Britisk Journal of Haernatology, 1975,30, 265.
Investigations on Antithrombin 111in Normal Plasma and Serum G ~ Z SAS," A DUNCAN S. PEPPER AND JOHN D. CASH S-E Scotland Regional Blood Transfusion Centre, R o y a l Infirmary, Edinburgh (Received 4 December 1974; accepted for publication
12
December 1974)
SUMMARY. Studies on antithrombin I11 (AT-111) were made by a modification of the two dimensional crossed immunoelectrophoresis technique and gel filtration. Mixing various quantities of heparin with agarose in the first phase of electrophoresis, AT-I11 from normal human plasma and serum revealed a heterogeneity which depended on the heparin concentration in the agarose gel. At heparin concentrations higher than 16 u/ml, AT-I11 displayed three components with different electrophoretic mobilities. The component with the highest mobility (designated immunoantithrombin 111' :IAT-111') dominated in plasma. In normal serum, however, the quantity of this component was decreased and the two other peaks with a slower electrophoretic mobility (IAT-111' and IAT-1113) became more evident. Normal human plasma and serum were filtered on Sephadex G-200 and the ATI11 concentration measured in the fractions by rocket immunoelectrophoresis. The peaks of AT-111 were found in the same fractions for both plasmaandserumand were coincident with the albumin peak of the plasma proteins. However, in the case of serum the AT-I11 concentration decreased less sharply in those fractions with higher molecular weight than in the corresponding plasma fractions. Analysis of these fractions by crossed immunoelectrophoresis revealed that the two components with slower electrophoretic mobility (IAT-1112 and IAT-1113) had higher molecular size than IAT-111' , that the concentration of IAT-1112 and IAT-1113 was significantly higher in serum, and that the high molecular weight components in plasma and serum were qualitatively identical. It is concluded that high molecular weight complexes between AT-I11 and activated coagulation factors may be present in normally circulating blood and that their detection and possibly quaiititatioii caii be achieved using the heparinlagarose crossed immunoelectrophoresis system. Although recent data have suggested that antithrombin 111 (AT-111) plays an important role in the inactivation of activated blood coagulation factors such as thrombin (Dombrose et al, 1971), factor Xa (Biggs et d , 1970; Yin et al, 1971), factors IXa and XIa (Damus et al, 1973), and is essential for the anticoagulant effect of heparin, the mechanisms are not yet fully elucidated. The clinical significance of AT-111 has been highlighted by reports of thrombophilic families with partial deficiency of AT-I11 (Egeberg, 1965 ; Van der Meer etnl, 1973;Marciniak et al, 1974).
* Present address: Postgraduate Medical School, First Department of Medicine, Budapest. Correspondence : D r John D. Cash, S-E Scotland Regional Blood Transfusion Centre, Royal Infirmary, Edinburgh EH3 gHB. 265
266
G. Sas, D. S . Pepper a n d l . D. Cask
Previous studies, reported from this laboratory, suggested that when heparin is incorporated into the agarose for cross immunoelectrophoresis there were marked differences in the AT-I11 patterns between plasma and serum (Sas et al, 1975). The following communication describes a series of studies in which this new technical approach was further explored, using heparin cofactor assays, ‘rocket’ immunoelectrophoresis and gel filtration. MATERIALS AND METHODS
Test samples. Citrated venous blood was obtained from healthy subjects (25 donors) using plastic syringes and stainless steel needles, and a solution of 3.8% sodium citrate (I ml anticoagulant to 9 ml blood). Blood without citrate was collected in glass tubes and with anticoagulant in polystyrene tubes. Plasma was obtained by centrifugation at 3000 g for 20 min at 4°C. ‘Stored’ serum was prepared from blood incubated at 37°C for 2 h, centrifuged at 2000 g for 20 min, and the supernatant incubated at 37°C for a further 6 h in a glass tube, dispensed into plastic vials and stored at - 3 5°C. Serum samples in which the thrombin generation had taken place to different degrees were obtained as follows : 20 ml of fresh venous blood was distributed in I .8 ml aliquots in plastic tubes without anticoagulant, incubated at 3 7°C and at various times after venepuncture (0, 0.5, I , 2,4, 8, 16, 32, 64, 128 and 256 min) 0.2 ml of 3.8% sodium citrate solution was added to each appropriate aliquot. Each sample was then further incubated for 15 min at 37”C, centrifuged at 2000 g for 15 rnin and the supernatant studied (vide infya). ‘Artificial’ sera were prepared by mixing various thrombin preparations or snake venom with citrated plasma. 0.2 ml of human (kindly supplied by Dr D. Ellis, Lister Institute, Elstree) and bovine (Parke Davies, Detroit, U.S.A.) thrombin solutions were mixed with 0.8 ml of normal pooled plasma and incubated in plastic tubes at 37°C for I h. After centrifugation (2000 g for 20 inin) the supernatant was either used immediately or stored at - 35°C. Serum was also obtained by mixing 0.1nil of various saline dilutions of Tiger Snake venom (V-0251 Snake Venom, Mainland Tiger Snake, Sigma Chemical Corp., St Louis, U.S.A.) with 0.9 ml of normal pooled citrated plasma. After 2 h incubation at 37°C the serum was removed by centrifugation (2000 g for 20 min) and was either used immediately or stored at - 35°C. Finally, a serum was prepared by adding 0.1ml Reptilase (Pentapharm Ltd, Basle, reconstituted as per instructions) to clot 0.3 ml of citrated pooled normal plasma, incubated at 37°C for 3 0 min and the liquor removed for study. Electrophoresis bufer. Barbitone buffer (pH 8.6,o.o~M) was used in the agarose gel and buffer tanks. Agarose. A 1% agarose (Indubiosem A 37, L’Industrie Biologique FranGaise, S.A., France) solution was prepared with barbitone buffer. Heparin. Heparin (Weddel Pharmaceuticals Ltd, London) solution 5000 I.U./ml was diluted in saline to the required concentration. Fibrinogen was Grade L, human, lyophilized with a clottability greater than 90% (A.B. Kabi, Stockholm). One gram of fibrinogen was dissolved in IOO mi of distilled water. Anti-A T-III immune serum. Human AT-I11 antiserum, prepared in rabbits, was obtained from Nyegaard and Co., A/S Oslo, Norway. Heparin cofacto~activity. A fibrinogen solution of 4 mg/ml was prepared from the stock solution (containing IOO u hepariniml) and kept at room temperature, 0.1 ml of this fibrino-
Antithronzbin III
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gen-heparin mixture was incubated with 0.1 ml of the AT-I11 containing solution at 37°C for I miii. 0.1 ml of thrombin solution (Parke Davies, Detroit, U.S.A., 20 ujml activity) was then added to the mixture and the clotting time recorded. From the clotting times of a series of dilutions of normal pooled plasma, the heparin cofactor activity of the invcstigated sample was expressed as a per cent of normal plasma. Detcrnzination of the A 7’-IIl concentvation by electvoivnnzunoassay (Laurell, 1966). A mixture of 12 in1 1% agarose and 0.1 nil anti-AT-I11 immune serum was poured on to a glass plate (80 x 80 mm) a t 56°C. Eighteen wells (of 3 mm diameter) were prepared in the agarose layer by a standard pattern. 10 pl of AT-111 containing solutions were placed in each well. Electrophoresis was carried out in a Shandon SAE 2761 electrophoresis apparatus at 20 V/cm for 4 h. After I h of rinsing in distilled water, the plates were covered with filter paper (Whatman No. I) and left overnight to dry. Coonlassie Brilliant Blue (1% w/v, British Drug Houses, Poole) staining was used. The length of the ‘rockets’was measured and the AT-111 concentrations of a sample expressed as a per cent of normal plasma from a calibration curve which was prepared from the dilutions of normal plasma ( 5 , 2.5 and 1.25%) on each plate. Two diwiensional cvossed irnmunoelectvophoresir (Laurell, 1965) was used for the investigation of AT-I11 but with modification, using heparin in the agarose for the first electrophoresis (Sas et d, 1975). 12 nil of a 1% agarose solution, containing 16.6 u heparin per ml agarose, were poured on to a glass plate (80 x 80 mm) in the routine assays. In those experiments in which the effect ofvarious heparin concentrationswcre measured, 0,0.8, 1.6,2.5,4.2, 5.8, 8.4, 16.6, 42.0 and 84.0 unit of heparin per ml agarose were applied. After solidification of the agarose, a well of 3 mm diameter was prepared in one corner of the plate (11 min from both edges) and 5 pl of sample was measured into the well. Electrophoresis was carried out in a Shandon SAE 2761 electrophoresis apparatus at 4°C. After the first electrophoresis (at 20 V/cm for 2 h) the agarose gel was cut in two parts, the direction of the cut was parallel with that of the electrophoresis and was located 14 mm from the edge of the plate. The smaller part of the agarose layer (which contained the proteins separated by the first electrophoresis) was kept, while the wider part was removed. 10 ml of 1% agarose was then mixed with 0.1 ml anti-AT-111 immune serum at 56”C, poured into the site of the discarded gel, the platc rotated through 900, and electrophoresis continued at 20 V/cm for 4 h at 4°C. After electrophoresis the plates remained in a moist chamber overnight and were then rinsed in saliiic three times for 2 h. After a final I h rinse in distilled water the plates were covered with filter paper (Whatman No. I) and left to dry overnight. Coomassie Brilliant Blue staining was used. The electrophoretic mobility of AT-111 was expressed as the distance between the centre of the well and the projected point of the peak on to the base line. Geljiltrution. 5 ml samples were applied to a column of G-200 Sephadcx (medium grade) 2.5 x IOO cm and eluted with a solution containing NaCl 150 mmolar, Tris 25 mmolar, boric acid 3.8 mmolar, EDTA I mmolar and sodium azide 3 mmolar, pH 8.8. Glucose (100mg) was added to all samples to increase the density and serve as a marker of total internal columii volume. The presence of glucose in fractions was estimated semiquantitatively with ‘Clinistix’ (Ames, Slough). Fractions of 5-7 ml were collected on a time based fraction collector at a flow rate of 25 ml/h. Exact fraction volumes were determined by weighing. The eluatc was rnonitorcd for protein at 280 nm in a spectrophotometer. In a second series of experiments normal citrated plasma was heparinized (final concentration IOO u/ml) and gel filtered using an eluant buffer containing heparin (100 u/nil).
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G. Sas, D. S. Pepper a n a l . D. Cash
The distribution coefficient (K,) was defined as KD = (V,- V,)/Vi, where V, the void volume was determined from the elution position of chylomicrons, V, was observed elution position of the substance under investigation and Vi was the difference between V, and the elution position of glucose. Fractions were concentrated in Minicon B 15 cells (Amicon Corp., Lexington, U.S.A.) for further investigation by crossed immunoelectrophoresis.
RESULTS
The Efect of Various Concentrationsof Heparin on the ElectrophoreticMobility of AT-III in Plasma Various quantities of heparin were mixed with the agarose in the first dimension of crossed imniunoelectrophoresis (see Materials and Methods). Fig I shows AT-I11 of normal pooled plasma when the agarose contained 0,1.6,2.5,4.2, 8.4, and 16.6 u heparin per ml agarose, and it was observed that the electrophoretic mobility of AT-I11 increased with increasing heparin concentration. At a heparin concentration of 1.6 u/ml a minor part of the AT 111 moved faster than the major part. On increasing the heparin concentration to 16.6 u per ml agarose, three components were differentiated clearly in both plasma and serum. At this concentration the main component (immuno-antithrombin 111‘ = IAT-1111) had the highest electrophoretic mobility and during the 2 h of electrophoresis its mean transit distance (12 samples) for both plasma and serum was 3 3 1.2 mm. The second and the third components had lower electrophoretic mobilities (IAT-1112 : 22.4 +_ 1.0 mm, and IAT-III’ : 16.8 k0.6 mm). At higher heparin concentrations (42 and 84 u/ml agarose) the results were similar to those obtained with 16.6 u/ml agarose. Accordingly 16.6 u heparin per ml agarose was used in all subsequent experiments. Twelve normal plasma showed the same three components as normal pooled plasma. Investigation of AT-III in Normal Serum by Crossed lmmunoelectrophoresis Serum samples were prepared (as described in Materials and Methods) in which thrombin generation had taken place to different degrees. AT-I11 was investigated in these samples by crossed immunoelectrophoresis with the standard heparin concentration of 16.6 u/ml. Coagulation did not take place in the samples in which the citrate was mixed with the blood 0.5, I, 2,4, 8 and 16 min after blood collection. In these samples the crossed immunoelectrophoresis revealed the same three components as plasma AT-111. When citrate was added after the onset of coagulation (about 25 min after venepuncture) there was a gradual decrease of IAT-IF and a concomitantincreaseofIAT-111’ and IAT-III’ components in the 32, 64, 128 and 256 min samples. In the 256 min samples the IAT-111’ and IAT-1113 components fused and gave a ‘spike’like precipitation line. In the ‘stored’ serum this latter phenomenon was even more evident (see Fig .a). When citrated plasma was clotted with various quantities of human or bovine thrombin, similar precipitation arcs appeared as those found in native serum; Fig z(b) shows the result when normal pooled plasma was clotted by human thrombin (50 u/ml plasma). At this thrombin concentration, a minor part of the IAT 111‘ component remained detectable. When the thrombin concentration was increased, the IAT-111’ component disappeared completely and a single precipitation arc was seen corresponding to the fused IAT-111’ and IAT-III’ components.
Aiitithrorrrbiiz III
FIG I . Effect of various concentrations of heparin on the electrophoretic mobility of AT-I11 in nornial plasma. Various concentrations of heparin were mixed with thc agarosc (a: o, b: 1.6, c : 2.5, d : 4.2, c: 8.4, f: 16.6 ulml) in the first phase ofthe crossed imniunoelectrophoresis. At 16.6 u hcpariri per nil agarose three different peaks are observed (IAT-1111, IAT-IIIZ and IAT-II13).
FIG 2. AT-I11 patterns with crossed imniunoelectrophoresis (agarose/heparin) in: (a) native ‘stored’ serum, (b) liquor of thrombin clotted citratcd plar:ma, (c) liquor of Tiger Snake venom clotted citrated plasma.
FIG 5 . Crossed imniuiioelcctrophoretic (agaroselheparin) AT-I11 patterns in gel filtered serum fractions: (a) AT-I11 in a concentrated pool of fractions 45 and 46, (b) AT-I11 in a concentrated pool of fractions 52 and 53.
Antithrombin 1Il
B"b
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-N
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FIG3 . Immunoreactive AT-I11 concentration (AT-111: -)
and heparin cofactor activity (HCA: 0 ) in the gel filtered fractions of normal plasma. AT-I11 and HCA were detected in the albumin peak fractions and a good correlation recorded. 0,OD1,:.
1.6 1.4
I.2
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0.4 0.2
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45 50 Fractions
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FIG 4. Immunological AT-111 concentration (AT-111: 0 ) and heparin cofactor activity (HCA: 0 )in the gel filtered fractions of 'stored' serum. The AT-I11 concentration was strikingly higher than the HCA in fractions 45,46 and 47. 0,OD.:
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G. Sas, D. S. Pepper and]. D. Cash
When plasma was clotted with Tiger Snake venom, the AT-111 showed similar changes; increasing the venom concentration decreased the IAT-111’ component progressively, whilst the component(s) with lower electrophoretic mobility increased (Fig 2c). However, the serum obtained from Reptilase clotted citrated plasma had an identical pattern to that of normal plasma.
Gel Filtration of Normal Plasma and Serum AT-III 5 in1 of normal pooled plasma and 5 ml of normal ‘stored’serum were filtered on Sephadex gel as described (see Materials and Methods). AT-111 was quantitated using ‘rocket’ immuiioelectrophoresis and the heparin cofactor activity was determined in the resultant fractions (see Materials and Methods). The results are summarized in Figs 3 and 4 which show that AT-I11 in the plasma was detected by the immunologic method mainly in the fractions corresponding to the ‘albumin peak’ (K, = 0.44), although a small quantity of AT-I11 antigen was present in the void volume. Heparin cofactor activity was detectable only in the albumin peak fractions. The correlation between the quantity of AT-I11 detected by the immunologic method and heparin cofactor assay was good in this region. Immunologically reactive AT-I11 in the serum fractions was also detected mainly in the albumin peak, but some differences were observed between serum and plasma. The serum fractions showed a definite ‘shoulder’ in the higher molecular weight region, although the peak of the immunoreactive AT-I11 had not changed position (K, = 0.44), compared to that of plasma. Another difference was the presence of higher quantities of AT-111 in the void volume than in the corresponding plasma fractions. Although the heparin cofactor activity was only present in the albumin rich fractions it was absent from the ‘shoulder’ of the curve as defined by immuno-AT-I11 assays. On the basis of the observed discrepancy between the immunologic AT-I11 values and heparin cofactor activities in the fractions 45-48, we assumed there was some heterogeneity of serum AT-111. This was supported by the observation that AT-I11 in serum fractions 45,46 and 47 appeared in a molecular weight region in which no AT-111 was detected in the plasma fractions. Further confirmation was obtained by running two Ioo-fold concentrated pools (consisting of fractions 45-46 and 52-5 3 respectively) on agaroseiheparin crossed immunoelectrophoresis technique and showing that the two slower components (IAT-111’ and IAT-1113) were present mainly in fractions 45 and 46, whilst fractions 52 and 53 contained a higher concentration of the IAT-111’ component (Fig 5 ) . Similar analysis of the gel filtered plasma fractions revealed that IAT-111’ and IAT-1113 were present in fractions 49 and 50 but in very small amounts compared to equivalent fractions of serum. 6-200
Gel Filtration Studies on Heparinized Normal Plasma 5 ml of normal pooled citrated plasma was heparinized with 500 units of heparin and gel filtered as above, but using heparinized buffer as the eluant. The AT-I11 concentration of the fractions was measured using ‘rocket’immunoelectrophoresis.The results are summarized in Fig 6 which shows that in the presence of heparin the immunoreactive AT-I11 eluted off the column in a higher molecular weight region (K, = 0.29) than that run in the absence of heparin (K, = 0.44: see Fig 3).
Antithrombin III
0
0
0 -
.\" El c
o a
3
Fractions
FIG 6. Immunoreactive AT-111 concentration (AT-111: 0 )in the gel filtered plasma fractions of nornial heparinized plasma. AT-I11 elutes off thc column in a higher molecule size region than that run in the absence of heparin (cf. Fig 3). 0,OD hi:.
DISCUSSION In the course of early immunological investigations on various coagulation factors we observed that improved definition of 'rocket patterns' was obtained by incorporating heparin into the agarose gel. This modification was iiicluded in studies on AT-I11 and it was noted that when heparin was added to thc gel for the first phase in crossed iminunoelectrophoresisthree separate peaks were identified (designated IAT-111' , IAT-Ill2 and IAT-II13) and that their individual concentrations in citrated plasma and native serum was significantly different (Sas et ul, 1975). In the present study the optimal concentration for the development of this phenomenon has been defined (16.6 u heparin per in1 of agarose) and the mobility of the three peaks in normal plasma and serum was found to be identical. Serum prepared by the addition of human thrombin and Tiger Snake venom to normal citrated plasma gavc crossed iinmunoelectrophoretic patterns that were similar to those of native serum. However, serum obtained without thrombin generation (Reptilase clots) was identical to norinal plasma. The gel filtration studies were primarily designed to ascertain the nature of the three peaks observed in crossed immunoelectrophoresis. The results of these investigations and those of the snake venoms referred to above, suggest that IAT-III' are higher molecular weight complexes, presumably with at least thrombin and Xa. This latter conclusion corresponds to those of Abilgaard (1969) and Binder (1973) who noted in gcl filtration studies that AT-I11 in serum had a higher molecular weight than that in plasma. The present gel filtration experiments also added further evidence to support the hypothesis that normal citrated plasma contains small but detectable quantities of complexed AT-I11 (IAT-111' and IAT-Ill3). Whether
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G. Sas, D. S. Pepper and]. D. Cash
this is a normal in vivo phenomenon, and thus reflects evidence of continuous low-grade activation of the coagulation mechanism, as postulated by Astrup (1956)~or an artefact arising during blood withdrawal, is not yet known. Damus et al (1973) have reported the neutralizing effect of AT-I11 on XIa, and it is possible that the generation of XIa cannot be avoided during venepuncture. We believe this explanation for the presence of trace quantities of IAT-1112 and IAT-1113 in normal plasma is unlikely as we have not observed a change in the AT-I11 crossed immunoelectrophoresis patterns when normal citrated plasma is filtered through glass bead columns. However, that other, as yet unknown, reactionts) in the coagulation mechanism during venepuncture may have given rise to this observation in plasma cannot be excluded at present. This problem is currently under investigation. The AT-111 gel filtration studies on heparinized normal plasma were of particular interest, for they indicate that when heparin binds to AT-I11 higher molecular weight complexes are formed. It therefore seems probable that the explanation of the difference in crossed immunoelectrophoresis AT-I11 patterns originally observed between simple agarose and agarosei heparin gels (Sas et al, 197s) lies in the fact that the AT-I11 heparin complexes formed in the latter gel are highly charged (negatively) and thus migrate a greater distance toward the anode than AT-I11 in plain agarose gels. This interpretation would lead us to conclude that because the binding sites for IAT-1112 and IAT-1113 are wholly or partially occupied by activated coagulation factors their heparin binding activity is reduced sufficiently to diminish their electrophoretic mobility in the agaroseiheparin gel. This phenomenon, therefore, permits the observed separation of IAT-111’ , IAT-111’ and IAT-II13. ACKNOWLEDGMENT
This work was funded by a Research Grant award by the Scottish Home and Health Department. REFERENCES ABILDGAARD, U. (1969)Binding of thrombin to antithrombin 111. Scandinavian Journal of Clinical and Laboratory Investigation, 24, 23. ASTRUP,T. (1956)The biological significance of fibrinolysis. Lancet, ii, 565. BIGGS,R., DENSON, K.W.E., AXMAN, N., BORRETT, R. & HADDEN,M. (1970)Antithrombin 111, antifactor Xa and heparin. British Journal afHaematology, 1% 283.
BINDER,B. (1973)On the complex formation of antithrombin I11 with thrombin. Gelfiltration studies on human plasma and serum. Thrombosis et Diathesir Haemorrhagica, 30, 280. DAMUS,P.S., HICKS, M. & ROSENBERG, R.D. (1973) Anticoagulant action of heparin. Nature, 246, 355.
DOMBROSE, F.A., SEEGERS, W.H. & SEDENSKY, J.A. (1971)Antithrombin. Inhibition of thrombin and autoprothrombin (F-Xa) as a mutual depletion system. Thrombosis et Diathesis Haemorrhagica, 26, 103.
EGEBERG, 0. (1965)Inherited antithrombin deficiency
causing thrombophilia. Thrombosis et Diathesis Haemorrhagica, 13, 516. LAURELL,C.-B. (1965) Antigen-antibody crossed electrophoresis. Analytical Biochemistry, 10, 3 58. LAURELL, C.-B. (1966)Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analytical Biochemistry, 15,45. MARCINIAK, E., FAFSEY, C.H. & DESIMONE,P.A. (1974)Familial thrombosis due to antithrombin 111 deficiency. Blood, 43, 219. SAS,G., PEPPER, D.S. & CASH,J.D. (1975)Plasma and serum antithrombin 111: Differentiation by crossed immunoelectrophoresis. Thrombosis Research, 6, 87. VAN DER MEER,J., STOEPMAN-VAN DALE”, E.A. & JANSEN, J.M.S. (1973)Antithrombin 111deficiency in a Dutch family. In: IVth International Congress on Thrombosis and Haemostasis, Vienna, Abstracts, p 226. YIN,E.T., WESSLER, S. & STOLL,P.J, (1971) Identity of plasma-activated factor X inhibitor with antithrombin 111 and heparin cofactor. Journal .f Biological Chemistry, 246, 3712.
Investigations on Antithrombin 111in Normal Plasma and Serum G ~ Z SAS," A DUNCAN S. PEPPER AND JOHN D. CASH S-E Scotland Regional Blood Transfusion Centre, R o y a l Infirmary, Edinburgh (Received 4 December 1974; accepted for publication
12
December 1974)
SUMMARY. Studies on antithrombin I11 (AT-111) were made by a modification of the two dimensional crossed immunoelectrophoresis technique and gel filtration. Mixing various quantities of heparin with agarose in the first phase of electrophoresis, AT-I11 from normal human plasma and serum revealed a heterogeneity which depended on the heparin concentration in the agarose gel. At heparin concentrations higher than 16 u/ml, AT-I11 displayed three components with different electrophoretic mobilities. The component with the highest mobility (designated immunoantithrombin 111' :IAT-111') dominated in plasma. In normal serum, however, the quantity of this component was decreased and the two other peaks with a slower electrophoretic mobility (IAT-111' and IAT-1113) became more evident. Normal human plasma and serum were filtered on Sephadex G-200 and the ATI11 concentration measured in the fractions by rocket immunoelectrophoresis. The peaks of AT-111 were found in the same fractions for both plasmaandserumand were coincident with the albumin peak of the plasma proteins. However, in the case of serum the AT-I11 concentration decreased less sharply in those fractions with higher molecular weight than in the corresponding plasma fractions. Analysis of these fractions by crossed immunoelectrophoresis revealed that the two components with slower electrophoretic mobility (IAT-1112 and IAT-1113) had higher molecular size than IAT-111' , that the concentration of IAT-1112 and IAT-1113 was significantly higher in serum, and that the high molecular weight components in plasma and serum were qualitatively identical. It is concluded that high molecular weight complexes between AT-I11 and activated coagulation factors may be present in normally circulating blood and that their detection and possibly quaiititatioii caii be achieved using the heparinlagarose crossed immunoelectrophoresis system. Although recent data have suggested that antithrombin 111 (AT-111) plays an important role in the inactivation of activated blood coagulation factors such as thrombin (Dombrose et al, 1971), factor Xa (Biggs et d , 1970; Yin et al, 1971), factors IXa and XIa (Damus et al, 1973), and is essential for the anticoagulant effect of heparin, the mechanisms are not yet fully elucidated. The clinical significance of AT-111 has been highlighted by reports of thrombophilic families with partial deficiency of AT-I11 (Egeberg, 1965 ; Van der Meer etnl, 1973;Marciniak et al, 1974).
* Present address: Postgraduate Medical School, First Department of Medicine, Budapest. Correspondence : D r John D. Cash, S-E Scotland Regional Blood Transfusion Centre, Royal Infirmary, Edinburgh EH3 gHB. 265
266
G. Sas, D. S . Pepper a n d l . D. Cask
Previous studies, reported from this laboratory, suggested that when heparin is incorporated into the agarose for cross immunoelectrophoresis there were marked differences in the AT-I11 patterns between plasma and serum (Sas et al, 1975). The following communication describes a series of studies in which this new technical approach was further explored, using heparin cofactor assays, ‘rocket’ immunoelectrophoresis and gel filtration. MATERIALS AND METHODS
Test samples. Citrated venous blood was obtained from healthy subjects (25 donors) using plastic syringes and stainless steel needles, and a solution of 3.8% sodium citrate (I ml anticoagulant to 9 ml blood). Blood without citrate was collected in glass tubes and with anticoagulant in polystyrene tubes. Plasma was obtained by centrifugation at 3000 g for 20 min at 4°C. ‘Stored’ serum was prepared from blood incubated at 37°C for 2 h, centrifuged at 2000 g for 20 min, and the supernatant incubated at 37°C for a further 6 h in a glass tube, dispensed into plastic vials and stored at - 3 5°C. Serum samples in which the thrombin generation had taken place to different degrees were obtained as follows : 20 ml of fresh venous blood was distributed in I .8 ml aliquots in plastic tubes without anticoagulant, incubated at 3 7°C and at various times after venepuncture (0, 0.5, I , 2,4, 8, 16, 32, 64, 128 and 256 min) 0.2 ml of 3.8% sodium citrate solution was added to each appropriate aliquot. Each sample was then further incubated for 15 min at 37”C, centrifuged at 2000 g for 15 rnin and the supernatant studied (vide infya). ‘Artificial’ sera were prepared by mixing various thrombin preparations or snake venom with citrated plasma. 0.2 ml of human (kindly supplied by Dr D. Ellis, Lister Institute, Elstree) and bovine (Parke Davies, Detroit, U.S.A.) thrombin solutions were mixed with 0.8 ml of normal pooled plasma and incubated in plastic tubes at 37°C for I h. After centrifugation (2000 g for 20 inin) the supernatant was either used immediately or stored at - 35°C. Serum was also obtained by mixing 0.1nil of various saline dilutions of Tiger Snake venom (V-0251 Snake Venom, Mainland Tiger Snake, Sigma Chemical Corp., St Louis, U.S.A.) with 0.9 ml of normal pooled citrated plasma. After 2 h incubation at 37°C the serum was removed by centrifugation (2000 g for 20 min) and was either used immediately or stored at - 35°C. Finally, a serum was prepared by adding 0.1ml Reptilase (Pentapharm Ltd, Basle, reconstituted as per instructions) to clot 0.3 ml of citrated pooled normal plasma, incubated at 37°C for 3 0 min and the liquor removed for study. Electrophoresis bufer. Barbitone buffer (pH 8.6,o.o~M) was used in the agarose gel and buffer tanks. Agarose. A 1% agarose (Indubiosem A 37, L’Industrie Biologique FranGaise, S.A., France) solution was prepared with barbitone buffer. Heparin. Heparin (Weddel Pharmaceuticals Ltd, London) solution 5000 I.U./ml was diluted in saline to the required concentration. Fibrinogen was Grade L, human, lyophilized with a clottability greater than 90% (A.B. Kabi, Stockholm). One gram of fibrinogen was dissolved in IOO mi of distilled water. Anti-A T-III immune serum. Human AT-I11 antiserum, prepared in rabbits, was obtained from Nyegaard and Co., A/S Oslo, Norway. Heparin cofacto~activity. A fibrinogen solution of 4 mg/ml was prepared from the stock solution (containing IOO u hepariniml) and kept at room temperature, 0.1 ml of this fibrino-
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gen-heparin mixture was incubated with 0.1 ml of the AT-I11 containing solution at 37°C for I miii. 0.1 ml of thrombin solution (Parke Davies, Detroit, U.S.A., 20 ujml activity) was then added to the mixture and the clotting time recorded. From the clotting times of a series of dilutions of normal pooled plasma, the heparin cofactor activity of the invcstigated sample was expressed as a per cent of normal plasma. Detcrnzination of the A 7’-IIl concentvation by electvoivnnzunoassay (Laurell, 1966). A mixture of 12 in1 1% agarose and 0.1 nil anti-AT-I11 immune serum was poured on to a glass plate (80 x 80 mm) a t 56°C. Eighteen wells (of 3 mm diameter) were prepared in the agarose layer by a standard pattern. 10 pl of AT-111 containing solutions were placed in each well. Electrophoresis was carried out in a Shandon SAE 2761 electrophoresis apparatus at 20 V/cm for 4 h. After I h of rinsing in distilled water, the plates were covered with filter paper (Whatman No. I) and left overnight to dry. Coonlassie Brilliant Blue (1% w/v, British Drug Houses, Poole) staining was used. The length of the ‘rockets’was measured and the AT-111 concentrations of a sample expressed as a per cent of normal plasma from a calibration curve which was prepared from the dilutions of normal plasma ( 5 , 2.5 and 1.25%) on each plate. Two diwiensional cvossed irnmunoelectvophoresir (Laurell, 1965) was used for the investigation of AT-I11 but with modification, using heparin in the agarose for the first electrophoresis (Sas et d, 1975). 12 nil of a 1% agarose solution, containing 16.6 u heparin per ml agarose, were poured on to a glass plate (80 x 80 mm) in the routine assays. In those experiments in which the effect ofvarious heparin concentrationswcre measured, 0,0.8, 1.6,2.5,4.2, 5.8, 8.4, 16.6, 42.0 and 84.0 unit of heparin per ml agarose were applied. After solidification of the agarose, a well of 3 mm diameter was prepared in one corner of the plate (11 min from both edges) and 5 pl of sample was measured into the well. Electrophoresis was carried out in a Shandon SAE 2761 electrophoresis apparatus at 4°C. After the first electrophoresis (at 20 V/cm for 2 h) the agarose gel was cut in two parts, the direction of the cut was parallel with that of the electrophoresis and was located 14 mm from the edge of the plate. The smaller part of the agarose layer (which contained the proteins separated by the first electrophoresis) was kept, while the wider part was removed. 10 ml of 1% agarose was then mixed with 0.1 ml anti-AT-111 immune serum at 56”C, poured into the site of the discarded gel, the platc rotated through 900, and electrophoresis continued at 20 V/cm for 4 h at 4°C. After electrophoresis the plates remained in a moist chamber overnight and were then rinsed in saliiic three times for 2 h. After a final I h rinse in distilled water the plates were covered with filter paper (Whatman No. I) and left to dry overnight. Coomassie Brilliant Blue staining was used. The electrophoretic mobility of AT-111 was expressed as the distance between the centre of the well and the projected point of the peak on to the base line. Geljiltrution. 5 ml samples were applied to a column of G-200 Sephadcx (medium grade) 2.5 x IOO cm and eluted with a solution containing NaCl 150 mmolar, Tris 25 mmolar, boric acid 3.8 mmolar, EDTA I mmolar and sodium azide 3 mmolar, pH 8.8. Glucose (100mg) was added to all samples to increase the density and serve as a marker of total internal columii volume. The presence of glucose in fractions was estimated semiquantitatively with ‘Clinistix’ (Ames, Slough). Fractions of 5-7 ml were collected on a time based fraction collector at a flow rate of 25 ml/h. Exact fraction volumes were determined by weighing. The eluatc was rnonitorcd for protein at 280 nm in a spectrophotometer. In a second series of experiments normal citrated plasma was heparinized (final concentration IOO u/ml) and gel filtered using an eluant buffer containing heparin (100 u/nil).
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The distribution coefficient (K,) was defined as KD = (V,- V,)/Vi, where V, the void volume was determined from the elution position of chylomicrons, V, was observed elution position of the substance under investigation and Vi was the difference between V, and the elution position of glucose. Fractions were concentrated in Minicon B 15 cells (Amicon Corp., Lexington, U.S.A.) for further investigation by crossed immunoelectrophoresis.
RESULTS
The Efect of Various Concentrationsof Heparin on the ElectrophoreticMobility of AT-III in Plasma Various quantities of heparin were mixed with the agarose in the first dimension of crossed imniunoelectrophoresis (see Materials and Methods). Fig I shows AT-I11 of normal pooled plasma when the agarose contained 0,1.6,2.5,4.2, 8.4, and 16.6 u heparin per ml agarose, and it was observed that the electrophoretic mobility of AT-I11 increased with increasing heparin concentration. At a heparin concentration of 1.6 u/ml a minor part of the AT 111 moved faster than the major part. On increasing the heparin concentration to 16.6 u per ml agarose, three components were differentiated clearly in both plasma and serum. At this concentration the main component (immuno-antithrombin 111‘ = IAT-1111) had the highest electrophoretic mobility and during the 2 h of electrophoresis its mean transit distance (12 samples) for both plasma and serum was 3 3 1.2 mm. The second and the third components had lower electrophoretic mobilities (IAT-1112 : 22.4 +_ 1.0 mm, and IAT-III’ : 16.8 k0.6 mm). At higher heparin concentrations (42 and 84 u/ml agarose) the results were similar to those obtained with 16.6 u/ml agarose. Accordingly 16.6 u heparin per ml agarose was used in all subsequent experiments. Twelve normal plasma showed the same three components as normal pooled plasma. Investigation of AT-III in Normal Serum by Crossed lmmunoelectrophoresis Serum samples were prepared (as described in Materials and Methods) in which thrombin generation had taken place to different degrees. AT-I11 was investigated in these samples by crossed immunoelectrophoresis with the standard heparin concentration of 16.6 u/ml. Coagulation did not take place in the samples in which the citrate was mixed with the blood 0.5, I, 2,4, 8 and 16 min after blood collection. In these samples the crossed immunoelectrophoresis revealed the same three components as plasma AT-111. When citrate was added after the onset of coagulation (about 25 min after venepuncture) there was a gradual decrease of IAT-IF and a concomitantincreaseofIAT-111’ and IAT-III’ components in the 32, 64, 128 and 256 min samples. In the 256 min samples the IAT-111’ and IAT-1113 components fused and gave a ‘spike’like precipitation line. In the ‘stored’ serum this latter phenomenon was even more evident (see Fig .a). When citrated plasma was clotted with various quantities of human or bovine thrombin, similar precipitation arcs appeared as those found in native serum; Fig z(b) shows the result when normal pooled plasma was clotted by human thrombin (50 u/ml plasma). At this thrombin concentration, a minor part of the IAT 111‘ component remained detectable. When the thrombin concentration was increased, the IAT-111’ component disappeared completely and a single precipitation arc was seen corresponding to the fused IAT-111’ and IAT-III’ components.
Aiitithrorrrbiiz III
FIG I . Effect of various concentrations of heparin on the electrophoretic mobility of AT-I11 in nornial plasma. Various concentrations of heparin were mixed with thc agarosc (a: o, b: 1.6, c : 2.5, d : 4.2, c: 8.4, f: 16.6 ulml) in the first phase ofthe crossed imniunoelectrophoresis. At 16.6 u hcpariri per nil agarose three different peaks are observed (IAT-1111, IAT-IIIZ and IAT-II13).
FIG 2. AT-I11 patterns with crossed imniunoelectrophoresis (agarose/heparin) in: (a) native ‘stored’ serum, (b) liquor of thrombin clotted citratcd plar:ma, (c) liquor of Tiger Snake venom clotted citrated plasma.
FIG 5 . Crossed imniuiioelcctrophoretic (agaroselheparin) AT-I11 patterns in gel filtered serum fractions: (a) AT-I11 in a concentrated pool of fractions 45 and 46, (b) AT-I11 in a concentrated pool of fractions 52 and 53.
Antithrombin 1Il
B"b
4.0
0 Q 1
I.o
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s?
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-N
n
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05 I.o
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50
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60
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Fractions
FIG3 . Immunoreactive AT-I11 concentration (AT-111: -)
and heparin cofactor activity (HCA: 0 ) in the gel filtered fractions of normal plasma. AT-I11 and HCA were detected in the albumin peak fractions and a good correlation recorded. 0,OD1,:.
1.6 1.4
I.2
5p 1.0 --N
n 0
0.0 0.6
0.4 0.2
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30
35
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45 50 Fractions
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60
65
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FIG 4. Immunological AT-111 concentration (AT-111: 0 ) and heparin cofactor activity (HCA: 0 )in the gel filtered fractions of 'stored' serum. The AT-I11 concentration was strikingly higher than the HCA in fractions 45,46 and 47. 0,OD.:
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When plasma was clotted with Tiger Snake venom, the AT-111 showed similar changes; increasing the venom concentration decreased the IAT-111’ component progressively, whilst the component(s) with lower electrophoretic mobility increased (Fig 2c). However, the serum obtained from Reptilase clotted citrated plasma had an identical pattern to that of normal plasma.
Gel Filtration of Normal Plasma and Serum AT-III 5 in1 of normal pooled plasma and 5 ml of normal ‘stored’serum were filtered on Sephadex gel as described (see Materials and Methods). AT-111 was quantitated using ‘rocket’ immuiioelectrophoresis and the heparin cofactor activity was determined in the resultant fractions (see Materials and Methods). The results are summarized in Figs 3 and 4 which show that AT-I11 in the plasma was detected by the immunologic method mainly in the fractions corresponding to the ‘albumin peak’ (K, = 0.44), although a small quantity of AT-I11 antigen was present in the void volume. Heparin cofactor activity was detectable only in the albumin peak fractions. The correlation between the quantity of AT-I11 detected by the immunologic method and heparin cofactor assay was good in this region. Immunologically reactive AT-I11 in the serum fractions was also detected mainly in the albumin peak, but some differences were observed between serum and plasma. The serum fractions showed a definite ‘shoulder’ in the higher molecular weight region, although the peak of the immunoreactive AT-I11 had not changed position (K, = 0.44), compared to that of plasma. Another difference was the presence of higher quantities of AT-111 in the void volume than in the corresponding plasma fractions. Although the heparin cofactor activity was only present in the albumin rich fractions it was absent from the ‘shoulder’ of the curve as defined by immuno-AT-I11 assays. On the basis of the observed discrepancy between the immunologic AT-I11 values and heparin cofactor activities in the fractions 45-48, we assumed there was some heterogeneity of serum AT-111. This was supported by the observation that AT-I11 in serum fractions 45,46 and 47 appeared in a molecular weight region in which no AT-111 was detected in the plasma fractions. Further confirmation was obtained by running two Ioo-fold concentrated pools (consisting of fractions 45-46 and 52-5 3 respectively) on agaroseiheparin crossed immunoelectrophoresis technique and showing that the two slower components (IAT-111’ and IAT-1113) were present mainly in fractions 45 and 46, whilst fractions 52 and 53 contained a higher concentration of the IAT-111’ component (Fig 5 ) . Similar analysis of the gel filtered plasma fractions revealed that IAT-111’ and IAT-1113 were present in fractions 49 and 50 but in very small amounts compared to equivalent fractions of serum. 6-200
Gel Filtration Studies on Heparinized Normal Plasma 5 ml of normal pooled citrated plasma was heparinized with 500 units of heparin and gel filtered as above, but using heparinized buffer as the eluant. The AT-I11 concentration of the fractions was measured using ‘rocket’immunoelectrophoresis.The results are summarized in Fig 6 which shows that in the presence of heparin the immunoreactive AT-I11 eluted off the column in a higher molecular weight region (K, = 0.29) than that run in the absence of heparin (K, = 0.44: see Fig 3).
Antithrombin III
0
0
0 -
.\" El c
o a
3
Fractions
FIG 6. Immunoreactive AT-111 concentration (AT-111: 0 )in the gel filtered plasma fractions of nornial heparinized plasma. AT-I11 elutes off thc column in a higher molecule size region than that run in the absence of heparin (cf. Fig 3). 0,OD hi:.
DISCUSSION In the course of early immunological investigations on various coagulation factors we observed that improved definition of 'rocket patterns' was obtained by incorporating heparin into the agarose gel. This modification was iiicluded in studies on AT-I11 and it was noted that when heparin was added to thc gel for the first phase in crossed iminunoelectrophoresisthree separate peaks were identified (designated IAT-111' , IAT-Ill2 and IAT-II13) and that their individual concentrations in citrated plasma and native serum was significantly different (Sas et ul, 1975). In the present study the optimal concentration for the development of this phenomenon has been defined (16.6 u heparin per in1 of agarose) and the mobility of the three peaks in normal plasma and serum was found to be identical. Serum prepared by the addition of human thrombin and Tiger Snake venom to normal citrated plasma gavc crossed iinmunoelectrophoretic patterns that were similar to those of native serum. However, serum obtained without thrombin generation (Reptilase clots) was identical to norinal plasma. The gel filtration studies were primarily designed to ascertain the nature of the three peaks observed in crossed immunoelectrophoresis. The results of these investigations and those of the snake venoms referred to above, suggest that IAT-III' are higher molecular weight complexes, presumably with at least thrombin and Xa. This latter conclusion corresponds to those of Abilgaard (1969) and Binder (1973) who noted in gcl filtration studies that AT-I11 in serum had a higher molecular weight than that in plasma. The present gel filtration experiments also added further evidence to support the hypothesis that normal citrated plasma contains small but detectable quantities of complexed AT-I11 (IAT-111' and IAT-Ill3). Whether
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G. Sas, D. S. Pepper and]. D. Cash
this is a normal in vivo phenomenon, and thus reflects evidence of continuous low-grade activation of the coagulation mechanism, as postulated by Astrup (1956)~or an artefact arising during blood withdrawal, is not yet known. Damus et al (1973) have reported the neutralizing effect of AT-I11 on XIa, and it is possible that the generation of XIa cannot be avoided during venepuncture. We believe this explanation for the presence of trace quantities of IAT-1112 and IAT-1113 in normal plasma is unlikely as we have not observed a change in the AT-I11 crossed immunoelectrophoresis patterns when normal citrated plasma is filtered through glass bead columns. However, that other, as yet unknown, reactionts) in the coagulation mechanism during venepuncture may have given rise to this observation in plasma cannot be excluded at present. This problem is currently under investigation. The AT-111 gel filtration studies on heparinized normal plasma were of particular interest, for they indicate that when heparin binds to AT-I11 higher molecular weight complexes are formed. It therefore seems probable that the explanation of the difference in crossed immunoelectrophoresis AT-I11 patterns originally observed between simple agarose and agarosei heparin gels (Sas et al, 197s) lies in the fact that the AT-I11 heparin complexes formed in the latter gel are highly charged (negatively) and thus migrate a greater distance toward the anode than AT-I11 in plain agarose gels. This interpretation would lead us to conclude that because the binding sites for IAT-1112 and IAT-1113 are wholly or partially occupied by activated coagulation factors their heparin binding activity is reduced sufficiently to diminish their electrophoretic mobility in the agaroseiheparin gel. This phenomenon, therefore, permits the observed separation of IAT-111’ , IAT-111’ and IAT-II13. ACKNOWLEDGMENT
This work was funded by a Research Grant award by the Scottish Home and Health Department. REFERENCES ABILDGAARD, U. (1969)Binding of thrombin to antithrombin 111. Scandinavian Journal of Clinical and Laboratory Investigation, 24, 23. ASTRUP,T. (1956)The biological significance of fibrinolysis. Lancet, ii, 565. BIGGS,R., DENSON, K.W.E., AXMAN, N., BORRETT, R. & HADDEN,M. (1970)Antithrombin 111, antifactor Xa and heparin. British Journal afHaematology, 1% 283.
BINDER,B. (1973)On the complex formation of antithrombin I11 with thrombin. Gelfiltration studies on human plasma and serum. Thrombosis et Diathesir Haemorrhagica, 30, 280. DAMUS,P.S., HICKS, M. & ROSENBERG, R.D. (1973) Anticoagulant action of heparin. Nature, 246, 355.
DOMBROSE, F.A., SEEGERS, W.H. & SEDENSKY, J.A. (1971)Antithrombin. Inhibition of thrombin and autoprothrombin (F-Xa) as a mutual depletion system. Thrombosis et Diathesis Haemorrhagica, 26, 103.
EGEBERG, 0. (1965)Inherited antithrombin deficiency
causing thrombophilia. Thrombosis et Diathesis Haemorrhagica, 13, 516. LAURELL,C.-B. (1965) Antigen-antibody crossed electrophoresis. Analytical Biochemistry, 10, 3 58. LAURELL, C.-B. (1966)Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analytical Biochemistry, 15,45. MARCINIAK, E., FAFSEY, C.H. & DESIMONE,P.A. (1974)Familial thrombosis due to antithrombin 111 deficiency. Blood, 43, 219. SAS,G., PEPPER, D.S. & CASH,J.D. (1975)Plasma and serum antithrombin 111: Differentiation by crossed immunoelectrophoresis. Thrombosis Research, 6, 87. VAN DER MEER,J., STOEPMAN-VAN DALE”, E.A. & JANSEN, J.M.S. (1973)Antithrombin 111deficiency in a Dutch family. In: IVth International Congress on Thrombosis and Haemostasis, Vienna, Abstracts, p 226. YIN,E.T., WESSLER, S. & STOLL,P.J, (1971) Identity of plasma-activated factor X inhibitor with antithrombin 111 and heparin cofactor. Journal .f Biological Chemistry, 246, 3712.
