Vol.
THROMBOSIS RESEARCH Printed in the United States
9, PP- 9-23,
1976
Pel-gamonPress, Inc.
RADIOLABELLED AT111 AS A PROBE FOR THE DETECTION OF ACTIVATION OF BLOOD COAGULATION -IN VIVO
S. Chandra,* N. U. Bang+ and C. Marks Lilly Laboratory for Clinical Research, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A.
(Received 12.3.1976: in revised form 5.5.1976. Accepted by Editor A.P. Fletcher) ABSTRACT Purified radiolabelled rabbit antithrombin III (ATIII) was used as a.probe for in-vivo activation of blood coagulation. Molecular weight ._._distribuflos _ _ radiolabelled AT111 and its complexes were studied in vitro and in vivo by two techni ues: gel exclusion chromatoeaphynd sofiuiiiiiiecylsulfate 9SDS) gel electrophoresis. The radiolabelled ourified AT111 readily formed radiolabelled complexes with activated procoagulants thrombin and factor Xa in vitro, as evidenced by SDS gel electrophoresis analysis. Gel exclus~n~atography separated AT111 from thrombin-AT111 complexes but not from Xa-AT111 complexes. The mean T-1/2 of AT111 in rabbits was 42 hrs. The molecular weight distribution of infused radiolabelled antithrombin III was analyzed by gel exclusion chromatography and SDS gel electrophoresis. Circulating 1261-labelled AT111 displayed an apparent molecular weight of 30,000 by gel exclusion chromatograohy as opposed to the apparent molecular weight of 68,000 for the purified protein. The gel chromatograms obtained at 10 minutes and 24 hrs displayed skewness toward higher molecular weight regions, suggesting comolex formation of part of the circulating radiolabelled protein. Purified and circulating AT111 displayed identical molecular weights of 62,000 Daltons by SDS gel electrophoresis. enty-four hrs after in-vivo administration, approximately 43% of Ts SIlabelled Ammsplayed molecular weights greater than 62,000 .Daltonsby SDS gel electrophoresis, suggesting complex formation between AT111 and activated orocoagulants under baseline physiological conditions. Thus, radiolabelled AT111 may represent a useful probe for the detection of physiological variations of in-vivo thrombin generation. -*Dr. Chandra is presently a Research Scientist with Blood Research Laboratories, American National Red Cross, Bethesda, Maryland, U.S.A. +Reprint requests: Lilly Laboratory for Clinical Research, Wishard Memorial Hospital, 960 Locke Street, Indianapolis, Indiana, 46202, U.S.A. 9
10
1251-LABELLED ATIII AS A PROBE
Vol.9,No.l
INTRODUCTION
It is now firmly established that antithrombin III (ATIII), or heparin cofactor, inactivates several activated procoagulants, in addition to kallikrein and plasmin, through the formation of tight stoichiometric complexes (l-6). Based on these observations, we have attempted to use purified radiolabelled AT111 as a probe for in-viva activation of blood coagulation. We hyoothesized that an increase in the in-vivo alar size of radiolabelled. circulating AT111 would be indicative of -complex formation between this inhibitor protein and activated.orocoagulants and/or additional plasma serine proteases. In this communication we report on experiments to determine molecular weights of infused radiolabelled AT111 by two techniques, gel exclusion chromatography and sodium dodecyl sulfate (SDS) gel electrophoresis. MATERIALS AND METHODS Human olasma was fresh, frozen ACD nlasma. Citrated rabbit plasma was obtained from Pel-Freeze Biologicals, Inc., Box 68, Rogers, Arkansas. Heparin (152 u/ml) from Sigma Chemical Company, St. Louis, Missouri, was used without further purif/cation. Trasylol (10,000 KIU/ml) was from FBA Pharmaceuticals Co., New York, N.Y. Agarose A 15-M was from Bio-Rad Laboratories, Richmond, California; and Sephadex G-150 was from Pharmacia Fine Chemicals, Piscataway, New Jersey. All other chemicals were of the highest purity available cornnercially,usually reagent grade. Human thrombin, purified according to Fenton et al. (7). was a generous gift from Dr. J. W. Fenton, New York State Department omath Laboratories, Albany, New York, Bovine factor X, purified according to Jackson and coworkers (8), was activated by purified factor X activator from Russell's viper venom. Bovine factor Xa was a generous gift from Dr. C. M. Jackson, Washington University School of Medicine, Department of Biological Chemistry, St. Louis, Missouri. Some experiments utilized factor Xa prepared in our laboratory according to Jackson (8). The preparation was identical to that supplied by Dr. Jackson by SDS polyacrylamide gel electrophoresis (see below) and oossessed a specific activity of 588 plasma equivalence units (PEUtmg protein, 1 PEU being the activity of activated factor X demonstrable in 1 ml of standard pooled human plasma in our laboratory, Purification of AT111 AT111 was purified by a simple two-step procedure of heparin agarose affinit chromatography and Sephadex G-150 gel filtration. Heparin was coupled to Bio Ge db A-15m by the cyanogen branide method. Cyanogen bromide activation of the agarose was performed according to Porath et al. (g), and heparin was coupled to the activated agarose essentially accoFdi* to Thaler and Schmer (10). Briefly, 500 ml of activated agarose was washed rapidly under suction in a Buchner funnel with ice-cold, distilled water (about 3-4 liters within 2 min). Two hundred ml of 0.1 M sodium biocarbonate containing 5 g of sodium heparin was sucked into the activated agarose gel to insure a high degree of binding due to the high concentration of heparin within the gel. The resulting heparin agarose gel was washed extensively with 2 M NaCl buffered with 0.02 M imidazole, pH 7.35, until no further heparin was demonstrable in the wash, as estimated by the method of Yin et al. (11). Purification orocedures were carried out at room temperature. One hundrermr of heparin agarose gel was packed and equilibrated with 0.15 M NaCl, 0.02 M imidazole, DH 7.35 (standard buffered saline), in a 2.5 X 3D-cm siliconized glass column. One
vol.9,No.l
125
l-LAUELLED
AT111
AS
A PRODE
11
liter of human or rabbit olasma was applied to the column. The column was washed with standard buffered saline until an O.D. 280 reaging of 0 was achieved. The column was further washed with a linear gradient of 400 ml each of 0.02 M imidazole buffered NaCl (pH 7.35) of 0.2 M and 0.6 M. The protein eluted during the gradient was discarded, AT111 was eluted with 2 M NaCl, 0.02 M imidazole, pH 7.35. A sharp symmetrical orotein peak was regularly obtained with this step. The protein in the peak was pooled and concentrated through dtrlysis against PEG 4,000. The concentrated protein was dialyzed against standard buffered saline. The concentrated dialyzed material was anplied to a Sephadex G-150, 2.5 X 30-cm siliconized glass column. Equilibration and elution of the protein was accomplished with standard buffered saline. The major symnetrical protein peak was pooled, concentrated against PEG, and dialyzed against standard buffered saline. The purified concentrated oreparations were stored either at +5OC or at -700C after increasing the NaCl concentration to 0.5 M. The purification factor regularly achieved was 1,000 and the recovery, 40%. The resultant purified preparations possessed specific activities of l2.8to ti.2plasma equivalence units (PEU)/mg protein, 1 PEU being the AT111 activity demonstrable in 1 ml of normal pooled human plasma from our laboratory. Radiolabelling of AT111 AT111 was labelled with 125 I, using the chloramine-T technique, according to Greenwood et al. (12) limiting the incubation time before adding the reducing agent The resultant preparation incorporated less than 0.5 atoms to less thzrseconds. of iodine/molecule of ATIII. Analytical Gel Exclusion Chromatography Senhadex G-150 was poured over a bed of 0.5-cm siliconized glass beads into 2.5 X lOO-cm siliconized glass columns, as proposed by Sachs and Painter (13). The buffer used for equilibration and elution was 0.02 M imidazole, 0.3 M NaCl, pH 7.35, containing 0.1% SDS. Determination of AT111 Molecular Weight by Gel Exclusion Chromatography and Activity Measurements Gel exclusion chromatography in these experiments were performed on Sephadex G-150 in a 2.5 X lOO-cm siliconized glass column which did not contain glass beads. The column was equilibrated and eluted with 0.02 M imidazole, 0.3 M NaCl, pH 7.35. Five ml of titrated rabbit plasma was chromatographed and effluent fractions assayed for AT111 activity essentially according to Yin et al. (11). Aliquots (0.5 ml) of column effluent fractions were incubated at 3nCfor 5 minutes with 0.24 PEU of factor Xa in a 0.5-ml volume of 0.02 M tris maleate buffer, pH 7.5, containing 1% bovine serum albumin and 0.008 units of heparin. Following 5 minutes' preincubation, the mixture was added to O.5-ml standard dialyzed bovine plasma, cephalin and CaC12 and the clotting time recorded in the assay of Yin et al. (11). Clotting times were converted to AT111 PEU using a calibration curve prepared from several dilutions of purified rabbit AT111 of known potency preincubated at 37OC for 5 minutes with 0.24 PEU of factor Xa, 0.008 units of heparin under assay conditions identical to those used for column fractions. SDS Polyacrylamide Gel Electronhoresis In -in-vitro experiments the method of Laemli (14) was In-Vitro EXDerimetItS. 1 ide). To assess the degree of complex formation between AT111 used 13% d 8% and activi:ed priELig?ants, radiolabelled AT111 was employed. After completion of electrophoresis, staining and electrical destaining, the gels were cut into segments according to the band oatterns. The radioactivity of each segment was expressed as percentage of total gel radioactivity. In-Viva Experiments.
SDS gel electrophoresis was done according to Fairbanks
12
1251-LA3ELLED ATIII AS A PROBE
Vol.9,No.l
et al. (15) in 1% SDS and in 5% polyacrylamide. After electrophoresis, the gels wereinediately placed in fixative (25% isopropyl alcohol, 10% acetic acid) and left overnight. The fixed gels were placed in a gel-slicing device manufactured in our workshops and cut in 2-m slices. The slices were counted for radioactivity. To estimate molecular weights by gel exclusion chromatography and SDS gel electrophoresis, Blue Dextran, Aldolase, Catalase, Ovalbumin, Chymotrypsinogen A and Ribonuclease A (Pharmacia Fine Chemicals, Piscataway, N.J.) were utilized as calibration reference standards. Protein Determinations Protein was quantified using OD absorption and assuming extinction coefficients of 6 for AT111 (16), 19.5 for thrombfn8'(17)and 8 for factor Xa (18). Animal Experiments Male and female albino rabbits weighing 2-3 kg were used in all experiments. Radiolabelled rabbit AT111 was injected i.v. and blood samples withdrawn at appropriate time intervals. Several precautions were taken to guard against minimal activation of blood coagulation during blood drawing and the subsequent handling of the sample. The rabbit's ear was shaved and hyperemia produced by rubbing the ear with xylene. A 19-gauge, lB-bore needle, the inner surface of which was electropolished (Becton-Dickinson, Rutherford, N.J.) was inserted into the marginal ear vein. With this technique blood flowed freely and rapidly, and 5 cc of blood was regularly obtained within 20-30 seconds. Siliconized glassware was used throughout. The anticoagulant used throughout the experiments (1 part anticoagulant, 9 parts blood) was 3.8% trisodium citrate and 10% benzamidine. RESULTS Human and rabbit radiolabelled AT111 readily formed complexes with activated procoagulants, as illustrated in the series of SDS polyacrvlamide gel electrophoresis oatterns depicted in Figure 1. Gel No. 1 is radiolabelled AT111 (1.74 mg/ml in standard buffered saline; molecular weight: 62,000). Gel No. 2 is 0.246 mg/ml of purified human thrombin in the same buffer (molecular weight: 38,500). Gel No. 3 depicts a mixture of 174 Pg AT111 and 24.6 Pg thrombin incubated in 0.2 ml standard buffered saline for 5 min in the presence of 10 u of heparin. The molar ratio of AT111 to thrombin is 4.4 to 1. The electrophoretic pattern shows higher molecular weight complexes: Cl (molecularweiqht: 100,000) and faint secondary complexes, C2 and C3). In this experiment the excised band representing residual uncomplexed AT111 contained 82% of the total gel radioactivity, and the excised bands representing Cl, C , and C3 accounted for 16%. Based on the molar ratio of enzyme/inhibitor in the m sxture, a maximum of 22% of available AT111 would be converted to radiolabelled complexes, orovided all available thrombin was inactivated through complex formation. Thus, AT111 appeared to readily form radiolabelled complexes with thrombin. Gel No. 4 is a preparation of purified activated factor X (0.435 mg/ml standard buffered saline) consistin of equal parts of Xa alpha and Xa beta (molecular weight: 44,000 and 41,0003 . As previously described by Fujikawa et al. (19), the activation of factor X by Russell's viper venom can result in two fo?&%f activated enzyme, Xa alpha and Xa beta. In the Xa beta molecule, a 3,000 Dalton peptide is cleaved from the carboxyl terminal end of the heavy chain resulting in no loss of-biological activity. Gel No. 5 represents a mixture of 174 ug of 1251-labelled AT111 incubated with 43.5 pg of factor Xa in 0.2 ml standard buffered saline for 5 min in the Note the presence of 10 u of heparin. The molar ratio of AT111 to Xa is 2.8. apoearance of several higher molecular weight complexes, primary Cl and C2 (molecular weights: 108,000 and 103,000) and secondary complexes C3 and C4. In this exoeriment the excised band of residual uncomplexed AT111 represented 68% of the total gel
Vol.9,No.l
125 I-LABELLED
AT111
AS A PROBE
13
radioactfvity and the excised bands representing Cl. C2, C3 and C4 accounted for 28%. Based on the molar ratio of enzyme/inhibitor in the mixture, a maximum of 35% of available AT111 would be converted to radiolabelled complexes, provided all available Xa was inactivated through complex formation. Thus, AT111 appeared to readily form radiolabelled comolexes with factor Xa.
4
AT III
t
Xb
AT Ill +t
FIG.
5
AT III+Xo
1
SDS gel electronhoresis patterns of unreduced proteins and protein ourified thrombin; mixtures. Gel No. 1: ATIII; Gel No, 2: Gel No. 3: mixture of AT111 and thrombin; Gel No. 4: factor Xa; Gel No. 5: mixture of factor Xa and ATIII. Fiqure 2 summarizes experiments to determine the biological half-life of radiolabelled AT111 in rabbits. The mean T-1/2 was 42 hr. The data points represent means and standard deviations for 10 different rabbit exaeriments utilizing two different batches of radiolabelled rabbit ATIII. When we attemoted to determine the molecular weight distribution of radiolabelled AT111 by gel exclusion chromatography on Sephadex G-150, we encountered unexpected difficulties. Surorisingly, qel exclusion chromatography did not distinguish between AT111 and Xa-AT111 complexes. When human or rabbit radiolabelled AT111 was incubated with nurified factor Xa in molar excess and the mixture chromatographed, the effluent nattern was indistinguishable from that of ourified AT111 (Figure 3). In contrast, gel exclusion chromatography readily distinguished between AT111 and ATIII-thrombin complexes. In Figure 4 the stippled line represents the effluent pattern of Durified radiolabelled rabbit ATIII. The position of the peak corresponds to a molecular weight of 68,000 Daltons. The solid tracing represents the effluent pattern of a mixture of purified radiolabelled AT111 and thrunbin in molar excess with a neak consistent with a molecular weight of 104,000, in good agreement with a comolex of one molecule of AT111 and one molecule of thrombin. However, AT111 in vivo displays a very different effluent pattern. The heavy solid tracing in Figure-represents the pattern of rabbit plasma AT111 from a
Vol. 9,No. 1
=:I-,LABELLED AT111 AS A PROBE
14
T
OF RABBIT
AT III
FIG. 2 Biological half-life (mean t SD) for 1 51-labelled AT111 infused into rabbits.
SEPHADEX G-150 GEL EXCLUSION CHROMATOGRAPHY
.
,
. . ........ &
110
+AT
m
LTUI
100
-
90
cpm x
30
lo
00
110 I30
150
II0
100
ELUTION VOLULlE (ml) ---
0
10
a0
30
40
FRACTION
50
PurY*d R&bit ATrn PurnbdIabbUATm+nuombla
60
NO.
FIG. 3 Gel exclusion chromatograms of 1251labelled AT111 (150 ~g of protein applied to the column) and of a mixture of 150 ~g of AT111 and 300 ug of activated factor X preincubated for 60 min before chromatography.
Gel exclusion chromate rams: (1) purified rabbit AT111 150 wg aoplied to the column); (2) 12I I-labelled purified rabbit AT111 (150 ~g preincubated with 325 ug of thrombin for 60 min prior to chromatography); (3) rabbit plasma 125Ilabelled AT111 from a blood sample obtained 10 min; and (4) a blood sample obtained 24 hr after injection of the radiolabelled material.
125x-LA.:ELLED A:PIII
AS A PZO~E
1'
snmnlc withdrawn lo twin and the stinplcd tracinrl, the pattern of a smplf! ol~taincd after injection of the radiolahelled orotein. In both samoles the major oeak of radioactivity is consistent with a molecular weight of 3n,%lO naltons; however. both tracinqs show considerable skewness to the left.
24 hrs
Similar effluent natterns were obtained in a rabbit oretreated with lr),OOqu of Trasylol/kq to block nlasma serine nroteases. klhen 1251-labelled AT111 was admixed ----_ in vitro with rabbit olasma containing lqflu of Trasvlol/ml and the mixture chromatogranhed on G-153, the radiolahel emerqed as a sharp symmetrical oeak consistent with a molecular weight of 31,Oqr)but displaved no skewness toward higher molecular weight reqions as observed for circulating radiolabelled ATIII. In an attemot to investigate these discreoancies between the molecular weiqhts of nurified AT111 and AT111 circulatino in vivo, we conducted the followinq exoeriment. Radiolabelled AT111 was injected ztorabbit. Thirty min later, the rabbit was exsanquinated. The yield was fX ml of plasma, which was anplied to a henarin aqarose column. The nonadsorbed nlasma radioactivity coming through at an ionic strenrlthof 1.15 Y, and the radioactivitv elutinq after aonlying 2.0 M NaCl to the column were analyzed seoarately b.yagarose gel chromatography.
FIG. 5 Gel exclusion chromatograms of ourified 1251-lahelled rabbit AT111 and rabbit plasma AT111 not adsorbed and adsorbed onto heparin agarose.
Fiqure 5 is a comnosite of three gel exclusion chromatograms. The solid line is the effluent nattern of an aliouot of the radiolabelled Durified rabbit AT111 utilized for this oarticular exneriment. The dotted line represents that nart of the infused circulating AT111 which upon chromatography came through under ionic strength conditions of 3.15 !1with the bulk of the plasma proteins, i.e., AT111 not adsorbed onto immobilized heparin. The stippled line is the effluent pattern for the radiolabelled nrotein desorbed from the henarin agarose qel with 2.0 !4 NaCl. The nonadThe oeak for this sorbed plasma ATI. behaves exactly like circulating ATIII. nonadsorbed orotein is consistent with a molecular weioht of 39,000; and, in analogy the effluent pattern of this material shows significant with circulating ATIII, skewness to the left. The reisolated rabbit ATIII, on the other hand, displays an effluent pattern which is suoerimoosable on the effluent nattern of the original nurified oreoaration. Roth materials disnlay an apparent molecular weight of 69,OOr) by this technique.
16
l%-LABELLED
SEPHADEX
G-150
AT111
GEL
AS .A PROBE
EXCLUSION
Vol.9,No.l
CHROhtATOGRAPHY
cpm x 10-3
io
do
9’0 ii0 ELUTION
50
VOLUME
lill
li0
(ml)
-
Rabbit
Plasma
ATIII
-
Rabbit
Plasma
AT III + Thrombtn
FIG. 6 Gel exclusion chromatograms of 12%-labelled rabbit plasma AT111 from a blood samnle obtained 10 min after the injection of the radiolabelled nrotein and from the same olasma sample following heat defibrination and treatment with 100 u of thrombin.
Circulating radiolabelled AT111 with an aoparent molecular weight of 30,000 proved fully caoable of complexing with thrombin, as evidenced by the qel exclusion chromatograms disolayed in Figure 6. In this experiment, AT111 was injected into a rabbit. A plasma samplewas obtained 10 min after the administration of the radiolabelled nrotein and heat-defibrinated at 56OC for 5 minutes. An aliquot of the heat-defibrinated sample was incubated with 100 u of thrombin. The plasma sample again displays an effluent pattern consistent with a molecular weight of 30,000, and the thrombin-treated plasma samole displays a peak consistent with a molecular weiqht of 65,Or)D, in qood agreement with a complex consisting of one molecule of AT111 (molecular weiqht: 30,000) and one molecule of thrombin. To further investigate whether the anomalous gel filtration behavior of radiolabelled AT111 in nlasma could be artifactual, we determined the molecular weight of native circulating rabbit AT111 by gel exclusion chromatography and AT111 activity measurements on effluent fractions. The gel exclusion chromatogram in Figure 7 shows AT111 activity to elute in a sharp, symmetrical peak. The effluent position for this peak for this column is consistent with a molecular weight of 66,000 for circulating rabbit ATIII. Thus, there is a clear discrepancv between molecular weiqht estimates of native circulating rabbit AT111 hv activity measurements and by radioactivity measurements on effluent fractions containinq nurified radiolabelled ATIII.
An alternative approach to molecular weight estimations of radiolabelled ATIII in the circulation is SDS qel electrophoresis. The three tracings depicted in Fiqure 8 reoresent the distribution of radioactivity over the lenath Of SDS gels after electrophoresis of purified, radiolabelled AT111 (light solid line). and the distribution for radiolahelled AT111 added to plasma in vitro (heavy solid line), both radiolabels displayinq an identical miqration CGamwith a molecular weiqht of 62,000 Daltons. The stippled line represents a distribution of radio-
iol_.y,r\T9.1
12:i--.LA-:;ELLED ATIII AS A P1?03E
m
0.5-
I
0.4-
ATUl
17
ACTlVlTY 0.3-
PEU/ml 0.2 -
O.l-
EFFLUENTVOLUME
FIG.
(ml)
7
G-150 gel exclusion chromatograohy of rabbit plasma showinq the effluent position of AT111 activity. Five ml of titrated rabbit plasma was chromatographed on a 2.5 X lOO-cm column. The effluent oeak of AT111 activity corresponds to a molecular weight of 66,000 Oaltons for this orotein according to the calibration of this column with marker proteins. SDSGEL
ELECTROPHORESB
(5% unreduced)
25
15
5
4 cpm x 10-3 3
1
A
/+J_ I
I\
/
o-
0
10
::.
30
I
30
MIGRATION
-
Purified
R&bit
-
Rnbblt
----
Rnbblt Phsma
Plasma
FIG.
40
50
(mm)
AT III AT Ill AT ,U + Thrombt,,
a
The distribution of radioactivity over the length of 5% SOS gels of Durified rabbit ATIII, rabbit plasma ATIII, and defibrinated rabbit plasma AT111 treated with 50 u of thrombin.
12%-~Ac~~~~~
18
ATIII
Vol.9,No.l
AS A PROBE
activitv for thromhin-treated rahbit olasma containinn radiolabelled AT111 showinrl tw0 neaks of radioactivity--one comioratinq with purified AT111 at 62,000 Daltons and the second ncak representinq thromhin-antithromhin complexes of molecular weights 0f iw,ono. The data summarized in Table I indicates that it is possible through separation of the radiolahel bv SDS qel electrophoresis to demonstrate complex formation between 4TIII and activated plasma serine 0roteases under baseline physiological in-vivo conditions. The table compares th;?distribution of radioactivity over thesh of SnS nels for four different preparations--purified ATIII, AT111 added to plasma in vitro, and AT111 from plasma samples obtained 3r)min and 24 hrs after injection Otthedinl~helled protein into six rahhits. TABLE I rlolecular Veiqht Distribution of 1251-Labelled ATIII by SDS Gel Electrophoresis (5% Unreduced) Percent Total Gel Radioactivity 62,000 Dalton Peak
MW ~62,000
4.2
82.2
8.6
10.9 14.8
80.1 78.1
23.5 f 3.2
66.5 1 4.1
10.0 + 1.5
44.8 f 6.8
47.2 f 5.8
8.0 f 1.1
YW >62,OOD Pure AT111
ATT11 added to
nlasma .in vitro: Exn. 1 Exn. 2
circulating (3n min after i.v. injection)*
YTIII
AT111 circulating
(24 hrs after i.v. injection)*
*Mean + SD (6 experiments) The radioactivity contained in the oortions of the gels representing molecular weiqits greater than 62,r)OO, the radioactivity in the 62,000 Dalton peaks, and the radioactivity contained in nortions of the gel representing molecular weights of less tClan62,ODO are exoressed as percent of the total gel radioactivity. Eighty-two percent of the ourified AT111 is in the 62,000 Dalton peak with only 4 percent in hiqher molecular weight regions. There is a slight increase in higher molecular weight material upon addition of radiolahelled AT111 to plasma in vitro. We hynothesize that this is due to the high viscosity of the sampl~a~ossly overloading t+e qel with protein. The majority of circulating ,ATIII is still contained in the 62,9OO Dalton peak 30 min after i.v. injectioQ of the radiolabelled protein. 'lowever, after 24 hrs there is sharp decrease in the radioactivity in the 62,000 Dalton neak with a concomitant increase in higher molecular weight material with a qean of 45 oercent of the total gel radiolabel exhibiting molecular weights of q-eater than 62,nVl Daltons.
Vol.
9,No.l
1251-LABELLED
A%111
Drsrussm
-_-L--e.-
AS A PROBE
19
-
The demonstration of raoid turnover rates of many clotting factors led several authors in the lq4D's and '50's to the belief that blood coagulation and fibrin deoosition are continuous ongoing nrocesses. The concent of a dynamic equilibrium between clotting and fibrinolysis has won sunporters, since such a hynothesis would seem to fit a number of experimental observations (2D,21). 4 critical review by Hjort and Hasselback (22) correctly concluded that the tools to definitively Drove such concepts were unavailable at that time. !dithin the last five years the develonment of new biochemical approaches toward the study of in-vivo blood coaqulation has set the stage for a reooening of an investigation of i%exiditv of the Duquid-4strup theories of continuous low-grade activation of blood coagulation under haseline physiological conditions. Fletcher et al. (23), having develoned the techninue of gel exclusion chromatoqranhy of olasafibrinogen, were able to detect with this techniqtrethe nresence of soluble macromolecular fibrin comnlexes in the nlasma of a wide variety of natients with thrombotic states. Soluble fihrin comnlexes are thought to be an indirect reflection of thrombin generation, since one essential comnonent in the generation of soluble fibrin complexes is fibrin monomer.
A comnuter nroqram developed by nlkiaersiq et al. (24), using chromatograohic nlate-theorv annlvsis, made it norsible to arrivi?ar-a semi-quantitative estimate of the contents of macromolecular soluhle fibrin complexes, fibrinogen, and fibrinogen. fibrin snlit products from poorly resolved chromatoqranhic natterns. Utilizinq this annroach, Fletcher and Alkiaersiq have estimated that as much as 2% of circulating olasma fibrinonen may be circulating as macromolecular soluble fibrin comnlexes in normal individuals under nhysiological conditions. Yossel et al. (25), havino develoned an elegant, highly specific radioimnunoassay for fib??%entide 4, similarly were able to detect small, but significant, quantities of this material in nlasma from normal peonle, stronqly suggesting that minute, but significant, quantities of thrombin mav be generated normally. In the nresent series of exneriqents we have utilized a different anoroach to investiqate this orohlem. The observations in l?osenberg's laboratory (26). demonstrating that inactivation of thrombin and other activated arocoagulants results in the formation of stable, stoichiometric comnlexes, oromoted us to investigate whether such comnlexes could be demonstrated -in vlvo. Isle concluded from in-vitro experiments that radiolabelled AT111 was suitable for SIP.?-FM. in-vivo studies,= we demonstrated that the ourified orotein radiolabelled under mild conditions was fully capable of forming labelled complexes with thromhin and factor Xa. We further concluded that the mean biological half-life of the orotein of 42 hrs made it suitable for -in-vivo studies. The technique of eel exclusion chromatogranhy for the determination of the molecular weiaht of radiolabelled antithrombin and its comolexes in vitro and in vivo nroved to have unexpected difficulties. Whereas, thrombin_Amcomplexes .____-were readilv senarable bv nel exclusion chromatoqranhy, complexes formed between factor Xa and 4TIII were not. The chronatographic behavior of uncomolexed AT111 and ST111 comolexes with factor Xa was identical on Senhadex G-150. Since radiolabelled comnlexes between AT111 and factor Xa were readily demonsand/or factor Xa undergo trable bv SDS qel electronhoresis, we conclude that !TIII substantial conformational changes during their interaction producing a comolex with an anomalous diffusion constant. Furthermore, circulating radiolahelled AT111 disnlayed an apparent molecular weight of 30,OOD bv nel exclusion chromatography, as opposed to the apparent
20
1251-LABELLED
AT111
AS A PROBE
Vol.9,No.l
moTecular weight of 613,000 of the purified protein. This phenomenon could not be attributed to proteolytic degradation of circulating ATIII, since its apparent molecular weight was 30,000 in an animal pretreated with massive quantities of Trasylol, a broad spectrum serine protease inhibitor. The aoparent molecular weight shift from 68,000 to 30,000 was also observed when this radiolabelled ourified protein was added in vitro to rabbit plasma containing Trasylol. Upon reisolation of the radiolabmed protein from plasma by heparin agarose chromatography, it reverted to the apoarent molecular weight of 68,000 of the purified protein, These observations suggest that radiolabelled AT111 is a molecule of considerable plasticity which readily changes its shape and, hence, its diffusion constant. We hypothesize that this peculair anomalous diffusion constant of radiolabelled AT111 in plasma is the consequence of subtle conformational changes of the protein introduced during iodination. This contention is supported by the experiment depicted in Figure 7 illustrating that the molecular weight of native AT111 in normal rabbit plasma by gel exclusion chromatography and activity measurements is 66,000 Daltons. Additional data from our laboratory (unpublished) in which the gel exclusion chromatographic behavior of human plasma AT111 was studied using imnunochemical assays for quantification demonstrated this protein to elute with a major neak consistent with a molecular weight of 67,000. Similarly, Rosenberg and Damus (1). in one of the steps of their purification for human ATIII, noted that this protein cochromatographed with albumin on Sephadex G-200. The apparent change in molecular weight of radiolabelled ATIII, after its introduction into the circulation, had no anparent effect on its biological activity, since gel exclusion chromatography of a plasma sample obtained from a rabbit after administration of the radiolabelled nrotein upon treatment with thrombin displayed a two-peak pattern, one peak consistent with a molecular weight of 30,OOD and a second peak at a molecular weight of 65,000 Daltons, in good agreement with a thrombin AT111 comolex, assuming a molecular weight of 30,000 for ATIII. Whereas, the gel chromatograms for AT111 added to rabbit plasma in vitro were oerfectly symnetrical, the gel chromatograms of circulating ATIIfwere consistently asymnetrical, displaying skewness toward higher molecular weight regions. These observations provide further evidence for biological activity of radiolabelled ATIII, suggesting that the radiolabelled protein does indeed form complexes with activated serine proteases in vivo. The technique of SDS gel electrophoresis orovided more reliable molecular weight estimates for AT111 and its complexes in plasma, since purified AT111 and AT111 in plasma displayed identical migrations comoatible with a molecular weight of the nrotein of 62,000 Daltons and since thrombin-antithrombin complexes in olasma by SDS gel electrophoresis appeared to possess a molecular weight of 100,000 Daltons in exact agreement with the estimated molecular weight of the comolex formed through the interaction between purified AT111 and purified thrombin. The demonstration by SDS gel electrophoresis of a shift within 24 hrs of 45% of the radiolabel of circulating AT111 to molecular sizes greater than 62,000 Daltons can, therefore, be viewed as good evidence for complex formation between the circulating radiolabelled protein and activated olasma serine proteases. Since AT111 has been reported to inhibit kallikrein and plasmin, in addition to activated. procoagulants. the question arises whether the AT111 complexes, which seem to arise under the conditions of our experiments, represent complexes between AT111 and activated nrocoagulants, or complexes between AT111 and kallikrein or plasmin. In other words, can the results of these experiments be interpreted to reflect continuous activation of blood coagulation in the normal rabbit? The nrotein concentrations in human plasma of the three major zymogens under consideration. nrothromhin, nlasminogen and prekallikrein, are very similar.
Vol.9,No.l
1251-LABELLED
AT111
AS A PROBE
21
Calculated from maximal thrombin activity generated from 1 ml of human plasma (140 NIH u), the purity of tiiethrombin preparations prepared by Fenton et al. (7). and assuming molecular weights for human prothrombin and'thrombin of;rb;OOO and 36,500, respectively, human plasma contains approximately 95 vg/ml prothrombin (27). Based on our normal laboratory values of 2-3 CTA u/ml plasma and the specific activity for the most highly ourified plasminogen of 25-30 CTA u/ml (28), human plasma contains approximately 100 ug/ml plasminogen. The published mean value for human plasma orekallikrein is 103 vg/ml (29). However, in exoeriments to be reported elsewhere (30) we have quantified the radioactive comnlexes formed through the interaction between radiolabelled AT111 and purified thrombin, factor Xa, and olasmin. Whereas, thrombin and factor Xa will react with greater than 90% of AT111 to form radiolabelled complexes, no greater than 5% of AT111 formed radiolabelled complexes with plasmin in several plasmin-AT111 molar ratios. Recent kinetical data similarly demonstrated that antithrombin is a very slow and ineffective inhibitor of kallikrein, as CornDaredto its capability to inhibit thrombin and factor Xa (31). The substantially qreater caoability of AT111 to form comolexes with thrombin and activated factor X than with plasmin and kallikrein, therefore, makes activation of blood coaqulation the most likely exnlanation for our experimental results at the present time.
Ne conclude that radiolabelled AT111 may represent a useful probe for the detection of ohysiological variations of activation of blood coagulation and in-vivo thrombin generation. --
The authors are indebted to Drs. Craig M. Jackson and John W. Fenton for their qifts of nurified factor Xa and thrombin, their advice and criticism and to Mr. Robert 0. Heidenreich for his skillful technical assistance. REFERENCES 1.
ROSENBERG, R.D. and DAMUS, P.S. The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem., 248, 6490, 1973.
2.
HIGHSMITH, R.F. and ROSENBERG, R.D. The inhibition of human plasmin by human antithrombin-heparin cofactor. J. Biol. Chem., 249, 4335, 1974.
3.
DAMUS, P.S., HICK, M. and ROSENBERG, R.D. Anticoagulant action of heparin. Nature, 246, 355, 1473.
4.
ROSENBERG, R.D. The effect of heparin on factor XIa and plasmin. Thromb. Diath. ~_rrh_, 33, 51, 1974.
5.
LAHIRI, R. ROSENBERG, R.D., TALAMO, R.L., BAGDASARIAN, A. and COLMAN, R.W. an inhibitor of human plasma kallikrein. Fed. Proc., 33, Antithrombin III: 642, 1974.
6.
YIN, E.T. Identity of plasma activated factor X inhibitor with antithrombin III and heparin cofactor. J. Biol. Chem., 246, 3712, 1971.
7.
FENTON, J.W. II, CAMPBELL, W.P., HARRINGTON, J.C. and MILLER, K.D. Large-scale nreparation and preliminary characterization of human thromhin. Riochim. Riophys. Acta. 229, 26. 1971.
1251-LABELLED
22
AT111
AS A PROBE
Vol.9,No.l
8.
JqCKSON, C.!l.,JOHNSON, T.F. and HANAHAN, D.J. Studies on bovine factor X. Large-scale purification of the bovine plasma protein possessing factor X I. activity, Biochemistry_, 7, 4492, 1968.
9.
PQRATH, J.. AXEN, R. and ERNBACK, S. Clinical coupling of prote'ins to agarose. Nature. 215, 1491. 1967.
10.
THALER. E. and SCH?ER, 6. A simple two-step isolation orocedure for human and bovine antithrombin II/III (heoarin cofactor): a comparison of two methods. Brit. J. Haematol., (ii pre&), 1976.
11.
YIN, E.T., WESSLER, S. and BUTLER, J.V. Plasma heparin: a unique, practical, submicrogram-sensitive assay. J. Lab. Clin. Med., 81, 298, 1973.
12.
GREENWOOD, F.C., HUNTER, W.M. and GLDVER, J.S. The oreparation of 1311-labelled human growth hormone of high specific radioactivity. Biochem. J., 89, 114, 1963.
13.
SACHS. D.H. and PAINTER, E. Imoroved flow rates with porous sephadex gels. Science, 175, 781. 1972.
14.
LAEMLI, M.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680, 1970.
15.
FAIRSANKS, G., STECK, T.L. and WALLACH, D.F.H. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10, 2606, 1971
16.
4NDERSSON. M.M., BORG, H. and ANDERSSON, L.-O. Purification of antithrombin III by affinity chromatography. Thromb. Res., 5, 439, 1974.
17.
WINZOR, D.G. and SCHERAGA, H.A. Studies of chemically reacting systems on Molecular weights of monomers in rapid association equilibrium. Sephadex. II. J. Physical Chem., 68, 338, 1964.
15.
JACKSON, C.M. Studies on bovine factor X:
19.
FUJIKALIA. K., LEG4Z, M.E. and DAVIE, E.W. Bovine factor X (Stuart factor): mechanism of activation by a protein from Russell's viper venom. Biochemistry, 11, 4892, 1972.
20.
!lUGUID, J.B.
21.
ASTRUP, T. In: Connective Tissue Thrombosis and Atherosclerosis. New York and London, Academic Press, 1959, D. 223.
22.
HJORT, P. and H4SSELBACK, R. 4 critical review of the evidence for a continuous hemostasis _in vivo. Thromb. Diath. Haemorrh., 6, 580, 1961.
23.
FLETCHER, A.P., ALKJAERSIG, N., O'BRIEN, J. and TULEVSKI, V.G. Blood hypercoagulability and thrombosis. Trans. Assoc. Amer. Phys., 83, 159, 1970.
24.
4LKJAERSIG, N., ROY, L. and FLETCHER, A. Analysis of gel exclusion chromatographic data by chromatogranhic plate theory analysis: application to plasma fibrinogen chromatogranhy. Thromb. Res., 3, 525, 1973.
25.
NOSSEL, H.L., YUDELMAN. I., CANFIELD. R.E., BUTLER, V.P. JR., SPANONDIS, K., WILNER, G.D. and (IURESHI,G.D. Measurement of fibrinopeptide A in human blood. J. Clin. Invest., 54, 43. 1974.
isolation, purification and partial characterization. Ph.D. Dissertation, University of Washington, Seattle, Washinqton, 1967.
Connective Tissue Thrombosis and Atherosclerosis, New York In: and London, Academic Press, 1959, p. 13.
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1251-LABELLED AT111 AS A PROBE
23
26.
JRDS:;iERG, R.D. Actions and interactions of antithrombin and heparin. New Eng. . ., 292, 146, 1975.
27.
FENTr)N,J. Personal communication, 1975.
25.
SUMMARIA. L., ARZADDN, L., RERNARE, P. and ROBBINS, K.C. Studies on the isolation of the multiole molecular forms of human plasminogen and plasmin by isoelectric focusing methods. J. Biol. Chem., 248, 2934, 1973.
29.
BAGDASARIAN, A., LAHIRI, R., TALAMO, K.C., WONG, P. and COLMAN, R.W. Imnunochemical studies of olasma kallikrein. J. Clin. Invest., 54, 1444, 1974.
30.
CHANDRA, S. and BANG, N.U. Yanuscriot in preparation, 1976.
31.
COLMAN, R. Personal communication, 1975.
THROMBOSIS RESEARCH Printed in the United States
9, PP- 9-23,
1976
Pel-gamonPress, Inc.
RADIOLABELLED AT111 AS A PROBE FOR THE DETECTION OF ACTIVATION OF BLOOD COAGULATION -IN VIVO
S. Chandra,* N. U. Bang+ and C. Marks Lilly Laboratory for Clinical Research, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A.
(Received 12.3.1976: in revised form 5.5.1976. Accepted by Editor A.P. Fletcher) ABSTRACT Purified radiolabelled rabbit antithrombin III (ATIII) was used as a.probe for in-vivo activation of blood coagulation. Molecular weight ._._distribuflos _ _ radiolabelled AT111 and its complexes were studied in vitro and in vivo by two techni ues: gel exclusion chromatoeaphynd sofiuiiiiiiecylsulfate 9SDS) gel electrophoresis. The radiolabelled ourified AT111 readily formed radiolabelled complexes with activated procoagulants thrombin and factor Xa in vitro, as evidenced by SDS gel electrophoresis analysis. Gel exclus~n~atography separated AT111 from thrombin-AT111 complexes but not from Xa-AT111 complexes. The mean T-1/2 of AT111 in rabbits was 42 hrs. The molecular weight distribution of infused radiolabelled antithrombin III was analyzed by gel exclusion chromatography and SDS gel electrophoresis. Circulating 1261-labelled AT111 displayed an apparent molecular weight of 30,000 by gel exclusion chromatograohy as opposed to the apparent molecular weight of 68,000 for the purified protein. The gel chromatograms obtained at 10 minutes and 24 hrs displayed skewness toward higher molecular weight regions, suggesting comolex formation of part of the circulating radiolabelled protein. Purified and circulating AT111 displayed identical molecular weights of 62,000 Daltons by SDS gel electrophoresis. enty-four hrs after in-vivo administration, approximately 43% of Ts SIlabelled Ammsplayed molecular weights greater than 62,000 .Daltonsby SDS gel electrophoresis, suggesting complex formation between AT111 and activated orocoagulants under baseline physiological conditions. Thus, radiolabelled AT111 may represent a useful probe for the detection of physiological variations of in-vivo thrombin generation. -*Dr. Chandra is presently a Research Scientist with Blood Research Laboratories, American National Red Cross, Bethesda, Maryland, U.S.A. +Reprint requests: Lilly Laboratory for Clinical Research, Wishard Memorial Hospital, 960 Locke Street, Indianapolis, Indiana, 46202, U.S.A. 9
10
1251-LABELLED ATIII AS A PROBE
Vol.9,No.l
INTRODUCTION
It is now firmly established that antithrombin III (ATIII), or heparin cofactor, inactivates several activated procoagulants, in addition to kallikrein and plasmin, through the formation of tight stoichiometric complexes (l-6). Based on these observations, we have attempted to use purified radiolabelled AT111 as a probe for in-viva activation of blood coagulation. We hyoothesized that an increase in the in-vivo alar size of radiolabelled. circulating AT111 would be indicative of -complex formation between this inhibitor protein and activated.orocoagulants and/or additional plasma serine proteases. In this communication we report on experiments to determine molecular weights of infused radiolabelled AT111 by two techniques, gel exclusion chromatography and sodium dodecyl sulfate (SDS) gel electrophoresis. MATERIALS AND METHODS Human olasma was fresh, frozen ACD nlasma. Citrated rabbit plasma was obtained from Pel-Freeze Biologicals, Inc., Box 68, Rogers, Arkansas. Heparin (152 u/ml) from Sigma Chemical Company, St. Louis, Missouri, was used without further purif/cation. Trasylol (10,000 KIU/ml) was from FBA Pharmaceuticals Co., New York, N.Y. Agarose A 15-M was from Bio-Rad Laboratories, Richmond, California; and Sephadex G-150 was from Pharmacia Fine Chemicals, Piscataway, New Jersey. All other chemicals were of the highest purity available cornnercially,usually reagent grade. Human thrombin, purified according to Fenton et al. (7). was a generous gift from Dr. J. W. Fenton, New York State Department omath Laboratories, Albany, New York, Bovine factor X, purified according to Jackson and coworkers (8), was activated by purified factor X activator from Russell's viper venom. Bovine factor Xa was a generous gift from Dr. C. M. Jackson, Washington University School of Medicine, Department of Biological Chemistry, St. Louis, Missouri. Some experiments utilized factor Xa prepared in our laboratory according to Jackson (8). The preparation was identical to that supplied by Dr. Jackson by SDS polyacrylamide gel electrophoresis (see below) and oossessed a specific activity of 588 plasma equivalence units (PEUtmg protein, 1 PEU being the activity of activated factor X demonstrable in 1 ml of standard pooled human plasma in our laboratory, Purification of AT111 AT111 was purified by a simple two-step procedure of heparin agarose affinit chromatography and Sephadex G-150 gel filtration. Heparin was coupled to Bio Ge db A-15m by the cyanogen branide method. Cyanogen bromide activation of the agarose was performed according to Porath et al. (g), and heparin was coupled to the activated agarose essentially accoFdi* to Thaler and Schmer (10). Briefly, 500 ml of activated agarose was washed rapidly under suction in a Buchner funnel with ice-cold, distilled water (about 3-4 liters within 2 min). Two hundred ml of 0.1 M sodium biocarbonate containing 5 g of sodium heparin was sucked into the activated agarose gel to insure a high degree of binding due to the high concentration of heparin within the gel. The resulting heparin agarose gel was washed extensively with 2 M NaCl buffered with 0.02 M imidazole, pH 7.35, until no further heparin was demonstrable in the wash, as estimated by the method of Yin et al. (11). Purification orocedures were carried out at room temperature. One hundrermr of heparin agarose gel was packed and equilibrated with 0.15 M NaCl, 0.02 M imidazole, DH 7.35 (standard buffered saline), in a 2.5 X 3D-cm siliconized glass column. One
vol.9,No.l
125
l-LAUELLED
AT111
AS
A PRODE
11
liter of human or rabbit olasma was applied to the column. The column was washed with standard buffered saline until an O.D. 280 reaging of 0 was achieved. The column was further washed with a linear gradient of 400 ml each of 0.02 M imidazole buffered NaCl (pH 7.35) of 0.2 M and 0.6 M. The protein eluted during the gradient was discarded, AT111 was eluted with 2 M NaCl, 0.02 M imidazole, pH 7.35. A sharp symmetrical orotein peak was regularly obtained with this step. The protein in the peak was pooled and concentrated through dtrlysis against PEG 4,000. The concentrated protein was dialyzed against standard buffered saline. The concentrated dialyzed material was anplied to a Sephadex G-150, 2.5 X 30-cm siliconized glass column. Equilibration and elution of the protein was accomplished with standard buffered saline. The major symnetrical protein peak was pooled, concentrated against PEG, and dialyzed against standard buffered saline. The purified concentrated oreparations were stored either at +5OC or at -700C after increasing the NaCl concentration to 0.5 M. The purification factor regularly achieved was 1,000 and the recovery, 40%. The resultant purified preparations possessed specific activities of l2.8to ti.2plasma equivalence units (PEU)/mg protein, 1 PEU being the AT111 activity demonstrable in 1 ml of normal pooled human plasma from our laboratory. Radiolabelling of AT111 AT111 was labelled with 125 I, using the chloramine-T technique, according to Greenwood et al. (12) limiting the incubation time before adding the reducing agent The resultant preparation incorporated less than 0.5 atoms to less thzrseconds. of iodine/molecule of ATIII. Analytical Gel Exclusion Chromatography Senhadex G-150 was poured over a bed of 0.5-cm siliconized glass beads into 2.5 X lOO-cm siliconized glass columns, as proposed by Sachs and Painter (13). The buffer used for equilibration and elution was 0.02 M imidazole, 0.3 M NaCl, pH 7.35, containing 0.1% SDS. Determination of AT111 Molecular Weight by Gel Exclusion Chromatography and Activity Measurements Gel exclusion chromatography in these experiments were performed on Sephadex G-150 in a 2.5 X lOO-cm siliconized glass column which did not contain glass beads. The column was equilibrated and eluted with 0.02 M imidazole, 0.3 M NaCl, pH 7.35. Five ml of titrated rabbit plasma was chromatographed and effluent fractions assayed for AT111 activity essentially according to Yin et al. (11). Aliquots (0.5 ml) of column effluent fractions were incubated at 3nCfor 5 minutes with 0.24 PEU of factor Xa in a 0.5-ml volume of 0.02 M tris maleate buffer, pH 7.5, containing 1% bovine serum albumin and 0.008 units of heparin. Following 5 minutes' preincubation, the mixture was added to O.5-ml standard dialyzed bovine plasma, cephalin and CaC12 and the clotting time recorded in the assay of Yin et al. (11). Clotting times were converted to AT111 PEU using a calibration curve prepared from several dilutions of purified rabbit AT111 of known potency preincubated at 37OC for 5 minutes with 0.24 PEU of factor Xa, 0.008 units of heparin under assay conditions identical to those used for column fractions. SDS Polyacrylamide Gel Electronhoresis In -in-vitro experiments the method of Laemli (14) was In-Vitro EXDerimetItS. 1 ide). To assess the degree of complex formation between AT111 used 13% d 8% and activi:ed priELig?ants, radiolabelled AT111 was employed. After completion of electrophoresis, staining and electrical destaining, the gels were cut into segments according to the band oatterns. The radioactivity of each segment was expressed as percentage of total gel radioactivity. In-Viva Experiments.
SDS gel electrophoresis was done according to Fairbanks
12
1251-LA3ELLED ATIII AS A PROBE
Vol.9,No.l
et al. (15) in 1% SDS and in 5% polyacrylamide. After electrophoresis, the gels wereinediately placed in fixative (25% isopropyl alcohol, 10% acetic acid) and left overnight. The fixed gels were placed in a gel-slicing device manufactured in our workshops and cut in 2-m slices. The slices were counted for radioactivity. To estimate molecular weights by gel exclusion chromatography and SDS gel electrophoresis, Blue Dextran, Aldolase, Catalase, Ovalbumin, Chymotrypsinogen A and Ribonuclease A (Pharmacia Fine Chemicals, Piscataway, N.J.) were utilized as calibration reference standards. Protein Determinations Protein was quantified using OD absorption and assuming extinction coefficients of 6 for AT111 (16), 19.5 for thrombfn8'(17)and 8 for factor Xa (18). Animal Experiments Male and female albino rabbits weighing 2-3 kg were used in all experiments. Radiolabelled rabbit AT111 was injected i.v. and blood samples withdrawn at appropriate time intervals. Several precautions were taken to guard against minimal activation of blood coagulation during blood drawing and the subsequent handling of the sample. The rabbit's ear was shaved and hyperemia produced by rubbing the ear with xylene. A 19-gauge, lB-bore needle, the inner surface of which was electropolished (Becton-Dickinson, Rutherford, N.J.) was inserted into the marginal ear vein. With this technique blood flowed freely and rapidly, and 5 cc of blood was regularly obtained within 20-30 seconds. Siliconized glassware was used throughout. The anticoagulant used throughout the experiments (1 part anticoagulant, 9 parts blood) was 3.8% trisodium citrate and 10% benzamidine. RESULTS Human and rabbit radiolabelled AT111 readily formed complexes with activated procoagulants, as illustrated in the series of SDS polyacrvlamide gel electrophoresis oatterns depicted in Figure 1. Gel No. 1 is radiolabelled AT111 (1.74 mg/ml in standard buffered saline; molecular weight: 62,000). Gel No. 2 is 0.246 mg/ml of purified human thrombin in the same buffer (molecular weight: 38,500). Gel No. 3 depicts a mixture of 174 Pg AT111 and 24.6 Pg thrombin incubated in 0.2 ml standard buffered saline for 5 min in the presence of 10 u of heparin. The molar ratio of AT111 to thrombin is 4.4 to 1. The electrophoretic pattern shows higher molecular weight complexes: Cl (molecularweiqht: 100,000) and faint secondary complexes, C2 and C3). In this experiment the excised band representing residual uncomplexed AT111 contained 82% of the total gel radioactivity, and the excised bands representing Cl, C , and C3 accounted for 16%. Based on the molar ratio of enzyme/inhibitor in the m sxture, a maximum of 22% of available AT111 would be converted to radiolabelled complexes, orovided all available thrombin was inactivated through complex formation. Thus, AT111 appeared to readily form radiolabelled complexes with thrombin. Gel No. 4 is a preparation of purified activated factor X (0.435 mg/ml standard buffered saline) consistin of equal parts of Xa alpha and Xa beta (molecular weight: 44,000 and 41,0003 . As previously described by Fujikawa et al. (19), the activation of factor X by Russell's viper venom can result in two fo?&%f activated enzyme, Xa alpha and Xa beta. In the Xa beta molecule, a 3,000 Dalton peptide is cleaved from the carboxyl terminal end of the heavy chain resulting in no loss of-biological activity. Gel No. 5 represents a mixture of 174 ug of 1251-labelled AT111 incubated with 43.5 pg of factor Xa in 0.2 ml standard buffered saline for 5 min in the Note the presence of 10 u of heparin. The molar ratio of AT111 to Xa is 2.8. apoearance of several higher molecular weight complexes, primary Cl and C2 (molecular weights: 108,000 and 103,000) and secondary complexes C3 and C4. In this exoeriment the excised band of residual uncomplexed AT111 represented 68% of the total gel
Vol.9,No.l
125 I-LABELLED
AT111
AS A PROBE
13
radioactfvity and the excised bands representing Cl. C2, C3 and C4 accounted for 28%. Based on the molar ratio of enzyme/inhibitor in the mixture, a maximum of 35% of available AT111 would be converted to radiolabelled complexes, provided all available Xa was inactivated through complex formation. Thus, AT111 appeared to readily form radiolabelled comolexes with factor Xa.
4
AT III
t
Xb
AT Ill +t
FIG.
5
AT III+Xo
1
SDS gel electronhoresis patterns of unreduced proteins and protein ourified thrombin; mixtures. Gel No. 1: ATIII; Gel No, 2: Gel No. 3: mixture of AT111 and thrombin; Gel No. 4: factor Xa; Gel No. 5: mixture of factor Xa and ATIII. Fiqure 2 summarizes experiments to determine the biological half-life of radiolabelled AT111 in rabbits. The mean T-1/2 was 42 hr. The data points represent means and standard deviations for 10 different rabbit exaeriments utilizing two different batches of radiolabelled rabbit ATIII. When we attemoted to determine the molecular weight distribution of radiolabelled AT111 by gel exclusion chromatography on Sephadex G-150, we encountered unexpected difficulties. Surorisingly, qel exclusion chromatography did not distinguish between AT111 and Xa-AT111 complexes. When human or rabbit radiolabelled AT111 was incubated with nurified factor Xa in molar excess and the mixture chromatographed, the effluent nattern was indistinguishable from that of ourified AT111 (Figure 3). In contrast, gel exclusion chromatography readily distinguished between AT111 and ATIII-thrombin complexes. In Figure 4 the stippled line represents the effluent pattern of Durified radiolabelled rabbit ATIII. The position of the peak corresponds to a molecular weight of 68,000 Daltons. The solid tracing represents the effluent pattern of a mixture of purified radiolabelled AT111 and thrunbin in molar excess with a neak consistent with a molecular weight of 104,000, in good agreement with a comolex of one molecule of AT111 and one molecule of thrombin. However, AT111 in vivo displays a very different effluent pattern. The heavy solid tracing in Figure-represents the pattern of rabbit plasma AT111 from a
Vol. 9,No. 1
=:I-,LABELLED AT111 AS A PROBE
14
T
OF RABBIT
AT III
FIG. 2 Biological half-life (mean t SD) for 1 51-labelled AT111 infused into rabbits.
SEPHADEX G-150 GEL EXCLUSION CHROMATOGRAPHY
.
,
. . ........ &
110
+AT
m
LTUI
100
-
90
cpm x
30
lo
00
110 I30
150
II0
100
ELUTION VOLULlE (ml) ---
0
10
a0
30
40
FRACTION
50
PurY*d R&bit ATrn PurnbdIabbUATm+nuombla
60
NO.
FIG. 3 Gel exclusion chromatograms of 1251labelled AT111 (150 ~g of protein applied to the column) and of a mixture of 150 ~g of AT111 and 300 ug of activated factor X preincubated for 60 min before chromatography.
Gel exclusion chromate rams: (1) purified rabbit AT111 150 wg aoplied to the column); (2) 12I I-labelled purified rabbit AT111 (150 ~g preincubated with 325 ug of thrombin for 60 min prior to chromatography); (3) rabbit plasma 125Ilabelled AT111 from a blood sample obtained 10 min; and (4) a blood sample obtained 24 hr after injection of the radiolabelled material.
125x-LA.:ELLED A:PIII
AS A PZO~E
1'
snmnlc withdrawn lo twin and the stinplcd tracinrl, the pattern of a smplf! ol~taincd after injection of the radiolahelled orotein. In both samoles the major oeak of radioactivity is consistent with a molecular weight of 3n,%lO naltons; however. both tracinqs show considerable skewness to the left.
24 hrs
Similar effluent natterns were obtained in a rabbit oretreated with lr),OOqu of Trasylol/kq to block nlasma serine nroteases. klhen 1251-labelled AT111 was admixed ----_ in vitro with rabbit olasma containing lqflu of Trasvlol/ml and the mixture chromatogranhed on G-153, the radiolahel emerqed as a sharp symmetrical oeak consistent with a molecular weight of 31,Oqr)but displaved no skewness toward higher molecular weight reqions as observed for circulating radiolabelled ATIII. In an attemot to investigate these discreoancies between the molecular weiqhts of nurified AT111 and AT111 circulatino in vivo, we conducted the followinq exoeriment. Radiolabelled AT111 was injected ztorabbit. Thirty min later, the rabbit was exsanquinated. The yield was fX ml of plasma, which was anplied to a henarin aqarose column. The nonadsorbed nlasma radioactivity coming through at an ionic strenrlthof 1.15 Y, and the radioactivitv elutinq after aonlying 2.0 M NaCl to the column were analyzed seoarately b.yagarose gel chromatography.
FIG. 5 Gel exclusion chromatograms of ourified 1251-lahelled rabbit AT111 and rabbit plasma AT111 not adsorbed and adsorbed onto heparin agarose.
Fiqure 5 is a comnosite of three gel exclusion chromatograms. The solid line is the effluent nattern of an aliouot of the radiolabelled Durified rabbit AT111 utilized for this oarticular exneriment. The dotted line represents that nart of the infused circulating AT111 which upon chromatography came through under ionic strength conditions of 3.15 !1with the bulk of the plasma proteins, i.e., AT111 not adsorbed onto immobilized heparin. The stippled line is the effluent pattern for the radiolabelled nrotein desorbed from the henarin agarose qel with 2.0 !4 NaCl. The nonadThe oeak for this sorbed plasma ATI. behaves exactly like circulating ATIII. nonadsorbed orotein is consistent with a molecular weioht of 39,000; and, in analogy the effluent pattern of this material shows significant with circulating ATIII, skewness to the left. The reisolated rabbit ATIII, on the other hand, displays an effluent pattern which is suoerimoosable on the effluent nattern of the original nurified oreoaration. Roth materials disnlay an apparent molecular weight of 69,OOr) by this technique.
16
l%-LABELLED
SEPHADEX
G-150
AT111
GEL
AS .A PROBE
EXCLUSION
Vol.9,No.l
CHROhtATOGRAPHY
cpm x 10-3
io
do
9’0 ii0 ELUTION
50
VOLUME
lill
li0
(ml)
-
Rabbit
Plasma
ATIII
-
Rabbit
Plasma
AT III + Thrombtn
FIG. 6 Gel exclusion chromatograms of 12%-labelled rabbit plasma AT111 from a blood samnle obtained 10 min after the injection of the radiolabelled nrotein and from the same olasma sample following heat defibrination and treatment with 100 u of thrombin.
Circulating radiolabelled AT111 with an aoparent molecular weight of 30,000 proved fully caoable of complexing with thrombin, as evidenced by the qel exclusion chromatograms disolayed in Figure 6. In this experiment, AT111 was injected into a rabbit. A plasma samplewas obtained 10 min after the administration of the radiolabelled nrotein and heat-defibrinated at 56OC for 5 minutes. An aliquot of the heat-defibrinated sample was incubated with 100 u of thrombin. The plasma sample again displays an effluent pattern consistent with a molecular weight of 30,000, and the thrombin-treated plasma samole displays a peak consistent with a molecular weiqht of 65,Or)D, in qood agreement with a complex consisting of one molecule of AT111 (molecular weiqht: 30,000) and one molecule of thrombin. To further investigate whether the anomalous gel filtration behavior of radiolabelled AT111 in nlasma could be artifactual, we determined the molecular weight of native circulating rabbit AT111 by gel exclusion chromatography and AT111 activity measurements on effluent fractions. The gel exclusion chromatogram in Figure 7 shows AT111 activity to elute in a sharp, symmetrical peak. The effluent position for this peak for this column is consistent with a molecular weight of 66,000 for circulating rabbit ATIII. Thus, there is a clear discrepancv between molecular weiqht estimates of native circulating rabbit AT111 hv activity measurements and by radioactivity measurements on effluent fractions containinq nurified radiolabelled ATIII.
An alternative approach to molecular weight estimations of radiolabelled ATIII in the circulation is SDS qel electrophoresis. The three tracings depicted in Fiqure 8 reoresent the distribution of radioactivity over the lenath Of SDS gels after electrophoresis of purified, radiolabelled AT111 (light solid line). and the distribution for radiolahelled AT111 added to plasma in vitro (heavy solid line), both radiolabels displayinq an identical miqration CGamwith a molecular weiqht of 62,000 Daltons. The stippled line represents a distribution of radio-
iol_.y,r\T9.1
12:i--.LA-:;ELLED ATIII AS A P1?03E
m
0.5-
I
0.4-
ATUl
17
ACTlVlTY 0.3-
PEU/ml 0.2 -
O.l-
EFFLUENTVOLUME
FIG.
(ml)
7
G-150 gel exclusion chromatograohy of rabbit plasma showinq the effluent position of AT111 activity. Five ml of titrated rabbit plasma was chromatographed on a 2.5 X lOO-cm column. The effluent oeak of AT111 activity corresponds to a molecular weight of 66,000 Oaltons for this orotein according to the calibration of this column with marker proteins. SDSGEL
ELECTROPHORESB
(5% unreduced)
25
15
5
4 cpm x 10-3 3
1
A
/+J_ I
I\
/
o-
0
10
::.
30
I
30
MIGRATION
-
Purified
R&bit
-
Rnbblt
----
Rnbblt Phsma
Plasma
FIG.
40
50
(mm)
AT III AT Ill AT ,U + Thrombt,,
a
The distribution of radioactivity over the length of 5% SOS gels of Durified rabbit ATIII, rabbit plasma ATIII, and defibrinated rabbit plasma AT111 treated with 50 u of thrombin.
12%-~Ac~~~~~
18
ATIII
Vol.9,No.l
AS A PROBE
activitv for thromhin-treated rahbit olasma containinn radiolabelled AT111 showinrl tw0 neaks of radioactivity--one comioratinq with purified AT111 at 62,000 Daltons and the second ncak representinq thromhin-antithromhin complexes of molecular weights 0f iw,ono. The data summarized in Table I indicates that it is possible through separation of the radiolahel bv SDS qel electrophoresis to demonstrate complex formation between 4TIII and activated plasma serine 0roteases under baseline physiological in-vivo conditions. The table compares th;?distribution of radioactivity over thesh of SnS nels for four different preparations--purified ATIII, AT111 added to plasma in vitro, and AT111 from plasma samples obtained 3r)min and 24 hrs after injection Otthedinl~helled protein into six rahhits. TABLE I rlolecular Veiqht Distribution of 1251-Labelled ATIII by SDS Gel Electrophoresis (5% Unreduced) Percent Total Gel Radioactivity 62,000 Dalton Peak
MW ~62,000
4.2
82.2
8.6
10.9 14.8
80.1 78.1
23.5 f 3.2
66.5 1 4.1
10.0 + 1.5
44.8 f 6.8
47.2 f 5.8
8.0 f 1.1
YW >62,OOD Pure AT111
ATT11 added to
nlasma .in vitro: Exn. 1 Exn. 2
circulating (3n min after i.v. injection)*
YTIII
AT111 circulating
(24 hrs after i.v. injection)*
*Mean + SD (6 experiments) The radioactivity contained in the oortions of the gels representing molecular weiqits greater than 62,r)OO, the radioactivity in the 62,000 Dalton peaks, and the radioactivity contained in nortions of the gel representing molecular weights of less tClan62,ODO are exoressed as percent of the total gel radioactivity. Eighty-two percent of the ourified AT111 is in the 62,000 Dalton peak with only 4 percent in hiqher molecular weight regions. There is a slight increase in higher molecular weight material upon addition of radiolahelled AT111 to plasma in vitro. We hynothesize that this is due to the high viscosity of the sampl~a~ossly overloading t+e qel with protein. The majority of circulating ,ATIII is still contained in the 62,9OO Dalton peak 30 min after i.v. injectioQ of the radiolabelled protein. 'lowever, after 24 hrs there is sharp decrease in the radioactivity in the 62,000 Dalton neak with a concomitant increase in higher molecular weight material with a qean of 45 oercent of the total gel radiolabel exhibiting molecular weights of q-eater than 62,nVl Daltons.
Vol.
9,No.l
1251-LABELLED
A%111
Drsrussm
-_-L--e.-
AS A PROBE
19
-
The demonstration of raoid turnover rates of many clotting factors led several authors in the lq4D's and '50's to the belief that blood coagulation and fibrin deoosition are continuous ongoing nrocesses. The concent of a dynamic equilibrium between clotting and fibrinolysis has won sunporters, since such a hynothesis would seem to fit a number of experimental observations (2D,21). 4 critical review by Hjort and Hasselback (22) correctly concluded that the tools to definitively Drove such concepts were unavailable at that time. !dithin the last five years the develonment of new biochemical approaches toward the study of in-vivo blood coaqulation has set the stage for a reooening of an investigation of i%exiditv of the Duquid-4strup theories of continuous low-grade activation of blood coagulation under haseline physiological conditions. Fletcher et al. (23), having develoned the techninue of gel exclusion chromatoqranhy of olasafibrinogen, were able to detect with this techniqtrethe nresence of soluble macromolecular fibrin comnlexes in the nlasma of a wide variety of natients with thrombotic states. Soluble fihrin comnlexes are thought to be an indirect reflection of thrombin generation, since one essential comnonent in the generation of soluble fibrin complexes is fibrin monomer.
A comnuter nroqram developed by nlkiaersiq et al. (24), using chromatograohic nlate-theorv annlvsis, made it norsible to arrivi?ar-a semi-quantitative estimate of the contents of macromolecular soluhle fibrin complexes, fibrinogen, and fibrinogen. fibrin snlit products from poorly resolved chromatoqranhic natterns. Utilizinq this annroach, Fletcher and Alkiaersiq have estimated that as much as 2% of circulating olasma fibrinonen may be circulating as macromolecular soluble fibrin comnlexes in normal individuals under nhysiological conditions. Yossel et al. (25), havino develoned an elegant, highly specific radioimnunoassay for fib??%entide 4, similarly were able to detect small, but significant, quantities of this material in nlasma from normal peonle, stronqly suggesting that minute, but significant, quantities of thrombin mav be generated normally. In the nresent series of exneriqents we have utilized a different anoroach to investiqate this orohlem. The observations in l?osenberg's laboratory (26). demonstrating that inactivation of thrombin and other activated arocoagulants results in the formation of stable, stoichiometric comnlexes, oromoted us to investigate whether such comnlexes could be demonstrated -in vlvo. Isle concluded from in-vitro experiments that radiolabelled AT111 was suitable for SIP.?-FM. in-vivo studies,= we demonstrated that the ourified orotein radiolabelled under mild conditions was fully capable of forming labelled complexes with thromhin and factor Xa. We further concluded that the mean biological half-life of the orotein of 42 hrs made it suitable for -in-vivo studies. The technique of eel exclusion chromatogranhy for the determination of the molecular weiaht of radiolabelled antithrombin and its comolexes in vitro and in vivo nroved to have unexpected difficulties. Whereas, thrombin_Amcomplexes .____-were readilv senarable bv nel exclusion chromatoqranhy, complexes formed between factor Xa and 4TIII were not. The chronatographic behavior of uncomolexed AT111 and ST111 comolexes with factor Xa was identical on Senhadex G-150. Since radiolabelled comnlexes between AT111 and factor Xa were readily demonsand/or factor Xa undergo trable bv SDS qel electronhoresis, we conclude that !TIII substantial conformational changes during their interaction producing a comolex with an anomalous diffusion constant. Furthermore, circulating radiolahelled AT111 disnlayed an apparent molecular weight of 30,OOD bv nel exclusion chromatography, as opposed to the apparent
20
1251-LABELLED
AT111
AS A PROBE
Vol.9,No.l
moTecular weight of 613,000 of the purified protein. This phenomenon could not be attributed to proteolytic degradation of circulating ATIII, since its apparent molecular weight was 30,000 in an animal pretreated with massive quantities of Trasylol, a broad spectrum serine protease inhibitor. The aoparent molecular weight shift from 68,000 to 30,000 was also observed when this radiolabelled ourified protein was added in vitro to rabbit plasma containing Trasylol. Upon reisolation of the radiolabmed protein from plasma by heparin agarose chromatography, it reverted to the apoarent molecular weight of 68,000 of the purified protein, These observations suggest that radiolabelled AT111 is a molecule of considerable plasticity which readily changes its shape and, hence, its diffusion constant. We hypothesize that this peculair anomalous diffusion constant of radiolabelled AT111 in plasma is the consequence of subtle conformational changes of the protein introduced during iodination. This contention is supported by the experiment depicted in Figure 7 illustrating that the molecular weight of native AT111 in normal rabbit plasma by gel exclusion chromatography and activity measurements is 66,000 Daltons. Additional data from our laboratory (unpublished) in which the gel exclusion chromatographic behavior of human plasma AT111 was studied using imnunochemical assays for quantification demonstrated this protein to elute with a major neak consistent with a molecular weight of 67,000. Similarly, Rosenberg and Damus (1). in one of the steps of their purification for human ATIII, noted that this protein cochromatographed with albumin on Sephadex G-200. The apparent change in molecular weight of radiolabelled ATIII, after its introduction into the circulation, had no anparent effect on its biological activity, since gel exclusion chromatography of a plasma sample obtained from a rabbit after administration of the radiolabelled nrotein upon treatment with thrombin displayed a two-peak pattern, one peak consistent with a molecular weight of 30,OOD and a second peak at a molecular weight of 65,000 Daltons, in good agreement with a thrombin AT111 comolex, assuming a molecular weight of 30,000 for ATIII. Whereas, the gel chromatograms for AT111 added to rabbit plasma in vitro were oerfectly symnetrical, the gel chromatograms of circulating ATIIfwere consistently asymnetrical, displaying skewness toward higher molecular weight regions. These observations provide further evidence for biological activity of radiolabelled ATIII, suggesting that the radiolabelled protein does indeed form complexes with activated serine proteases in vivo. The technique of SDS gel electrophoresis orovided more reliable molecular weight estimates for AT111 and its complexes in plasma, since purified AT111 and AT111 in plasma displayed identical migrations comoatible with a molecular weight of the nrotein of 62,000 Daltons and since thrombin-antithrombin complexes in olasma by SDS gel electrophoresis appeared to possess a molecular weight of 100,000 Daltons in exact agreement with the estimated molecular weight of the comolex formed through the interaction between purified AT111 and purified thrombin. The demonstration by SDS gel electrophoresis of a shift within 24 hrs of 45% of the radiolabel of circulating AT111 to molecular sizes greater than 62,000 Daltons can, therefore, be viewed as good evidence for complex formation between the circulating radiolabelled protein and activated olasma serine proteases. Since AT111 has been reported to inhibit kallikrein and plasmin, in addition to activated. procoagulants. the question arises whether the AT111 complexes, which seem to arise under the conditions of our experiments, represent complexes between AT111 and activated nrocoagulants, or complexes between AT111 and kallikrein or plasmin. In other words, can the results of these experiments be interpreted to reflect continuous activation of blood coagulation in the normal rabbit? The nrotein concentrations in human plasma of the three major zymogens under consideration. nrothromhin, nlasminogen and prekallikrein, are very similar.
Vol.9,No.l
1251-LABELLED
AT111
AS A PROBE
21
Calculated from maximal thrombin activity generated from 1 ml of human plasma (140 NIH u), the purity of tiiethrombin preparations prepared by Fenton et al. (7). and assuming molecular weights for human prothrombin and'thrombin of;rb;OOO and 36,500, respectively, human plasma contains approximately 95 vg/ml prothrombin (27). Based on our normal laboratory values of 2-3 CTA u/ml plasma and the specific activity for the most highly ourified plasminogen of 25-30 CTA u/ml (28), human plasma contains approximately 100 ug/ml plasminogen. The published mean value for human plasma orekallikrein is 103 vg/ml (29). However, in exoeriments to be reported elsewhere (30) we have quantified the radioactive comnlexes formed through the interaction between radiolabelled AT111 and purified thrombin, factor Xa, and olasmin. Whereas, thrombin and factor Xa will react with greater than 90% of AT111 to form radiolabelled complexes, no greater than 5% of AT111 formed radiolabelled complexes with plasmin in several plasmin-AT111 molar ratios. Recent kinetical data similarly demonstrated that antithrombin is a very slow and ineffective inhibitor of kallikrein, as CornDaredto its capability to inhibit thrombin and factor Xa (31). The substantially qreater caoability of AT111 to form comolexes with thrombin and activated factor X than with plasmin and kallikrein, therefore, makes activation of blood coaqulation the most likely exnlanation for our experimental results at the present time.
Ne conclude that radiolabelled AT111 may represent a useful probe for the detection of ohysiological variations of activation of blood coagulation and in-vivo thrombin generation. --
The authors are indebted to Drs. Craig M. Jackson and John W. Fenton for their qifts of nurified factor Xa and thrombin, their advice and criticism and to Mr. Robert 0. Heidenreich for his skillful technical assistance. REFERENCES 1.
ROSENBERG, R.D. and DAMUS, P.S. The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem., 248, 6490, 1973.
2.
HIGHSMITH, R.F. and ROSENBERG, R.D. The inhibition of human plasmin by human antithrombin-heparin cofactor. J. Biol. Chem., 249, 4335, 1974.
3.
DAMUS, P.S., HICK, M. and ROSENBERG, R.D. Anticoagulant action of heparin. Nature, 246, 355, 1473.
4.
ROSENBERG, R.D. The effect of heparin on factor XIa and plasmin. Thromb. Diath. ~_rrh_, 33, 51, 1974.
5.
LAHIRI, R. ROSENBERG, R.D., TALAMO, R.L., BAGDASARIAN, A. and COLMAN, R.W. an inhibitor of human plasma kallikrein. Fed. Proc., 33, Antithrombin III: 642, 1974.
6.
YIN, E.T. Identity of plasma activated factor X inhibitor with antithrombin III and heparin cofactor. J. Biol. Chem., 246, 3712, 1971.
7.
FENTON, J.W. II, CAMPBELL, W.P., HARRINGTON, J.C. and MILLER, K.D. Large-scale nreparation and preliminary characterization of human thromhin. Riochim. Riophys. Acta. 229, 26. 1971.
1251-LABELLED
22
AT111
AS A PROBE
Vol.9,No.l
8.
JqCKSON, C.!l.,JOHNSON, T.F. and HANAHAN, D.J. Studies on bovine factor X. Large-scale purification of the bovine plasma protein possessing factor X I. activity, Biochemistry_, 7, 4492, 1968.
9.
PQRATH, J.. AXEN, R. and ERNBACK, S. Clinical coupling of prote'ins to agarose. Nature. 215, 1491. 1967.
10.
THALER. E. and SCH?ER, 6. A simple two-step isolation orocedure for human and bovine antithrombin II/III (heoarin cofactor): a comparison of two methods. Brit. J. Haematol., (ii pre&), 1976.
11.
YIN, E.T., WESSLER, S. and BUTLER, J.V. Plasma heparin: a unique, practical, submicrogram-sensitive assay. J. Lab. Clin. Med., 81, 298, 1973.
12.
GREENWOOD, F.C., HUNTER, W.M. and GLDVER, J.S. The oreparation of 1311-labelled human growth hormone of high specific radioactivity. Biochem. J., 89, 114, 1963.
13.
SACHS. D.H. and PAINTER, E. Imoroved flow rates with porous sephadex gels. Science, 175, 781. 1972.
14.
LAEMLI, M.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680, 1970.
15.
FAIRSANKS, G., STECK, T.L. and WALLACH, D.F.H. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10, 2606, 1971
16.
4NDERSSON. M.M., BORG, H. and ANDERSSON, L.-O. Purification of antithrombin III by affinity chromatography. Thromb. Res., 5, 439, 1974.
17.
WINZOR, D.G. and SCHERAGA, H.A. Studies of chemically reacting systems on Molecular weights of monomers in rapid association equilibrium. Sephadex. II. J. Physical Chem., 68, 338, 1964.
15.
JACKSON, C.M. Studies on bovine factor X:
19.
FUJIKALIA. K., LEG4Z, M.E. and DAVIE, E.W. Bovine factor X (Stuart factor): mechanism of activation by a protein from Russell's viper venom. Biochemistry, 11, 4892, 1972.
20.
!lUGUID, J.B.
21.
ASTRUP, T. In: Connective Tissue Thrombosis and Atherosclerosis. New York and London, Academic Press, 1959, D. 223.
22.
HJORT, P. and H4SSELBACK, R. 4 critical review of the evidence for a continuous hemostasis _in vivo. Thromb. Diath. Haemorrh., 6, 580, 1961.
23.
FLETCHER, A.P., ALKJAERSIG, N., O'BRIEN, J. and TULEVSKI, V.G. Blood hypercoagulability and thrombosis. Trans. Assoc. Amer. Phys., 83, 159, 1970.
24.
4LKJAERSIG, N., ROY, L. and FLETCHER, A. Analysis of gel exclusion chromatographic data by chromatogranhic plate theory analysis: application to plasma fibrinogen chromatogranhy. Thromb. Res., 3, 525, 1973.
25.
NOSSEL, H.L., YUDELMAN. I., CANFIELD. R.E., BUTLER, V.P. JR., SPANONDIS, K., WILNER, G.D. and (IURESHI,G.D. Measurement of fibrinopeptide A in human blood. J. Clin. Invest., 54, 43. 1974.
isolation, purification and partial characterization. Ph.D. Dissertation, University of Washington, Seattle, Washinqton, 1967.
Connective Tissue Thrombosis and Atherosclerosis, New York In: and London, Academic Press, 1959, p. 13.
Vol.9,No.l
1251-LABELLED AT111 AS A PROBE
23
26.
JRDS:;iERG, R.D. Actions and interactions of antithrombin and heparin. New Eng. . ., 292, 146, 1975.
27.
FENTr)N,J. Personal communication, 1975.
25.
SUMMARIA. L., ARZADDN, L., RERNARE, P. and ROBBINS, K.C. Studies on the isolation of the multiole molecular forms of human plasminogen and plasmin by isoelectric focusing methods. J. Biol. Chem., 248, 2934, 1973.
29.
BAGDASARIAN, A., LAHIRI, R., TALAMO, K.C., WONG, P. and COLMAN, R.W. Imnunochemical studies of olasma kallikrein. J. Clin. Invest., 54, 1444, 1974.
30.
CHANDRA, S. and BANG, N.U. Yanuscriot in preparation, 1976.
31.
COLMAN, R. Personal communication, 1975.
