THROMBOSIS RESEARCH 61; l-10, 1991 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

A PURIFIED ANTITHROMBIN III - HEPARIN COMPLEX AS A POTENT INHIBITOR OF THROMBIN IN PORCINE ENDOTOXIN SHOCK M. Spannagl, H. Hoffmann 1, M. Siebeckl, J. Weipertl, H. P. Schwarz2* W. Schrm Departments of Medicine, Clinical Chemistry and Clinical Biochemistryl, Klinikum Innebstadt, Ludwig-Maximilians University, Munich, FRG Immuno AG, Vienna, Austria2 (Received 6.2.1990; accepted in revised form 1 .lO.i990 by EditorA. Hens&en)

ABSTRACT Inhibition of activated clotting factors is an important therapeutic approach in disseminated intravascular coagulation (DIC)~. We examined the possible protective effect of a purified complbx of human antithrombin III (AT III) and heparin in endotoxin-induced DIC in pigs. Two groups of endotoxemic pigs were studied. AT III heparin group pigs (n=8) were pretreated with a bolus injection of 500 units AT III - heparin complex, followed by a continuous infusion of 1000 units of the complex for 6 hours given simultaneously with the infusion of 10 pg/kgh of S. abortus equi endotoxin. Controls (n=9) were given saline in addition to the continuous infusion of endotoxin. AT III activity, prothrombin and soluble fibrin in plasma were determined by chromogenic substrate methods. Fibrinogen was measured turbidimetrically. Human AT III antigen in the treated group was 64 f 3% at 2 hours and increased to 84 f 4% until the end of the experiment. AT III activity in the AT III - heparin group was elevated throughout the whole observation period (> lOO%), whereas it was significantly lower in the controls. Prothrombin decreased similarly in both groups by approximately 35% until the end of the experiment. AT III - heparin treatment significantly attenuated the endotoxin-induced consumption of fibrinogen and completely prevented the increase in soluble fibrin in plasma. However, no significant effect of AT III - heparin was observed on endotoxininduced mortality and dysfunction in pulmonary gas exchange. Therefore we conclude that the purified AT III - heparin complex inhibits thrombin effects and prevents development of DIC, but fails to significantly influence clinical outcome in endotoxin shock of the Pig* Keywords:

DIC, endotoxin, complex, pig

thrombin,

soluble

fibrin,

AT III - heparin

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INTRODUCTION The consumption of hemostatic factors as well as of platelets plays a major role in disseminated intravascular coagulation (DIC) associated with septicemia (1,2,3). Thrombin enhances the consumption by a positive feedback mechanism with humoral and cellular constituents of the hemostatic system (4). Therefore, the inhibition of activated clotting factors, in particular the inhibition of thrombin, is a therapeutic approach to interrupt DIC ($6). Antithrombin III (AT III) is an important endogenous inhibitor of serin proteases. Its slow progressive inhibition of thrombin is markedly accelerated by complex formation with heparin due to the promotion of a ternary intermediate complex between thrombin, AT III and distinct heparin domains (7,8). Heparin binds less strongly to this complex than to free AT III; therefore it is able to function as a catalyst by initiating multiple rounds of complex formation (9,10,11). Since AT III levels in plasma of patients with DIC are low (12,13), administration of AT III has been proposed as a therapeutic approach (14,15). However, clinical experience with AT III substitution in DIC is still limited. Moreover, AT III substitution without heparin therapy was ineffective in experimental DIC (16). Besides bleeding complications, the administration of native heparin may also have side effects on platelets, endothelium or on the immune system (17,18). In addition, AT III may be cleaved by thrombin into inactive fragments in the presence of heparin (19). Therefore, it seems more promising to use AT III and heparin as a purified complex to interupt DIC. Preliminary experiments in rabbits with intravenous administration of a purified AT III - heparin complex demonstrated a comparable anticoagulant effect in plasma with a longer half life time than with heparin alone (20). The mortality in endotoxin-induced DIC in rats was reduced as compared to the administration of heparin or AT III alone (21). We examined the effect of the administration of a purified AT III - heparin complex in a porcine model of endotoxin-induced DIC.

METHODS Animals Anesthetized weaned miniature pigs of either sex (Medical Service GmbH, Munich, FRG), weighing 18-23 kg were used. The animals were fasted overnight, except for unlimited access to water. Only animals without signs of infection and with a baseline body temperature below 38.6”C, Hb above 7.0 g/lOOml, WBC 5000-8000 /pl and lung compliance/kg bodyweight greater than 1 were included.

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Anesthesia procedures The animals were premeditated with azaperone (3 mg/kg) (Stresnil Janssen, Neuss, FRG) and metomidate-hydrochloride (3.75 mg/kg) (Hyponodil Janssen, Neuss, FRG), injected intramuscularly. Anesthesia was induced with pentobarbital 24 mg/kg (Narcoren, Iffa Merieux, Laupheim, FRG), injected intravenously, and maintained throughout the experiment by a continuous infusion of piritramide (0.5 mg/kgh) (Dipidolor Janssen, Neuss, FRG), pancuronium bromide (0.3 mg/kgh) and pentobarbital (4 mg/kgh). The animals were orally intubated and mechanically ventilated (volume cycled) with Pi02 = 0.4. Oxygen saturation in arterial blood was measured with a hemoximeter OSM 2 (Radiometer Copenhagen) from heparinized blood. Arterial blood gases were measured with a model 168 pH/blood gas analyzer (Coming Medical GmbH, Femwald, FRG). Experimental protocol After baseline measurements 17 animals were randomly assigned into 2 groups: the control group (n=9) received a continuous infusion of 10 pg/lqgh of Salmonella abortus equi endotoxin (LPS) (batch: M6-Nl TEN, 5 mg/ml, kindly provided by Dr. C. Galanos, Max-Planck-Institut fur Immunologie, F&burg, FRG) and placebo (saline) treatment for 6 hours. The drug-treated groups (n=8) was given a bolus injection of 500 Inh U of AT III - heparin complex before endotoxin followed by a continuous infusion of 1000 Inh U AT III - heparin complex over 6 hours simultaneously with LPS. During the experiment a total of 17 ml/kgh of cristalloid solution was administered. All animal procedures were approved by the Tierschutzreferat, Regierung Oberbayem. AT III - heparin complex The purified complex was prepared by IMMUNO AG, Vienna, Austria. Vials containing approximately 500 mg lyophilized protein material dissolved in’ 10 ml of distilled water were used. Each vial contained 500 IU of AT III and ‘20003500 IU of a special fraction of bovine heparin with high affinity for AT III as declared by the manufacturer (calibrated by WHO Reference preparation). One inhibitor unit (Inh U) of the complex preparation was arbitrarily assumed to represent the amount containing 1 IU AT III. Blood samples Blood was sampled from a short large bore cannula in the left external jugular vein. Citrated blood (0.11 mol Na-titrated 1:lO) was immediately centrifuged at room temperature at 1500 rpm for 15 min. The plasma was aliquoted; snap frozen in liquid nitrogen and stored at -80°C until measurement. Immuno-electrophoresis Immuno-electrophoresis using rabbit antiserum against human AT III (BehringWerke Marburg, FRG) was performed in agarose gel (20 g a arose (LitexHSB, Paesel Frankfurt, FRG) in 200 ml barbital buffer pH 8.6 and f 00 ~1

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AT III-antiserum) according to Laurell. The standard curve was prepared with a human normal plasma pool. Thrombin time 1.5 units bovine thrombin (BehringWerke Marburg, FRG) were added to 200 pl titrated plasma. Clotting time was determined manually. Antithrombin activity Citrated plasma was diluted 1:lOO with Tris buffer (0.1 mol/l Tris, 0.15 mol/l NaCl, 2 USP units/ml of heparin, EDTA 0.01 mol/l, 1% PEG 6000) and bovine thrombin (0.5 U/ml) was added in excess. After 3 minutes of incubation the thrombin activity was measured by a chromogenic substrate (S 2238 KabiVitrum, Munich, FRG). Assay calibration was performed in percent employing a human normal plasma pool as reference. Prothrombin activity Tris Buffer-diluted plasma (1:lOO) was incubated with factor Xa, phospholipid and CaC12 solution for 120 s. Thrombin generation was measured after addition of soybean trypsin inhibitor (SBTI) by a chromogenic substrate (S2238, KabiVitrum, Munich, FRG). Assay calibration was performed in percent employing a human normal plasma pool as reference (22). Fibrinogen Fibrinogen cleavage by batroxobin (final concentration 9 U/l) was measured in titrated plasma diluted 1:lO with buffer (Tris 0.1 mol/l, pH 7.5 2% PEG 6000, 1% Brij 35, 0.01% aprotinine, CaC12 5 mmol/l). The change in absorbance/min was turbidimetrically determined at 340 nm. Calibration was performed using a human plasma standard (Boehringer Mannheim, FRG) (23). Soluble Fibrin Plasma samples were 40-fold diluted with Tris buffer (Tris 0.063 mol/l, pH 8.5 , 0.01% Tween 80) and incubated with 200 pl of plasminogen (0.2 mg/ml) and 100 pl of a mixture of t-PA (6.6 pgJm1) and S 2390 (2.9 mmol/l). After a lag phase the increase in absorbance is dependent on the fibrin concentration of the plasma sample. Soluble fibrin concentration was standardized by reference amounts of batroxobin digested human fibrinogen in plasma (KabiVitrum Munich, FRG). Statistics Data are presented as mean f 95% confidence intervals. Normality was confirmed for each parameter by normal distribution plots. Missing values due to mortality were replaced by the last valid measurement.

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5

RESULTS AT III antigen and activity are presented as percent of a human normal plasma pool, because human AT III was administered by means of the complex preparation. During the infusion of endotoxin the AT III activity in plabma of the control group decreased from baseline levels of 99.8 + 5.8% to 84.1 zk 4.2% at 6 hours; the AT III activity in plasma in the drug-treated group increasqd after 2 hours (126.6 f 7.3%) and then slightly declined, but remained elevated throughout the 6 h experimental period (fig la). The plasma concentration of the human AT III antigen in the treated animals as assessed by immuno-electrophoresis was 64 zk 3% at 2 hours and continued to increase until the end of the experiment reaching 84 + 4% (fig. lb). The rabbit antiserum showed no cross-reactivity with pig AT III. Infusion of AT III - heparin complex produced a prolongation of thrombin time within 2 hours and the thrombin time remained above baseline until the lend of the experiment (6 h). In control animals no change in thrombin tinie was observed (fig. lc).

a

AT III activity

5

b z

Human AT III antigen

C

Thrombln time

0123456 hours -

hours LpS with AT Ill_hepsrin complex

hours

G--Z LPS with saline

m (a) AT Ill activity in pig plasma increased after pretreatment with 25 Inh U complex prepaiatiotikg body weight and a continuous infusion of 8.5 Inh U/kgh until 2 hours after the start of the exp riment. The time course of both groups thereafter paralleled the decrease in total protein concentration. (b) Concentrations of human AT Ill antigen in the treated group increased until the end of the ex riment. Calibration of AT III tests was done by a human normal plasma pool, representing 100% on the a scissa. (c) Thrombin inhibition, as determined by thrombin time, paralleled the AT Ill activity in the druf -treated animals and was unchanged in the control group.

AT III IN ENDOTOXIN SHOCK

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a

b

Prothrombln

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C

Fibrinogen

Soluble fibrin

600% 100%

0

2

3 4 hours

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6

I I I 0123456

I

I

,

I

I , I 0123456

I

LPS with AT Ill-heparin complex

I

I

hours

hours

IM

I

C+

LPS with saline

I

(a) Prothrombin in plasma decreased similarly in both groups by approximately 35% until the end of the experiment. (b) In the control group a progressive consumption of ‘plasma fibrinogen was noted during the LPS infusion. (c) Soluble fibrin in plasma increased in the control animals, whereas in

Fia. 2;

the drug-treated animals the increase was prevented. All data are expressed in percent of the individual baseline levels.

Because of the calibration with human plasma material prothrombin, fibrinogen and soluble fibrin data are presented as percent of the individual baseline levels. Prothrombin plasma levels showed a continous decrease by 32 k 10.1 % in the controls and 34 + 7.9 % in the drug-treated animals until the end of the experiment (fig. 2a) In controls fibrinogen in plasma decreased to 65.3 + 10.1% after 6 hours. The AT III - heparin complex attenuated the decline of plasma fibrinogen to 88.5 + 10.1% (fig. 2b). Fibrinogen consumption in the control group was associated with an increase in soluble fibrin in plasma to 443.3 + 107.6 nmol/l at 6 hours. The AT III - heparin complex completely prevented this increase (fig 2b). Platelets decreased from 395.000 /pl to 165.000 /l.rl in the control group. Thrombocytopenia was less pronounced in the drug-treated group (418.000 + 160.000 /pl to 213.000 + 68.000 /pl). Total protein concentration in plasma decreased from 4.3 + 0.3 g/d1 to 3.6 + 0.3 g/d1 in the control group and from 4.7 +_0.5 g/d1 to 3.6 + 0.4 g/d1 in the drug-treated group. Oxygen saturation in arterial blood decreased in the control group steadily to 81.4 +, 10.2 % of baseline until the end of the experiment. Administration of the AT III - heparin complex slightly attenuated this decrease to 87.1 f 10.1 % (fig. 3a). Four out of nine animals of the control group and five out of eight of the drugtreated group survived the 6 hour experimental period (fig. 3b). However, this difference was not statistically significant.

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AT III IN ENDOTOXIN

0

1

2 3 hours

4

5

6

SHOCK

0123456 hours

-

LPS with AT Ill-heparin complex

---

LPS with saline

1

Fia. 3: (a) Decrease of oxygen saturation in arterial blood was attenuated in the treated animals as compared to the controls. (b) Kaplan-Meier plot for the mortality during endotoxin infusion. Percentage of survival on the abscissa. Although survival was prolonged and oxygen saturation was improved in the treated animals, these differences were not statistically significant.

DISCUSSION In the present study we have demonstrated that an intravenous infusiqn of a purified AT III - heparin complex into anesthetized pigs prevented endotoxininduced DIC. The dose of endotoxin used caused a mortality rate of 50%~in the control group after 6 hours of endotoxin infusion. Laboratory diagnosis of DIC was suggested by the consumption of plasma fibrinogen paralleled by an increase in soluble fibrin in plasma and a decrease in platelet counts. Pretreatment with 25 Inh U/kg of a purified AT III - heparin complex foIlowed by a continuous infusion of 8.5 Inh U/kgh resulted in a significant inhibition of thrombin in plasma. This was demonstrated by the lack of specific consumption of plasma fibrinogen and the lack of an increase in soluble fibrin in plasma compared to LPS controls. The slight decrease in plasma fibrinogen in the drugtreated animals (fig. 2b) was probably caused by other factors (e.g. protein extravasation, plasma sequestration, non-specific cleavage), since it was paralleled by a similar decrease in serum total protein concentration. The tremendous increase in soluble fibrin in the control animals may in part be due to the generation of FDP during DIC. High plasma levels of FDP, in particular early fibrinogen- and fibrin-degradation-products, may influence the assay system. The late FDP fragment E has been shown to be about 40 times less stimulative than fibrin (24). Prothrombin consumption was similar in both groups, indicating that thrombin generation was not influenced by the AT III heparin complex (fig. 2a). This could be due to endotoxin-induced factor release (25) and subsequent thrombin generation that is not inhibitable by AT III heparin complex administration. Factor Xa could be protected from AT III when it is bound to cell membranes (26) or factor V / calcium / phospholipid complex (19).

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The AT III used in the complex preparation was isolated from human plasma and complexed in vitro to heparin. The concentration in plasma after administration could be assessed by rabbit antiserum employing an immuno-electrophoretic assay against human AT III. 64% of the concentration of a human reference plasma pool (equal 0.64 Inh U/ml) were detected 2 hours after the onset of the experiment and administration of about 800 Inh U of the complex preparation. Assuming a total plasma volume of 1200 ml, these findings suggest a good recovery. However, in part the complex may dissociate because of the relatively weak binding constant of 8~107M-1 between AT III and heparin (27). AT III activity in the AT III - heparin group also showed no specific decrease from 2 to 6h after onset of the experiment compared to the total protein content. Therefore, we assume that the applied dosage of 25 Inh U/kgh AT III - heparin complex preparation followed by a continuous infusion of 8.5 Inh U/kgh is enough to compensate the markedly reduced half life time of AT III plasma activity in septicemia (28, 29). The discrepancies between increasing AT III - heparin concentrations and decreasing AT III activity in plasma can be explained both by proteolytic breakdown and by complex formation of the degraded AT III - heparin complex with proteases. Degraded or protease complexed human AT III may increase until the end of the experiment due to the lack of metabolic turnover and excretion. Thrombin generation during septicemia has been related to lung dysfunction (4). The endotoxin-induced pulmonary dysfunction, as evidenced by a decrease in arterial oxygen saturation, was slightly improved in the treated animals. Also, the mortality rate was reduced in the AT III - heparin group. However, these differences were not statistically significant. This may be explained by the strong stimulus (high amount of endotoxin) used. Furthermore, it seems possible that the purified AT III - heparin complex does not inhibit thrombin effects, which are not related to the proteolytic breakdown of fibrinogen. Thrombin has been shown to exert direct cellular effects on blood and endothelial cells in vitro and in vivo (30,31). In conclusion, the AT III - heparin complex preparation is a potent inhibitor of thrombin effects on fibrinogen, prevents development of DIC, but fails to improve lung injury and to prolong survival time in porcine endotoxin shock under the applied conditions.

ACKNOWLEDGEMENTS The authors wish to thank Prof. Dr. Hans Fritz for supporting this project and Dr. Renate Kammerer for valuable assistance. Off print requests to Prof. Dr. W. Schramm, Med. Klinik, Ziemssenstr.1, 8000 Mtinchen 2, FRG

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