGACTIVATORSAND INHIBITORS (a)
[58]
(b)
2OOO
I ooo ,-,
600
400 2OO "6
I00
/
6O 40 2O 0% tO Incubation time (rain)
Fie. 1. The straight-line relation between the logarithm of the clotting time and the thrombin-antithrombin incubation time. (a) With varying dilutions of defibrinated plasma as the source of antithrombin, and (b) dilution of purified antithrombin. times. The slope of this line is the rate constant K, and varies directly with the antithrombin concentration. Validity of this statement is illustrated by Fig. 1. The simplest and best method was introduced by Gerendas. 8 Porcelain spot plates are used in place of test tubes and a fine glass hook is used to detect the first signs of coagulation. To 0.3 ml of plasma (or antithrombin fraction), 0.2 ml of 0.05 M phosphate buffer pH 7.8 and 0.3 ml of thrombin solution (10 / m l ) are added. At 30second and minute intervals thereafter, 0.1 ml of this incubation mixture is added to 0.2 ml of a fibrinogen solution and the clotting time noted. Clotting times over a period of 6 minutes are plotted and the best-fitting straight line is drawn. Antithrombin activity is expressed in terms of frozen pooled defibrinated beef plasma stored in small aliquots at --20 °, and prepared fresh at 6-month intervals. Specific activity refers to the activity per milligram of protein as compared to this standard beef plasma. When it is desirable to use units to give a quantitative comparison rather than percent of standard, 1 ml of standard beef plasma is considered to contain 100 units of antithrombin. In our hands this has proven to be much more satisfactory than attempting to express antis M. Gerendas, Nature 157, 837 (1946).
[68]
ASSAY OF PLASMA ANTITHROMBIN
917
thrombin in terms of units of thrombin destroyed, since a thrombin unit is itself a somewhat arbitrary figure.
Method B If one wishes to use larger quantities of thrombin and to avoid clotting time as a measure, then the following method is recommended. Two hundred units of thrombin are placed in each of a number of siliconized test tubes. Varying amounts of the antithrombin solution to be assayed are then added. At least one tube has no antithrombin added, and serves as a control. The volumes are then adjusted to 1 ml by the addition of Tris buffer and the tubes are allowed to incubate at 28 ° for 1 hour. The amount of residual thrombin is then estimated by adding 0.2-ml aliquots of the incubated thrombin-antithrombin mixture to 0.2 ml of a 0.4 M TAME (p-toluenesulfonyl arginine methyl ester) solution. The volumes are adjusted to 2 ml with Tris buffer, pH 8.5, the tubes are incubated at 37 ° for 30 minutes and the extent of TAME hydrolysis determined. A curve is then made by plotting volume of antithrombin solution against units of thrombin neutralized. For further details of the method, consult the publication by Monkhouse et al? Purification The first stage in the purification of antithrombin is adsorption on aluminum hydroxide, and since the preparation of the aluminum hydroxide is the key to successful purification, details are given.
Preparation o] Aluminum Hydroxide Gel ]or Adsorption o] Plasma Antithrombin Note: This procedure should be carried out as rapidly as possible, never over more than 21/~ hours. 1. Weigh out 22 g of ammonium sulfate. Dissolve in approximately 750 ml of distilled water. Bring to 63 °. 2. Weigh out 76.5 g of aluminum ammonium sulfate. Dissolve in approximately 2¼ liters of distilled water. Bring to 58 °. 3. Prepare a 100-ml quantity of a 50% solution of absolutely fresh ammonium hydroxide (ammonia analysis--minimum 28%, maximum 30%) from a previously unopened bottle. This is important. 4. Quickly pour 50% ammonium hydroxide in one lot into ammonium sulfate solution. The temperature will drop from 63 ° to approximately 60 ° . Immediately pour this solution in one lot into the aluminum ammonium sulfate solution. A precipitate will form immediately. Begin F. C. Monkhouse, E. S. France, and W. H. Seegers, Circulation Res. 3, 397 (1955).
918
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[58]
stirring the gel very vigorously, maintaining the temperature between 58 ° and 60 °, and continue for 10 minutes. 5. Centrifuge the gel rapidly (2000 rpm for 5 minutes sufficient). Use the centrifuge brake to speed up the process, and pour off the supernatant. 6. Suspend the precipitated gel by stirring or shaking in 1 liter of distilled water to which 0.44 ml of concentrated ammonium hydroxide has been added. 7. Centrifuge as before, suspending the precipitated gel in 1 liter of distilled water to which has been added 0.88 ml of ammonium hydroxide. 8. Centrifuge three times more, suspending the precipitated gel each time in 1 liter of distilled water (no ammonium hydroxide added). 9. Centrifuge and suspend gel in the least amount of distilled water which still allows the gel to be pipetted. Store at 4 °.
Adsorption o] Antithrombin Defibrinate the plasma by heating to 53 °, maintaining that temperature for 3 minutes and cooling quickly to room temperature. Remove the denatured fibrinogen 'by filtering the plasma through several layers of gauze. To remove the prothrombin, add 50 mg of BaCOs powder for each milliliter of plasma, stir gently for 10 minutes, and centrifuge. To each milliliter of supernatant add 0.2 ml of aluminum hydroxide, stir gently for 10 minutes, centrifuge, and discard the supernatant. Elute the antithrombin from the precipitate with 0.3 ml of 0.05 M sodium phosphate buffer (pH 7.8) per milliliter or original plasma.
Preparation ]or Chromatography Each liter of original plasma yields approximately 300 ml of eluate. For further purification, this eluate is reduced in volume to approximately 40 ml. Lyophilization frequently results in loss of specific activity. In our hands, pervaporation in cellophane bags has proven to give the most consistent results. Following the reduction in volume, dialyze the material ai~ainst a constantly stirred 0.05 M phosphate buffer for 5 hours, with a change of buffer every hour. The material is now referred to as crude antithrombin and usually contains a concentration of antithrombin activity 9-12 times that of normal defibrinated plasma. It can be maintained without loss of activity at this stage for an indefinite period of time if stored at --20 °. Before applying to a chromatographic column for further purification, dialyze the crude antithrombin for a further 2 hours against 0.065 M Tris buffer, pH 8.6.
Preparation o] Chromatographic Columns Suspend 120 g of N,N-diethylaminoethyl ether (DEAE) cellulose in 3.5 liters of distilled water to which is added approximately 5 g of so-
[68]
ASSAY OF PLASMA ANTITHROMBIN
919
dium hydroxide pellets. Stir thoroughly with a magnetic stirrer (pH will rise to 11 or higher) and filter. Add concentrated HC1 to the cellulose suspended in water until the p H is reduced to 2. Following this, add sufficient 5 N sodium hydroxide to raise the p H to 3. Filter and wash with distilled water and reconstitute in Tris buffer at pH 8.6. When the cellulose has been equilibrated to the buffer (this takes three to four changes of 2-liter quantities of buffer with 1 hour of equilibration per change), run the column for 3 hours with 0.065 M tris buffer, p H 8.6. The above amount of cellulose will normally prepare a column 40 mm in diameter and 120 cm in length.
Separation by a DEAE-Cellulose Column With the stopcock at the bottom of the column closed, pipette 25 ml of crude antithrombin (concentrated eluate) carefully onto the surface of the column. When the eluate has covered the column with an even layer, partially open the stopcock and allow the eluate to flow into the cellulose. Allow a head of 30 to 40 mm of the first eluting solution, Tris buffer containing 0.075 M sodium chloride, to build up, and seal the attached head. Adjust the flow rate to 3 ml per minute. Apply a one-bed volume of each of the following solutions in the order given: (1) 0.075 M, (2) 0.125 M, and (3) 0.200 M sodium chloride, all in Tris buffer at p H 3.0 A
_0 0.
0
2.5
2.C 1.5 1.0
p
05 0 1
2
3 Liters of eluate
4
FIG. 2. Chromatography on DEAE-cellulose of crude antithrombin from beef plasma. The column was equilibrated with Tris buffer, 0.065M, pH 8.6. The proteins were eluted from the column with gradients of 0.075M, 0.125M, 0200 M, and 2.0 M sodium chloride in Tris buffer. Arrows indicate where changes of concentration at the top of the column began. The shaded area indicates the area where antithrombin activity was recovered. The small numbers over the bars indicate specific fractions which were subjected to eleetrophoresis on cellulose acetate strips shown in Fig. 3.
920
NATURALLY OCCURRING ACTIVATORS AND INHIBITORS
[68]
Antithrombin % specific activity
38
17
35
4
457
2885
6
3421 1-79
FI(~. 3. Electrophoretic patterns on cellulose acetate strips run 75 minutes at 200 V in 0.03 M phosphate buffer, pH 7~. Numbers 1-7 refer to the samples from the DEAE column in the regions indicated by the numbers over the bars in Fig. 2. 8.6. Figure 2 illustrates the pattern of fractionation and the shaded area indicates where the antithrombin activity is eluted. In Fig. 3, electrophoretic patterns of aliquots from this area are shown along with the antithrombin activity. '
Further Purification with Starch Gel Electrophoresis Small quantities of antithrombin with high specific activity can be obtained by subjecting the best material from the chromatographic column ~ starch gel electrophoresis according to the method of Smithies.1° By making the gel double the standard thickness, and using a slot double the thickness used by Smithies, up to 2 ml of elua~e can be processed a~ one time. The buffer used for both starch suspension and the bridge soluO. Smithies, Biochem. J. 61, 629 (1955).
[58]
ASSAY OF PLASMA ANTITHROMBIN
921
tion should be 0.006M in respect to phosphate and contain 1.85 g of NaH2PO, per liter. Best results have been obtained at pH 8.5 with runs for 17 hours at 280 V. During this time the current gradually increases from approximately 20 to 50 mA. Following the electrophoresis, a strip of gel cut longitudinally is stained with amido black and the remainder cut in sections perpendicular to the direction of flow, as indicated by bands on the stained portion. The sections are frozen and thawed and the fluid expressed by pressure. They are washed once with 0.05 M phosphate buffer. The extracts from a number of runs can be pooled and reduced in % specific activity Antithrombin
Cofactor
6625
175
2
5484
539
5
2217
0
4
4003
802
FXG.4. Electrophoretic patterns of material recovered from starch gel after electrophoresis. (1) Trailing edge of protein band after elution. (2) Center of band. (3) Leading edge of band. (4) Material applied to starch gel. (5) Standard plasma. Cofactor refers to heparin cofactor activity. Percent specific activity refers to the activity per milligramof protein compared to a standard plasma. volume. In Fig. 4, electrophoretic patterns of material prepared by the starch gel method are illustrated as they appear on cellulose acetate strips. Properties of Antithrombin Optimal activity of antithrombin occurs between pH 8 and 8.5.11 At pH's below 5.7, antithrombin loses its activity and if maintained at this pH for 1 hour or more, this loss of activity is irreversible. Antithrombin 11F. C. Monkhouse, Thromb. Diath. Hemorrhag. 9, 387 (1963).
922
NATURALLYOCCURRING ACTIVATORS AND INHIBITORS
[68]
I'00i 80" 60" 4020-
0
20
4o
60
8'0
Incubationat 60° (rain) (0
FIa. 5. The decrease in antithrombin activity of plasma when incubated at 60 °. O ) Dog plasma; ( O O ) human plasma.
loses activity at low ionic concentration. 11 Full activity can be restored/ by additions of di- or trivalent anions, with phosphate ion the most active. Loss of activity occurs if antithrombin is kept in a low ionic solution for any length of time, thus it should be stored in a 0.05 M phosphate solution. Antithrombin is relatively stable below 60 ° but loses activity rapidly above 60 °. There is some species difference, with human antithrombin being most labile (Fig. 5)~ Human antithrombin is more labile than that of dog, rabbit, beef, or rat. Many organic solvents destroy antithrombin activity. Ethyl alcohol, anesthetic ~ether, and chloroform are particularly destructive, but petroleum ether has little effect. If anything, it tends to increase activity. At least three washes with 3 volumes of anesthetic ether are necessary to remove all antithrombin activity from plasma. When this is accomplished, the thrombin formed from the prothrombin in the ether-extracted plasma remains stable for several hours. 12 F. C. Monkhouse, Can. J. Physiol. Pharmacol. 43, 819 (1965).
[68]
ASSAY OF PLASMA ANTITHROMBIN
923
One of the oddities of the antithron~bin-thrombin relationship is that they mutually destroy each other. 13 This accounts for the somewhat lower antithrombin activity in serum than in plasma, and the greater the amount of thrombin generated during coagulation, the lower the antithrombin activity in the resulting serum.13 Similarly, the addition of thrombin to plasma lowers the antithrombin level in proportion to the amount of thrombin added. Highly purified fractions of antithrombin have been found by Seegers et al. to quantitatively inactivate their purified autoprothrombin C 14 and purified autoprothrombin I (probably equivalent to factor VII).1~ More recently, work in our laboratory has shown that highly purified antithrombin inactivates plasmin (Fig. 6), However, while the antithrombin fractions lose their ability to inactivate plasmin on freezing and storage at --20 °, they retain full antithrombin activity. No change in
95OI
1450
850 L
•~
25o
#
.c
.
c:
E
g
~ 150 .=:
E 150
5O
50 1
4
5 Fraction
6
7
number
FIG. 6. The antithrombin and antiplamnin activity of fractions of crude antithrombin subjected to purification by DEAE-cellulose chromatography. The antiplasmin is expressed in terms of micromoles of e-amino-N-caproic acid which would give an equivalent degree of inhibition. Solid bars represent antiplasmin activity, open bars antithrombin activity. F. C. Monkhouse, Am. J. Physiol. 197, 984 (1959). ~4W. H. Seegers, E. R. Cole, C. R. Harmison, and F. C. Monkhouse, Can. J. Biochem. 42, 359 (1964). W. H. Seeger~, H. Schroer, and K. Mitsyasu, Can. J. Biochem. 42, 1425 (1964).
924
NATURALLY OCCURRING ACTIVATORS AND INI-IIBITORS
[69]
protein pattern following the freezing has been demonstrable so far. It may be, therefore, that the two activities are functions of different parts of the same molecule. An association between antithrombin and antiplasrain has been reported by other workers. 16,1~ While the main antithrombin activity is associated with the alpha globulins, it has 'been shown that some antithrombin activity is associated with preparations of alpha macroglobulins, is This antithrombin apparently does not inhibit the esterase activity of antithrombin, la The main antithrombin, that associated with the ~.pha globulins, inactivates both the clotting activity and the esterase activity of thrombin. The exact relationship of these closely associated proteolytic activities cannot be settled until more chemically pure fractions are available. To obtain this purity without loss of activity is proving extremely difficult. N. Heimburger, First International Symposium on Tissue Factors in the Homeostasis o] the Coagulation,-Fibrinolysis System. Florence, Italy, May, 1967 p. 353. ~*H. Gans and B. It. Tan, Clin. Chim. Acta 17, 111 (1967). M. Steinbuch, C. Blatrix, and F. Josso, Rev. Franc. Etudes Clin. Biol. 13, 179 (1968). "M. Steinbuch, C. Blatrix, and F. Josso, Nature 2!6, 500 (1967).
[ 6 9 ] H i r u d i n a s a n I n h i b i t o r of T h r o m b i n B y :FRITZ MARKWhRDT
Introduction The salivary glands (also called neck glands or pharyngeal glands) of the leech Hirudo medicinalis contain a substance with anticoagulant properties which has been named hirudin. During leeching, the blood sucker secretes this anticoagulant into the wound in order to keep the blood from clotting. Hirudin was first isolated in 1955 and identified as a polypeptide. 1'2 The following amino acid composition was found (number of residues per mole): 10 Asp, 13 Glu, 6 Cys, 4 Ser, 9 Gly, 4 Thr, 1 Ala, 3 Val, 4 Leu, 2 Ile, 3 Pro, 2 Phe, 2 Tyr, 1 His, and 4 Lys. The amino acid sequence from the C-terminal end of the molecule is -Ala-Gly-Ser-Glu-Leu? The molecular weight based on the amino acid composition was 10,800. This is in agreement with a value of 9060 obtained by measurements with the analytical ultracentrifuge. The sedi1F. Markwardt, Natururissenscha]ten 42, 587 (1955). F. Markwardt, Hoppe-Seylers Z. Physiol. Chem. 308, 147 (1957). P. de la Llosa, C. Tertrin, and M. Jutisz, Biochim. Biophys. Acta 93, 40 (1964).