Further studies of the turnover of dog antithrombin III. Study of 131I-labelled antithrombin protease complexes

THROMBOSIS RESEARCH 30; 165-177, 1983 0049-3848/83/080165-13$03.00/O Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.

FURTHER STUDIES OF THE TURNOVER OF DOG ANTITHROMBIN III. STUDY OF l3lI-LABELLED ANTITHROMBIN PROTEASE COMPLEXES B. Leonard, R. Bies, T. Carlson", E.B. Reeve Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, USA 80262; *Department of Biochemistry, University of New Mexico, Albuquerque, NM, USA 87131.

(Received 4.2.1982; in revised form 8.2.1983. Accepted by Editor G.A. Jamieson)

ABSTRACT Fresh plasma containing 131I-antithrombin III (*I-AT) was coagulated and incubated at 37'C for 2 hr. A "complex peak," separated on heparin-agarose contained AT and *I-AT antigen but no heparin cofactor activity. Crossed immunoelectrophoresis showed only AT complexes. SDS PAGE showed 80% of the *I-AT in a major band (Q80,OOO daltons), 15% in a minor band (QlOO,OOO daltons) and the rest in trace bands (~60,000 and/or 115,000 daltons). Ammonia treatment of the complex peak released a-thrombin. After i.v. injection 80% of the complexed *I-AT, chiefly as the major band, left the plasma with t l/2 sl.5min and was almost immediately catabolized to low molecular weight breakdown products. A major catabolic site was the liver. A simple kinetic model describes the findings approximately.

INTRODUCTION Antithrombin III (AT) forms inactive complexes with the intrinsic coagulation proteases (1) particularly thrombin (2) and factor Xa (3). -In vitro one antithrombin binds to one thrombin molecule and without excess thrombin the reverse reaction is very slow (4). With excess thrombin the complexes release altered antithrombin and thrombin products (5,6). Little is known about the behavior of antithrombin-coagulation protease complexes -in vivo. The availability (7) of a satisfactory purified dog AT labelled with radioactive iodine (*I) allowed studies of the disposal of *I-AT coagulation protease complexes in dogs. The complexes were formed by adding *I-AT to plasma and activating coagulation. A "complex fraction" was separated by heparinagarose chromatography and contained *I-labelled protein fragments ranging between 60,000 and 115,000 daltons. When injected i.v. into dogs 80% or more of the *I-AT complexes were removed from the plasma very rapidly and rapidly catabolized to low molecular weight fragments. This provides a powerful mechanism for disposing of circulating coagulation proteases. Key Words: Anti-thrombin III, 131I-AT III-protease, heparin, crossed immunoelectrophoresis, a-thrombin. 165

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MATERIALS AND METHODS Chemicals were reagent grade or the highest grade available. Animal experiments, blood sampling and radioactive counting were performed as described previously (7). Polyacrylamide gel electrophoresis was by the method of Weber and Osborn (8). Heparin cofactor activity was measured with S-2160 or S-2238 (9,lO) from Ortho Diagnostics, Raritan, NJ. The heparin used for assays or crossed immunoelectrophoresis was Riker Laboratories Lipo-Hepin. "Rocket" immunoelectrophoresis was performed according to Laurel1 (11) using an antibody against our pure dog AT raised in rabbits, and crossed immunoelectrophoresis was according to Ganrot (12) with modifications of Andersson and coworkers (13). Thrombin Measurement. This was by p-nitroanilide release from S-2238 and by fibrinogen clotting time. For the former (10) 50 U of thrombincontaining solution or blank were mixed with 400 pl of heparin buffer (0.25 mol/l Tris pH 8.4, 0.05 mol/l NaCl, 0.25 mol/l K2EDTA containing 3 units heparinlml) and incubated at 37'C for 30 or 60 sec. Then 300 pl of 0.75 mmol/l S-2238 in water were added with rapid mixing and after 2.00 min incubation the S-2238 digestion was stopped by rapidly injecting 400 ~1 50% glacial acetic acid. Thrombin standards were prepared from dilutions of Parke-Davis bovine thrombin topical (10 u/ml in .15 mol/l NaCl at 4°C). Released p-nitroanilide was measured from absorbance at 405 nm. For the latter 200 ~1 of fibrinogen solution in a 12 x 75 mm glass tube were warmed at 37'C for 2 min and then 50 pl of thrombin-containing sample or standard were added. After rapid mixing the tube was repeatedly tilted until a clot was first observed. The thrombin standards were as above. Calibration curves were prepared by plotting on log-log paper coagulation times against units of the thrombin standards. The fibrinogen solution was 1% w/v bovine fibrinogen (Sigma, %,95Xclottable) freshly dissolved in 0.026 mol/l imidazole buffer pH 7.4 with 0.66% (w/v) polyethylene glycol 6000 (Baker), spun free from particles and kept on ice. Ammonia Treatment and Thrombin Assay of AT Complexes. Samples from Peak 3, or preparations of AT-thrombin complexes, were diluted 9/l (v/v) with 5 mol/l NH4OH and incubated for 15 min at 37'C. The solutions were then diluted with the appropriate buffers and assayed for thrombin with S-2238 and fibrinogen. After dialysis against buffer they were examined by SDS PAGE electrophoresis. Distribution of *I-AT Complexes in Tissues. Peak 3 *I-AT complex fraction free from Peak 2 *I-AT was injected i.v. and plasma samples were withdrawn over 2.5 hrs. The dog was then anesthetized and exsanguinated. The liver, spleen, kidney, heart and lung were immediately removed and weighed. The radioactivity in representative tissue samples from each organ was measured. PREPARATIONS Buffers. Tris buffer (TB) was 0.01 mol/l in Tris (hydroxymethyl) aminomethane, while Tris-Citrate buffer (TCB) was 0.01 mol/l in both Tris and trisodium citrate. Salt was added to the buffers as follows:

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Buffer 1, TCB pH 7.5, NaCl 0.145 mol/l; buffer 2 TCB pH 8.3, NaCl .225 mol/l; buffer 3, TB pH 7.5, NaCl .145 mol/l; buffer 4, TB pH 7.5, NaCl 1.30 mol/l; buffer 5, TB pH 8.5, NaCl 0.6 molfl. In some studies, as noted below, pH 7.5 0.025 mol/l phosphate buffer was substituted for TB. *I-AT and AT. Labelling of AT with 1311 was as described previously (7). AT was prepared as described previously (7) or by the following small scale modification: Thirty ml of fresh heat defibrinated (7) titrated plasma diluted l/l with buffer 1 was loaded at 1 ml/min on 14 ml of heparin-agarose (7) in a 1 cm diam glass column. After washing with 20 ml buffer 1 and 30 ml buffer 2 at 0.5 mlfmin the AT was eluted with a linear salt gradient at 0.09 ml/min using equal (55 ml) volumes of buffers 3 and 4. Chromatography was at room temperature. Serum *I-AT Complexes.

a> Separation by Heparin-Agarose Chromatography. Fresh titrated plasma (30-35 ml) from a well-fasted dog was mixed in a 50 ml polycarbonate centrifuge tube with 0.1 to 0.3 mg purified *I-AT in 2 to 5 ml buffer 5. Then 0.017 ml of 1.5 mol/l CaC12 and .05 ml of rabbit (Bacto) or dog thromboplastin solution were added for each ml of plasma with rapid mixing. Dog thromboplastin was prepared as described in (15). After 2 hr at 37°C the coagulated plasma was spun for 20 min at 18,000 rpm (RCF-27,000) at room temperature. The separated serum was diluted with an equal volume of buffer 1 or phosphate buffer 1. The dilute serum (60-70 ml) was loaded on a 14 ml heparin-agarose column equilibrated with Tris or phosphate buffer 1. The column was washed with 30-40 ml buffer 1 and then eluted at .09 ml/min with a linear salt gradient obtained with 60 ml buffer 3 and 60 ml buffer 4. Figure la shows typical elution patterns from serum of AT and *I-AT complexes (Peak 3) and of native AT and *I-AT (Peak 1). b) Separation by Crossed Agarose Electrophoresis. The separated serum, prepared as above, was electrophoresed in 2 mm thick agarose gels for 90 min at 14 volts/cm (14). A circular hole, 5 mm in diameter, was punched 2 mm above the region of the electrophoresis track containing *I-AT and complexes and was filled with the buffer. The *I-AT containing proteins were electrophoresed into this hole by passing a current at right angles to the first current. Serial buffer samples from the hole were scanned for radioactivity. Serum with Raised Peak 3 Complex Levels. Five vols of fresh titrated plasma were freed from AT by batch absorption at room temperature with 1 vol of heparin-agarose equilibrated with buffer 1. Then Peak 1 *I-AT freed from Peak 2 *I-AT was added to the centrifuged absorbed plasma followed by sufficient unabsorbed plasma to reduce the final (native) AT concentration to one-third normal. The plasma was coagulated with calcium chloride and thromboplastin as described above and the serum, which contained both Peak 3 *I-AT complexes and Peak 1 uncomplexed *I-AT, was separated and injected i.v. without further purification. Dog AT-Human Thrombin Complexes. Purified dog AT, .35 mg/ml in pH 7.4 phosphate buffer (.Ol mol/l in phosphate, .145 mol/l NaCl, 0.66% polyethylene glycol 6000) was added to an equal volume of 0.09 mg/ml human thrombin (2000 U/mg, NIH Lot H-l) in the same buffer. After incubation in a glass tube for 10 min at 37°C the mixture (AT/thrombin molar ratio %2/l) was dialyzed against the buffer and analyzed on SDS PAGE (8).

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Peak 2 *I-AT Contamination of Peak 3 *I-AT. Labelling AT with 1311 (7) may result in the appearance of a small peak (Peak 2) of *I-AT eluting from heparin-agarose between Peaks 1 and 3 (Fig. lb). This was either removed from Peak 3 preparations by very slow salt gradient elution from heparinagarose (see Serum *I-AT Complexes a) above) or corrected for as described below. Corrections of Turnover Studies a) for Presence of Peak 2 Overlabelled *I-AT in the Peak 3 *I-AT Complex Fraction. The fraction of Peak 2 *I-AT in the injected *I-AT complexes of Peak 3 was obtained from E/0.537 as described in Appendix 1. Given this and our unreported observations that the turnover of Peak 2 and Peak 1 *I-AT are very similar, the expected radioactivities over time of Peak 2 in the plasma, *I-p2, in the breakdown products, *I-z2 and in the whole body, *I-WB2 can be calculated from our previous studies (7) as described in Appendix 1. These values were then subtracted from the total plasma *I-AT, "I-p, the total plasma *I-catabolites, *I-z, and the total whole body radioactivity, *I-WB, obtained in the animals receiving the mixtures of Peak 3 *I-AT complexes and Peak 2 overlabelled *I-AT. b) for Presence of Peak 1 *I-AT in Serum with Raised Peak 3 Complex Levels. The fraction of Peak 1 *I-AT was determined as under a) above and the expected radioactivities caused by Peak 1 *I-AT, *I-pl, and Peak 1 *Ilabelled catabolites, *I-zl, were calculated from the previous studies (7) as described in Appendix 1. The observed values of *I-p and *I-z after injecting the serum were then corrected by subtraction of these values. RESULTS Separation of Serum AT Complexes. Figure la shows the fractions obtained by heparin-agarose chromatography using phosphate buffer from serum prepared from titrated plasma containing *I-AT. Three absorbance peaks at 280 nm are seen, Peak 1 of native AT, Peak 3 of AT complexes and a lipid peak. The lipid peak is further defined by A320. Also seen are two *I-peaks, Peak 1 *I-AT and Peak 3 complexed *I-AT (14). Sometimes, as shown in Figure lb, a shoulder appeared on the left hand (ascending) side of radioactive Peak 1. Studies, not reported here, showed this to be caused by the presence of "Peak 2" *I-AT. This consists of AT labelled with 2 or 3 *I atoms per molecule with reduced affinity for heparin (Figure lb) but turnover behavior indistinguishable from that of Peak 1 *I-AT (7).

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FIGURE 1

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Constituents of the AT Complex Fraction (Peak 3) and Distribution of Radioactivity. Heparin cofactor activity was very low; irmnunoelectrophoresis showed the presence of AT but crossed immunoelectrophoresis showed the presence of AT complexes with negligible free AT (Fig. 2). By SDS polyacrylamide gel electrophoresis (Fig. 3), Peak 3 contained a major band of %80,000, a minor band of Q~100,000and trace bands of Q15,OOO and sometimes of ~60,000 daltons. These bands are compared with gels of Peak 1 dog AT and *I-AT, of human thrombin and of dog AT-human thrombin complexes. Human thrombin was used as our dog thrombin preparations were unsatisfactory. The dog AT-human thrombin complexes ran at ~100,000 daltons. By slicing at 1 mm intervals and counting the *I-AT radioactivity in the gel bands the major 80,000 dalton band contained 83% of the radioactivity, the minor 100,000 dalton band contained 15% and the trace 115,000 dalton band contained 2%.

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FIGURE 2

Plate A Fig. 2 Heparin Crossed Immunoelectrophoresis (CIE). Plate A is titrated plasma, Plate B (CIE of Peak 1, pure AT) is superimposed on Plate C (CIE of Peak 3 AT-complexes).

Plate B and C

FIGURE 3

Fig. 2. PAGE SDS Gels of A, Peak 3; B, Pure Thrombin-AT Complexes and Peak 1 AT; C, Ovalbumin and Bovine Albumin Standards; D, Peak 1 AT and *I-AT; E, NIH Human Thrombin, Lot Hl.

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To exclude the possibility that passage through heparin-agarose was responsible for the formation of the major 80,000 dalton *I-AT complex band the *I-AT complexes were separated from serum by crossed electrophoresis into buffer-filled wells in agarose gels, as described under Methods. PAGE SDS gels of the fractions electrophoresed into the buffer wells showed the same major distribution of radioactivity in the 80,000 dalton band. TABLE I Thrombin Content of Peak 3 AT-Complex Fraction Before and After Incubation with 0.5 mol/l NH4OH Fibrinogen Clotting Assay S-2238 Assay Thrombin Thrombin u/ml u/ml Sample 10 10 Thrombin Solution 5 5 Thrombin Treated with NH40H 1 Peak 3 Untreated 3.5 26* '36.0* Peak 3 Treated with NH4OH _1.7* Peak 3 Treated with NH40H then with AT *Corrected for losses resulting from NH,,OH Table I summarizes studies of the effects of 15 min of ammonia digestion. In control studies this reduced S-2238 and fibrinogen clotting activity of 0.5 unit bovine thrombin to one-half. Digestion of the Peak 3 complex fraction increased initial low thrombin activity lo- to 20-fold and 95% of this activity was immediately removed by adding purified AT. The fibrinogen assay on ammonia treated Peak 3 samples measured about 213 of the thrombin activity measured by S-2238. Unreduced SDS PAGE gels showed that the ammonia digestion replaced the *I-AT bands initially present with bands of s68,OOO and "~56,000 daltons containing 95% and 5% of the *I radioactivity respectively. Turnover of the *I-AT Complex Fraction. Peak 3 *I-AT complex fraction, free from Peak 2 *I-AT, was injected i.v. and plasma samples and whole body counts were obtained over the next 3 days, Findings in dog BL are shown in Fig. 4 by symbols connected by straight lines. The dashed lines indicate predictions obtained from the model outlined in Appendix 2 and pictured in Fig. 4. Initially, the TCA precipitable radioactivity of the plasma, *I-p, left the plasma very rapidly with only 10% remaining 5 hr later. After 5 hr the removal rate slowed. The plasma *I-p curve, representpEt the circulating *I-AT complexes, is described by *I-p = .77 e-30t + ,18 e- * + .05 e-*435t with t in days. *I-z is the TCA soluble radioactivity in the plasma, multiplied by 7 to adjust for the volume of distribution (16) and represents the low molecular weight radioactive breakdown products of Peak 3 *I-AT. *I-z rose rapidly to a maximum in 5 hr and then declined steadily. The whole body radioactivity, *I-WB, declined as the radioactivity was excreted. The dashed lines calculated from *I-A(O) = .77, *I-B(O) = .18, *I-C(O) = .05, r-1= 30, r2 = 5.4, r3 = .435, h = 1.9 day-l, k = 1.6 day-l fit *I-WB and *I-Z approximately and show the predicted course of the radioactivity in the breakdown compartment *I-b. The r's and k are defined in the Appendix. Fig. 5A shows the *I-p, *I-Z and *I-WB curves for two other animals corrected for the presence of Small amounts of contaminating Peak 2 *I-AT as described under Methods. The Peak 2 *I-AT at t = 0 is given in Fig. 5A by 1.0 - *I-p(O).

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FIGURE 4

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Fig. 4. Courses of Peak 3 *I-AT in the Plasma, *I-p, Low Molecular Weight Radioactive Breakdown Products, *I--z,and Whole Body Radioactivity *I-WB (all continuous lines) Compared with Predictions of the Simple Model (Inset and *I-b is the predicted radioactivity in Appendix 2) Shown by Dashed Lines. compartment b, see Appendix 2.

To test if the very rapid catabolism was due to damage to the *I-AT complexes by the chromatography, serum containing increased *I-AT complexes was prepared (see under Methods) and injected without further purification into 3 dogs. The radioactivity in the injected serum consisted of 45% Peak 3 *I-AT and 55% Peak 1 *I-AT. Correction for the latter was made as described under Methods and the Peak 3 values were normalized by dividing by .45. Figure 5B shows that the unpurified Peak 3 *I-AT complexes were removed just as rapidly as purified Peak 3 complexes.

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FIGURE 5

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Fig. 5. Disappearance of Peak 3 *I-AT, *I-P, from the Plasma and Appearance of Low Molecular Weight Radioactive Breakdown Products, *I-Z in 4 Dogs. *I-WB is whole body radioactivity. Disappearances of purified *I-AT complexes (A) and *I-AT complexes not separated from serum (B) are similar. See text.

Rates of Removal of Peak 3 *I-AT Gel Bands from the Plasma. Peak 3 *I-AT complexes were prepared from pure Peak 1 *I-AT and injected into a fasting dog. Measurements of *I-p in samples obtained 3 min, 1 hr and 2.5 hr later showed the usual rapid loss of Peak 3 *I-AT complexes from the plasma. Peak 3 *I-AT complexes were separated from the 2.5 hr plasma sample by heparinagarose chromatography as described under Methods and this fraction and the injected Peak 3 *I-AT were run on SDS PAGE and the radioactivity in the separated bands was determined. The injected material contained 77.5% of the *I-AT in the 80,000 dalton band (A), 15.5% in the 100,000 (B) and 7% in the 60,000 (C) band, Table II. Peak 3 separated from the 2.5 hr sample contained 21% of the *I-AT in the 80,000, 45% in the 100,000 and 34% in the 60,000 dalton bands. Table II also shows the percentages of radioactivity in bands A, B and C at 2.5 hr calculated from the *I-p e uation of dog BL, Fig. 4. Thus A(t=O) of .772 was assumed to decay at am38t, B(t=O) of .178 at e-5.5t and C(t=O) of .05 at e-0*5t, with t in days. Agreement between the values calculated from dog BL and the observed values is reasonable and the findings in Table II rule out equal fractional decay rates for all 3 bands.

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TABLE II Distribution of Radioactivity in SDS PAGE Bands A, B and C at t = 0 and t = 2.5 hr After Peak 3 Injection Compared with Values Calculated from Dog BL Bands Time, hr

A 'L80,OOOMW, c/min,(SD),%

0 2.5

Observed Per Cent Radioactivity in Bands 2378,(17),77.5% 478,(10),15.5% 214,(8),7% 15,(7),21% 32,(11),45% 25,(5),34%

B "100,000 MW c/min,(SD),%*

C "~60,000MW c/min,(SD),%*

Calculated Per Cent Radioactivity in Bands from Plasma Decay Curve of Dog BL 77.2% 17.8% 5% 18.6% 55.4% 26%

0 2.5 * % is

Band Radioactivity at time t x 100 Band A+B+C Radioactivity at time t

The Appendix gives equations of a minimal model of *I-AT complex catabolism derived from these studies and Fig. 4 shows a block diagram of the model with some predictions for dog BL.

Distribution of *I-AT Complex Radioactivity in Tissues. At 2.5 hr after i.v. injection of purified Peak 3 *I-AT 23% of the total *I was in the plasma (one-third of this-was bound to AT), 58% of the total *I was estimated to be distributed through the plasma and tissue fluids as low molecular weight breakdown products, 8.5% of the total *I was in the liver and the spleen, kidneys and lungs each contained 1.0%. The liver appears to be a main catabolic site. DISCUSSION To prepare biologically-formed *I-AT-protease complexes we coagulated plasma containing purified *I-AT and separated an *I-AT "complex fraction" by heparin-agarose chromatography. We found Tris and phosphate buffers equally effective in the separations. In initial studies this Peak 3 contained some Peak 2 *I-AT labelled with 2 or more iodine atoms, but in later studies this was removed. The presence of complexes in pure Peak 3 was demonstrated by crossed immunoelectrophoresis, loss of antithrombin activity, the increased MW of the *I-AT bands separated on gels and the release of thrombin by Tonia treatment. Fibrinogen clotting activity measured about 213 of the NH4 released activity determined by S-2238 digestion. SDS PAGE analysis of Peak 3 usually showed 3 radioactive bands, a main band (%80X) of about 80,000 daltons, a subsidiary band (%15%) of about 100,000 daltons and a trace band (%5X) sometimes of about 115,000, sometimes of about 60,000. The subsidiary fraction ran identically with l/l complexes of dog AT and human thrombin made -in vitro. Calculations show that it could account for all the thrombin released by ammonia. The 80,000 daltons of the main fraction, however, suggests extensive partial digestion of AT-protease complexes or formation of complexes of AT with 8 or y thrombin (17,18). Rosenberg and Damus (.2)observed the formation of complexes of this size in their -in vitro studies of purified components. The 115,000 dalton band might represent factor Xa-AT while the 60,000 dalton band might represent altered AT (19). We dfd not see polymers of AT-protease complexes of ~200,000

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daltons (20). Since 95% or more of *I-AT added to plasma was recovered after coagulation in Peaks 1, 2 and 3, the 3 bands must represent the only radioactive complex species present in significant amounts. The important finding in the turnover studies was the great rapidity with which 80% or more of injected Peak 3 *I-AT left the plasma and was catabolized into low molecular weight radioactive fragments. For instance, in 7 experiments (4 shown in Fig. 5) 85 to 90% left the plasma with a t l/2 of 10 to 30 min. Clearly this forms a very powerful mechanism for removing coagulation proteases from the circulation and destroying them. The main sites of destruction seem to be the liver, and so probably the reticuloendothelial system. The finding of 3 labelled bands on SDS PAGE of Peak 3 poses interesting questions. How does the major 80,000 dalton band arise? Does it result from digestion of 100,000 dalton AT-thrombin complexes by absorbed proteases during passage through heparin-agarose? Our studies with crossed agarose electrophoresis exclude this. Does it result from digestion in the serum of 100,000 dalton AT-thrombin complexes, or from combination of partially digested protease and AT in the serum? Is the neutralization of coagulation proteases by AT in the dog representative of what happens in man? If a similar multiplicity of AT-coagulation protease bands with different removal rates is seen in human plasma, how does this affect the interpretation of AT-protease neoantigen levels in human coagulation disease (21,22)? If the simple kinetic model given in the Appendix describes the behavior of AT-protease complexes in man it should help in determining the significance in disease of different neoantigen levels. Vogel and collaborators (23) reported findings that seem to disagree with those reported here. They injected *I-labelled rabbit thrombin, rabbit antithrombin and rabbit antithrombin-thrombin complexes i.v. and followed their behavior in the plasma. In interpreting their findings they neglected the initial very rapid loss of 80% of the labelled complexes from the plasma shown in their Figure 3 and drew attention to the slow later loss of radioactivity with half life of about 8 hr. In the absence of measurements of *I-z and *I-WB the cause of the latter is uncertain, but it may in part represent the excretion rate of *I-labelled catabolic products. We think their findings in rabbits agree with our findings in dogs. APPENDIX 1 The disappearance of i.v. injected Peak 1 *I-AT from the plasma is described by Clesalt + C2e-a2t with mean values of .537 e-.294t +' .463 .-3.816t (7). As noted above the disappearance of Peak 2 *I-AT is very similar. By one to two days after injecting mixtures of Peak 1 or Peak 2 with Peak 3 *I-AT, levels of Peak 3 *I-AT become negligible and only the tails of Peak 1 or Peak 2 *I-AT, described by f.Clemalt, remain. Here f is the fraction of total radioactivity injected as Peak 1 or Peak 2. E, the zero time intercept, is obtained by extrapolation through the natural logs of the tail nI-AT values plotted over several days, Since E = f.Cl, f = E/0.537. Given mean values of plasma disappearance (7), f and mean fractional excretion rate of *Icatabolites (16) the equations of plasma protein Model 2 (24) allow calculation of the expected values of *I-z1 or *I-z2 and *I-WBl or *I-WB2 resulting from injecting Peak 1 or Peak 2 *I-AT with the Peak 3 *I-AT complexes.

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APPENDIX 2 (See Fig. 4) Let the time course of Peak 3 plasma *I&AT complexes, *I-p - Aevrlt + Be-r2t + Ce-r3t where A is the fraction at t = 0 of Q~80,000 dalton *I-AT complexes, B of the ~100,000 and C of the ~60,000. Then from the model in Fig. 4 inset, 1) A(t) = Aewrlt, B(t) = Be-r2t, C(t) = Ce-r3t where r is the fractional transfer rate of 80,000 dalton, r2 the fractional transfe3 rate of 100,000 dalton and r3 the fractional transfer rate of 60,000 dalton complexes to the common breakdown compartment, b. If *I-b is the radioactivity of *I-AT complexes in b and h is the fractional breakdown rate of *I-b d*I-b rlA(t) + r2B(t) + r3C(t) - h*I-b 2) dt= If *I-z is the radioactivity of low molecular weight breakdown products released instantaneously on breakdown of complexes in compartment b to enter compartment z.and these are then excreted at fractional rate k into compartment u d*I-z 3) 7 = h*I-b - k*I-z Solutions to equation 2) and 3) are readily obtained. ACKNOWLEDGEMENTS These studies were supported by Grant HL25477 from the U.S. Public Health Services, National Heart, Lung and Blood Institute, NIH; Grant RR00051, Division of Research Resources, DHHS, and by the Colorado Heart Association. REFERENCES 1.

ROSENBERG, R.D. Chemistry of the hemostatic mechanism and its relationship to the action of heparin. Fed. Proc. 36,10-18, 1977.

2.

DAMUS, P.S. and ROSENBERG, R.D. Antithrombin-Heparin Cofactor. In: Methods in Enzymology XLV; Proteolytic Enzymes B. Academic Press, New York, 1976, 653-669. L. Lorand (Ed.).

3.

YIN, E.T., WESSLER, S. and STOLL, P.J. Biological properties of the naturally occurring plasma inhibitor to activated factor X. J. Biol. Chem. 246,3703-3711, 1971.

4.

JESTY, J. Dissociation of complexes and their derivatives formed during inhibition of bovine thrombin and activated factor X by antithrombin III. J. Biol. Chem. =,1044-1049, 1979.

5.

SEEGERS, W.H., YOSHINARI, M. and LANDABURU, R.H. Antithrombin as substrate for the enzyme thrombin. Thromb. Diath. Haemorrh. (Stuttgart) 4, 293-298, 1960.

6.

FISH, W.W., ORRE, K. and BJORK, I. Routes of thrombin action in the production of proteolytically modified secondary forms of antithrombinthrombin complex. -Eur. J. Riochun. lE,39-44, 1979.

7.

REEVE, E.B., LEONARD, B., WENTLAND, S.H. and DAMUS, P. Studies with 131-I-labelledantithrombin III in dogs. Thromb. Res. 20,375-389, 1980.

8.

WEBER, K. and OSBORN, M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol.

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9.

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177

Chem. 244,4406-4412, 1969. -ODEGARD, O.R., LIE, M. and ABILDGAARD, U. Heparin cofactor activity measured with an amidolytic method. Thromb. Res. 6_,287-294,1975.

10.

ABILGAARD, U., LIE, M. and ODEGARD, O.R. Antithrombin (heparin cofactor) assay with "new" chromogenic substrates (S-2238 and Chromozym TH). Thromb. Res. 11,549-553, 1977.

11.

LAURELL, C.B. Electroimmunoassay. Suppl. 124,21-37, 1972.

12.

GANROT, P.O. Crossed immunoelectrophoresis. Invest. 29, Suppl. 124,34, 1972.

13.

ANDERSSON, L.D., ENGMAN, L., HENNINGSON, E. Crossed immunoeelectrophoresis as applied to studies on complex formation, the binding of heparin to antithrombin III and the antithrombin III-thrombin complex. J. Immunol. Methods 12:271-281, 1977.

14.

JOHANSSON, B.G. Agarose gel electrophoresis. Invest. ZJ, Suppl. 124,7-19, 1972.

15.

Human Blood Coagulation Haemostasis and Thrombosis, 2nd Ed., BIGGS, R. ) Blackwell Scientific, Oxford, 1976, Appendix 1, Section 17, pp 663, 664.

16.

TAKEDA, Y. Hormonal effects on distribution and excretion of iodide1311 in the dog. Am. J. Physiol. .206,1237-1243, 1964.

17.

MACHOVICH, R., BORSODI, A., BLASKO, G. and ORAKZAI, S.A. Inactivation of o.-and B-thrombin by antithrombin III, a2-macroglobulin and alproteinase inhibitor. Biochem. J. 167,393-398, 1977.

18.

CHANG, T., FEINMAN, R.D., LANDIS, B.H. and FENTON, J.W. Antithrombin reactions with a- and y-thrombins. Biochemistry 12,113-119, 1979.

19.

FISH, W.W. and BJORK, I. Release of a two-chain form of antithrombin from the antithrombin-thrombin complex. Eur. J. Biochem. 1%,31-38, 1979,

20.

PEPPER, D.S., BENHEGYI, D. and CASH, J.D. The different forms of antithrombin III in serum. Thromb. Haemos. 32,494-503, 1977.

21.

COLLEN, D., DE COCK, F. and VERSTRAETE, M. antithrombin III complexes in human blood. 407-411, 1977.

22.

LAU, H.K. and ROSENBERG, R.D. The isolation and characterization of a specific antibody population directed against the thrombin-antithrombin complex. J. Biol. Chem. =,5885-5894, 1980.

23.

VOGEL, C.N., KINGDON, H.S. and LUNDBLAD, R.L. Correlation of in vivo and in vitro inhibition of thrombin by plasma inhibitors. J. Lab.Clr -Med. %,661-673, 1979.

24.

REEVE, E.B., LEONARD, B. and CARLSON, T. Kinetic studies -in vivo of antithrombin III. Ann. N.Y. Acad. Sci. =,680-694, 1981.

Stand. J. Clin. Lab. Invest. -' 29 Stand. J. Clin. Lab.

Stand. J. Clin. Lab.

Quantitation of thrombinEur. J. Clin. Invest. 7_,