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Preparation and immunological determination of rat antithrombin III
THROMBOSIS RESEARCH 17; 789-799 (c) Pergamon Press Ltd. 1980. Printed
in the U.S.A.
0049~3848/80/06078g-11$02.00/o
PREPARATION AND IMMUNOLOGICAL DETERMINATION OF RAT ANTITHROMBIN III
Toshihiko Kumada and Yasushi Abiko Laboratory of Biochemistry,Research Institute Daiichi Seiyaku Co., Ltd. Edogawa-ku,Tokyo 132, Japan (Received 31.8.1979; in revised form 4.12.1979. Acce.pted by Editor K.M. Brinkhous) ABSTRACT
AntithrombinIII was purified from rat plasma to a single component by the procedures Including polyethyleneglycol precipitation,affinity chromatographyon heparin-Sepharose,isoelectric focusing and polyacrylamidegel disc electrophoresis. Rat antithrombinIII was found to be a single polypeptide chain with a molecular weight of about 6.4 x 104 daltons and a p1 of 4.85. The purified rat antithrombinIII was injected to rabbits to produce specific antibody, which was found to cross-reactwith a plasma component, probably antithrombinIII, of mice, monkeys and humans but not with guinea pig and dog plasmas. The specific antiserum was applied to immunologicaldeterminationof plasma antithrombinIII levels in rats to find that antlthrombinIII was significantlyincreasedwith a minor increase in its complexed form during thrombus formation in an experimentalvenous thrombosis which was induced by insertion of a stainless steel wire coil into the inferior vena cava. The increase in antithrombinIII, most of which was in free form, was thought to be a response to local inflammation due to the operation, because a similar elevation of plasma antithrombinIII was observed with sham-operatedrats.
INTRODUCTION Antithrombin III is one of the important physiologicalanti-thrombogenic factors as suggested by many clinical findings that hypercoagulablestate, recurrent thromboembolicdisorders and high incidence of post-operativevenous thrombosiswere associatedwith low plasma concentratfonsof antithrombinIII (1 - 4). Key Words: AntithrombinIII, Purification,Immunologicaldetermination, Experimentalvenous thrombosis,Rat. 789
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For further understandingof the pathophysiologicalrole of antithrombin III in the animal study, specific antibody against this protein is a useful tool. There have been some reports concerningantithrombinIII in experimental animals, dogs (5 - 8) and rabbits (9, lo), but only few dealing with rat antithrombin III (11). The present report describes purificationof rat plasma antithrombinIII, preparationof specific antiserumagainst rat antithrombinIII and its application to immunologicaldeterminationof free and complexed antithrombinIII in rat plasma during thrombus formation in the vein. The cross-reactivityof the antiserum to plasma componentsof other animal species was also examined.
MATERIALS AND METHODS Rat plasma. Blood was obtained into a plastic syringe containing 0.1 volume of 3.8 X Na3-citratedihydrate from the carotid artery of about 60 male STD:Wistarrats weighing about 250 g under pentobarbitalanesthesia (40 mg/kg, ip). The titrated blood (10 ml/rat) was centrifugedat 4“C and at 1500 g for 10 min to obtain platelet-poorplasma (about 360 ml in total). The plasma was stored at -2O'C until use. Determinationof antithrombinactivity. This was performed according to Miller-Andersson(12), with a slight modification.The reaction mixture contained 20 p1 of a-sample solution; 100 ~1 of 0.05 M Tris-BCl containing0.1 M NaCl (pH 7.5) and 100 N of bovine thrombin (Parke-Davis,10 NIB units/ml Tris NaCl buffer). After preincubationat 37*C for 30 min, 100 pl of bovine fibrinogen (DaiichiPure Chemicals Co., Tokyo, 5 mg/ml Tris-NaCl buffer) was added to the mixture, and clotting time was recorded at 37°C. A unit of antithrombin III was defined as the antithrombinactivity contained in 1 ml of normal rat plasma, according to Miller-Andersson(12). In addition, for clarificationof enzymologicalpotency of the antithrombinactivity, an enzymologicaiunit was defined as the activity which inactivatedone unit of thrombln under the above conditions.The units of thrombin inactivatedwere calculatedfrom a standard clotting time curve as a function of thrombin unit. Determinationof heparin cofactor activity. This was carried out accordingtoMiller?Andersron(l2), with a slight modification.The reaction mixture contained 10 pl of plasma to be tested, 100 pl of heparin sodium (DaiichiPure Chemicals Co., 20 units/ml Tris-NaClbuffer) and 100 pl of bovine thrombin (15 NIH units/ml Tris-NaCl buffer). After preincubationat 37'C for 1 min, 100 pl of bovine fibrinogen (5 mg/ml Tris-NaCl buffer) was added to the mixture, and clotting time was recorded at 37OC. Determinationof protein. Protein was determinedby the method of Lowry et al. (13) using bovine serum albumin as a standard or, in some cases, arbitrarily by absorbancyat 280 nm (A280). Heparin-Sepharose. Heparin was purified from a commercialpreparation (DaiichiPure Chem. Co.) by gel filtrationthrough Sephdex 6150, and coupled to Sepharose 4B according to the method of Cuatrecasas (14). Beparin content of the heparin-Sepharoseobtained was 15.6 mg/ml gel. Preparationof crude antithrombinIII. Rat antithrombinIII was isolated from 360 ml of the titrated plasma, essentiallyaccording to the method "B" of Thaler and Schmer (15), by the procedures including defibrination(56'C,3 min), fractionalprecipitationby polyethyleneglycol (PEG 4000, 13 - 28 X) and
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affinity chromatographyon a heparin-Sepharosecolumn (16 x 640 mm) with aNaC1 gradient elution (the activity eluted with NaCl at 0.95 - 1.25 M). These procedures yielded about 25 units (9000 enzymologicalunits) of a crude antithrombin III preparation (140 ml) with a specific activity of 1.8 units (645 i enzymologicalunits)/A280 (Table 1). Isoelectricfocusing. This was carried out according to Vesterverg and Svensson (16) for further purificationof rat antithrombinIII. Electrofocusing was performed in a LKR 8101 column (110 ml, LRB Co., Stockholm)with a mixture of carrier ampholytes (LRR ampholine),pH 4-6 (4 volumes) and pH 3.5-10 (1voL) at a final concentrationof 1 X. After electrophoresisat 0-1°C for 24 hr at 250 volts, 2 hr at 500 volts and 48 hr at 700 volts, the column content was drained in fractions of 1.8 ml each at a flow rate of 1.8 ml/min. Polyacrylamidegel disc electrophoresis. This was carried out, according to Davis (17). for final nurificationof rat antithrombinIII and for determination of molecular weight with 5 or 7.5 % gel (5 mm diameter) in the presence or absence of 1 % sodium dodecylsulfateand 2-mercaptoethanolat 6 or 3 mA/gel for 1.5 hr. Preparation of antiserum. As described in Results, antithrombinIII was electrophoreticallyseparated from some contaminantproteins in polyacrylamide gels, and the gel slices from 6 runs containingantithrombinIII was homogenized with 1 ml of complete Freund's adjuvant. Two Japanese white rabbits were immunizedwith the homogenate (about 0.39 mg protein of antithrombinIII in 1 ml per a rabbit) 3 times at 2 week intervals.The rabbits were bled 8 days after the last injection of the antigen, and the serum was stored frozen after inactivationat 56'C for 30 min. Immunologicaltests. Double diffusionwas carried out according to Ouchterlony (18) on agar plates with 1.5 % Agar Noble (Difco Labs., Michigan) in barbital buffer (pH 8.6, I * 0.05) at 37'C for 48 hr. Rocket immunoelectrophoresls was performed for assay of antithrombinIII-relatedantigen according to Laurel1 (19) with a 1 X agarose plate of 90 x 90 mm (14 ml) containing0.17 ml of anti-rat antithromblnIII rabbit serum at 180 volts (30 mA/2 plates) for 2.5 hr. Barbital buffer, pH 8.6 and I 0.02, was used. Sample volume was 5 ~1. For differentialassay of the free and complexed forms of antithrombinIII, according to Andersson et al. (20), samples (10 fl each) were first electrophoresed on a 1 X agarose plate of 74 x 110 mm (14 ml) containing 0.3 ml of heparin-la (1000 units/ml) at 110 volts for 1.5 hr. The plate was cut longitudinally into strips with 20 mm wide to separate each run. The gel strip was transferredonto one end of a glass plate of 90 x 90 mm. The remaining part of the glass plate was covered with 10 ml of 1 % agarose containing0.12 ml of the antiserum. Then, electrophoreslswas carried out in the second dimension at 40 volts for about 16 hr. Induction of vein thrombosis in rats. According to the method reported elsewhere (21), experimentalvenous thrombosiswas produced in rats by insertion of a small stainless steel wire coil (a dental-pastecarrier, 20 nanlong) into the inferior vena cava just below the left renal vein branching. The thrombus was formed and grew up around the wire and onto the vein wall with a maximum size of about 1 mg protein 2-3 days after the wire coil insertion(U). Control rats received similar laparotomywithout wire coil insertion.
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FEWLTS Purificationof Rat AntlthromblnIII The crude preparationof rat antlthrombinIII (140 ml) obtained by the heparln-Sepharosechromatographywas dialyzed against 2.5 1 of 0.05 M Tris-HCl (pH 7.5) and concentratedto 24 ml by the use of a collodlon bag (Sartorlus Membrane Co., Goettlngen,Germany). The concentratedsolution was subjected to isoelectricfocusing at pH range of 3.5 - 10. Fig. 1 Illustratesthe lsoelectric fractionationprofile with a main peak of the antlthromblnactivity in the fraction Nos. 24 - 31 (PI, 4.85). The active fractionswere combined, dialyzed against 0.05 M Trls-HCI containing0.1 M NaCl (pH 7.5) and concentrated to 18.4 ml.
I
I
1.6 -
!
1.2 -
t i7
0.8 -
c) 1 2 0" 5 2 *
0.4
FIG. 1 Isoelectricfractionationof rat antlthromblnIII obtained by heparln-Sepharosechromatography.Column size, 110 ml (LKB8101); carrier ampholyte,a mixture of 1 % LKB amphollne,pH 4-6 and pH 3.5-10 at 4:l; fraction size, 1.8 ml/tube.
Polyacrylamldegel disc electrophoreslsrevealed 2 minor contaminating proteins in this preparation. To remove these contsmlnants,the concentrated fraction was subjected to polyacrylamldegel disc electrophoresls(130 pg protein/gel). The gels, marked with a stained marker gel, were cut and antlthrombln III was extractedwith the Trls-NaCl buffer or, for immunization, with complete Preund's adjuvant (6 gels/ml). The result of purificationis shown in Table 1. Molecular weight of the purified rat antlthromblnIII was estimated to be about 6.4 x lo4 daltons by SDS-polyacrylamldegel disc electrophoreslsusing ovalbumln (4.5 x 104) and bovine serum albumin (6.7 x 104) as references.
RAT ANTITHROMBIN
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TABLE1 Summary of Purificationof AntithrombinIII from Rat Plasma Volume
Total protein
Step
Antithrorrbin activity Total Specific (Unit) (Unit/A280) (UnithId
(ml)
(*280)
Defibrination
340
17340
22720
340 (113600)
0.0196 (6.55)
PEGprecipitation
188
8573
-
254 (79000)
0.0296 (9.21)
-
25.4 (9130)
1.80 (645)
21.7 (7250)
1.99 (718)
k3)
HeparinSepharose
24
14.2
Isoelectric focusing
18.4
10.9 16.1
0.015 (5.0)
1.35 (450)
Polyacrylamide gel electrophoresis
(500)
Figures in the parentheses are enzymologicalunits of the antithrombinactivity.
FIG. Control 2
Heparin cofactor nature of the purified rat antithrombinIII. The incubationmixture contained 100 pl of thrombin (20 NIH units/ ml), 50 ~1 of the purified antithrombin III (64 pg/ml> and 50 ~1 of the Tris-NaCl buffer (a, 0) or heparin (0.4 unit/ml;?? , 0). Control runs contained the buffer solution in place of antithrombinIII. After incubationat 37'C for the indicated period, the remaining thrombln activity was determinedby the fibrinogen clotting assay.
80
ii 60
+ Antithrombin III
O1
’
0
8
I
I
10 20 30 Incubation time (min)
2
1
40
Rat antithrombinIII did not dissociate in reduced SDS-polyacrylamidegel electrophoresis,indicating a single polypeptide chain. As shown in Fig. 2, the purified rat antithrombinIII inactivatedthromhin progressivelyby itself and did so instantaneouslyin the presence of heparin, indicatingheparin cofactor activity of the purified protein.
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FIG. 3 IPnaunodouble diffusion of the anti-rat antithrombinIII rabbit serum against plasmas of various species including rat (a, e), mouse (b), rabbit (cl, dog (d), man (f), monkey (8) and guinea pig (h).
FIG. 4 Rocket ismtunoelectrophoresis of normal rat plasma for antithrombinIII. Normal rat plasma, from right to left, undiluted, diluted to 75, 50 and 25 2 with normal rabbit plasma. Purified rat antithrdiabin III, from right to left, 200, 150, 100, 50 and 20 &ml Tris-NaCl.The sample volume was 5 pl each.
ImmunologicalExaminationof Rat AntithrombinIII Antiserumwas produced in rabbits against the purified rat.antithrombin III. The antiserum gave a single precipitin line against normal rat plasma on immunodoublediffusion and immunoelectrophoresis. As shown in Fig. 3, antirat antithrombinIII rabbit serum was found to cross-reactwith a plasma component,probably antithrombinIII, in plasmas of mice, humans and, to a minor extent, monkeys (Macacusrhesus), but not with guinea pig and dog plasmas. Determinationof AntithrombinIII in Rat Plasma Changes in the plasma level of antithrombinIII-relatedantigen and the heparin cofactor activity were studied during thrombus formation in the vein thrombosismodel in rats. Plasma samples were obtained from the jugular vein of the rats before and 6, 24, 48 and 72 hr after the wire coil insertion into the inferior vena cava. Total antithrombinIII was assayed by the rocket immunoelectrophoresisshown in Fig. 4. Plasma samples from the sham-operated control rats were also assayed. Normal plasma level of antithrombinIII was found to be around 200 &ml. As shown in Table 2, total amount of anti-
Two dimensional electrophoresisof rat plasma and serum. The first run on a heparin-agaroseplate and the second run on an agarose plate containing anti-rat antithrombinIII rabbit serum. A: Normal rat plasma (complexedform, 2 xl),B: pooled thrombosed rat plasma obtained 48 hr after the wire coil insertion (complexedform, 9 X), C: pooled shamoperated rat plasma obtained 48 hr after operation (complexedform, 6 X), and D: normal rat serum (complexedform, 61 X).
FIG. 5
u
-4 W
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thrombin III-relatedantigen was considerablyincreased after the wire coil insertion,while a similar increase in antithrombinIII-relatedantigen was also observedwith the sham-operatedrats. The heparin cofactor activity behaved in parallel with the level of aatithrombinIII-relatedantigen. But some discrepancywas observed in the plasma samples at 24 hr: increase in the cofactor activity was less than that in antithrombinIII-relatedantigen. This was more clearly shown by decrease in the ratio of the heparln cofactor activity to antithrombinIII-relatedantigen in the 24 hr-plasma samples from the thrombosedrats (Table 2). Differentialanalysis of antithrombinIII-relatedantigen in the pooled plasmas of each group by the two dimensionalelectrophoresisindicated that most of the increment of antithromblnIII-relatedantigen was due to increase in free antithrombinIII regardlessof accompanyingthrombus formation (Fig.51, and that there was a minor increase in the complexed form of antithrombinIII in the thromboeedrats when compared with the sham-operatedrats (Fig. 5). The ratios of the complexed form to total antithrombinIII were 2-3 X in normal plasmas, 6 X (6 hr) and 9 X (24, 48 and 72 hr after operation) in plasmas from the thrombosedrats, 3 X (6 hr) and 6 X (48 hr) in the sham-operatedrat plaaza55, and 60 X in normal rat sera (Fig. 5).
TABLE 2 Changes in Plasma AntithrombinIII Levels during Thrombus Formation in Eats
Before
Time (hr) after operation 6 48 24
72
AT-III antigen (%) Control Thrombosed
104 ??4 103 f 4
110 * 3* 105 * 4
134 f 3** 151 * 9#
142 i 41 150 f 81
151 f 41 143 * 5fi
AT-III activity (%) Control Thrombosed
100 i-3 102 ??5
103 i 2 100 * 4
142 f 5# 147 f 6**
Ratio (activity/antigen) Control 0.97 * 0.03 0.94 ??0.03 0.88 * 0.02 1.01 f 0.03 1.04 f 0.03 Thrombosed 1.00 f 0.03 0.96 f 0.03 0.80 f 0.0W0.99 i 0.07 1.12 * 0.07 AntithrombinIII (AT-III)related antigen and AT-III activity (heparincofactor activity)were assayed after operation and presented as % of normal. The ratio of heparin cofactor activity to AT-III antigen was calculated. * ~(0.05, ** p
DISCUSSION In the present study, an antithrombincomponentwas highly purified from rat plasma by the proceduresanalogous to those used for human antithrombinIII. The purified antithrombincomponent satisfied the definitionof antithrombin
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III (heparin cofactor),"progressiveneutralizationof thrombin by itself and instantaneousinactivationof the enzyme in the presence of heparin" (22). There are some molecular similaritiesbetween rat antithrombinIII and the counterpartsof other animal species. Molecular weight of rat antithrombin III, 6.4 x 104, was similar to that reported with human (12, 231, rabbits (9), dogs (7) and bovine (24), although some higher and lower values were reported (5, 25). The isoelectricpoint of rat antithrombinIII, 4.85, was close to p1 of human antithrombinIII, 5.11 (23). Immunologicalcross-reactionof antithrombin III was observed between rats and other animals ineludingmice,humans and monkeys, but not guinea pigs and dogs. Normal plasma level of antithrombin III-relatedantigen in rats was around 200 pg/ml in agreementwith thereported value (220 i 10 )Ig/ml,Ref. 11) and close to the correspondingnormal plasma level In humans (199 - 191 pg/ml, Ref. 26). Many clinical studies have suggested a close relationshipbetween lowered plasma antithrombinIII levels and hypercoagulablestates or high incidence of thromboembolicdisorders (1 - 4). On the other hand, experimentallyinduced thrombophlebitis(6) and disseminatedintravascularcoagulation (8) in dogs were reported to be not associatedwith any significantchanges in the plasma level and the turnover rate of antithrombinIII, even inthe casesaccompanying significantconsumptivedecrease in platelets and plasma fibrinogen (8). They did not refer to the plasma level of complexed antithrombinIII. The present study, however, indicated a remarkable increase in antithrombin III-relatedantigen as well as in the heparin cofactor activity in plasmas from the rats with venous thrombosis.The similar changes were also observed in the sham-operatedcontrol rats. Most of the increased antithrombinIII was in its free form and a minor increase was detected in the complexed form which was thought to be composed of some activated coagulationfactors and antithrombin III. These findings suggest that such elevation of the plasma antithrombin III levels may be a response to local inflammationdue to operation injury, as reported by Koj and his colleagueswith the turpentine-induced inflammationmodels (lO,.ll),and that the elevationwas too great to detect any definite changes in antithromblnIII levels related to thrombus formation. Simultaneousincrease in plasma fibrinogenwas observed in both the thrombosed and sham-operatedrats (21), also suggestingsome inflammatoryreaction. However, a significantdecrease was observed in the ratio of the heparin cofactor activity to antithromblnIII-relatedantigen in the thrombosedrats 24 hr after the operation, suggesting some consumptionof functionalantithrombin III associatedwith thrombus formation.This may relate to a slight but larger increase in the complexed form of antithrombinIII-relatedantigen in the thrombosedrats than in the control rats (Fig. 5). In this experimental vein thrombosismodel, thrombus formation actively progreesed during 2 days after the wire coil insertionwith prolongationof partial thromboplastintime and decrease in the circulatingplatelet count, suggestingactivation and consumptionof coagulationfactors (21).
REFERENCES 1. EGEBERG, 0. Inherited antithrombindeficiency causing thrombophilia. Thrombos. Diathes. Haemorrh. 13, 516-529, 1965. 2. MARCINIAK, E., ERLEY, C.H. and DE SIMONE, P.A. Familial thrombosisdue to antithrombinIII deficiency.Blood, 43, 219-231, 1974.
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3. FAGERHOL,M.K. and ABILDGAARD,U. Immunologicalstudies on human antithrombin III - Influenceof age, sex and use of oral contraceptiveson serum concentration.Stand. J. Haemat. 7, 10-17, 1970. 4. SAGAR, S., STAMATAKIS,J.D., THOMAS, D.P. and KAKKAR, V.V. Oral contraceptives,antithrombinIII activity, and postoperativedeep-vein thrombosis Lancet, i, 509-511, 1976. 5. DAMUS, P.S. and WALLACE, G.A. Purificationof canine antithrombinIIIheparin cofactor using affinity chromatography.Biochem. Biophys. Res. Conuz.61, 1147-1153,1974. 6. KOBAYASHI,N. and TAKEDA, Y. Studies of the effects of estradiol,progesterone, cortisol, thrombophlebitisand typhoid vaccine on synthesis and catabolismof antithrombinIII in the dog. Thrombos. Haemoetas. 37, 111-122, 1977.. 7. KOBAYASHI,N., TANAKA, H., UENO, T., GONMORI, H. and MAEKAWA, T. Studies on antithrombinIII. Purificationof canine antithrombinIII and its properties.Blood and Vessel, 9, 139-144, 1978. 8. TANAKA, H., UENG, T., GONMORI, H., TAKEUCHI, T., TANAKA, S., ARAI, H., KOBAYASHI,N. and MAEKAWA, T. Studies on antithrombinIII (AT III). IV. Effect of thrombin administrationon metabolism of canine AT III. Blood and Vessel, 10, 59-62, 1979. 9. YIN, E.T., WESSLER, S. and STOLL, P.J. Rabbit plasma inhibitorof the activated species of blood coagulationfactor X. Purificationand some properties.J. Biol. Chem. 246, 3694-3702,1971. 10. KOJ, A. and REGOECZI, E. Effect of experimentalinflammationon the synthesisand distributionof antithrombinIII andtil-antitrypsinin rabbits. Br. J. exp. Path. 59, 473-481, 1978. 11. KOJ, A., REGOECZI, E., TOEWS, C.J., LEVEILLE, R. and GAULDIE, J. Synthesis of antithrombinIII and alpha-1-antitrypsin by the perfused rat liver. Biochim. Biophys. Acta, 539, 496-504, 1978. 12. MILLER-ANDERSSON,M., BORG, H. and ANDBRSSON, L.-O. Purificationof antithrombin III by affinity chromatography.ThrombosisRes. 5, 439-452, 1974. 13. LGWRY, O.H., ROSEBROUGH,N.J., FARR, A.L. and RANDALL, R.J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275, 1951. 14. CUATRECASAS,P. Protein purificationby affinity chromatographyderivatizationsof agarose and polyacrylamidebeads. J. Biol. Chem. 245, 3059-3065,1970. 15. THALER, E. and SCHMER, G. A simple two-step isolationprocedure for human and bovine antithrombinII/III (heparincofactor):a comparisonof two methods. Brit. J. Haemat. 31, 233-243, 1975. 16. VESTERBERG,0. and SVENSSON,H. Isoelectricfractionations,analysis, and characterizationof ampholytesin neutral pH gradients.IV. Resolving power in connectionwith separationof myoglobins.Acta Chem. Stand. 20, 820-834, 1966.
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17. DAVIS, B.J. Disc electrophoresis- II. Method and applicationto human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427, 1964. 18. OIJCHTERLONY, 0. Diffusion in gel methods for immunologicalanalysis. Progr. Allergy, 5, l-78, 1958. 19. LAURKLL, C.-B. Quantitativeestimationof proteins by electrophoresisin agarose gel containingantibodies.Analytical Biochem. 15, 45-52, 1966. 20. ANDERSSON, L.-O., ENGMAN, L. and HENNINGSSON,E. Crossed immunoelectrophoresis as applied to studies on complex formation. The binding of heparin to antithrombinIII and the antithrombinIII-thrombincomplex. J. Immunol. Methods, 14, 271-281, 1977 21. KUMADA, T., ISHIHARA,M., OGAWA, H. and ABIKO, Y. Experimentalmodel of venous thrombosis in rats and effect of some agents. ThrombosisRes. in press. 22. YIN, E.T., WESSLKR, S. and STOLL, P.J. Identity of plasma-activated factor X inhibitorwith antithrombinIII and heparin cofactor. J. Biol. w. 246, 3712-3719, 1971. .
23. ROSENBERG, R.D. and DAMNS, P.S. The purificationand mechanism of action of human antithrombin-heparincofactor. J. Biol. Chem. 248, 6490-6505,1973. 24. ABILDGAARD,U. Binding of thrombin to antithrombinIII. Scand. J. Clin. Lab. Invest. 24, 23-27, 1969. 25. KURACHI, K., SCHMBR, G., HERMODSON,M.A., TELLER, D.C. and DAVIE, E.W. Characterizationof human, bovine and horse antithrombinIII. Biochemistry,15, 368-373, 1976. 26: CHAN, V., CHAN, T.K., WONG, V., TSO, S.C. and TODD, D. The determination of antithrombinIII by radioimmunoassayand its clinical application. Brit. J. Haemat. 41,.563-572,1979.
in the U.S.A.
0049~3848/80/06078g-11$02.00/o
PREPARATION AND IMMUNOLOGICAL DETERMINATION OF RAT ANTITHROMBIN III
Toshihiko Kumada and Yasushi Abiko Laboratory of Biochemistry,Research Institute Daiichi Seiyaku Co., Ltd. Edogawa-ku,Tokyo 132, Japan (Received 31.8.1979; in revised form 4.12.1979. Acce.pted by Editor K.M. Brinkhous) ABSTRACT
AntithrombinIII was purified from rat plasma to a single component by the procedures Including polyethyleneglycol precipitation,affinity chromatographyon heparin-Sepharose,isoelectric focusing and polyacrylamidegel disc electrophoresis. Rat antithrombinIII was found to be a single polypeptide chain with a molecular weight of about 6.4 x 104 daltons and a p1 of 4.85. The purified rat antithrombinIII was injected to rabbits to produce specific antibody, which was found to cross-reactwith a plasma component, probably antithrombinIII, of mice, monkeys and humans but not with guinea pig and dog plasmas. The specific antiserum was applied to immunologicaldeterminationof plasma antithrombinIII levels in rats to find that antlthrombinIII was significantlyincreasedwith a minor increase in its complexed form during thrombus formation in an experimentalvenous thrombosis which was induced by insertion of a stainless steel wire coil into the inferior vena cava. The increase in antithrombinIII, most of which was in free form, was thought to be a response to local inflammation due to the operation, because a similar elevation of plasma antithrombinIII was observed with sham-operatedrats.
INTRODUCTION Antithrombin III is one of the important physiologicalanti-thrombogenic factors as suggested by many clinical findings that hypercoagulablestate, recurrent thromboembolicdisorders and high incidence of post-operativevenous thrombosiswere associatedwith low plasma concentratfonsof antithrombinIII (1 - 4). Key Words: AntithrombinIII, Purification,Immunologicaldetermination, Experimentalvenous thrombosis,Rat. 789
790
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For further understandingof the pathophysiologicalrole of antithrombin III in the animal study, specific antibody against this protein is a useful tool. There have been some reports concerningantithrombinIII in experimental animals, dogs (5 - 8) and rabbits (9, lo), but only few dealing with rat antithrombin III (11). The present report describes purificationof rat plasma antithrombinIII, preparationof specific antiserumagainst rat antithrombinIII and its application to immunologicaldeterminationof free and complexed antithrombinIII in rat plasma during thrombus formation in the vein. The cross-reactivityof the antiserum to plasma componentsof other animal species was also examined.
MATERIALS AND METHODS Rat plasma. Blood was obtained into a plastic syringe containing 0.1 volume of 3.8 X Na3-citratedihydrate from the carotid artery of about 60 male STD:Wistarrats weighing about 250 g under pentobarbitalanesthesia (40 mg/kg, ip). The titrated blood (10 ml/rat) was centrifugedat 4“C and at 1500 g for 10 min to obtain platelet-poorplasma (about 360 ml in total). The plasma was stored at -2O'C until use. Determinationof antithrombinactivity. This was performed according to Miller-Andersson(12), with a slight modification.The reaction mixture contained 20 p1 of a-sample solution; 100 ~1 of 0.05 M Tris-BCl containing0.1 M NaCl (pH 7.5) and 100 N of bovine thrombin (Parke-Davis,10 NIB units/ml Tris NaCl buffer). After preincubationat 37*C for 30 min, 100 pl of bovine fibrinogen (DaiichiPure Chemicals Co., Tokyo, 5 mg/ml Tris-NaCl buffer) was added to the mixture, and clotting time was recorded at 37°C. A unit of antithrombin III was defined as the antithrombinactivity contained in 1 ml of normal rat plasma, according to Miller-Andersson(12). In addition, for clarificationof enzymologicalpotency of the antithrombinactivity, an enzymologicaiunit was defined as the activity which inactivatedone unit of thrombln under the above conditions.The units of thrombin inactivatedwere calculatedfrom a standard clotting time curve as a function of thrombin unit. Determinationof heparin cofactor activity. This was carried out accordingtoMiller?Andersron(l2), with a slight modification.The reaction mixture contained 10 pl of plasma to be tested, 100 pl of heparin sodium (DaiichiPure Chemicals Co., 20 units/ml Tris-NaClbuffer) and 100 pl of bovine thrombin (15 NIH units/ml Tris-NaCl buffer). After preincubationat 37'C for 1 min, 100 pl of bovine fibrinogen (5 mg/ml Tris-NaCl buffer) was added to the mixture, and clotting time was recorded at 37OC. Determinationof protein. Protein was determinedby the method of Lowry et al. (13) using bovine serum albumin as a standard or, in some cases, arbitrarily by absorbancyat 280 nm (A280). Heparin-Sepharose. Heparin was purified from a commercialpreparation (DaiichiPure Chem. Co.) by gel filtrationthrough Sephdex 6150, and coupled to Sepharose 4B according to the method of Cuatrecasas (14). Beparin content of the heparin-Sepharoseobtained was 15.6 mg/ml gel. Preparationof crude antithrombinIII. Rat antithrombinIII was isolated from 360 ml of the titrated plasma, essentiallyaccording to the method "B" of Thaler and Schmer (15), by the procedures including defibrination(56'C,3 min), fractionalprecipitationby polyethyleneglycol (PEG 4000, 13 - 28 X) and
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affinity chromatographyon a heparin-Sepharosecolumn (16 x 640 mm) with aNaC1 gradient elution (the activity eluted with NaCl at 0.95 - 1.25 M). These procedures yielded about 25 units (9000 enzymologicalunits) of a crude antithrombin III preparation (140 ml) with a specific activity of 1.8 units (645 i enzymologicalunits)/A280 (Table 1). Isoelectricfocusing. This was carried out according to Vesterverg and Svensson (16) for further purificationof rat antithrombinIII. Electrofocusing was performed in a LKR 8101 column (110 ml, LRB Co., Stockholm)with a mixture of carrier ampholytes (LRR ampholine),pH 4-6 (4 volumes) and pH 3.5-10 (1voL) at a final concentrationof 1 X. After electrophoresisat 0-1°C for 24 hr at 250 volts, 2 hr at 500 volts and 48 hr at 700 volts, the column content was drained in fractions of 1.8 ml each at a flow rate of 1.8 ml/min. Polyacrylamidegel disc electrophoresis. This was carried out, according to Davis (17). for final nurificationof rat antithrombinIII and for determination of molecular weight with 5 or 7.5 % gel (5 mm diameter) in the presence or absence of 1 % sodium dodecylsulfateand 2-mercaptoethanolat 6 or 3 mA/gel for 1.5 hr. Preparation of antiserum. As described in Results, antithrombinIII was electrophoreticallyseparated from some contaminantproteins in polyacrylamide gels, and the gel slices from 6 runs containingantithrombinIII was homogenized with 1 ml of complete Freund's adjuvant. Two Japanese white rabbits were immunizedwith the homogenate (about 0.39 mg protein of antithrombinIII in 1 ml per a rabbit) 3 times at 2 week intervals.The rabbits were bled 8 days after the last injection of the antigen, and the serum was stored frozen after inactivationat 56'C for 30 min. Immunologicaltests. Double diffusionwas carried out according to Ouchterlony (18) on agar plates with 1.5 % Agar Noble (Difco Labs., Michigan) in barbital buffer (pH 8.6, I * 0.05) at 37'C for 48 hr. Rocket immunoelectrophoresls was performed for assay of antithrombinIII-relatedantigen according to Laurel1 (19) with a 1 X agarose plate of 90 x 90 mm (14 ml) containing0.17 ml of anti-rat antithromblnIII rabbit serum at 180 volts (30 mA/2 plates) for 2.5 hr. Barbital buffer, pH 8.6 and I 0.02, was used. Sample volume was 5 ~1. For differentialassay of the free and complexed forms of antithrombinIII, according to Andersson et al. (20), samples (10 fl each) were first electrophoresed on a 1 X agarose plate of 74 x 110 mm (14 ml) containing 0.3 ml of heparin-la (1000 units/ml) at 110 volts for 1.5 hr. The plate was cut longitudinally into strips with 20 mm wide to separate each run. The gel strip was transferredonto one end of a glass plate of 90 x 90 mm. The remaining part of the glass plate was covered with 10 ml of 1 % agarose containing0.12 ml of the antiserum. Then, electrophoreslswas carried out in the second dimension at 40 volts for about 16 hr. Induction of vein thrombosis in rats. According to the method reported elsewhere (21), experimentalvenous thrombosiswas produced in rats by insertion of a small stainless steel wire coil (a dental-pastecarrier, 20 nanlong) into the inferior vena cava just below the left renal vein branching. The thrombus was formed and grew up around the wire and onto the vein wall with a maximum size of about 1 mg protein 2-3 days after the wire coil insertion(U). Control rats received similar laparotomywithout wire coil insertion.
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792
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FEWLTS Purificationof Rat AntlthromblnIII The crude preparationof rat antlthrombinIII (140 ml) obtained by the heparln-Sepharosechromatographywas dialyzed against 2.5 1 of 0.05 M Tris-HCl (pH 7.5) and concentratedto 24 ml by the use of a collodlon bag (Sartorlus Membrane Co., Goettlngen,Germany). The concentratedsolution was subjected to isoelectricfocusing at pH range of 3.5 - 10. Fig. 1 Illustratesthe lsoelectric fractionationprofile with a main peak of the antlthromblnactivity in the fraction Nos. 24 - 31 (PI, 4.85). The active fractionswere combined, dialyzed against 0.05 M Trls-HCI containing0.1 M NaCl (pH 7.5) and concentrated to 18.4 ml.
I
I
1.6 -
!
1.2 -
t i7
0.8 -
c) 1 2 0" 5 2 *
0.4
FIG. 1 Isoelectricfractionationof rat antlthromblnIII obtained by heparln-Sepharosechromatography.Column size, 110 ml (LKB8101); carrier ampholyte,a mixture of 1 % LKB amphollne,pH 4-6 and pH 3.5-10 at 4:l; fraction size, 1.8 ml/tube.
Polyacrylamldegel disc electrophoreslsrevealed 2 minor contaminating proteins in this preparation. To remove these contsmlnants,the concentrated fraction was subjected to polyacrylamldegel disc electrophoresls(130 pg protein/gel). The gels, marked with a stained marker gel, were cut and antlthrombln III was extractedwith the Trls-NaCl buffer or, for immunization, with complete Preund's adjuvant (6 gels/ml). The result of purificationis shown in Table 1. Molecular weight of the purified rat antlthromblnIII was estimated to be about 6.4 x lo4 daltons by SDS-polyacrylamldegel disc electrophoreslsusing ovalbumln (4.5 x 104) and bovine serum albumin (6.7 x 104) as references.
RAT ANTITHROMBIN
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TABLE1 Summary of Purificationof AntithrombinIII from Rat Plasma Volume
Total protein
Step
Antithrorrbin activity Total Specific (Unit) (Unit/A280) (UnithId
(ml)
(*280)
Defibrination
340
17340
22720
340 (113600)
0.0196 (6.55)
PEGprecipitation
188
8573
-
254 (79000)
0.0296 (9.21)
-
25.4 (9130)
1.80 (645)
21.7 (7250)
1.99 (718)
k3)
HeparinSepharose
24
14.2
Isoelectric focusing
18.4
10.9 16.1
0.015 (5.0)
1.35 (450)
Polyacrylamide gel electrophoresis
(500)
Figures in the parentheses are enzymologicalunits of the antithrombinactivity.
FIG. Control 2
Heparin cofactor nature of the purified rat antithrombinIII. The incubationmixture contained 100 pl of thrombin (20 NIH units/ ml), 50 ~1 of the purified antithrombin III (64 pg/ml> and 50 ~1 of the Tris-NaCl buffer (a, 0) or heparin (0.4 unit/ml;?? , 0). Control runs contained the buffer solution in place of antithrombinIII. After incubationat 37'C for the indicated period, the remaining thrombln activity was determinedby the fibrinogen clotting assay.
80
ii 60
+ Antithrombin III
O1
’
0
8
I
I
10 20 30 Incubation time (min)
2
1
40
Rat antithrombinIII did not dissociate in reduced SDS-polyacrylamidegel electrophoresis,indicating a single polypeptide chain. As shown in Fig. 2, the purified rat antithrombinIII inactivatedthromhin progressivelyby itself and did so instantaneouslyin the presence of heparin, indicatingheparin cofactor activity of the purified protein.
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FIG. 3 IPnaunodouble diffusion of the anti-rat antithrombinIII rabbit serum against plasmas of various species including rat (a, e), mouse (b), rabbit (cl, dog (d), man (f), monkey (8) and guinea pig (h).
FIG. 4 Rocket ismtunoelectrophoresis of normal rat plasma for antithrombinIII. Normal rat plasma, from right to left, undiluted, diluted to 75, 50 and 25 2 with normal rabbit plasma. Purified rat antithrdiabin III, from right to left, 200, 150, 100, 50 and 20 &ml Tris-NaCl.The sample volume was 5 pl each.
ImmunologicalExaminationof Rat AntithrombinIII Antiserumwas produced in rabbits against the purified rat.antithrombin III. The antiserum gave a single precipitin line against normal rat plasma on immunodoublediffusion and immunoelectrophoresis. As shown in Fig. 3, antirat antithrombinIII rabbit serum was found to cross-reactwith a plasma component,probably antithrombinIII, in plasmas of mice, humans and, to a minor extent, monkeys (Macacusrhesus), but not with guinea pig and dog plasmas. Determinationof AntithrombinIII in Rat Plasma Changes in the plasma level of antithrombinIII-relatedantigen and the heparin cofactor activity were studied during thrombus formation in the vein thrombosismodel in rats. Plasma samples were obtained from the jugular vein of the rats before and 6, 24, 48 and 72 hr after the wire coil insertion into the inferior vena cava. Total antithrombinIII was assayed by the rocket immunoelectrophoresisshown in Fig. 4. Plasma samples from the sham-operated control rats were also assayed. Normal plasma level of antithrombinIII was found to be around 200 &ml. As shown in Table 2, total amount of anti-
Two dimensional electrophoresisof rat plasma and serum. The first run on a heparin-agaroseplate and the second run on an agarose plate containing anti-rat antithrombinIII rabbit serum. A: Normal rat plasma (complexedform, 2 xl),B: pooled thrombosed rat plasma obtained 48 hr after the wire coil insertion (complexedform, 9 X), C: pooled shamoperated rat plasma obtained 48 hr after operation (complexedform, 6 X), and D: normal rat serum (complexedform, 61 X).
FIG. 5
u
-4 W
RAT ANTITHROMBIN
‘796
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thrombin III-relatedantigen was considerablyincreased after the wire coil insertion,while a similar increase in antithrombinIII-relatedantigen was also observedwith the sham-operatedrats. The heparin cofactor activity behaved in parallel with the level of aatithrombinIII-relatedantigen. But some discrepancywas observed in the plasma samples at 24 hr: increase in the cofactor activity was less than that in antithrombinIII-relatedantigen. This was more clearly shown by decrease in the ratio of the heparln cofactor activity to antithrombinIII-relatedantigen in the 24 hr-plasma samples from the thrombosedrats (Table 2). Differentialanalysis of antithrombinIII-relatedantigen in the pooled plasmas of each group by the two dimensionalelectrophoresisindicated that most of the increment of antithromblnIII-relatedantigen was due to increase in free antithrombinIII regardlessof accompanyingthrombus formation (Fig.51, and that there was a minor increase in the complexed form of antithrombinIII in the thromboeedrats when compared with the sham-operatedrats (Fig. 5). The ratios of the complexed form to total antithrombinIII were 2-3 X in normal plasmas, 6 X (6 hr) and 9 X (24, 48 and 72 hr after operation) in plasmas from the thrombosedrats, 3 X (6 hr) and 6 X (48 hr) in the sham-operatedrat plaaza55, and 60 X in normal rat sera (Fig. 5).
TABLE 2 Changes in Plasma AntithrombinIII Levels during Thrombus Formation in Eats
Before
Time (hr) after operation 6 48 24
72
AT-III antigen (%) Control Thrombosed
104 ??4 103 f 4
110 * 3* 105 * 4
134 f 3** 151 * 9#
142 i 41 150 f 81
151 f 41 143 * 5fi
AT-III activity (%) Control Thrombosed
100 i-3 102 ??5
103 i 2 100 * 4
142 f 5# 147 f 6**
Ratio (activity/antigen) Control 0.97 * 0.03 0.94 ??0.03 0.88 * 0.02 1.01 f 0.03 1.04 f 0.03 Thrombosed 1.00 f 0.03 0.96 f 0.03 0.80 f 0.0W0.99 i 0.07 1.12 * 0.07 AntithrombinIII (AT-III)related antigen and AT-III activity (heparincofactor activity)were assayed after operation and presented as % of normal. The ratio of heparin cofactor activity to AT-III antigen was calculated. * ~(0.05, ** p
DISCUSSION In the present study, an antithrombincomponentwas highly purified from rat plasma by the proceduresanalogous to those used for human antithrombinIII. The purified antithrombincomponent satisfied the definitionof antithrombin
v01.17,~0.6
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797
III (heparin cofactor),"progressiveneutralizationof thrombin by itself and instantaneousinactivationof the enzyme in the presence of heparin" (22). There are some molecular similaritiesbetween rat antithrombinIII and the counterpartsof other animal species. Molecular weight of rat antithrombin III, 6.4 x 104, was similar to that reported with human (12, 231, rabbits (9), dogs (7) and bovine (24), although some higher and lower values were reported (5, 25). The isoelectricpoint of rat antithrombinIII, 4.85, was close to p1 of human antithrombinIII, 5.11 (23). Immunologicalcross-reactionof antithrombin III was observed between rats and other animals ineludingmice,humans and monkeys, but not guinea pigs and dogs. Normal plasma level of antithrombin III-relatedantigen in rats was around 200 pg/ml in agreementwith thereported value (220 i 10 )Ig/ml,Ref. 11) and close to the correspondingnormal plasma level In humans (199 - 191 pg/ml, Ref. 26). Many clinical studies have suggested a close relationshipbetween lowered plasma antithrombinIII levels and hypercoagulablestates or high incidence of thromboembolicdisorders (1 - 4). On the other hand, experimentallyinduced thrombophlebitis(6) and disseminatedintravascularcoagulation (8) in dogs were reported to be not associatedwith any significantchanges in the plasma level and the turnover rate of antithrombinIII, even inthe casesaccompanying significantconsumptivedecrease in platelets and plasma fibrinogen (8). They did not refer to the plasma level of complexed antithrombinIII. The present study, however, indicated a remarkable increase in antithrombin III-relatedantigen as well as in the heparin cofactor activity in plasmas from the rats with venous thrombosis.The similar changes were also observed in the sham-operatedcontrol rats. Most of the increased antithrombinIII was in its free form and a minor increase was detected in the complexed form which was thought to be composed of some activated coagulationfactors and antithrombin III. These findings suggest that such elevation of the plasma antithrombin III levels may be a response to local inflammationdue to operation injury, as reported by Koj and his colleagueswith the turpentine-induced inflammationmodels (lO,.ll),and that the elevationwas too great to detect any definite changes in antithromblnIII levels related to thrombus formation. Simultaneousincrease in plasma fibrinogenwas observed in both the thrombosed and sham-operatedrats (21), also suggestingsome inflammatoryreaction. However, a significantdecrease was observed in the ratio of the heparin cofactor activity to antithromblnIII-relatedantigen in the thrombosedrats 24 hr after the operation, suggesting some consumptionof functionalantithrombin III associatedwith thrombus formation.This may relate to a slight but larger increase in the complexed form of antithrombinIII-relatedantigen in the thrombosedrats than in the control rats (Fig. 5). In this experimental vein thrombosismodel, thrombus formation actively progreesed during 2 days after the wire coil insertionwith prolongationof partial thromboplastintime and decrease in the circulatingplatelet count, suggestingactivation and consumptionof coagulationfactors (21).
REFERENCES 1. EGEBERG, 0. Inherited antithrombindeficiency causing thrombophilia. Thrombos. Diathes. Haemorrh. 13, 516-529, 1965. 2. MARCINIAK, E., ERLEY, C.H. and DE SIMONE, P.A. Familial thrombosisdue to antithrombinIII deficiency.Blood, 43, 219-231, 1974.
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3. FAGERHOL,M.K. and ABILDGAARD,U. Immunologicalstudies on human antithrombin III - Influenceof age, sex and use of oral contraceptiveson serum concentration.Stand. J. Haemat. 7, 10-17, 1970. 4. SAGAR, S., STAMATAKIS,J.D., THOMAS, D.P. and KAKKAR, V.V. Oral contraceptives,antithrombinIII activity, and postoperativedeep-vein thrombosis Lancet, i, 509-511, 1976. 5. DAMUS, P.S. and WALLACE, G.A. Purificationof canine antithrombinIIIheparin cofactor using affinity chromatography.Biochem. Biophys. Res. Conuz.61, 1147-1153,1974. 6. KOBAYASHI,N. and TAKEDA, Y. Studies of the effects of estradiol,progesterone, cortisol, thrombophlebitisand typhoid vaccine on synthesis and catabolismof antithrombinIII in the dog. Thrombos. Haemoetas. 37, 111-122, 1977.. 7. KOBAYASHI,N., TANAKA, H., UENO, T., GONMORI, H. and MAEKAWA, T. Studies on antithrombinIII. Purificationof canine antithrombinIII and its properties.Blood and Vessel, 9, 139-144, 1978. 8. TANAKA, H., UENG, T., GONMORI, H., TAKEUCHI, T., TANAKA, S., ARAI, H., KOBAYASHI,N. and MAEKAWA, T. Studies on antithrombinIII (AT III). IV. Effect of thrombin administrationon metabolism of canine AT III. Blood and Vessel, 10, 59-62, 1979. 9. YIN, E.T., WESSLER, S. and STOLL, P.J. Rabbit plasma inhibitorof the activated species of blood coagulationfactor X. Purificationand some properties.J. Biol. Chem. 246, 3694-3702,1971. 10. KOJ, A. and REGOECZI, E. Effect of experimentalinflammationon the synthesisand distributionof antithrombinIII andtil-antitrypsinin rabbits. Br. J. exp. Path. 59, 473-481, 1978. 11. KOJ, A., REGOECZI, E., TOEWS, C.J., LEVEILLE, R. and GAULDIE, J. Synthesis of antithrombinIII and alpha-1-antitrypsin by the perfused rat liver. Biochim. Biophys. Acta, 539, 496-504, 1978. 12. MILLER-ANDERSSON,M., BORG, H. and ANDBRSSON, L.-O. Purificationof antithrombin III by affinity chromatography.ThrombosisRes. 5, 439-452, 1974. 13. LGWRY, O.H., ROSEBROUGH,N.J., FARR, A.L. and RANDALL, R.J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275, 1951. 14. CUATRECASAS,P. Protein purificationby affinity chromatographyderivatizationsof agarose and polyacrylamidebeads. J. Biol. Chem. 245, 3059-3065,1970. 15. THALER, E. and SCHMER, G. A simple two-step isolationprocedure for human and bovine antithrombinII/III (heparincofactor):a comparisonof two methods. Brit. J. Haemat. 31, 233-243, 1975. 16. VESTERBERG,0. and SVENSSON,H. Isoelectricfractionations,analysis, and characterizationof ampholytesin neutral pH gradients.IV. Resolving power in connectionwith separationof myoglobins.Acta Chem. Stand. 20, 820-834, 1966.
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17. DAVIS, B.J. Disc electrophoresis- II. Method and applicationto human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427, 1964. 18. OIJCHTERLONY, 0. Diffusion in gel methods for immunologicalanalysis. Progr. Allergy, 5, l-78, 1958. 19. LAURKLL, C.-B. Quantitativeestimationof proteins by electrophoresisin agarose gel containingantibodies.Analytical Biochem. 15, 45-52, 1966. 20. ANDERSSON, L.-O., ENGMAN, L. and HENNINGSSON,E. Crossed immunoelectrophoresis as applied to studies on complex formation. The binding of heparin to antithrombinIII and the antithrombinIII-thrombincomplex. J. Immunol. Methods, 14, 271-281, 1977 21. KUMADA, T., ISHIHARA,M., OGAWA, H. and ABIKO, Y. Experimentalmodel of venous thrombosis in rats and effect of some agents. ThrombosisRes. in press. 22. YIN, E.T., WESSLKR, S. and STOLL, P.J. Identity of plasma-activated factor X inhibitorwith antithrombinIII and heparin cofactor. J. Biol. w. 246, 3712-3719, 1971. .
23. ROSENBERG, R.D. and DAMNS, P.S. The purificationand mechanism of action of human antithrombin-heparincofactor. J. Biol. Chem. 248, 6490-6505,1973. 24. ABILDGAARD,U. Binding of thrombin to antithrombinIII. Scand. J. Clin. Lab. Invest. 24, 23-27, 1969. 25. KURACHI, K., SCHMBR, G., HERMODSON,M.A., TELLER, D.C. and DAVIE, E.W. Characterizationof human, bovine and horse antithrombinIII. Biochemistry,15, 368-373, 1976. 26: CHAN, V., CHAN, T.K., WONG, V., TSO, S.C. and TODD, D. The determination of antithrombinIII by radioimmunoassayand its clinical application. Brit. J. Haemat. 41,.563-572,1979.