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Effect of thrombin and endotoxin on the in vivo metabolism of antithrombin III (at III) in dogs
THROMBOSIS
RESEARCH 40; 291-306, 1985 Printed in the USA. (c) 1985 Pergamon Press Ltd. All rights reserved.
0049-3848/85 $3.00 t .OO Copyright
%FFECT OF THROMBIN AND ENDOTOXIN ON THE -IN VIVO METABOLISM OF ANTITHROMBIN III (AT III) IN DOGS
H.Tanaka, N.Kobayashi and T.Maekawa The Third Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma, 371, Japan.
(Received 24.5.1985; Accepted in revised form 1.8.1985 by Editor M. Matsuda)
AHSTRACT Effect of thrombin and endotoxin on the metabolism of 1-125labelled canine AT III was studied in mongrel dogs. Under control condition, mean total amount of intravascular AT III with standard deviation was 23.4 t 2.4 mg/kg, plasma half life of i.v. injected I-125-AT III was 1.7 t 0.2 days, and the fractional catabolic flux ( j3x ) was 16.3 t 1.6 mg/kg/day. The total amount of intra- and extra-vascular AT III was 36.0 + 0.34 mglkg. Neither a 3 hour infusion of a small dose (30 units/kg/hr) of thrombin nor i.v. injection of a large amount of thrombin (5,000 - 15,000 units/day) with heparin significantly affected AT III metabolism except for a transient decrease in AT III concentration in the latter case, although decrease in plasma fibrinogen concentration and platelet count was observed in both cases. Two injections with 200 pg/kg of endotoxin resulted in an evident acceleration of AT III metabolism with significant decrease in the plasma AT III, fibrinogen concentrations and platelet count. More marked changes in AT III metabolism were induced by a single infusion with 1 nrg/kgof endotoxin. Changes in hemostatic system coincided with those observed in DIC.
INTRODUCTION
Plasma antithrombin TJI (AT III) is an important.inhibitor in blood coagulation, inhibiting not only clot promoting serine proteases such as thrombin and factor Xa ( 1,2 ) but also plasmin ( 3 ). Reduction in plasma AT 114 activity has often been observed during the course of tiisser 'l$at~IS.ntrnv:?rrl;!,lr roagul:jtior(PTC)( 4 - 15 ), although the -.. ____ _.__-___ KEY VORDS: Antithrombin IIT ( AT III ), Metabolism of AT TV, 1-125-antithrombin TIT, Thrombin, Endotoxin, DIC. 291
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underlying mechanism of the reduction has not been resolved. Studies were therefore designed to investigate the metabolism of AT III in dogs with DIC experimentally induced by administration of either bovine thrombin or endotoxin from Escherichia coli using I-125-labelled canine AT III as a tracer.
MATERIALS AND METHODS
Purification of canine antithrombin III (AT III). Canine AT III was purified according to the method described by Damus and Wallace (16) with a minor modification using heparin-Sepharose affinity chromatography. Nine volumes of blood were withdrawn directly from the heart of a healthy adult canine to a centrifuge tube containing 1 volume of ACD solution. Fresh canine plasma separated by centrifugation at 4' C was defibrinated by heating to 54'C for 3 min. After centrifugation at 3000 rpm for 20 min at 4' C, 100 ml of the defibrinated plasma was applied on a heparin-Sepharose 4B ( Pharmacia, Uppsala, Sweden ) column, 2.5 x 45 cm in size, and washed with 100 ml of 0.145 M NaCl-0.01 M Tris-HCl buffer ( pH 7.5 > followed by 60 ml of 0.3 M NaCl-0.01 M Tris-HCl buffer ( pH 7.5 ). The protein was then eluted with a linear gradient of 500 ml of 0.3 M NaCl-0.01 M Tris-HCl buffer ( pH 7.5 ) in a mixing chamber and 500 ml of 1.0 M NaCl-0.01 M TrisHCl buffer( pH 7.5 ) in a reservoir at 20' C. The flow rate was 30 ml/hr and fraction volume was 5 ml/fraction. The active fraction was obtained as a small peak at an optical density ( OD ) of 280 nm after a large amount of protein was eluted. About 80 ml of the active fraction was collected into a dialyzing tube and then concentrated to 12-15 ml by drying under cold air. This concentrated fraction was dialyzed against 0.05 M phosphate buffer ( pH 8.0 ) overnight at 4'C. The obtained AT III fraction contained a trace amount of impurity according to the 10 % analytical polyacrylamide gel electrophoresis ( PAGE ) method of Davis ( 17 ); therefore, this active fraction was further purified using preparative PAGE. Gels of 10 % polyacrylamide 0.8 x 5 cm in size were prepared in the following buffer: 70.7 mg ammonium persulfate, 0.726 % Tris, 29 mg TEMED ( N,N,N',N'tetramethylene diamine ) and 0.263 % methylene bis acrylamide in 100 ml of distilled water ( pH 8.6 ). The chamber buffer consisted of 0.6 g Tris and 2.88 g glycine in 100 ml of H20 ( pH 8.3 ). Electrophoresis usually ran for about five hours at 5 mA per gel. After electrophoresis, the gels were cut into 10 pieces ( 5 mm wide ) and the one containing AT III, which was recognized by the reference gel stained with 1 g% amide black 10B ( Merck, Darmstadt, Germany ) was crushed into fine pieces by passing it through a 10 ml plastic syringe. The crushed gels were mixed with 5 ml of 0.05 M phosphate buffer ( pH 8.0 ) and kept at 4'C overnight. The suspension was finally centrifuged at 3000 rpm for 3 min at 4'C, then the supernatant contgining purified AT III was divided into 1 ml aliquots and stored at - 20 C. AS shown in Fig.1, the purified AT III showed a single band on 10 % analytical PAGE. Measurement of antithrombin and heparin-cofactor activity. AT III activity was determined bv the method of Rosenberg and Damus and heuarin cofactor activity was determined by the same methid, except for the-use of 0.05 M phosphate buffer ( pH 8.0 ) and bovine thrombin in the place of 150 mM of NaCl in 10 mM of Tris-HCl ( pH 7.5 ) buffer and human thrombin ( 18 ). Specific activity of the purified AT III preparation was 400 units/mg when the activity in 1 ml of defibrinated healthy canine plasma was arbitrarily assigned as 100 units.
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FIGURE 1.
a
b
c
d,
FIG.1. Ten percent polyacrylamide gel electrophoresis of purified canine antithrombin III. About (a) ZOOpg, (b) 100 pg, (c) 5Opg and (d) 25yg were analyzed. Purified AT III consisted of a single component.
Production of the antibody against AT III. About 200 ug of purified canine AT III was mixed with Freund's complete adjuvant ( Difco Laboratories, Detroit, MI, USA ) and injected intramuscularly into each of two rabbits once a week for 4 weeks. The blood was harvested within one week after the last injection and thus obtained antisera against AT III was divided into aliquots of 1 ml, then stored at -2O'C. Immunoelectrophoresis of purified AT III with the antiserum revealed a single precipitin line ( Fig. 2 ). Amount of AT III in the test Determination of amount of AT III. samples was determined by single radial immunodiffusion ( 19 ). One g% of agarose plate containing-4 % of anti-AT III antiserum was prepared. Buffer containing 0.55 g barbital, 3.50 g sodium barbital, 0.51 g calcium lactate and 0.1 g sodium azide per liter of distilled water ( pH8.6 ) was used as gel buffer. Next, 3 ul of the test samples was applied to each of twelve wells, 2 mm in diameter, which were filled with the agarose plate. After 48 hours, the diameter of the precipitate ring was measured and an AT IIJ concentration obtained from the standard curve. Purified AT III solution of 160 mg/dl of concentration was used as standard. Determination of extinction coefficient and molecular weight of purified AT III. The extinction coefficient was determined by measuring the weight of well-dried lyophilized materials which were dialyzed against distilled water, and by measuring their optical density dissolved in 0.05 M phosphate buffer ( pH 8.0 ) at 280 nm using a 1 cm wide quartz cuvette; this latter was 6.2 for 10 mg of the purified AT III per ml of solution. The molecular weight of the purified AT III determined by the method of Andrews ( 20 ) using a Sephadex G-200 column 2.5 x 100 cm in size was 70,000 daltons.
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FIG.2. Immunoelectrophoresis of purified canine AT III. The central trough was filled with anti-AT III antiserum and the upper well and lower well were filled with purified AT III and canine plasma, respectively. The canine AT III showed a single precipitin line against anti-AT III antiserum.
Labelling of AT III with I-125. AT III was labelled with I-125 by the iodine monochloride method of McFarlane ( 21 ). Free I-125 was removed by passing it through a Sephadex G-25 column 1.5 x 20 cm in size equilibrated with 0.05 M phosphate buffer ( pH 8.0 >. The free I-125 remaining in the preparation was usually less than 1 % of total radioactivity. Specific activity of I-125-AT III was about 1 mCi/mg. 1-125AT III was further tested with respect to AT III activity, heparin cofactor activity and its electrophoretic mobility in 10 % polyacrylamide gels. These results were compared with those of unlabelled AT III and no appreciable differences were found. The prepared I-125-AT III was injected intravascularly into a puppy to remove the denatured molecules, and plasma containing undenatured I-125-AT III was harvested about 30 min after the initial injection. This was then used for studies of the AT III metabolism. A comparison of the metabolism of screened and unscreened I-125-AT III showed no significant differences between them ( data not shown ). Studies on the metabolism of AT III in healthy dogs. Mongrel dogs with 7 to 8 kg of body weight were used for studies on metabolism of AT III. After i.v. injection with 10 to 15 pCi of I-125-AT III, blood samples were obtained serially for 5 days using ethylene diamine tetraacetic acid ( EDTA > or 3.8 % sodium citrate solution as anticoagulant. The radioactivity of 1 ml of each plasma separated from the blood samples was measured, the data were depicted on a semilogarithmic scale, and the disappearance curve of the radioactivity from the plasma was analyzed. As the plasma disappearance of radioiodine was described by a biexponential curve within an observation period of 120 hours, the data were analyzed by the two compartment model reported by Atencio et a1.(22).
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METABOLISM ~F~FPfMINE AT III
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Studies of effect of thrombin and endotoxin on I-125-AT III metabolism. On the second day after I-125-AT III injection, ( 1 ) bovine thrombin solution ( 2 u/ml of normal saline ), ( Parke Davis, Lot No. 656, USA ) was i.v. infused at a rate of 30 u/kg/hour or 5,000 units - 15,000 units of thrombin with 5,000 units- 15,000 units of heparin was i.v. injected at one time: ( 2 ) or two hundred rg/kg of endotoxin ( E.coli, 026:B6, Sigma Chemicals, St.Louis, MO, USA ) were i.v. injected into dogs twice with an interval of 24 hours, ( 3 ) or 1 mg/kg of endotoxin was infused into dogs for 3 hours. Determination of various clotting factors. For the determination of activities of various clotting factors, conventional methods using deficient plasma ( Dade, Miami, FL, USA ) as a substrate were adopted.
RESULTS
Metabolism of AT III in healthy dogs under the control condition. In vivo behavior of i.v. administered I-125-AT III in control dogs is shown -in Fig.3. The plasma clearance curve of the i.v. injected I-125-AT III is closely expressed by two exponential functions: X=0.52e-0'40gt + 0.48e-3'02t. Since the plasma AT III packed cell volume were maintained relatively constant the dogs were in a steady state with respect to AT III tracer data calculated from the analysis of the plasma are shown in Table 1 a,b,c.
concentration and during the study, metabolism. The disappearance curve
FIGURE 3.
mg/ 1 OOml Plasma AT
III
on
concentratl
___..--r_-.____.....~____________~__---__~.__--r---------...~
40
-30
byr 0
1
2
3
4
f 5
FIG.3. Plasma clearance of I-125-canine AT III under control condition. The plasma behavior of injected I-125-AT III was closely expressed by two -0.409t exponential functions: X= 0.52e t 0.48e-3'02t'
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IN DIC
TABLE la,b,c. Metabolic Parameters of AT III in Normal Control Dogs. TABLE la.
Dog No.
Weight Kg
Ht %
Plasma Vol.
Plasma AT III
Plasma AT III(x)
mg/lOOml
ml/kg
mg/kg
1 2 3 4 5
10.5 12.8 10.5 9.5 10.5
36.0 35.2 37.0 34.0 37.0
58.7 64.5 57.4 54.6 51.8
42.6 37.1 46.3 35.9 42.5
25.0 23.9 26.6 19.6 22.0
Mean SD
10.8 1.1
35.8 1.1
57.4 4.3
40.8 3.8
23.4 2.4
TABLE lb. -at t l/2 +C embt x=C e j, j jl days /day /day /day ' a2 No. c2 b c1 ~-__-------___-------___-_------__-----~~~---------_____-_---____
Dog
1 2 3 4 5
.48 .54 .57 .54 .47
.408 .433 .347 .433 .425
.52 .46 .43 .46 .53
3.01 2.77 2.48 3.85 2.97
1.7 1.6 2.0 1.6 1.6
1.02 .80 .71 1.27 1.00
Mean SD
.52 .04
.409 .03
.48 .04
3.02 .46
1.7 .2
.96 .19
1.66 1.70 1.56 2.28 1.62
.74 .71 .55 .73 .78
1.76 .70 .26 .08
TABLE lc. ---__------------------_-----------_-----------------______----__ Dog
jlx
j,x
Y
Y/x
x+y
No. mg/kg/day mg/kg mg/kg ~~~-----~~-~~----~--__^_________________-~~----_-___~-----------.62 40.4 18.5 15.4 1 25.5 .47 35.2 16.9 11.3 2 19.2 2 5
24.9 18.9 22.0
14.4 14.6 17.2
12.1 11.0 13.5
.46 .56 .61
38.7 30.6 35.5
Mean SD
22.1 2.8
16.3 1.6
12.7 1.6
.54 .07
36.0 .34
x: plasma AT III, mg per kg. t l/2: the half-life of plasma I-125-AT III. y: extravascular AT III, mg per kg. x+y: total amount of AT III, mg per kg. jl: fractional transcapillary transfer rate to extravascular space, per day. j : fractional return rate of y to plasma, per day. j,: fractional cataboI*ic rate, per day. jlx: transcapillary flux, mg/kg/day. j3x: catabolic flux, mg/kg/day.
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The radioactive fraction of the plasma obtained either 10 min or 2 days after I-125-AT III injection showed a single peak identical to the location of native AT III in Sephadex G-200 column chromatography ( Fig. 4 ).
FIG. 4. Sephadex G-200 chromatogram of defibrinated plasma obtained at 10 min and 2 days after the injection of 1-125AT III. The injected I-125-AT III was not split into several radioactive fragments -in vivo and the location of I-125-AT III was identical with that of native AT III determined by immunological method.
01,
20
40
00
’ ’2
Fraction ~0.
Effect of thrombin administration on the metabolism of AT III. To study the effects of thrombin on the metabolism of AT III, 30 units/kg/hr of bovine thrombin dissolved in physiological saline at a concentration of two units/ml were intravenously infused to each of 5 dogs on the second day after the intravenous injection of I-125-AT III. Thrombin infusion on the 2nd day of I-125-AT III injection resulted in a reduction of the plasma fibrinogen concentration and the platelet count to 60 % of the initial level, although it significantly affected neither the disappearance curve of I-125-AT III i.v. injected 1 day before nor the plasma AT III concentration ( Fig. 5a >. The effect of a large amount of thrombin administration with heparin on canine AT III metabolism was then studied. From 5,000 to 15,000 units of thrombin were intravenously injected with 5,000 to 15,000 units of heparin one day after the injection of I-125-AT III into dogs. Although the plasma fibrinogen concentration was not changed significantly, after the injection of such a large amount of thrombin with heparin the plasma radioactivity of I-125-AT III and the concentration of AT III were decreased to 70 % and 60 % of the initial concentration,respectively,within 6 hours. It was, however, noted in these dogs that the disappearance rate of the injected I125-AT III was not changed even after i.v. injection of a large amount of thrombin with heparin ( Fig. 5b ). Two Effect of endotoxin administration on the metabolism of AT III. hundredpg/kg of endotoxin (E.coli 026:B6) was injected intravenously
METABOLISM OF CANINE AT III
298
IN DIC
a
lhrombln
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b
X)u/hg/hr,3h,. hcprrm
50’ Plasma
AT III concentration
~q&...*_.__..~....*.....* 30. Plasma LOO.
3oocy
500045ooOU
. . . .. -.
flbiinoeon concentration
:
200. 100 0.
Days
t 0
1
2
3
P
1
‘
I
5
E
lcw
, 0
I 1
1 2
DIYS i 3
1 4
1 5
FIG. 5a. -In vivo behavior of i.v. administered I-125-AT III under the influence of a small dose of bovine thrombin. Although the plasma fibrinogen concentration was decreased, the plasma clearance of I-125-AT III was not affected. FIG. 5b. -In vivo behavior of i.v. administered I-125-AT III under the influence of a large dose of bovine thrombin with simultaneous heparin. In this case also the plasma clearance of I-125-AT III was not affected.
into the right jugular vein of five dogs both one and two days after 1-125AT III injection. The second injection resulted in a reduction of plasma AT III concentration, plasma fibrinogen concentration and platelet count to 80 %, 60 % and 60 X of the initial levels, respectively. Plasma half-life of I-125-AT III was shortened to 1.4 days ( control value: 1.7 + 0.2 days) with significant acceleration of the metabolism of I-125-AT III. The catabolic rate, j , increased to 1.25/day whereas normal control value was 0.70 + O.O8/day. ?t is suggested from these results that the decrease in plasma AT III concentration resulted mainly from the acceleration of AT III catabolism ( Fig. 6a ).
026:B6) was infused intravenously for three hours through a catheter which was indwelled in the right jugular vein of each of 5 dogs. The plasma AT III concentration was decreased to 70 % of the initial level within 3 to 24 hours after the start of endotoxin infusion, and then was gradually restored to the initial level within 48 to 72 hours. The plasma fibrinogen concentration and the platelet count were also decreased to 70 % and 20 %
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of the initial levels, respectively, within three hours after completion of the endotoxin infusion. The disappearance of I-125-AT III from the plasma was accelerated and the mean plasma half-life of I-125-AT III was shortened to 1.4 days ( Fig. 6b >.
b
a
‘:P
Endotoxln
200~1g/kg
Endotorln
I.v. Injoctlon
t mg/kg
1.~. Infualon
to,
.6 .4 s z2 0’ Y 4 P
:A5 .05 .04
60
PIWM
50 Plasma 50 50
AT III
conc.ntratlon
1g/lOOml
g
40
0
30
___.____.. 4 ........__..____. _. ./.+-.. +
z 20 \ e 600
40’ 30
AT Ill concentration
. .._ __
.
Plwma
L flbrlnogen
f
f
i
j
concentration
5400
20 300 Phrmr 000
tIbr1nog.n
concentration 2004
yap
$/h.‘-i_.
-
~1
Platblet
B
500
count
400
71
1
2
3
4
5
i
i
2
j
FIG. 6a. -In vivo behavior of i.v. administered I-125-AT III under the influence of endotoxin. The metabolism of I-125-AT III was accelerated and the plasma half life of I-125-AT III was shortened to 1.4 days. FIG. 6b. -In vivo behavior of i.v. administered I-125-AT III under the influence of a large single dose of endotoxin. The plasma clearance of I-125-AT III was accelerated and the half life of AT III was shortened to 1.4 days. Changes in the plasma AT III concentration, plasma fibrinogen concentration, platelet count, fibrin/fibrinogen degradation products (FDP) levels, prothrombin time, activated partial thromboplastin time and plasma coagulation factors, such as factors II, V, VII, VIII, IX and X, were measured serially after i.v. infusion with 1 mg/kg of endotoxin. Plasma concentrations of AT III and fibrinogen and platelet count were decreased to 30 X, 40 % and 20 X of the initial levels, respectively, and FDP levels increased to 20 to 40 pg/ml 3 to 24 hours after the endotoxin infusion ( Fig. 7a ). Both prothrombin time and the activated partial thromboplastin time were significantly prolonged as compared with the initial value, and the activities of factors II and V in plasma were decreased to 60 % and 70 % of the initial levels, respectively, during the same time intervals
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( Fig. 7b ). The activities of factors VII, VIII, IX and X in plasma were also decreased to 60 X, 70 %, 80 % and 70 % of the initial levels, respectively ( Fig. 7c >. b
a
#
Endotoxin to, 3 bra.
1 me/kg
I.v. intwion
0 10 20
FIG. 7a. Effect of endotoxin on the plasma AT III, fibrinogen, fibrin/fibrinogen degradation product and platelet count. FIG. 7b. Effect of endotoxin on the prothrombin time ( PI ), activated partial thromboplastin time ( APTT ), plasma coagulation factors II and V. FIG. 7c. Effect of endotoxin on_the . plasma coagulation factors VII, VIII, IX and X ( see text for details ).
DISCUSSION Although there have been many reports indicating a certain decrease in AT III concentration in plasma during the course of disseminated intravascular coagulation ( DIC )( 4 - 15 ), it has not been known whether this decreased concentration is due to the consumption of or the decreased production of AT III. Kinetics of AT III in patients with DIC has not yet been studied, although the -in vivo kinetics of radiolabelled AT III have been extensively investigated in man ( 23 - 26 ) and animals (27-33). Therefore, the metabolism of AT III was investigated both in healthy control dogs and in dogs with DIC induced by either thrombin or endotoxin administration. The purity of the AT III used in the present study was established by its high specific activity, single band on analytical PAGE and a single precipitin line under immunoelectrophoretic analysis. The extinction
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METABOLISM OF CANINE AT III IN DIC
coefficient of the purified AT III preparation was 6.0 which coincides with the value previously reported ( 29 ), while different values of 10.5 for man( 18 ), 7.0 for rabbit ( 34 ) and 8.6 for canine AT III ( 16 ) have been reported. Although the reason for such different values is not known, it is possible that they are due to different measurement conditions. The molecular weight of the canine AT III was 70,000 which was generally in accord with those reported previously ( 29 ). After radiolabelling, no changes in the physicochemical properties or biological activities of AT III were observed. In healthy control dogs, the -in vivo metabolism of the i.v. injected I-125-AT III was essentially identical in "unscreened" and "screened" conditions, indicating that the I-125-AT III preparation did not contain denatured proteins. All these results support that the purified AT III used in the present study is an appropriate preparation for metabolic study on AT III. The results obtained should be highly reliable, In five healthy dogs in the present study, mean t standard deviation of plasma half-life of administered I-125-AT III was 1.7 + 0.2 days, Cl was 0.52 t 0.04, fractional catabolic rate (j,) was 0.70 _+0.08, catabolic flux was 16.3 t 1.6 mg/kg/day and extravascular/plasma AT III was 0.54 t 0.07. The plasma half-life previously reported ranged from 1.97 days in female dog ( 29 ) to 2.83 days in man ( 23 ). For fractional catabolic rate (j,), 0.509 in dog ( 32 )( 0.59 in males and 0.68 in females; 29 ), 0.716 in rabbit ( 33 ), and 0.554 ( 23 ) and 0.60 in man ( 24 ) have been reported. As compared with these results, in our study the plasma half life of AT III was shorter and the fractional catabolic rate (j ) was higher. The reason why such an accelerated result was obtained in t;I e present study is not clear; difference in the strain of dogs might be involved. It has been reported that AT III is bound in a 1:l molar ratio to thrombin to form the AT III-thrombin complex ( 2 ). The amount of thrombin administered to dogs was 900-15,000 units. Since the specific activity of the thrombin administered was 2,000-3,000 units/mg protein, the amount administered was calculated as 0.45-7.5 mg. While the mean value of the canine plasma AT III was about 253 mg/body, the amount of AT III which was bound to thrombin was at most 15 mg; this is only 6% of the total plasma AT III. The amount of thrombin which was administered may thus not have been enough to affect the metabolism of AT III. It has also been reported that thrombin is bound with high affinity to cultured human endothelial cells ( 35 ), and that the clearance of thrombin from the circulation depends upon the high affinity binding site of the endothelial surface ( 36 ). In experimental dogs given a small dose of thrombin, the plasma AT III concentration did not decrease significantly as shown in Figure 5a, whereas the plasma fibrinogen concentration did decrease. In this case, thrombin initially catalyzed fibrinogen to fibrin in plasma; thus, residual thrombin might bind to endothelial cells faster than to AT III through the mechanism of progressive inactivation. Therefore, thrombin injected might not be fully bound to AT III, and consequently the plasma concentration and the metabolism of AT III were not changed significantly. In dogs given a larger amount of thrombin, heparin was administered simultaneously to prevent the sudden death of the animals. As shown in Figure 5b, the decrease in plasma AT III concentration was larger, whereas the decrease in fibrinogen concentration was smaller in comparison to dogs given a small dose of thrombin. The presence of heparin might be the reason for such different results. Since AT III can inhibit the catalytic action of thrombin immediately in the presence of heparin, decrease in fibrinogen due
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to fibrin formation can also be inhibited. Heparin inhibits the binding of thrombin to the endothelial cells ( 37 >, therefore most of the thrombin administered may be bound to AT III. However, the metabolism of AT III was not affected significantly in our study because of the limited amount of thrombin administered. The effect of heparin on the catabolism of AT III contradicted some earlier studies, although results obtained in the present study that heparin administration with a larger amount of thrombin did not affect the AT III turnover significantly agreed with others( 28, 38 ). In the experiment with endotoxin, two endotoxin administration of 200 &body into dog caused an acceleration of AT III turnover with a shortening of plasma half-life of AT III to 1.4 days and a reduction in the concentrations of plasma AT III and fibrinogen. In these dogs, the intravascular coagulation process might have been accelerated by the endotoxin having been injected twice. It has been reported that endotoxin induces the detachment of cultured bovine aortic endothelial cells with loss of fibronectin cell surface but does not induce the detachment of cultured human umbilical vein endothelial cells ( 39 ). It has also been noted that endotoxin activates the complement system, resulting in a cleavage of C5 to C5-derived peptides which can damage cultured human endothelial cells through the adherence of polymorphonuclear leukocytes ( PMN ) to the cells ( 40 ). Free radicals from the endotoxin-stimulated PMNs may induce endothelial cell injury ( 41 ). In any event, endotoxin administration may damage canine vascular endothelial cells which trigger the intrinsic blood coagulation process through the activation of factor XII ( 42 )'.It is well known that the endothelial cells and monocytes synthesize the tissue thromboplastin upon endotoxin stimuli ( 43, 44 ). In addition the complement which is activated by endotoxin also stimulates the tissue thromboplastin synthesis ( 45 ), resulting in activation of the extrinsic blood coagulation process. Thus endotoxin administration triggers a continuous generation of thrombin. AT III is decreased due to the increased consumption through the progressive binding to thrombin. Single administration with a larger amount of endotoxin resulted in more marked changes in the hemostatic system which coincided with those observed in DIC. AT III turnover was evidently accelerated with a shortening of its plasma half-life to 1.4 days. It is suggested from these results that various plasma coagulation factors activated by the infusion of endotoxin were neutralized by forming a complex with AT III. It has already been reported that the AT III-thrombin complex is rapidly eliminated from the circulation ( 46 ). The complex formed in experimental dogs in the present study, mostly between AT III and thrombin and factor Xa, should also have been eliminated from the circulation quickly. This might have resulted in a significant acceleration of the metabolism of AT III. Thus, it is possible that changes in the hemostatic system in dogs induced by endotoxin injection bear closer resemblance to those observed in DIC than do those induced by thrombin, and that the former would provide the opportunity for detailed study of DIC as an animal model of a clinical case.
ACKNOWLEDGEMENT This study was supported by a Research Grant from the Intractable Disease Division of the Ministry of Health and Welfare of Japan.
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REFERENCES
1.
RIZZA, C.R. Naturally occurring inhibitors of blood coagulation. In: Human Blood Coagulation, Haemostasis and Thrombosis. 3rd. Ed. R. Biggs and C.R. Rizza ( Eds. ) Oxford, London, Edinburgh, Boston, Palo Alto, Melbourne: Blackwell Scientific Publications, 1984, pp.81-91.
2.
ROSENBERG, R.D. Heparin-antithrombin system. 1n:Hemostasis and Thrombosis: Basic principles and clinical practice. R.W. Coleman, J.Hirsh, V.J. Marder and E.W. Salzman ( Eds.) Philadelphia, Toronto: J.B. Lippincott Company, 1982, pp. 962-985.
3.
HARPEL, P.C. Blood proteolytic enzyme inhibitors: their role in modulating blood coagulation and fibrinolytic enzyme pathways. In: ibid. pp. 738-747.
4.
BICK, R.L., DUKES, M.L., WILSON, W.L. and FEKETE, L.F. Antithrombin III ( AT-III ) as a diagnostic aid in disseminated intravascular coagulation. Thromb. Res. 10, 721- 729, 1977.
5.
BICK, R.L., BICK, M.D. and FEKETE, L.F. Antithrombin III pattern in disseminated intravascular coagulation. Am. J. Clin. Pathol. 73, 577583, 1980.
6.
SCHIPPER, H.G., ROOS, J., V.D. MEULEN, F. and TEN CATE, J.W. Antithrombin III deficiency in surgical intensive care patients. Thromb. Res. 21, 73-80, 1981.
7.
BICK, R.L. Clinical relevance of antithrombin III. Sem. Thromb. Hemostas. & 276-287, 1982.
8.
RAYMOND, S.L. and DODDS, W.J. Plasma antithrombin activity: a comparative study in normal and diseased animals. Proc. Sot. Exp. Biol. Med. 161, 464-479, 1979.
9.
LXMMLE, B., TRAN, T.H., RITZ, R. and DUCKERT, F. Plasma prekallikrein, factor XII, antithrombin III, Cl-inhibitor and d2-macroglobulin in critically ill patients with suspected disseminated intravascular coagulation ( DIC >. Am. J. Clin. Pathol. 82,396-404, 1984.
10.
ABILDGAARD, U. Antithrombin and related inhibitors of coanulation. In: Recent-Advances in Blood Coagulation. L. Poller ( Ed. ) Edinburgh, London, Melbourne and New York: Churchill Livingstone, 1981, pp. 151-173.
11.
LAURSEN, B., MORTENSEN, J.Z., FROST, L. and HANSEN, K.B. Disseminated intravascular coagulation in hepatic failure treated with antithrombin III. Thromb. Res. 22, 701-704, 1981.
12.
JESPERSEN, J., RASMUSSEN, N.R. and TOFTGAARD, C. Observations during the treatment with antithrombin-III concentrate of a case of tampon-related toxic shock syndrome and disseminated intravascular coagulation. Discrepancies between functional and immunologic determinations of antithrombin. Thromb. Res. 26,457-462, 1982.
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13.
LAURSEN, B., FABER, V., BROCK, A., GORMSEN, J. and SgRENSEN, H. Disseminated intravascular coagulation, antithrombin III, and complement in meningococcal infections. Acta. Med. Stand. 209, 221-227, 1981.
14.
JESPERSEN, J. Liver disease in pregnancy with DIC and low levels of AT III. Stand. J. Haematol. 30,492-494, 1983.
15.
LIEBMAN, H.A.,MCGEHEE, W.G., PATCH, M.J. and FEINSTEIN, D.I. Severe depression of antithrombin III associated with disseminated intravascular coagulation in women with fatty liver of pregnancy. Ann. Intern. Med. 98,330-333, 1983.
16.
DAMUS, P.S. and WALLACE, G.A. Purification of canine antithrombin III-heparin cofactor using affinity chromatography. Biochem. Biophys. Res. Commun. 61, 1147-1153, 1974.
17.
DAVIS, B.J. Disc electrophoresis-II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427, 1964.
18.
ROSENBERG, R.D. and DAMUS, P.S. The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem. 248, 6490-6505, 1973.
19. MANCINI, G., CARBONARA, A.O. and HEREMANS, J.F. Immunochemical quantitation of antigens by single radial immunodiffusion. Internat. J. Immunochem. 2_L 235-254, 1965. 20.
ANDREWS, P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595-605, 1965.
21.
MCFARLANE, A.S. Efficient trace-labelling of proteins with iodine. Nature ( London ) 182, 53, 1958.
22.
ATENCIO, A.C., BAILEY, H.R. and REEVE, E.B. Studies on the metabolism and distribution of fibrinogen in young and older rabbits. I. Methods and models. J. Lab. Clin. Med. 66, 1-19, 1965.
23.
COLLEN, D., SCHETZ, J., COCK, F.D., HOLMER, E. and VERSTRAETE, M. Metabolism of antithrombin III ( heparin cofactor ) in man: Effects of venous thrombosis and of heparin administration. Eur. J. Clin. Invest. L 27-35, 1977.
24.
GONMORI, H., TSUKADA, H., TAKADA, M., TANAKA, H., KOBAYASHI, N. and MAEKAWA, T. Vitamin K dependent clotting factors and their inhibitors. - with special reference to prothrombin and antithrombin III. Acta Haematol. Jpn. 44, 1466-1478, 1981.
25.
SAS, G., KREMMER, T., SPIT, B. and PETI),I. Isotopic and immunological half-life determination of antithrombin III in various types of antithrombin III deficiency. Thrombos. Haemostas. 46, 370, 1981.
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METABOLISM OF CANINE AT III IN DIC
26.
DE SWART, C.A.M., NIJIMEYER, B., ANDERSSON, L.-O., HOLMER, E., SIXMA, J.J. and BOUMA, B.N. Elimination of intravenously administered radiolabelled antithrombin III and henarin in humans. Thrombos. Haemostas. 52, 66-70, 1984.
27.
KOBAYASHI, N. and TAKEDA, Y. Studies of the effects of estradiol, progesterone, cortisol, thrombophlebitis, and typhoid vaccine on synthesis and catabolism of antithrombin III in the dog. Thrombos. Haemostas. 37, 111-122, 1977.
28.
TAKEDA, Y. and KOBAYASHI, N. Studies of heparin affinity to antithrombin III and other proteins in vitro and in vivo. Thrombos. Haemostas. x685-695, 1977.
29.
KOBAYASHI, N. and TAKEDA, Y. Effects of a large dose of oestradiol on antithrombin III metabolism in male and female dogs. Eur. J. Clin. Invest. L 373-381, 1977.
30.
CHANDRA, S., BANG, N.U. and MARKS, C. Radiolabelled AT III as a probe for the detection of activation of blood coagulation in vivo. Thromb. Res. 9, 9-23, 1976.
31.
REEVE, E.B., LEONARD, B., WENTLAND, S.H. and DAMUS, P. Studies with I-131-labelled antithrombin III in dogs. Thromb. Res. 20, 375-389, 1980.
32.
REEVE, E.B., LEONARD, B. and CARLSON, T. Kinetic studies in vivo of antithrombin III. Ann. N.Y. Acad. Sci. 370,680-694, 1981.
33.
CARLSON, T.H., ATENCIO, A.C. and SIMON, T.L. In vivo behaviour of radioiodinated rabbit antithrombin III. Demonstration of a noncirculating vascular compartment. J. Clin. Invest. 74, 191-199, 1984.
34.
KOIDE, T. Isolation and characterization of antithrombin III from human, porcine and rabbit plasma, and rat serum. J. Biochem. 86, 1841-1850, 1979.
35.
AWBREY, B.J., HOAK, J.C. and OWEN, W.G. Binding of human thrombin to cultured human endothelial cells. J. Biol. Chem. 254, 4092-4095, 1979.
36.
LOLLAR, P. and OWEN, W.G. Clearance of thrombin from circulation in rabbits by high-affinity binding sites on endothelium. Possible role in the inactivation of thrombin by antithrombin III. J. Clin. Invest. 66, 1222-1230, 1980.
37.
BAUER, P.L., MACHOVICH, R., ARANYI, P., BUKI, K.G., CSONKA, E. and HORVATH, I. Mechanism of thrombin binding to endothelial cells. Blood 61, 368-372, 1983.
38.
SOULIER, J.P., GOZIN, D. and LERABLE, J. In vivo attempt to consume antithrombin III by i.v. injection of a thrombin-heparin mixture. Thromb. Res. x255-262, 1984.
39.
HARLAN, M., HARKER, L.A., STRIKER, G.E. and COUNTS, R.B. Endotoxinmediated endothelial injury in vitro. Thrombos. Haemostas. 46, 200, 1981.
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I.M.and PEREZ, H.D. Biologically active peptides derived 40. GOLDSTEIN, from the fifth component of complementr In: Progress in_Hemostasis and Thrombosis. Vol. 5. T.H. Spaet ( Ed.) New York, London, Toronto, Sydney, San Francisco : Grune & Stratton, 1980, pp. 41-79. 41.
YAMADA, O., MOLDOW, C.F., SACKS,T., CRADDOCK, P.R., BOOGAERTS, M.A. and JACOB. H.S. Deleterious effects of endotoxin on cultured endothelial cells: An in vitro model of vascular injury. Inflammation L 115-126, 1981.
42.
MULLER-BERGHAUS, G. Pathophysiology of generalized intravascular coagulation. Sem. Thromb. Hemostas. L 209-246, 1977.
43.
COLUCCI, M., BALCONI, G., LORENZET, R., PIETRA, A., LOCATI, D., DONATI. M.B. and SEMERARO. N. Cultured human endothelial cells generate tissue factor in.response to endotoxin. J. Clin. Invest. /1, 1893-1896, 1983.
44.
PRYTZ, H. and ALLISON, A.C. Tissue thromboplastin activity of isolated human monocytes. Thrombos. Haemostas. 39, 582-591, 1978.
45.
MUHLFELDER, T.W., NIEMETZ, J., KREUTZER, D., BEEBE, D., WARD, P.A. and ROSENFELD, S.I. C5 chemotactic fragment induces leukocyte production of tissue factor activity. A link between complement and coagulation. J. Clin. Invest. 63, 147-150, 1979.
46.
LEONARD, B., BIES, R., CARLSON, T. and REEVE, E.B. Further studies of the turnover of dog antithrombin III. Study of I-131-labelled antithrombin protease complexes. Thromb. Res. 30, 165-177, 1983.
RESEARCH 40; 291-306, 1985 Printed in the USA. (c) 1985 Pergamon Press Ltd. All rights reserved.
0049-3848/85 $3.00 t .OO Copyright
%FFECT OF THROMBIN AND ENDOTOXIN ON THE -IN VIVO METABOLISM OF ANTITHROMBIN III (AT III) IN DOGS
H.Tanaka, N.Kobayashi and T.Maekawa The Third Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma, 371, Japan.
(Received 24.5.1985; Accepted in revised form 1.8.1985 by Editor M. Matsuda)
AHSTRACT Effect of thrombin and endotoxin on the metabolism of 1-125labelled canine AT III was studied in mongrel dogs. Under control condition, mean total amount of intravascular AT III with standard deviation was 23.4 t 2.4 mg/kg, plasma half life of i.v. injected I-125-AT III was 1.7 t 0.2 days, and the fractional catabolic flux ( j3x ) was 16.3 t 1.6 mg/kg/day. The total amount of intra- and extra-vascular AT III was 36.0 + 0.34 mglkg. Neither a 3 hour infusion of a small dose (30 units/kg/hr) of thrombin nor i.v. injection of a large amount of thrombin (5,000 - 15,000 units/day) with heparin significantly affected AT III metabolism except for a transient decrease in AT III concentration in the latter case, although decrease in plasma fibrinogen concentration and platelet count was observed in both cases. Two injections with 200 pg/kg of endotoxin resulted in an evident acceleration of AT III metabolism with significant decrease in the plasma AT III, fibrinogen concentrations and platelet count. More marked changes in AT III metabolism were induced by a single infusion with 1 nrg/kgof endotoxin. Changes in hemostatic system coincided with those observed in DIC.
INTRODUCTION
Plasma antithrombin TJI (AT III) is an important.inhibitor in blood coagulation, inhibiting not only clot promoting serine proteases such as thrombin and factor Xa ( 1,2 ) but also plasmin ( 3 ). Reduction in plasma AT 114 activity has often been observed during the course of tiisser 'l$at~IS.ntrnv:?rrl;!,lr roagul:jtior(PTC)( 4 - 15 ), although the -.. ____ _.__-___ KEY VORDS: Antithrombin IIT ( AT III ), Metabolism of AT TV, 1-125-antithrombin TIT, Thrombin, Endotoxin, DIC. 291
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underlying mechanism of the reduction has not been resolved. Studies were therefore designed to investigate the metabolism of AT III in dogs with DIC experimentally induced by administration of either bovine thrombin or endotoxin from Escherichia coli using I-125-labelled canine AT III as a tracer.
MATERIALS AND METHODS
Purification of canine antithrombin III (AT III). Canine AT III was purified according to the method described by Damus and Wallace (16) with a minor modification using heparin-Sepharose affinity chromatography. Nine volumes of blood were withdrawn directly from the heart of a healthy adult canine to a centrifuge tube containing 1 volume of ACD solution. Fresh canine plasma separated by centrifugation at 4' C was defibrinated by heating to 54'C for 3 min. After centrifugation at 3000 rpm for 20 min at 4' C, 100 ml of the defibrinated plasma was applied on a heparin-Sepharose 4B ( Pharmacia, Uppsala, Sweden ) column, 2.5 x 45 cm in size, and washed with 100 ml of 0.145 M NaCl-0.01 M Tris-HCl buffer ( pH 7.5 > followed by 60 ml of 0.3 M NaCl-0.01 M Tris-HCl buffer ( pH 7.5 ). The protein was then eluted with a linear gradient of 500 ml of 0.3 M NaCl-0.01 M Tris-HCl buffer ( pH 7.5 ) in a mixing chamber and 500 ml of 1.0 M NaCl-0.01 M TrisHCl buffer( pH 7.5 ) in a reservoir at 20' C. The flow rate was 30 ml/hr and fraction volume was 5 ml/fraction. The active fraction was obtained as a small peak at an optical density ( OD ) of 280 nm after a large amount of protein was eluted. About 80 ml of the active fraction was collected into a dialyzing tube and then concentrated to 12-15 ml by drying under cold air. This concentrated fraction was dialyzed against 0.05 M phosphate buffer ( pH 8.0 ) overnight at 4'C. The obtained AT III fraction contained a trace amount of impurity according to the 10 % analytical polyacrylamide gel electrophoresis ( PAGE ) method of Davis ( 17 ); therefore, this active fraction was further purified using preparative PAGE. Gels of 10 % polyacrylamide 0.8 x 5 cm in size were prepared in the following buffer: 70.7 mg ammonium persulfate, 0.726 % Tris, 29 mg TEMED ( N,N,N',N'tetramethylene diamine ) and 0.263 % methylene bis acrylamide in 100 ml of distilled water ( pH 8.6 ). The chamber buffer consisted of 0.6 g Tris and 2.88 g glycine in 100 ml of H20 ( pH 8.3 ). Electrophoresis usually ran for about five hours at 5 mA per gel. After electrophoresis, the gels were cut into 10 pieces ( 5 mm wide ) and the one containing AT III, which was recognized by the reference gel stained with 1 g% amide black 10B ( Merck, Darmstadt, Germany ) was crushed into fine pieces by passing it through a 10 ml plastic syringe. The crushed gels were mixed with 5 ml of 0.05 M phosphate buffer ( pH 8.0 ) and kept at 4'C overnight. The suspension was finally centrifuged at 3000 rpm for 3 min at 4'C, then the supernatant contgining purified AT III was divided into 1 ml aliquots and stored at - 20 C. AS shown in Fig.1, the purified AT III showed a single band on 10 % analytical PAGE. Measurement of antithrombin and heparin-cofactor activity. AT III activity was determined bv the method of Rosenberg and Damus and heuarin cofactor activity was determined by the same methid, except for the-use of 0.05 M phosphate buffer ( pH 8.0 ) and bovine thrombin in the place of 150 mM of NaCl in 10 mM of Tris-HCl ( pH 7.5 ) buffer and human thrombin ( 18 ). Specific activity of the purified AT III preparation was 400 units/mg when the activity in 1 ml of defibrinated healthy canine plasma was arbitrarily assigned as 100 units.
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METABOLISM OF CANINE AT III
IN DIC
FIGURE 1.
a
b
c
d,
FIG.1. Ten percent polyacrylamide gel electrophoresis of purified canine antithrombin III. About (a) ZOOpg, (b) 100 pg, (c) 5Opg and (d) 25yg were analyzed. Purified AT III consisted of a single component.
Production of the antibody against AT III. About 200 ug of purified canine AT III was mixed with Freund's complete adjuvant ( Difco Laboratories, Detroit, MI, USA ) and injected intramuscularly into each of two rabbits once a week for 4 weeks. The blood was harvested within one week after the last injection and thus obtained antisera against AT III was divided into aliquots of 1 ml, then stored at -2O'C. Immunoelectrophoresis of purified AT III with the antiserum revealed a single precipitin line ( Fig. 2 ). Amount of AT III in the test Determination of amount of AT III. samples was determined by single radial immunodiffusion ( 19 ). One g% of agarose plate containing-4 % of anti-AT III antiserum was prepared. Buffer containing 0.55 g barbital, 3.50 g sodium barbital, 0.51 g calcium lactate and 0.1 g sodium azide per liter of distilled water ( pH8.6 ) was used as gel buffer. Next, 3 ul of the test samples was applied to each of twelve wells, 2 mm in diameter, which were filled with the agarose plate. After 48 hours, the diameter of the precipitate ring was measured and an AT IIJ concentration obtained from the standard curve. Purified AT III solution of 160 mg/dl of concentration was used as standard. Determination of extinction coefficient and molecular weight of purified AT III. The extinction coefficient was determined by measuring the weight of well-dried lyophilized materials which were dialyzed against distilled water, and by measuring their optical density dissolved in 0.05 M phosphate buffer ( pH 8.0 ) at 280 nm using a 1 cm wide quartz cuvette; this latter was 6.2 for 10 mg of the purified AT III per ml of solution. The molecular weight of the purified AT III determined by the method of Andrews ( 20 ) using a Sephadex G-200 column 2.5 x 100 cm in size was 70,000 daltons.
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FIG.2. Immunoelectrophoresis of purified canine AT III. The central trough was filled with anti-AT III antiserum and the upper well and lower well were filled with purified AT III and canine plasma, respectively. The canine AT III showed a single precipitin line against anti-AT III antiserum.
Labelling of AT III with I-125. AT III was labelled with I-125 by the iodine monochloride method of McFarlane ( 21 ). Free I-125 was removed by passing it through a Sephadex G-25 column 1.5 x 20 cm in size equilibrated with 0.05 M phosphate buffer ( pH 8.0 >. The free I-125 remaining in the preparation was usually less than 1 % of total radioactivity. Specific activity of I-125-AT III was about 1 mCi/mg. 1-125AT III was further tested with respect to AT III activity, heparin cofactor activity and its electrophoretic mobility in 10 % polyacrylamide gels. These results were compared with those of unlabelled AT III and no appreciable differences were found. The prepared I-125-AT III was injected intravascularly into a puppy to remove the denatured molecules, and plasma containing undenatured I-125-AT III was harvested about 30 min after the initial injection. This was then used for studies of the AT III metabolism. A comparison of the metabolism of screened and unscreened I-125-AT III showed no significant differences between them ( data not shown ). Studies on the metabolism of AT III in healthy dogs. Mongrel dogs with 7 to 8 kg of body weight were used for studies on metabolism of AT III. After i.v. injection with 10 to 15 pCi of I-125-AT III, blood samples were obtained serially for 5 days using ethylene diamine tetraacetic acid ( EDTA > or 3.8 % sodium citrate solution as anticoagulant. The radioactivity of 1 ml of each plasma separated from the blood samples was measured, the data were depicted on a semilogarithmic scale, and the disappearance curve of the radioactivity from the plasma was analyzed. As the plasma disappearance of radioiodine was described by a biexponential curve within an observation period of 120 hours, the data were analyzed by the two compartment model reported by Atencio et a1.(22).
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METABOLISM ~F~FPfMINE AT III
IN DIC
Studies of effect of thrombin and endotoxin on I-125-AT III metabolism. On the second day after I-125-AT III injection, ( 1 ) bovine thrombin solution ( 2 u/ml of normal saline ), ( Parke Davis, Lot No. 656, USA ) was i.v. infused at a rate of 30 u/kg/hour or 5,000 units - 15,000 units of thrombin with 5,000 units- 15,000 units of heparin was i.v. injected at one time: ( 2 ) or two hundred rg/kg of endotoxin ( E.coli, 026:B6, Sigma Chemicals, St.Louis, MO, USA ) were i.v. injected into dogs twice with an interval of 24 hours, ( 3 ) or 1 mg/kg of endotoxin was infused into dogs for 3 hours. Determination of various clotting factors. For the determination of activities of various clotting factors, conventional methods using deficient plasma ( Dade, Miami, FL, USA ) as a substrate were adopted.
RESULTS
Metabolism of AT III in healthy dogs under the control condition. In vivo behavior of i.v. administered I-125-AT III in control dogs is shown -in Fig.3. The plasma clearance curve of the i.v. injected I-125-AT III is closely expressed by two exponential functions: X=0.52e-0'40gt + 0.48e-3'02t. Since the plasma AT III packed cell volume were maintained relatively constant the dogs were in a steady state with respect to AT III tracer data calculated from the analysis of the plasma are shown in Table 1 a,b,c.
concentration and during the study, metabolism. The disappearance curve
FIGURE 3.
mg/ 1 OOml Plasma AT
III
on
concentratl
___..--r_-.____.....~____________~__---__~.__--r---------...~
40
-30
byr 0
1
2
3
4
f 5
FIG.3. Plasma clearance of I-125-canine AT III under control condition. The plasma behavior of injected I-125-AT III was closely expressed by two -0.409t exponential functions: X= 0.52e t 0.48e-3'02t'
295
METABOLISM OF CANINE AT III
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IN DIC
TABLE la,b,c. Metabolic Parameters of AT III in Normal Control Dogs. TABLE la.
Dog No.
Weight Kg
Ht %
Plasma Vol.
Plasma AT III
Plasma AT III(x)
mg/lOOml
ml/kg
mg/kg
1 2 3 4 5
10.5 12.8 10.5 9.5 10.5
36.0 35.2 37.0 34.0 37.0
58.7 64.5 57.4 54.6 51.8
42.6 37.1 46.3 35.9 42.5
25.0 23.9 26.6 19.6 22.0
Mean SD
10.8 1.1
35.8 1.1
57.4 4.3
40.8 3.8
23.4 2.4
TABLE lb. -at t l/2 +C embt x=C e j, j jl days /day /day /day ' a2 No. c2 b c1 ~-__-------___-------___-_------__-----~~~---------_____-_---____
Dog
1 2 3 4 5
.48 .54 .57 .54 .47
.408 .433 .347 .433 .425
.52 .46 .43 .46 .53
3.01 2.77 2.48 3.85 2.97
1.7 1.6 2.0 1.6 1.6
1.02 .80 .71 1.27 1.00
Mean SD
.52 .04
.409 .03
.48 .04
3.02 .46
1.7 .2
.96 .19
1.66 1.70 1.56 2.28 1.62
.74 .71 .55 .73 .78
1.76 .70 .26 .08
TABLE lc. ---__------------------_-----------_-----------------______----__ Dog
jlx
j,x
Y
Y/x
x+y
No. mg/kg/day mg/kg mg/kg ~~~-----~~-~~----~--__^_________________-~~----_-___~-----------.62 40.4 18.5 15.4 1 25.5 .47 35.2 16.9 11.3 2 19.2 2 5
24.9 18.9 22.0
14.4 14.6 17.2
12.1 11.0 13.5
.46 .56 .61
38.7 30.6 35.5
Mean SD
22.1 2.8
16.3 1.6
12.7 1.6
.54 .07
36.0 .34
x: plasma AT III, mg per kg. t l/2: the half-life of plasma I-125-AT III. y: extravascular AT III, mg per kg. x+y: total amount of AT III, mg per kg. jl: fractional transcapillary transfer rate to extravascular space, per day. j : fractional return rate of y to plasma, per day. j,: fractional cataboI*ic rate, per day. jlx: transcapillary flux, mg/kg/day. j3x: catabolic flux, mg/kg/day.
METABOLISM OF CANINE AT III IN DIC
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297
The radioactive fraction of the plasma obtained either 10 min or 2 days after I-125-AT III injection showed a single peak identical to the location of native AT III in Sephadex G-200 column chromatography ( Fig. 4 ).
FIG. 4. Sephadex G-200 chromatogram of defibrinated plasma obtained at 10 min and 2 days after the injection of 1-125AT III. The injected I-125-AT III was not split into several radioactive fragments -in vivo and the location of I-125-AT III was identical with that of native AT III determined by immunological method.
01,
20
40
00
’ ’2
Fraction ~0.
Effect of thrombin administration on the metabolism of AT III. To study the effects of thrombin on the metabolism of AT III, 30 units/kg/hr of bovine thrombin dissolved in physiological saline at a concentration of two units/ml were intravenously infused to each of 5 dogs on the second day after the intravenous injection of I-125-AT III. Thrombin infusion on the 2nd day of I-125-AT III injection resulted in a reduction of the plasma fibrinogen concentration and the platelet count to 60 % of the initial level, although it significantly affected neither the disappearance curve of I-125-AT III i.v. injected 1 day before nor the plasma AT III concentration ( Fig. 5a >. The effect of a large amount of thrombin administration with heparin on canine AT III metabolism was then studied. From 5,000 to 15,000 units of thrombin were intravenously injected with 5,000 to 15,000 units of heparin one day after the injection of I-125-AT III into dogs. Although the plasma fibrinogen concentration was not changed significantly, after the injection of such a large amount of thrombin with heparin the plasma radioactivity of I-125-AT III and the concentration of AT III were decreased to 70 % and 60 % of the initial concentration,respectively,within 6 hours. It was, however, noted in these dogs that the disappearance rate of the injected I125-AT III was not changed even after i.v. injection of a large amount of thrombin with heparin ( Fig. 5b ). Two Effect of endotoxin administration on the metabolism of AT III. hundredpg/kg of endotoxin (E.coli 026:B6) was injected intravenously
METABOLISM OF CANINE AT III
298
IN DIC
a
lhrombln
Vol. 40, No. 3
b
X)u/hg/hr,3h,. hcprrm
50’ Plasma
AT III concentration
~q&...*_.__..~....*.....* 30. Plasma LOO.
3oocy
500045ooOU
. . . .. -.
flbiinoeon concentration
:
200. 100 0.
Days
t 0
1
2
3
P
1
‘
I
5
E
lcw
, 0
I 1
1 2
DIYS i 3
1 4
1 5
FIG. 5a. -In vivo behavior of i.v. administered I-125-AT III under the influence of a small dose of bovine thrombin. Although the plasma fibrinogen concentration was decreased, the plasma clearance of I-125-AT III was not affected. FIG. 5b. -In vivo behavior of i.v. administered I-125-AT III under the influence of a large dose of bovine thrombin with simultaneous heparin. In this case also the plasma clearance of I-125-AT III was not affected.
into the right jugular vein of five dogs both one and two days after 1-125AT III injection. The second injection resulted in a reduction of plasma AT III concentration, plasma fibrinogen concentration and platelet count to 80 %, 60 % and 60 X of the initial levels, respectively. Plasma half-life of I-125-AT III was shortened to 1.4 days ( control value: 1.7 + 0.2 days) with significant acceleration of the metabolism of I-125-AT III. The catabolic rate, j , increased to 1.25/day whereas normal control value was 0.70 + O.O8/day. ?t is suggested from these results that the decrease in plasma AT III concentration resulted mainly from the acceleration of AT III catabolism ( Fig. 6a ).
026:B6) was infused intravenously for three hours through a catheter which was indwelled in the right jugular vein of each of 5 dogs. The plasma AT III concentration was decreased to 70 % of the initial level within 3 to 24 hours after the start of endotoxin infusion, and then was gradually restored to the initial level within 48 to 72 hours. The plasma fibrinogen concentration and the platelet count were also decreased to 70 % and 20 %
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METABOLISM OFzCANINE AT III
IN DIC
299
of the initial levels, respectively, within three hours after completion of the endotoxin infusion. The disappearance of I-125-AT III from the plasma was accelerated and the mean plasma half-life of I-125-AT III was shortened to 1.4 days ( Fig. 6b >.
b
a
‘:P
Endotoxln
200~1g/kg
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I.v. Injoctlon
t mg/kg
1.~. Infualon
to,
.6 .4 s z2 0’ Y 4 P
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60
PIWM
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AT III
conc.ntratlon
1g/lOOml
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___.____.. 4 ........__..____. _. ./.+-.. +
z 20 \ e 600
40’ 30
AT Ill concentration
. .._ __
.
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2
j
FIG. 6a. -In vivo behavior of i.v. administered I-125-AT III under the influence of endotoxin. The metabolism of I-125-AT III was accelerated and the plasma half life of I-125-AT III was shortened to 1.4 days. FIG. 6b. -In vivo behavior of i.v. administered I-125-AT III under the influence of a large single dose of endotoxin. The plasma clearance of I-125-AT III was accelerated and the half life of AT III was shortened to 1.4 days. Changes in the plasma AT III concentration, plasma fibrinogen concentration, platelet count, fibrin/fibrinogen degradation products (FDP) levels, prothrombin time, activated partial thromboplastin time and plasma coagulation factors, such as factors II, V, VII, VIII, IX and X, were measured serially after i.v. infusion with 1 mg/kg of endotoxin. Plasma concentrations of AT III and fibrinogen and platelet count were decreased to 30 X, 40 % and 20 X of the initial levels, respectively, and FDP levels increased to 20 to 40 pg/ml 3 to 24 hours after the endotoxin infusion ( Fig. 7a ). Both prothrombin time and the activated partial thromboplastin time were significantly prolonged as compared with the initial value, and the activities of factors II and V in plasma were decreased to 60 % and 70 % of the initial levels, respectively, during the same time intervals
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( Fig. 7b ). The activities of factors VII, VIII, IX and X in plasma were also decreased to 60 X, 70 %, 80 % and 70 % of the initial levels, respectively ( Fig. 7c >. b
a
#
Endotoxin to, 3 bra.
1 me/kg
I.v. intwion
0 10 20
FIG. 7a. Effect of endotoxin on the plasma AT III, fibrinogen, fibrin/fibrinogen degradation product and platelet count. FIG. 7b. Effect of endotoxin on the prothrombin time ( PI ), activated partial thromboplastin time ( APTT ), plasma coagulation factors II and V. FIG. 7c. Effect of endotoxin on_the . plasma coagulation factors VII, VIII, IX and X ( see text for details ).
DISCUSSION Although there have been many reports indicating a certain decrease in AT III concentration in plasma during the course of disseminated intravascular coagulation ( DIC )( 4 - 15 ), it has not been known whether this decreased concentration is due to the consumption of or the decreased production of AT III. Kinetics of AT III in patients with DIC has not yet been studied, although the -in vivo kinetics of radiolabelled AT III have been extensively investigated in man ( 23 - 26 ) and animals (27-33). Therefore, the metabolism of AT III was investigated both in healthy control dogs and in dogs with DIC induced by either thrombin or endotoxin administration. The purity of the AT III used in the present study was established by its high specific activity, single band on analytical PAGE and a single precipitin line under immunoelectrophoretic analysis. The extinction
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coefficient of the purified AT III preparation was 6.0 which coincides with the value previously reported ( 29 ), while different values of 10.5 for man( 18 ), 7.0 for rabbit ( 34 ) and 8.6 for canine AT III ( 16 ) have been reported. Although the reason for such different values is not known, it is possible that they are due to different measurement conditions. The molecular weight of the canine AT III was 70,000 which was generally in accord with those reported previously ( 29 ). After radiolabelling, no changes in the physicochemical properties or biological activities of AT III were observed. In healthy control dogs, the -in vivo metabolism of the i.v. injected I-125-AT III was essentially identical in "unscreened" and "screened" conditions, indicating that the I-125-AT III preparation did not contain denatured proteins. All these results support that the purified AT III used in the present study is an appropriate preparation for metabolic study on AT III. The results obtained should be highly reliable, In five healthy dogs in the present study, mean t standard deviation of plasma half-life of administered I-125-AT III was 1.7 + 0.2 days, Cl was 0.52 t 0.04, fractional catabolic rate (j,) was 0.70 _+0.08, catabolic flux was 16.3 t 1.6 mg/kg/day and extravascular/plasma AT III was 0.54 t 0.07. The plasma half-life previously reported ranged from 1.97 days in female dog ( 29 ) to 2.83 days in man ( 23 ). For fractional catabolic rate (j,), 0.509 in dog ( 32 )( 0.59 in males and 0.68 in females; 29 ), 0.716 in rabbit ( 33 ), and 0.554 ( 23 ) and 0.60 in man ( 24 ) have been reported. As compared with these results, in our study the plasma half life of AT III was shorter and the fractional catabolic rate (j ) was higher. The reason why such an accelerated result was obtained in t;I e present study is not clear; difference in the strain of dogs might be involved. It has been reported that AT III is bound in a 1:l molar ratio to thrombin to form the AT III-thrombin complex ( 2 ). The amount of thrombin administered to dogs was 900-15,000 units. Since the specific activity of the thrombin administered was 2,000-3,000 units/mg protein, the amount administered was calculated as 0.45-7.5 mg. While the mean value of the canine plasma AT III was about 253 mg/body, the amount of AT III which was bound to thrombin was at most 15 mg; this is only 6% of the total plasma AT III. The amount of thrombin which was administered may thus not have been enough to affect the metabolism of AT III. It has also been reported that thrombin is bound with high affinity to cultured human endothelial cells ( 35 ), and that the clearance of thrombin from the circulation depends upon the high affinity binding site of the endothelial surface ( 36 ). In experimental dogs given a small dose of thrombin, the plasma AT III concentration did not decrease significantly as shown in Figure 5a, whereas the plasma fibrinogen concentration did decrease. In this case, thrombin initially catalyzed fibrinogen to fibrin in plasma; thus, residual thrombin might bind to endothelial cells faster than to AT III through the mechanism of progressive inactivation. Therefore, thrombin injected might not be fully bound to AT III, and consequently the plasma concentration and the metabolism of AT III were not changed significantly. In dogs given a larger amount of thrombin, heparin was administered simultaneously to prevent the sudden death of the animals. As shown in Figure 5b, the decrease in plasma AT III concentration was larger, whereas the decrease in fibrinogen concentration was smaller in comparison to dogs given a small dose of thrombin. The presence of heparin might be the reason for such different results. Since AT III can inhibit the catalytic action of thrombin immediately in the presence of heparin, decrease in fibrinogen due
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to fibrin formation can also be inhibited. Heparin inhibits the binding of thrombin to the endothelial cells ( 37 >, therefore most of the thrombin administered may be bound to AT III. However, the metabolism of AT III was not affected significantly in our study because of the limited amount of thrombin administered. The effect of heparin on the catabolism of AT III contradicted some earlier studies, although results obtained in the present study that heparin administration with a larger amount of thrombin did not affect the AT III turnover significantly agreed with others( 28, 38 ). In the experiment with endotoxin, two endotoxin administration of 200 &body into dog caused an acceleration of AT III turnover with a shortening of plasma half-life of AT III to 1.4 days and a reduction in the concentrations of plasma AT III and fibrinogen. In these dogs, the intravascular coagulation process might have been accelerated by the endotoxin having been injected twice. It has been reported that endotoxin induces the detachment of cultured bovine aortic endothelial cells with loss of fibronectin cell surface but does not induce the detachment of cultured human umbilical vein endothelial cells ( 39 ). It has also been noted that endotoxin activates the complement system, resulting in a cleavage of C5 to C5-derived peptides which can damage cultured human endothelial cells through the adherence of polymorphonuclear leukocytes ( PMN ) to the cells ( 40 ). Free radicals from the endotoxin-stimulated PMNs may induce endothelial cell injury ( 41 ). In any event, endotoxin administration may damage canine vascular endothelial cells which trigger the intrinsic blood coagulation process through the activation of factor XII ( 42 )'.It is well known that the endothelial cells and monocytes synthesize the tissue thromboplastin upon endotoxin stimuli ( 43, 44 ). In addition the complement which is activated by endotoxin also stimulates the tissue thromboplastin synthesis ( 45 ), resulting in activation of the extrinsic blood coagulation process. Thus endotoxin administration triggers a continuous generation of thrombin. AT III is decreased due to the increased consumption through the progressive binding to thrombin. Single administration with a larger amount of endotoxin resulted in more marked changes in the hemostatic system which coincided with those observed in DIC. AT III turnover was evidently accelerated with a shortening of its plasma half-life to 1.4 days. It is suggested from these results that various plasma coagulation factors activated by the infusion of endotoxin were neutralized by forming a complex with AT III. It has already been reported that the AT III-thrombin complex is rapidly eliminated from the circulation ( 46 ). The complex formed in experimental dogs in the present study, mostly between AT III and thrombin and factor Xa, should also have been eliminated from the circulation quickly. This might have resulted in a significant acceleration of the metabolism of AT III. Thus, it is possible that changes in the hemostatic system in dogs induced by endotoxin injection bear closer resemblance to those observed in DIC than do those induced by thrombin, and that the former would provide the opportunity for detailed study of DIC as an animal model of a clinical case.
ACKNOWLEDGEMENT This study was supported by a Research Grant from the Intractable Disease Division of the Ministry of Health and Welfare of Japan.
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REFERENCES
1.
RIZZA, C.R. Naturally occurring inhibitors of blood coagulation. In: Human Blood Coagulation, Haemostasis and Thrombosis. 3rd. Ed. R. Biggs and C.R. Rizza ( Eds. ) Oxford, London, Edinburgh, Boston, Palo Alto, Melbourne: Blackwell Scientific Publications, 1984, pp.81-91.
2.
ROSENBERG, R.D. Heparin-antithrombin system. 1n:Hemostasis and Thrombosis: Basic principles and clinical practice. R.W. Coleman, J.Hirsh, V.J. Marder and E.W. Salzman ( Eds.) Philadelphia, Toronto: J.B. Lippincott Company, 1982, pp. 962-985.
3.
HARPEL, P.C. Blood proteolytic enzyme inhibitors: their role in modulating blood coagulation and fibrinolytic enzyme pathways. In: ibid. pp. 738-747.
4.
BICK, R.L., DUKES, M.L., WILSON, W.L. and FEKETE, L.F. Antithrombin III ( AT-III ) as a diagnostic aid in disseminated intravascular coagulation. Thromb. Res. 10, 721- 729, 1977.
5.
BICK, R.L., BICK, M.D. and FEKETE, L.F. Antithrombin III pattern in disseminated intravascular coagulation. Am. J. Clin. Pathol. 73, 577583, 1980.
6.
SCHIPPER, H.G., ROOS, J., V.D. MEULEN, F. and TEN CATE, J.W. Antithrombin III deficiency in surgical intensive care patients. Thromb. Res. 21, 73-80, 1981.
7.
BICK, R.L. Clinical relevance of antithrombin III. Sem. Thromb. Hemostas. & 276-287, 1982.
8.
RAYMOND, S.L. and DODDS, W.J. Plasma antithrombin activity: a comparative study in normal and diseased animals. Proc. Sot. Exp. Biol. Med. 161, 464-479, 1979.
9.
LXMMLE, B., TRAN, T.H., RITZ, R. and DUCKERT, F. Plasma prekallikrein, factor XII, antithrombin III, Cl-inhibitor and d2-macroglobulin in critically ill patients with suspected disseminated intravascular coagulation ( DIC >. Am. J. Clin. Pathol. 82,396-404, 1984.
10.
ABILDGAARD, U. Antithrombin and related inhibitors of coanulation. In: Recent-Advances in Blood Coagulation. L. Poller ( Ed. ) Edinburgh, London, Melbourne and New York: Churchill Livingstone, 1981, pp. 151-173.
11.
LAURSEN, B., MORTENSEN, J.Z., FROST, L. and HANSEN, K.B. Disseminated intravascular coagulation in hepatic failure treated with antithrombin III. Thromb. Res. 22, 701-704, 1981.
12.
JESPERSEN, J., RASMUSSEN, N.R. and TOFTGAARD, C. Observations during the treatment with antithrombin-III concentrate of a case of tampon-related toxic shock syndrome and disseminated intravascular coagulation. Discrepancies between functional and immunologic determinations of antithrombin. Thromb. Res. 26,457-462, 1982.
303
304
METABOLISM OF CANINEAT III
IN DIC
Vol. 40, No. 3
13.
LAURSEN, B., FABER, V., BROCK, A., GORMSEN, J. and SgRENSEN, H. Disseminated intravascular coagulation, antithrombin III, and complement in meningococcal infections. Acta. Med. Stand. 209, 221-227, 1981.
14.
JESPERSEN, J. Liver disease in pregnancy with DIC and low levels of AT III. Stand. J. Haematol. 30,492-494, 1983.
15.
LIEBMAN, H.A.,MCGEHEE, W.G., PATCH, M.J. and FEINSTEIN, D.I. Severe depression of antithrombin III associated with disseminated intravascular coagulation in women with fatty liver of pregnancy. Ann. Intern. Med. 98,330-333, 1983.
16.
DAMUS, P.S. and WALLACE, G.A. Purification of canine antithrombin III-heparin cofactor using affinity chromatography. Biochem. Biophys. Res. Commun. 61, 1147-1153, 1974.
17.
DAVIS, B.J. Disc electrophoresis-II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427, 1964.
18.
ROSENBERG, R.D. and DAMUS, P.S. The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem. 248, 6490-6505, 1973.
19. MANCINI, G., CARBONARA, A.O. and HEREMANS, J.F. Immunochemical quantitation of antigens by single radial immunodiffusion. Internat. J. Immunochem. 2_L 235-254, 1965. 20.
ANDREWS, P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595-605, 1965.
21.
MCFARLANE, A.S. Efficient trace-labelling of proteins with iodine. Nature ( London ) 182, 53, 1958.
22.
ATENCIO, A.C., BAILEY, H.R. and REEVE, E.B. Studies on the metabolism and distribution of fibrinogen in young and older rabbits. I. Methods and models. J. Lab. Clin. Med. 66, 1-19, 1965.
23.
COLLEN, D., SCHETZ, J., COCK, F.D., HOLMER, E. and VERSTRAETE, M. Metabolism of antithrombin III ( heparin cofactor ) in man: Effects of venous thrombosis and of heparin administration. Eur. J. Clin. Invest. L 27-35, 1977.
24.
GONMORI, H., TSUKADA, H., TAKADA, M., TANAKA, H., KOBAYASHI, N. and MAEKAWA, T. Vitamin K dependent clotting factors and their inhibitors. - with special reference to prothrombin and antithrombin III. Acta Haematol. Jpn. 44, 1466-1478, 1981.
25.
SAS, G., KREMMER, T., SPIT, B. and PETI),I. Isotopic and immunological half-life determination of antithrombin III in various types of antithrombin III deficiency. Thrombos. Haemostas. 46, 370, 1981.
Vol. 40, No. 3
METABOLISM OF CANINE AT III IN DIC
26.
DE SWART, C.A.M., NIJIMEYER, B., ANDERSSON, L.-O., HOLMER, E., SIXMA, J.J. and BOUMA, B.N. Elimination of intravenously administered radiolabelled antithrombin III and henarin in humans. Thrombos. Haemostas. 52, 66-70, 1984.
27.
KOBAYASHI, N. and TAKEDA, Y. Studies of the effects of estradiol, progesterone, cortisol, thrombophlebitis, and typhoid vaccine on synthesis and catabolism of antithrombin III in the dog. Thrombos. Haemostas. 37, 111-122, 1977.
28.
TAKEDA, Y. and KOBAYASHI, N. Studies of heparin affinity to antithrombin III and other proteins in vitro and in vivo. Thrombos. Haemostas. x685-695, 1977.
29.
KOBAYASHI, N. and TAKEDA, Y. Effects of a large dose of oestradiol on antithrombin III metabolism in male and female dogs. Eur. J. Clin. Invest. L 373-381, 1977.
30.
CHANDRA, S., BANG, N.U. and MARKS, C. Radiolabelled AT III as a probe for the detection of activation of blood coagulation in vivo. Thromb. Res. 9, 9-23, 1976.
31.
REEVE, E.B., LEONARD, B., WENTLAND, S.H. and DAMUS, P. Studies with I-131-labelled antithrombin III in dogs. Thromb. Res. 20, 375-389, 1980.
32.
REEVE, E.B., LEONARD, B. and CARLSON, T. Kinetic studies in vivo of antithrombin III. Ann. N.Y. Acad. Sci. 370,680-694, 1981.
33.
CARLSON, T.H., ATENCIO, A.C. and SIMON, T.L. In vivo behaviour of radioiodinated rabbit antithrombin III. Demonstration of a noncirculating vascular compartment. J. Clin. Invest. 74, 191-199, 1984.
34.
KOIDE, T. Isolation and characterization of antithrombin III from human, porcine and rabbit plasma, and rat serum. J. Biochem. 86, 1841-1850, 1979.
35.
AWBREY, B.J., HOAK, J.C. and OWEN, W.G. Binding of human thrombin to cultured human endothelial cells. J. Biol. Chem. 254, 4092-4095, 1979.
36.
LOLLAR, P. and OWEN, W.G. Clearance of thrombin from circulation in rabbits by high-affinity binding sites on endothelium. Possible role in the inactivation of thrombin by antithrombin III. J. Clin. Invest. 66, 1222-1230, 1980.
37.
BAUER, P.L., MACHOVICH, R., ARANYI, P., BUKI, K.G., CSONKA, E. and HORVATH, I. Mechanism of thrombin binding to endothelial cells. Blood 61, 368-372, 1983.
38.
SOULIER, J.P., GOZIN, D. and LERABLE, J. In vivo attempt to consume antithrombin III by i.v. injection of a thrombin-heparin mixture. Thromb. Res. x255-262, 1984.
39.
HARLAN, M., HARKER, L.A., STRIKER, G.E. and COUNTS, R.B. Endotoxinmediated endothelial injury in vitro. Thrombos. Haemostas. 46, 200, 1981.
305
306
METABOLISM OF CANINE AT III
IN DIC
Vol. 40, No. 3
I.M.and PEREZ, H.D. Biologically active peptides derived 40. GOLDSTEIN, from the fifth component of complementr In: Progress in_Hemostasis and Thrombosis. Vol. 5. T.H. Spaet ( Ed.) New York, London, Toronto, Sydney, San Francisco : Grune & Stratton, 1980, pp. 41-79. 41.
YAMADA, O., MOLDOW, C.F., SACKS,T., CRADDOCK, P.R., BOOGAERTS, M.A. and JACOB. H.S. Deleterious effects of endotoxin on cultured endothelial cells: An in vitro model of vascular injury. Inflammation L 115-126, 1981.
42.
MULLER-BERGHAUS, G. Pathophysiology of generalized intravascular coagulation. Sem. Thromb. Hemostas. L 209-246, 1977.
43.
COLUCCI, M., BALCONI, G., LORENZET, R., PIETRA, A., LOCATI, D., DONATI. M.B. and SEMERARO. N. Cultured human endothelial cells generate tissue factor in.response to endotoxin. J. Clin. Invest. /1, 1893-1896, 1983.
44.
PRYTZ, H. and ALLISON, A.C. Tissue thromboplastin activity of isolated human monocytes. Thrombos. Haemostas. 39, 582-591, 1978.
45.
MUHLFELDER, T.W., NIEMETZ, J., KREUTZER, D., BEEBE, D., WARD, P.A. and ROSENFELD, S.I. C5 chemotactic fragment induces leukocyte production of tissue factor activity. A link between complement and coagulation. J. Clin. Invest. 63, 147-150, 1979.
46.
LEONARD, B., BIES, R., CARLSON, T. and REEVE, E.B. Further studies of the turnover of dog antithrombin III. Study of I-131-labelled antithrombin protease complexes. Thromb. Res. 30, 165-177, 1983.