THROMBOSIS RESEARCH Printed in the United
Vol. States
7, pp* 555-566, Pergamon Press,
1975
Inc.
SIMULTANEOUS DETERMINATION OF PLASMA PROTHROMBIN AND ANTITHROMBIN III (HEPARIN COFACTOR)
Anthony A. Magnin" and William B. Lawson (with technical assistance of William W. Knox III) Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201, U.S.A.
(Received Received
Accepted by Editor 4.4.1975. by Executive Editorial Office
F. Markwardt. 20.8.1975)
ABSTRACT A method for the simultaneous determination of plasma prothrombin and antithrombin III has been developed. It involves activation of a dilute plasma solution with Taipan snake venom and measurement of the heparin-mediated loss of thrombin activity after short incubation. The antithrombin III content is quantitated on the basis of results with known mixtures of normal and antithrombin III-deficient plasmas.
INTRODUCTION Oxyuranus 5. scutellatus (Australian Taipan snake) venom is being used increasingly for specific assay of prothrombin, i.e., factor II (1,2), by conversion to thrombin (3,4). Although calcium is necessary, the venom does not require additional coagulation factors to activate prothrombin. It thus permits assay of the purified zymogen. We have adapted this assay to measure simultaneously prothrombin and the natural inhibitor antithrombin III (AT-III), also called heparin cofactor (5-8) or antifactor Xa (9-13). AT-III inhibits thrombin by reacting with it to yield a very tight molecular complex, the formation of which requires the active-center serine of thrombin (8,14). The formation of the complex is greatly accelerated by heparin, although its mode of action is still uncertain. Similar complexes are probably formed by AT-III with factors IXa, Xa, XIa, and plasmin (15-17) in an analogous fashion. A hereditary deficiency of AT-III has been reported in several families particularly susceptible to recurrent deep vein thromboses (18-25). Affected patients had AT-III levels between 30 and 60% of normal. This thrombotic tendency is surprising in view of the fact that normal plasma has sufficient AT-III to neutralize about 4 times the total potential plasma thrombin (14). By contrast, severe hemorrhagic complications of hereditary or acquired "'PresentAddress:
Connaught Laboratories Ltd., Steeles Ave. W., Willowdale, Ontario, Canada 555
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coagulopathies usually do not occur until the levels of clotting factors are reduced to less than lo-20% of normal. Low AT-III levels have been associated with liver cirrhosis and other forms of liver disease (26-30). Decreased levels in women on oral contraceptives have been reported by some workers (31,32) but not others (27). AT-III has been measured by immunologic techniques (23,27,31) or by coagulation assays (11,12,26,29,33-35). The latter assays measure either (a) the increase in the fibrinogen clotting time of a standard thrombin solution to which defibrinated plasma or, in some cases, serum has been added, or (b) the decrease in clotting activity of purified factor Xa after incubation with AT-III. The coagulation assay procedures used to date are time-consuming and require purified thrombin or factor Xa. For example, Yue et al. (36) added heat-defibrinated, barium sulfate-adsorbed plasma to purified bovine thrombin and found a rapid loss of thrombin activity, followed by a much slower loss due to other plasma antithrombins, such as a2-macroglobulin. The rapid, reliable AT-III assay reported here should be especially useful in the assessment of liver function (28) and would permit further investigation of the effect of oral contraceptives on AT-III. This assay should also facilitate the study of families having histories of recurrent venous thromboses.
MATERIALS Taipan snake venom was obtained from Eric Worrell's Australian Reptile Park, Gosford, NSW, Australia. Heparin-sodium was purchased from Sigma, St. Louis, MO. Bovine fibrinogen, 90% clottable, was purchased from Miles Laboratories, Kankakee, Ill. Inosithin was obtained from Associated Concentrates, Woodside, N.Y. Polyethylene glycol (PEG)-6000 was purchased from J. T. Baker, Phillipsburg, N.J. NIH standard thrombin (lot B-3) was obtained from the Bureau of Biologics, U. S. Food and Drug Administration. The standard human plasma used in these experiments was a pool of samples from 200 normal blood donors. Blood (4 ml) was drawn into acid-citrate-dextrose (ACD) anticoagulant (1 ml) in a Vacutainer tube (Becton-Dickinson) and centrifuged at 1,000 x g for 2 min. Supernatant plasma was aspirated with a plastic syringe. The plasma pool was -recentrifuged at 1,000 x a, dispensed in 0.5- and l.O-ml aliquots into polycarbonate tubes, and stored at -70°C. The prothrombin level remained constant after 10 months of storage. Plasma from individual donors was obtained in the same way. AT-III-deficient (AT-III-D) plasma was prepared from the standard plasma by gentle shaking with three volumes of isoamyl alcohol for 1 min according to Monkhouse et al. (16) and stored in polycarbonate tubes in 0.2-ml aliquots at -70°C. Although AT-III is almost entirely inactivated (C4%), the plasma retains all its prothrombin activity even after 6 months of storage. Buffer 1 contained 0.9% N&l and 0.068% Three buffers were used. -_ imidazole, adjusted with HCl to pH 7.4. The others were identical except for the following additions: Buffer 2> 0.88% PEG-6000; Buffer 3,)Q.88% PEG-6000 All quantities were w/v. and 0.4% CaC12 *2&0it Inosithin was suspended in Buffer 2 (10% W/V) and stored frozen in 0. l-ml aliquots. Before use, an aliquot was thawed and diluted l,OOO-fold in Buffer 2.
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Taipan venom was dissolved in Buffer 2 (1 mg/ml) and stored frozen in O.l-ml aliquots. This stock solution was diluted to 10 ml in Buffer 2 and then with an equal amount of Buffer 3 before use. Fibrinogen was stored frozen in lo-ml aliquots of a 1% (w/v) solution in Buffer 1. Heparin was prepared as a stock solution (500 U.S.P. units/ml) in Buffer 1 and stored frozen in O.l-ml aliquots. It was diluted for use to the appropriate concentration in Buffer 1. NIH standard thrombin was mixed thoroughly in Buffer 1 to a concentration of 100 units/ml and stored frozen in l-ml aliquots. These reagents were stored in polycarbonate tubes at -30°C.
METHODS Assay.of prothrombin To 0.5 ml of a human plasma dilution in Buffer 2 (usually 1:50) were added 0.5 ml of dilute inosithin suspension and 1.0 ml of Taipan venom solution containing calcium. This mixture was incubated for 2 min at 37°C in 0.3-ml aliquots; then 0.1 ml of fibrinogen solution was added, and the clot formation was timed with a Fibrometer (Baltimore Biological Laboratories). The assays were run in duplicate or triplicate, and the prothrombin concentration was determined from a clotting time curve. All prothrombin concentrations were corrected for anticoagulant. Our laboratory routinely substitutes PEG for acacia in the assay of highly purified thrombin solutions (37). A final concentration of 0.66% (w/v) PEG was chosen, although 0.4% was later found to approximate more closely the effect of acacia as reported by Ware and Seegers (38). One prothrombin unit was defined as the amount which, when activated, causes a 1% fibrinogen solution to clot in 15 sec. (Dilutions of NIH standard thrombin in this system with and without the venom give clotting times corresponding to about 1.5 times the NIH unit.) The different slope of the line obtained with this method necessitated the calculation of a new calibration chart (Table 1). Assay of plasma antithrombin III Solutions containing varying levels of AT-III were prepared by mixing of diluted (1:50) normal human and AT-III-D plasmas. To appropriate amounts 0.5-ml portion of each solution was added an equal volume of the dilute inosithin suspension described above, followed by 1.0 ml of the dilute venom solution. The mixture was incubated at 37°C for exactly 1 min. Ten (11 of either Buffer 3 or heparin (7.5 units/ml; final concentration, 3.75 x 10s3 units/ml) were then added, and the solution was mixed and incubated in 0.3-ml aliquots at 37'C for exactly 90 sec. Fibrinogen solution (0.1 ml) was introduced and the clotting time was measured. Duplicate assays were run simultaneo;sly by using two Fibrometers. The fraction of thrombin remaining was calculated from the mean clotting times in the presence and absence of heparin. A linear relationship was obtained between the percentage of thrombin remaining in solution and the percentage of AT-III present, based upon the serial dilution of normal human plasma with AT-III-D plasma (see Results). Individual plasma samples were diluted to give clotting times of 12-20 set in the absence of heparin (usually a 1:50 dilution). The percentage of thrombin remaining after incubation with heparin was calculated, and the AT-III
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TABLE 1 Concentration of Thrombih Related to Clotting Time
Seconds
Units
12.0 .2 .4 .6 .8 13.0 .2 .4 .6 .8 14.0 .2 .4 .6 .8
1.44 1.40 1.36 1.32 1.29 1.25 1.22 1.20 1.17 1.14 1.11 1.09 1.06 1.04 1.02
Seconds
15.0 .2 .4 .6 .8 16.0 .2 .4 .6 .8 17.0 .2 .4 .6 .8
Units
Seconds
1.00 0.98 0.96 0.94 0.92 0.91 0.89 0.87 0.86 0.84 0.83 0.81 0.80 0.79 0.78
18.0 .2 .4 .6 .8 19.0 .2 .4 .6 .8 20.0 .2 .4 .6 .8
Units
0.76 0.75 0.74 0.73 0.72 0.71 0.70 0.69 0.68 0.67 0.66 0.65 0.64 0.63 0.62
Seconds
21.0 .2 .4 .6 .8 22.0 .2 .4 .6 .8 23.0
TABLE 2 Clotting Time of Diluted Pooled Human Plasma Solutions (Corrected for Anticoagulant)
Final dilution of plasma
200 233 267 300 333 367 400 433 467 500
Clotting time (set)
Standard deviation
No. of assays
11.7 12.8 14.2 15.5 16.5 17.4 18.6 19.5 20.8 21.9
0.4 0.4 0.5 0.2 0.3 0.3 0.3 0.3 0.4 0.3
63 81 67 70 71 63 63 68 74 25
Units
0.62 0.61 0.60 0.59 0.59 0.58 0.57 0.57 0.56 0.55 0.55
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content was read from the graph. A second determination was carried out by diluting the sample with diluted normal or AT-III-D plasma.
RESULTS Prothrombin assays Table 2 summarizes the results of up to 81 replicate assays of the pooled human plasma. Over the range of 12-20 set, the clotting time is linear and inversely proportional to the prothrombin concentration. In 35 plasmas from individual healthy donors, the prothrombin concentration was found to be 287 f 41 units (mean f 1 SD; range 211-416); this is equivalent to a mean of 100 + 14% of the standard pool. To measure the rate of activation and the stability of the plasma-lipidvenom mixture, venom was added to plasma (initially diluted 1:50 in Buffer 1) and lipid mixtures as described under Methods. Aliquots were incubated for fixed intervals, and the clotting times were determined. Table 3 summarizes the data. Activation of prothrombin is complete within 2 min, and there is no measurable decrease in thrombin levels for up to 6.min. When incubated for more than 6 min, thrombin activity is lost at an approximately linear rate. The delay in inactivation of the formed thrombin forms the basis of the AT-III assay and, in addition, permits measurement of replicate clotting times when assaying for prothrombin.
TABLE 3 Rate of Activation of Plasma Prothrombin
Incubation time (min) Clotting time (set)
0.5 1
2
3
4
5
6
7
10
20
30
60
18.9 16.8 16.5 16.5 16.4 16.5 16.6 17.9 17.3 18.6 21.1 25.2
f SD
1.3 0.7 0.6 0.6 0.5 0.5 0.3 0.5 0.6 0.2 0.2
No. samples
3
29
35
28
17
16
6
3
4
2
2
1
A 1:50 dilution of pooled human plasma was activated with Taipan venom as described. The quantity of thrombin formed was determined following incubation at 37°C. Antithrombin assays The AT-III assay was performed in duplicate as described under Methods. The amount of thrombin remaining was calculated by dividing the mean clotting time in the absence of heparin by that in the presence of heparin. Figure 1 shows a linear relationship between the percentage of thrombin remaining and the percentage of AT-III in the test plasma. The action of other plasma
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z 5 40
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REMAINING
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FIGURE 1 Calibration of the AT-III assay. The percentage AT-III is based upon mixtures of diluted normal pooled (100%) and diluted isoamyl alcohol-treated (0%) human plasma. The percentage of thrombin remaining is calculated from the ratio of the clotting times in the absence and presence of heparin (see text). antithrombins, such as u2-macroglobulin is compensated for by the blank determination. The relatively short incubation time also precluded a major contribution by these more slowly acting inhibitors. The prothrombin concentration is readily calculated from the clotting time in the absence of heparin. Factors influencing antithrombin III assay To determine the optimum time for incubation of the formed thrombin with AT-III and heparin, the following experiment was performed: To plasma solutions containing known AT-III levels, lipid and Taipan venom were added as described. After exactly 1 min, 10 ~1 of a heparin solution (10 units/ml) were added, and the mixtures were further incubated at 37°C in 0.3-ml aliquots. After 2.5, 5.0, and 6.5 min from the start of the experiment, residual thrombin was assayed by the addition of fibrinogen. These results are plotted in Fig. 2. AS the incubation time increased the plots became progressively nonlinear. f 4.0,
The effect of incubation time on the rate of inactivation of formed thrombin in normal plasma, AT-III-D plasma, and an equal mixture of the two was tested after addition of varying amounts of heparin (5.0-10.0 units/ml). The least deviation from linearity was obtained with a short incubation time (90 set) and a low level of heparin.
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6C
2c )-
I-
20
60
40
THROMBIN
80
REMAINING
100
(%)
FIGURE 2 Effect of incubation time on plot of the percentage AT-III vs percentage thrombin remaining. Heparin (10 ~1, 10 units/ml) was added to activate dilute normal plasma to which varying amounts of dilute AT-III-D plasma had been added. The mixtures were incubated for 1.5 (o), 3.0 (A), 4.0 (I) and 5.5 (0) min.
To determine the optimum heparin concentration, stock solutions of plasma diluted 1:30, 1:50, 1:70, and 1:90, containing 50% levels of AT-III, were activated with Taipan venom for exactly 1 min. Heparin was then introduced in lo-~1 portions at four concentrations, and each mixture was assayed for thrombin after exactly 90-set incubation. The means of the four dilutions at each heparin level (units/ml) showed the following loss of thrombin activity 2.5 units, 24.9%; 5.0 units, 38.1%; 7.5 units, 48.7%; 10.0 units, 55.7%. A concentration of 7.5 units/ml was chosen as optimum. giving a significant loss of thrombin activity in the mixture, yet retaining maximum linearity over the 90-see incubation period. When the clotting time of each plasma dilution is plotted against the heparin concentration (Fig.3), the-percent of thrombin remaining at each heparin concentration is seen to be essentially constant, indicating that the plasma prothrombin content doesnot significantly influence the AT-III determination in the range studied. Blood from patients hospitalized with venous thrombosis was collected at local hospitals and assayed as described above. The plasmas from three patients known to be congenitally deficient in AT-III were also studied. Table 4 summarizes these results. When plasma containing low levels of AT-III is assayed, the change in clotting time on addition of heparin is small, resulting
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FIGURE 3 Effect of the initial concentration of prothrombin on the rate of inactivation of the formed thrombin. The plasma dilutions corrected for anticoagulant were (o), ZOO-fold; (B> 333-fold, (0) 467-fold, and (A) 600-fold. in relatively large errors in the calculation of the percentage of thrombin remaining. The AT-III level of such plasmas can, however, be readily adjusted with pooled human plasma to a level of activity which permits more accurate assay. Plasmas containing high levels ('100%) of AT-III were diluted with AT-III-D plasma to facilitate AT-III quantitation.
DISCUSSION' Under the conditions described above, the thrombin generated from dilute human plasma by Taipan snake venom is sufficiently stable to allow the assay of AT-III. This simple method involves comparing the fibrinogen clotting times of the activated plasma solutions in the presence of a low level of heparin or buffer. All reagents except the snake venom are inexpensive and readily available, and very little of the venom is used. Purified factor Xa or thrombin is not required, since thrombin is generated from plasma in the assay. PEG, which is available in fairly pure form, substitutes well for acacia (33) in accelerating the clotting time. In contrast to the results of van der Meer et al. (23), simultaneous immunologic determination of AT-III levels led to higher values than those obtained by coagulation.assay using our method or that of Marciniak et al. (25). This discrepancy was particularly evident with the congenitally AT-III-D
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TABLE 4 Antithrombin III and Prothrombin Assays of Individual Plasmas
Patient (age, sex)
Normal human plasma A (70,F) B (74,M) C (23,~) D (50,M) E (23,~) F (24,~) G (26,F)l H (42,~) I (65,M) J (42,M) K (40,F) L (39,F)2 M ( ?,?) N (34,M); 0 (14,M)
AT-III (%) Immunoassay* This method
100 102 94 105 81 103 66 110 85 101 76 99 108 35 34 42
* 7 f 11 + 1 z!z 2 f 11 * 5 f 5 + 3 f.4 + 6 f 6 + 3 + 2 * 2
100 113 103 114 101 115 111 106 107 105 115
83 90
Prothrombin Units+ %
287 253 267 307 240 416 380 453 113 277 337 337 366 284 302 279
100 88 93 107 84 145 132 128 39 97 117 117 128 99 105 97
Ji
Performed by Dr. Richard Propp of Albany Medical College by Laurel1 electroimmunodiffusion using antibody prepared by Alper from antigen provided +by Rosenberg (8) Corrected for anticoagulant. 1Patient on warfarin. 2Patient congenitally AT-III-D. Plasma a gift from Dr. S. S. Shapiro; AT-III 50% by immunoassay (personal communication). 3Patient congenitally AT-III-D. Gift of Dr. E. Marciniak. Reported AT-III levels were: Patient N, 35%; Patient 0, 40% (personal cormnunication).
plasmas (Table 4). Although no further data are presently available, the results raise the’ possibility of the presence of a nonfunctional antigen related to AT-III, analogous to the situation observed in hemophilia (39).
ACKNOWLEDGMENTS The authors wish to thank Dr. Sandor S. Shapiro, Cardeza Foundation, Jefferson Medical College, Philadelphia, Pa., and Dr. Ewa Marciniak, University of Kentucky Medical Center, Lexington, KY., for congenitally AT-III-D plasmas; Dr. Edwin Taft, Albany Medical Center Hospital, Albany, N.Y., and Dr. George Vlahides, Ellis Hospital, Schenectady, N.Y., for plasmas from patients with recurrent venous thromboses; Dr. Anthony F. H. Britten, American National Red Cross, Northeastern New York Chapter, Albany, N.Y., for the normal plasma pool and the plasma from normal people used in this study; and Dr. W. Jean Dodds of this Division for many helpful discussions.
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This research was supported in part by Grant AM 05299 from the National Institutes of Health.
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ABILDGAARD, U. Highly purified antithrombin III with heparin cofactor activity prepared by disc electrophoresis. Stand. J. Clin. Lab. Invest. 2 89, 1968.
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ROSENBERG, R. D. and DAMUS, P. S. The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem. 248. 6490, 1973.
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SEEGERS, W. H., COLE, E. R., HARMISON, C. R. and MONKHOUSE, F. C. Neutralization of autoprothrombin C activity with antithrombfn. Canad. J. Biochem. 42, 359, 1964.
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BIGGS, R., DENSON, K. W. E., AKMAN, N. BQRRETI, R. and HADDEN, M. Antithrombin III, antifactor Xa and hebarin. Brit. J. Haemat. 19. 283, 1970.
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MARCINIAK, E. and TSUKAMURA, S. Two progressive inhibitors of factor Xa in human blood. Brit. J. Haemat. 2 341, 1972.
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MARCINIAK, E. Factor-Xa inactivation by antithrombin III: Evidence for biological stabilization of factor Xa by factor V-phospholipid complex. Brit. J. Haemat. 24. 391, 1973.
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Vol. States
7, pp* 555-566, Pergamon Press,
1975
Inc.
SIMULTANEOUS DETERMINATION OF PLASMA PROTHROMBIN AND ANTITHROMBIN III (HEPARIN COFACTOR)
Anthony A. Magnin" and William B. Lawson (with technical assistance of William W. Knox III) Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201, U.S.A.
(Received Received
Accepted by Editor 4.4.1975. by Executive Editorial Office
F. Markwardt. 20.8.1975)
ABSTRACT A method for the simultaneous determination of plasma prothrombin and antithrombin III has been developed. It involves activation of a dilute plasma solution with Taipan snake venom and measurement of the heparin-mediated loss of thrombin activity after short incubation. The antithrombin III content is quantitated on the basis of results with known mixtures of normal and antithrombin III-deficient plasmas.
INTRODUCTION Oxyuranus 5. scutellatus (Australian Taipan snake) venom is being used increasingly for specific assay of prothrombin, i.e., factor II (1,2), by conversion to thrombin (3,4). Although calcium is necessary, the venom does not require additional coagulation factors to activate prothrombin. It thus permits assay of the purified zymogen. We have adapted this assay to measure simultaneously prothrombin and the natural inhibitor antithrombin III (AT-III), also called heparin cofactor (5-8) or antifactor Xa (9-13). AT-III inhibits thrombin by reacting with it to yield a very tight molecular complex, the formation of which requires the active-center serine of thrombin (8,14). The formation of the complex is greatly accelerated by heparin, although its mode of action is still uncertain. Similar complexes are probably formed by AT-III with factors IXa, Xa, XIa, and plasmin (15-17) in an analogous fashion. A hereditary deficiency of AT-III has been reported in several families particularly susceptible to recurrent deep vein thromboses (18-25). Affected patients had AT-III levels between 30 and 60% of normal. This thrombotic tendency is surprising in view of the fact that normal plasma has sufficient AT-III to neutralize about 4 times the total potential plasma thrombin (14). By contrast, severe hemorrhagic complications of hereditary or acquired "'PresentAddress:
Connaught Laboratories Ltd., Steeles Ave. W., Willowdale, Ontario, Canada 555
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coagulopathies usually do not occur until the levels of clotting factors are reduced to less than lo-20% of normal. Low AT-III levels have been associated with liver cirrhosis and other forms of liver disease (26-30). Decreased levels in women on oral contraceptives have been reported by some workers (31,32) but not others (27). AT-III has been measured by immunologic techniques (23,27,31) or by coagulation assays (11,12,26,29,33-35). The latter assays measure either (a) the increase in the fibrinogen clotting time of a standard thrombin solution to which defibrinated plasma or, in some cases, serum has been added, or (b) the decrease in clotting activity of purified factor Xa after incubation with AT-III. The coagulation assay procedures used to date are time-consuming and require purified thrombin or factor Xa. For example, Yue et al. (36) added heat-defibrinated, barium sulfate-adsorbed plasma to purified bovine thrombin and found a rapid loss of thrombin activity, followed by a much slower loss due to other plasma antithrombins, such as a2-macroglobulin. The rapid, reliable AT-III assay reported here should be especially useful in the assessment of liver function (28) and would permit further investigation of the effect of oral contraceptives on AT-III. This assay should also facilitate the study of families having histories of recurrent venous thromboses.
MATERIALS Taipan snake venom was obtained from Eric Worrell's Australian Reptile Park, Gosford, NSW, Australia. Heparin-sodium was purchased from Sigma, St. Louis, MO. Bovine fibrinogen, 90% clottable, was purchased from Miles Laboratories, Kankakee, Ill. Inosithin was obtained from Associated Concentrates, Woodside, N.Y. Polyethylene glycol (PEG)-6000 was purchased from J. T. Baker, Phillipsburg, N.J. NIH standard thrombin (lot B-3) was obtained from the Bureau of Biologics, U. S. Food and Drug Administration. The standard human plasma used in these experiments was a pool of samples from 200 normal blood donors. Blood (4 ml) was drawn into acid-citrate-dextrose (ACD) anticoagulant (1 ml) in a Vacutainer tube (Becton-Dickinson) and centrifuged at 1,000 x g for 2 min. Supernatant plasma was aspirated with a plastic syringe. The plasma pool was -recentrifuged at 1,000 x a, dispensed in 0.5- and l.O-ml aliquots into polycarbonate tubes, and stored at -70°C. The prothrombin level remained constant after 10 months of storage. Plasma from individual donors was obtained in the same way. AT-III-deficient (AT-III-D) plasma was prepared from the standard plasma by gentle shaking with three volumes of isoamyl alcohol for 1 min according to Monkhouse et al. (16) and stored in polycarbonate tubes in 0.2-ml aliquots at -70°C. Although AT-III is almost entirely inactivated (C4%), the plasma retains all its prothrombin activity even after 6 months of storage. Buffer 1 contained 0.9% N&l and 0.068% Three buffers were used. -_ imidazole, adjusted with HCl to pH 7.4. The others were identical except for the following additions: Buffer 2> 0.88% PEG-6000; Buffer 3,)Q.88% PEG-6000 All quantities were w/v. and 0.4% CaC12 *2&0it Inosithin was suspended in Buffer 2 (10% W/V) and stored frozen in 0. l-ml aliquots. Before use, an aliquot was thawed and diluted l,OOO-fold in Buffer 2.
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Taipan venom was dissolved in Buffer 2 (1 mg/ml) and stored frozen in O.l-ml aliquots. This stock solution was diluted to 10 ml in Buffer 2 and then with an equal amount of Buffer 3 before use. Fibrinogen was stored frozen in lo-ml aliquots of a 1% (w/v) solution in Buffer 1. Heparin was prepared as a stock solution (500 U.S.P. units/ml) in Buffer 1 and stored frozen in O.l-ml aliquots. It was diluted for use to the appropriate concentration in Buffer 1. NIH standard thrombin was mixed thoroughly in Buffer 1 to a concentration of 100 units/ml and stored frozen in l-ml aliquots. These reagents were stored in polycarbonate tubes at -30°C.
METHODS Assay.of prothrombin To 0.5 ml of a human plasma dilution in Buffer 2 (usually 1:50) were added 0.5 ml of dilute inosithin suspension and 1.0 ml of Taipan venom solution containing calcium. This mixture was incubated for 2 min at 37°C in 0.3-ml aliquots; then 0.1 ml of fibrinogen solution was added, and the clot formation was timed with a Fibrometer (Baltimore Biological Laboratories). The assays were run in duplicate or triplicate, and the prothrombin concentration was determined from a clotting time curve. All prothrombin concentrations were corrected for anticoagulant. Our laboratory routinely substitutes PEG for acacia in the assay of highly purified thrombin solutions (37). A final concentration of 0.66% (w/v) PEG was chosen, although 0.4% was later found to approximate more closely the effect of acacia as reported by Ware and Seegers (38). One prothrombin unit was defined as the amount which, when activated, causes a 1% fibrinogen solution to clot in 15 sec. (Dilutions of NIH standard thrombin in this system with and without the venom give clotting times corresponding to about 1.5 times the NIH unit.) The different slope of the line obtained with this method necessitated the calculation of a new calibration chart (Table 1). Assay of plasma antithrombin III Solutions containing varying levels of AT-III were prepared by mixing of diluted (1:50) normal human and AT-III-D plasmas. To appropriate amounts 0.5-ml portion of each solution was added an equal volume of the dilute inosithin suspension described above, followed by 1.0 ml of the dilute venom solution. The mixture was incubated at 37°C for exactly 1 min. Ten (11 of either Buffer 3 or heparin (7.5 units/ml; final concentration, 3.75 x 10s3 units/ml) were then added, and the solution was mixed and incubated in 0.3-ml aliquots at 37'C for exactly 90 sec. Fibrinogen solution (0.1 ml) was introduced and the clotting time was measured. Duplicate assays were run simultaneo;sly by using two Fibrometers. The fraction of thrombin remaining was calculated from the mean clotting times in the presence and absence of heparin. A linear relationship was obtained between the percentage of thrombin remaining in solution and the percentage of AT-III present, based upon the serial dilution of normal human plasma with AT-III-D plasma (see Results). Individual plasma samples were diluted to give clotting times of 12-20 set in the absence of heparin (usually a 1:50 dilution). The percentage of thrombin remaining after incubation with heparin was calculated, and the AT-III
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TABLE 1 Concentration of Thrombih Related to Clotting Time
Seconds
Units
12.0 .2 .4 .6 .8 13.0 .2 .4 .6 .8 14.0 .2 .4 .6 .8
1.44 1.40 1.36 1.32 1.29 1.25 1.22 1.20 1.17 1.14 1.11 1.09 1.06 1.04 1.02
Seconds
15.0 .2 .4 .6 .8 16.0 .2 .4 .6 .8 17.0 .2 .4 .6 .8
Units
Seconds
1.00 0.98 0.96 0.94 0.92 0.91 0.89 0.87 0.86 0.84 0.83 0.81 0.80 0.79 0.78
18.0 .2 .4 .6 .8 19.0 .2 .4 .6 .8 20.0 .2 .4 .6 .8
Units
0.76 0.75 0.74 0.73 0.72 0.71 0.70 0.69 0.68 0.67 0.66 0.65 0.64 0.63 0.62
Seconds
21.0 .2 .4 .6 .8 22.0 .2 .4 .6 .8 23.0
TABLE 2 Clotting Time of Diluted Pooled Human Plasma Solutions (Corrected for Anticoagulant)
Final dilution of plasma
200 233 267 300 333 367 400 433 467 500
Clotting time (set)
Standard deviation
No. of assays
11.7 12.8 14.2 15.5 16.5 17.4 18.6 19.5 20.8 21.9
0.4 0.4 0.5 0.2 0.3 0.3 0.3 0.3 0.4 0.3
63 81 67 70 71 63 63 68 74 25
Units
0.62 0.61 0.60 0.59 0.59 0.58 0.57 0.57 0.56 0.55 0.55
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content was read from the graph. A second determination was carried out by diluting the sample with diluted normal or AT-III-D plasma.
RESULTS Prothrombin assays Table 2 summarizes the results of up to 81 replicate assays of the pooled human plasma. Over the range of 12-20 set, the clotting time is linear and inversely proportional to the prothrombin concentration. In 35 plasmas from individual healthy donors, the prothrombin concentration was found to be 287 f 41 units (mean f 1 SD; range 211-416); this is equivalent to a mean of 100 + 14% of the standard pool. To measure the rate of activation and the stability of the plasma-lipidvenom mixture, venom was added to plasma (initially diluted 1:50 in Buffer 1) and lipid mixtures as described under Methods. Aliquots were incubated for fixed intervals, and the clotting times were determined. Table 3 summarizes the data. Activation of prothrombin is complete within 2 min, and there is no measurable decrease in thrombin levels for up to 6.min. When incubated for more than 6 min, thrombin activity is lost at an approximately linear rate. The delay in inactivation of the formed thrombin forms the basis of the AT-III assay and, in addition, permits measurement of replicate clotting times when assaying for prothrombin.
TABLE 3 Rate of Activation of Plasma Prothrombin
Incubation time (min) Clotting time (set)
0.5 1
2
3
4
5
6
7
10
20
30
60
18.9 16.8 16.5 16.5 16.4 16.5 16.6 17.9 17.3 18.6 21.1 25.2
f SD
1.3 0.7 0.6 0.6 0.5 0.5 0.3 0.5 0.6 0.2 0.2
No. samples
3
29
35
28
17
16
6
3
4
2
2
1
A 1:50 dilution of pooled human plasma was activated with Taipan venom as described. The quantity of thrombin formed was determined following incubation at 37°C. Antithrombin assays The AT-III assay was performed in duplicate as described under Methods. The amount of thrombin remaining was calculated by dividing the mean clotting time in the absence of heparin by that in the presence of heparin. Figure 1 shows a linear relationship between the percentage of thrombin remaining and the percentage of AT-III in the test plasma. The action of other plasma
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z 5 40
I
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0
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REMAINING
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FIGURE 1 Calibration of the AT-III assay. The percentage AT-III is based upon mixtures of diluted normal pooled (100%) and diluted isoamyl alcohol-treated (0%) human plasma. The percentage of thrombin remaining is calculated from the ratio of the clotting times in the absence and presence of heparin (see text). antithrombins, such as u2-macroglobulin is compensated for by the blank determination. The relatively short incubation time also precluded a major contribution by these more slowly acting inhibitors. The prothrombin concentration is readily calculated from the clotting time in the absence of heparin. Factors influencing antithrombin III assay To determine the optimum time for incubation of the formed thrombin with AT-III and heparin, the following experiment was performed: To plasma solutions containing known AT-III levels, lipid and Taipan venom were added as described. After exactly 1 min, 10 ~1 of a heparin solution (10 units/ml) were added, and the mixtures were further incubated at 37°C in 0.3-ml aliquots. After 2.5, 5.0, and 6.5 min from the start of the experiment, residual thrombin was assayed by the addition of fibrinogen. These results are plotted in Fig. 2. AS the incubation time increased the plots became progressively nonlinear. f 4.0,
The effect of incubation time on the rate of inactivation of formed thrombin in normal plasma, AT-III-D plasma, and an equal mixture of the two was tested after addition of varying amounts of heparin (5.0-10.0 units/ml). The least deviation from linearity was obtained with a short incubation time (90 set) and a low level of heparin.
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6C
2c )-
I-
20
60
40
THROMBIN
80
REMAINING
100
(%)
FIGURE 2 Effect of incubation time on plot of the percentage AT-III vs percentage thrombin remaining. Heparin (10 ~1, 10 units/ml) was added to activate dilute normal plasma to which varying amounts of dilute AT-III-D plasma had been added. The mixtures were incubated for 1.5 (o), 3.0 (A), 4.0 (I) and 5.5 (0) min.
To determine the optimum heparin concentration, stock solutions of plasma diluted 1:30, 1:50, 1:70, and 1:90, containing 50% levels of AT-III, were activated with Taipan venom for exactly 1 min. Heparin was then introduced in lo-~1 portions at four concentrations, and each mixture was assayed for thrombin after exactly 90-set incubation. The means of the four dilutions at each heparin level (units/ml) showed the following loss of thrombin activity 2.5 units, 24.9%; 5.0 units, 38.1%; 7.5 units, 48.7%; 10.0 units, 55.7%. A concentration of 7.5 units/ml was chosen as optimum. giving a significant loss of thrombin activity in the mixture, yet retaining maximum linearity over the 90-see incubation period. When the clotting time of each plasma dilution is plotted against the heparin concentration (Fig.3), the-percent of thrombin remaining at each heparin concentration is seen to be essentially constant, indicating that the plasma prothrombin content doesnot significantly influence the AT-III determination in the range studied. Blood from patients hospitalized with venous thrombosis was collected at local hospitals and assayed as described above. The plasmas from three patients known to be congenitally deficient in AT-III were also studied. Table 4 summarizes these results. When plasma containing low levels of AT-III is assayed, the change in clotting time on addition of heparin is small, resulting
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FIGURE 3 Effect of the initial concentration of prothrombin on the rate of inactivation of the formed thrombin. The plasma dilutions corrected for anticoagulant were (o), ZOO-fold; (B> 333-fold, (0) 467-fold, and (A) 600-fold. in relatively large errors in the calculation of the percentage of thrombin remaining. The AT-III level of such plasmas can, however, be readily adjusted with pooled human plasma to a level of activity which permits more accurate assay. Plasmas containing high levels ('100%) of AT-III were diluted with AT-III-D plasma to facilitate AT-III quantitation.
DISCUSSION' Under the conditions described above, the thrombin generated from dilute human plasma by Taipan snake venom is sufficiently stable to allow the assay of AT-III. This simple method involves comparing the fibrinogen clotting times of the activated plasma solutions in the presence of a low level of heparin or buffer. All reagents except the snake venom are inexpensive and readily available, and very little of the venom is used. Purified factor Xa or thrombin is not required, since thrombin is generated from plasma in the assay. PEG, which is available in fairly pure form, substitutes well for acacia (33) in accelerating the clotting time. In contrast to the results of van der Meer et al. (23), simultaneous immunologic determination of AT-III levels led to higher values than those obtained by coagulation.assay using our method or that of Marciniak et al. (25). This discrepancy was particularly evident with the congenitally AT-III-D
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TABLE 4 Antithrombin III and Prothrombin Assays of Individual Plasmas
Patient (age, sex)
Normal human plasma A (70,F) B (74,M) C (23,~) D (50,M) E (23,~) F (24,~) G (26,F)l H (42,~) I (65,M) J (42,M) K (40,F) L (39,F)2 M ( ?,?) N (34,M); 0 (14,M)
AT-III (%) Immunoassay* This method
100 102 94 105 81 103 66 110 85 101 76 99 108 35 34 42
* 7 f 11 + 1 z!z 2 f 11 * 5 f 5 + 3 f.4 + 6 f 6 + 3 + 2 * 2
100 113 103 114 101 115 111 106 107 105 115
83 90
Prothrombin Units+ %
287 253 267 307 240 416 380 453 113 277 337 337 366 284 302 279
100 88 93 107 84 145 132 128 39 97 117 117 128 99 105 97
Ji
Performed by Dr. Richard Propp of Albany Medical College by Laurel1 electroimmunodiffusion using antibody prepared by Alper from antigen provided +by Rosenberg (8) Corrected for anticoagulant. 1Patient on warfarin. 2Patient congenitally AT-III-D. Plasma a gift from Dr. S. S. Shapiro; AT-III 50% by immunoassay (personal communication). 3Patient congenitally AT-III-D. Gift of Dr. E. Marciniak. Reported AT-III levels were: Patient N, 35%; Patient 0, 40% (personal cormnunication).
plasmas (Table 4). Although no further data are presently available, the results raise the’ possibility of the presence of a nonfunctional antigen related to AT-III, analogous to the situation observed in hemophilia (39).
ACKNOWLEDGMENTS The authors wish to thank Dr. Sandor S. Shapiro, Cardeza Foundation, Jefferson Medical College, Philadelphia, Pa., and Dr. Ewa Marciniak, University of Kentucky Medical Center, Lexington, KY., for congenitally AT-III-D plasmas; Dr. Edwin Taft, Albany Medical Center Hospital, Albany, N.Y., and Dr. George Vlahides, Ellis Hospital, Schenectady, N.Y., for plasmas from patients with recurrent venous thromboses; Dr. Anthony F. H. Britten, American National Red Cross, Northeastern New York Chapter, Albany, N.Y., for the normal plasma pool and the plasma from normal people used in this study; and Dr. W. Jean Dodds of this Division for many helpful discussions.
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This research was supported in part by Grant AM 05299 from the National Institutes of Health.
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