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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1292-1297.)
© 1996 American Heart Association, Inc.

Articles

Thrombin Inhibition by Antithrombin III on the Subendothelium Is Explained by the Isoform ATß

Siw Frebelius; Sven Isaksson; Jesper Swedenborg

the Vascular Research Group, Department of Surgical Sciences, Karolinska Institute, and Pharmacia (S.I.), Stockholm, Sweden.

Correspondence to Jesper Swedenborg MD, Vascular Research Group, Department of Surgery, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail kjsg@kir.ks.se.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Balloon injury of the rabbit aorta results in thrombin coagulant activity on the injured vessel wall that causes fibrin formation. The anticoagulant activity of both the intact and injured vessel wall has been partly explained by glycosaminoglycans with heparin-like activity that augment the activity of antithrombin III (AT). AT exists in two isoforms, and ß. ATß, which constitutes only 5% to 10% of AT in plasma, lacks one carbohydrate side chain, has higher affinity for glycosaminoglycans, and associates more readily with the subendothelium. This study evaluated whether AT can inhibit thrombin on the injured vessel wall and, if so, whether one of the isoforms is more effective then the other. The two isoforms were isolated from human plasma by heparin-Sepharose chromatography, and the purity was investigated by isoelectric focusing and crossed immunoelectrophoresis. Rabbits were subjected to balloon injury of the aorta; 3 hours after injury the aorta was excised. Thrombin coagulant activity on the aorta was measured by exposure to fibrinogen and thereafter by measuring the generation of fibrinopeptide A. Injured animals were treated with AT, AT, or ATß and were compared with control animals. AT was demonstrated on the injured vessel wall by using an immunohistochemical method. Animals receiving crude AT had significantly lower amounts of thrombin coagulant activity on the injured aortic wall than control animals, but AT at a comparable dose had no effect. ATß was given in the same dose as crude AT and also at a dose (10%) proportional to its presence in plasma. Animals receiving ATß had significantly lower values of thrombin on the injured aortic wall than control animals. We conclude that the inhibitory effect of AT on thrombin coagulant activity on the injured vessel wall is explained by its ATß content.

Key Words: antithrombin III • antithrombin isoform • thrombin • vascular injury • fibrin

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Thrombosis on the injured arterial wall in response to surgery or balloon angioplasty is an important clinical problem in connection with vascular reconstruction. Since fibrin formation, which is caused by thrombin, is a major component of the thrombosis, inhibition of thrombin is a key event in the defense against vessel wall thrombosis.

The normal vessel wall has two primary systems, which involve TM¹ and heparan sulfate,² to prevent thrombin coagulant activity. When bound to TM, the specificity of thrombin is altered so that it no longer cleaves fibrinogen but instead inhibits factors V and VIII, which precede thrombin in the coagulation cascade.³ Thus, binding of thrombin to TM induces a negative feedback loop that stops the coagulation. Heparan sulfate, which is synthesized by endothelial cells and is present in the endothelial cell layer,⁴ acts similarly to injected heparin by binding and catalyzing the inhibitory effect of AT.^{5 6}

It could be assumed that the inhibition of thrombin coagulant activity by TM is lost after arterial injury since TM is present only in the endothelial layer.^{7 8} GAGs of slightly varying compositions exist in all layers of the arterial wall.^{9 10} GAGs in the deeper layers of the vessel wall can also support inhibition of thrombin by AT, although the inhibition of thrombin is reduced after deep vessel-wall injury.^{11 12}

Binding of AT to the deeper vascular layers exposed after vessel-wall injury occurs both in vitro¹¹ and after balloon injury in vivo.^{13 14} AT infused after arterial injury accumulates on the injured surface and results in decreased amounts of thrombin coagulant activity on the injured surface.¹³ Irrespective of the initiating mechanism, fibrin formation on the injured vessel wall is preceded by thrombin generation.^{15 16 17 18} Consequently, inhibition of the thrombin coagulant activity associated with the injured vessel wall also prevents fibrin formation.

AT is present in two isoforms in plasma, AT and ATß. The latter constitutes approximately 5% to 10% of the total content of plasma AT.^{19 20} ATß is less negatively charged and has a higher affinity for heparin and subendothelial GAGs.^{20 21}

The purpose of the present study was to examine the effect of AT and ATß on the inhibition of thrombin after balloon injury of the vessel wall.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Purification of AT and ATß
As starting material, a heparin-Sepharose eluate from Pharmacia's commercial production of AT was used. This eluate was rechromatographed on a heparin-Sepharose column (Pharmacia) and eluted with a linear gradient of 0.15 to 1.5 mol/L NaCl in 20 mmol/L Tris-HCl (pH 7.5) to give fractionation of AT and ATß.²¹ Each fraction was then concentrated by using an ultrafilter system (Sartorius Filtration AB) with a 10 000-kD cutoff point.

Screenelectrophoresis and IEF
The proteins in the sample solution were separated according to charge by electrophoresis with agarose as the stabilizing medium. After electrophoresis proteins were fixed and stained with Coomassie blue.

IEF, a high-resolution technique for separation of proteins that is based on their pIs, was performed by using the Pharmacia PhastSystem with precast gels (pI 4 to 6.5) according to the Pharmacia PhastSystem manual, Separation Technique File No. 100. IEF was followed by silver staining.

Heparin Cofactor Activity
The biological activity of AT was defined as heparin cofactor activity expressed in international units as defined by the international standard for AT concentrate (National Institute for Biological Standards and Control, Hertfordshire, UK). The assay is based on inactivation of thrombin by AT in the presence of excess heparin (Pharmacia). The remaining thrombin splits off the chromogenic paranitroaniline group from the chromogenic substrate S-2238 (Chromogenix AB). Thus, the AT activity is inversely proportional to the release of paranitroaniline, which is measured as a change in optical density at 405 nm.²²

Crossed Immunoelectrophoresis in the Presence of Heparin
The heparin-binding capacity of the two AT isoforms was determined by using crossed immunoelectrophoresis with agarose gels.²³ First-dimension electrophoresis was performed in the presence of heparin and the absence of antibodies. Heparin, which is negatively charged, binds to native AT, resulting in an increased negatively charged molecule and enhanced mobility compared with non–heparin-binding molecules. In the second-dimension electrophoresis AT molecules migrated into a gel containing antibodies against human AT (DAKO A/S).

Experimental Procedures
Vessel-Wall Injury
Forty-eight New Zealand White rabbits of both sexes (body weight, 2.5 to 5.5 kg) were anesthetized with sodium pentobarbital 40 mg/kg IV, and vessel-wall injury was obtained by pulling a No. 3 Fogarty embolectomy catheter with an inflated balloon along the aorta three times.

The rabbits were divided into five groups. Four groups received different preparations of AT, and a control group received physiological saline immediately after the vessel-wall injury. One group received AT (specific activity, 6 IU/mg protein; Pharmacia) 20 IU/kg in 0.15 mol/L NaCl as a bolus followed by 40 IU/kg IV over 1 hour. Two groups received AT or ATß (specific activity, 6 IU/mg protein) in equal doses as crude AT in milligrams per kilogram body weight. Since ATß constitutes only 10% of the total content of AT in plasma, the fifth group was given ATß in a dose 10% that given to the other groups. Blood samples were collected in 3.8% sodium citrate (9:1) before and 3 hours after injury to determine the plasma AT concentration.

The rabbits were killed after 3 hours, and the aorta was immediately excised,¹³ rinsed with 20 mL Tyrode's buffer, and placed in Tyrode's. Tyrode's buffer contained one vial Tyrode's powder (GIBCO) dissolved in distilled water, sodium bicarbonate 50 g/L, and HEPES 20 mmol/L (Merck); pH was adjusted with NaOH to 7.4. Excess tissue was removed, and the aorta was divided into 2.5-cm-long segments. Smaller segments were also taken for immunohistochemical examination. The longer segments were carefully everted by using a nontraumatic technique and mounted on a polyethylene rod for determination of thrombin on the injured surface. The ends of the segments were ligated to avoid contact between the outer surface of the vessel and the incubation solutions. The aortic segments were then rinsed with 20 mL Tyrode's solution and immediately rotated in a fibrinogen solution (Imco Ltd) to determine coagulantly active thrombin on the injured surface.

Injured aortic segments were also incubated in vitro with 1 mL human thrombin (5 NIH U/mL [70 pmol]; Sigma) in Tyrode's buffer containing 0.5% human serum albumin (Pharmacia) and 0.2% polyethylene glycol 6000 (KEBO). The concentration of the commercial thrombin was determined by comparisons with the international standard for thrombin (National Institute for Biological Standards and Control). Coagulantly active thrombin on the surface of the injured segment was determined.¹³

Assays of Plasma AT and Surface Thrombin
The plasma AT concentration was determined by using a heparin cofactor method with a chromogenic substrate as described above for heparin cofactor activity and was compared with the international standard for human AT.

Thrombin coagulant activity on the injured aortic surface was measured by rotating the aortic segments in 1 mL 0.2% human fibrinogen for 10 minutes. Fibrinopeptide A, which forms during such incubation, is a measure of thrombin coagulant activity. After the incubation, fibrinopeptide A production was interrupted by adding 0.1 mL of a protease inhibitor solution containing 1000 IU aprotinin (Bayer AG), 1000 IU heparin, and 11 IU AT/mL. The amount of fibrinopeptide A was determined by using a radioimmunoassay,^{24 25} and the corresponding thrombin coagulant activity on the aortic surface was calculated as units per centimeter squared. The background activity recorded on noninjured control segments by the substrate was subtracted from all determinations. The method for determination of thrombin on surfaces has been described in detail.²⁶

Immunohistochemistry
Segments were taken from the middle section of the injured aorta, frozen on dry ice, and stored at -70°C. Cryosections (5 µm) were thaw-mounted on SuperFrost/Plus slides and refrozen at -70°C. Specimens were air-dried and incubated for 2 hours at 20°C in normal goat serum (DAKO A/S) diluted 1:40 in Dulbecco's PBS with Ca²⁺ and Mg²⁺ (GIBCO). The sections were then incubated overnight at 4°C with a polyclonal primary antibody against human AT raised in rabbit (DAKO A/S) or a monoclonal mouse antibody against rabbit fibrin with no reactivity toward fibrinogen (a kind gift from Dr Tomofuni Kurokawa, Takeda Chemical Industries) diluted 1:50 together with normal goat serum (1:40) in PBS. After the overnight incubation the specimens were rinsed three times for 5 min/time in PBS and exposed to fluorescein isothiocyanate–conjugated goat anti-rabbit IgG (Sigma) or anti-mouse IgG (Sigma) diluted 1:50 in normal goat serum/PBS for 2 hours at 20°C. After being rinsed in PBS, the specimens were mounted in Vectashield (Vector Laboratories).

Background staining was determined either by omitting the primary antibody and staining with the secondary antibody only or by using nonimmune rabbit IgG as the primary antibody. Background staining of the secondary antibody used together with fibrin was also examined. The specimens were examined by using a Nikon Labophot microscope with epifluorescence optics, and photographs were taken on Kodak Tri-X-Pan film.

Statistical Analysis
ANOVA and multiple comparisons were used for statistical analysis to compare different groups.²⁷

	Results

Top Abstract Introduction Methods Results Discussion References

 
Isolation and Characterization of AT and ATß
The fractions of the two isoforms of AT from the gradient elution showed a difference in heparin affinity. AT was eluted at 0.8 and ATß at 1.2 mol/L NaCl. As evaluated by screenelectrophoresis, AT was more negatively charged than ATß, and IEF confirmed that the two isoforms had different pIs, 4.9 for AT and 5.1 for ATß (Fig 1). Heparin cofactor analysis revealed that both isoforms have biological AT activity (specific activity, 6 IU/mg protein for both isoforms). Crossed immunoelectrophoresis confirmed that the antigen-antibody complex is similar for the two isoforms.

View larger version (111K): [in this window] [in a new window]   Figure 1. The two isoforms of AT had different pIs compared with a pI standard as assessed by using an IEF technique. Lanes 1-3 show commercial AT, human serum albumin control, and eluate, respectively, from the first rechromatography with heparin-Sepharose. After further rechromatography on a heparin-Sepharose column with an NaCl linear gradient AT and ATß were eluted; lanes 4 and 5 show fractions from two different preparations of AT. Lanes 6 and 7 show ATß in two different concentrations, and lane 8, the pI standard (range, pI 4 to 6.5).

The plasma levels of AT increased after infusion of crude AT or AT. The lower dose of ATß did not affect plasma levels of AT, whereas the higher dose caused an increase of the plasma AT concentration (Fig 2).

View larger version (50K): [in this window] [in a new window]   Figure 2. Bar graph shows plasma levels (mean±SD) of AT (AT III) before () and 3 hours after () aortic injury. The AT infusion started at the time of injury and lasted for 1 hour. AT concentration was determined by using a heparin cofactor method with a chromogenic substrate and was compared with the international standard for AT.

Thrombin on the Aortic Surface After Injury
After balloon injury thrombin was measured on the excised aortic segments. Control animals had significantly higher levels of thrombin on the aortic surface than animals that received crude AT and ATß in both doses used (P=.001 by ANOVA ). The thrombin concentration on the injured aortic surface from animals that received AT, however, did not differ from the control animals. Thus, it can be concluded that the inhibitory effect of AT on the appearance of thrombin coagulant activity on the injured vessel wall is mainly explained by its ATß content (Fig 3).

View larger version (26K): [in this window] [in a new window]   Figure 3. Box plot presentation of thrombin coagulant activity on the surface of balloon-injured aortic segments from animals treated with crude AT, AT, or ATß for 1 hour. The animals were killed after 3 hours, and thrombin coagulant activity on the excised aorta was measured. Comparisons are with aortic segments from injured animals receiving physiological saline (control). Lines within the boxes indicate the 50th percentile (median) of thrombin coagulant activity; lower and upper ends of the boxes, 25th and 75th percentiles, respectively; and horizontal lines below and above the boxes, 10th and 90th percentiles, respectively. Significantly less thrombin was demonstrated on the injured aortic surface in animals treated with crude AT or ATß (*P=.001 by ANOVA).

Thrombin Inhibition and AT Binding and Fibrin Deposition
Thrombin on the vessel wall was measured on excised injured aortic segments after exposure to a thrombin solution in vitro. Thrombin segments obtained from animals receiving crude AT, AT, and ATß had significantly lower thrombin concentrations on the surface after incubation with thrombin than segments obtained from control animals (P=.008 by ANOVA). There seemed to be no difference between animals receiving AT and ATß. The animals that had received ATß in the higher dose had the lowest thrombin concentrations after incubation in vitro, but they were not significantly different from those that had received AT or ATß in the lower dose (Fig 4).

View larger version (21K): [in this window] [in a new window]   Figure 4. Box plot presentation of surface-bound in vitro thrombin coagulant activity after incubation of balloon-injured aortic segments with thrombin (70 pmol) in vitro for 5 minutes. Animals received an infusion of saline (Control), crude AT, AT, or ATß immediately after injury, and the aorta was excised 3 hours later. See Fig 3 for explanation of box plot. Aortic segments from animals treated with all forms of AT inhibited thrombin more effectively than control (*P=.008 by ANOVA).

AT was detected on the injured vessel wall with an anti–human AT antibody by using immunofluorescence microscopy. The antibody reacted with crude AT as well as AT and ATß. Fig 5 shows AT on the injured vessel wall and after infusion of the different human AT preparations. Fibrin was detected on the control aorta after injury as well as after treatment with crude AT or AT. Almost no fibrin could be detected on the injured aorta from animals treated with the higher dose of ATß (Fig 6). Fibrin was, however, detected on the aorta of animals receiving the lower dose of ATß (10%) (not shown in Fig 6).

View larger version (131K): [in this window] [in a new window]   Figure 5. Photomicrographs show AT detected by immunofluorescence with an anti–human AT antibody as an intense staining overlying the internal elastic lamina on the injured aortic surface from animals receiving (a) AT, (b) AT, or (c) ATß. No staining for AT was detected when the injured animals were treated with physiological saline (d) (original magnification x250).

View larger version (124K): [in this window] [in a new window]   Figure 6. Photomicrographs show fibrin detected by immunofluorescence with a monoclonal anti-rabbit fibrin antibody as an intense staining overlying the internal elastic lamina after balloon injury in control animals receiving physiological saline (a). Animals with a continuous infusion of ATß for 1 hour at the same dose as for AT and AT had almost no detectable staining for fibrin (b). Fibrin was also detected on the internal elastic lamina from animals treated with (c) crude AT or (d) AT (original magnification x250).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
AT, the major physiological inhibitor of thrombin, is present in plasma in concentrations ranging between 0.88 and 1.36 IU/mL (2.6 µmol/L).²⁸ The progressive inhibition of thrombin by AT can be increased approximately 2000-fold by heparin.²⁹ Heparin-like GAGs and heparin induce a conformational change in the AT molecule.^{2 30 31} Heparin is not present in plasma, but endothelial cells synthesize heparan sulfate, which acts as a cofactor for AT. The intact surface of blood vessels binds AT, a mechanism that is of major importance for the inhibition of thrombin on the vessel wall.³²

The initial event after arterial injury is accumulation of platelets; fibrin formation occurs later.³³ Thus, fibrin formation on the vessel wall after arterial injury in vivo has been demonstrated in experimental animals after vessel-wall injury and also on atherosclerotic plaques.^{13 34 35} Thrombosis after arterial injury in clinical situations, eg, percutaneous transluminal angioplasty either in coronary or peripheral arteries or vascular surgical procedures, is an important clinical problem. Fibrin binds further thrombin, and when bound to fibrin it is partially excluded from inhibition by the heparin-AT complex.^{36 37} Thus, inhibition of the initial fibrin formation may be clinically important.

TM is confined to the endothelial layer and is therefore not present after arterial injury with loss of endothelium.³⁸ Different GAGs with heparin-like activity, mainly dermatan sulfate, are also present in the deeper arterial layers; dermatan sulfate binds thrombin and can support thrombin inhibition by heparin cofactor II.³⁹ AT-mediated inhibition of thrombin has also been demonstrated after arterial injury with exposure of deeper arterial layers.^{12 13}

ATß is glycosylated to a lesser extent depending on the lack of one of four carbohydrate side chains, ie, the chain that originates at Asp 135.²¹ A similar type of recombinant AT that lacks Asp 135 and therefore has no carbohydrate side chain in this position has been expressed in a baculovirus system after site-directed mutagenesis.⁴⁰ ATß has a higher affinity for heparin as well as a higher affinity for both the intact and injured vessel wall.⁴¹ Because of its higher affinity for heparin compared with AT, ATß is cleared more rapidly by heparin from the vascular compartment.⁴²

In the present study binding of AT to the injured vessel wall was demonstrated for both isoforms by using immunohistochemistry. By using an infusion of human AT, binding of AT could be detected on the injured rabbit aorta without any interference with native AT since the antibody only reacts with human AT. Human AT is reported to have a distribution in rabbits that is equal to native AT.⁴³

There was a discrepancy between the in vitro and in vivo findings. ATß but not AT fully prevented the appearance of thrombin with coagulant activity after vessel-wall injury in vivo. When exposed to thrombin in vitro, thrombin was found in equal amounts on arterial segments irrespective of which isoform the animals had received. The amount of thrombin on the vessel wall after incubation in vitro is inversely proportional to the inhibitory capacity.²⁶ Both isoforms of AT were present on the injured vessel wall and both had equal heparin cofactor activity. It is therefore logical that arterial segments from animals receiving either isoform should behave equally when tested in vitro. The ATß isoform is probably more effective in vivo because it binds more rapidly to the injured vessel wall and prevents initial fibrin formation.

It has been reported that ATß binds more readily to both intact and injured vessels,⁴¹ and the present study confirms these findings in vivo. The amount of ATß present in normal AT could fully explain the inhibition of thrombin appearing on the injured vessel wall provided by crude AT. ATß is reported to inhibit thrombin more rapidly than AT,²⁰ and possibly it is mainly the ATß in plasma that is in equilibrium with vessel-wall AT. However, whether the relative amount of ATß on the vessel wall differs from that in plasma remains to be shown. In conclusion, ATß may be more effective than crude AT in preventing thrombosis on the injured vessel wall after interventional procedures for vascular occlusive disease.

	Selected Abbreviations and Acronyms

 

AT = antithrombin III GAG = glycosaminoglycan IEF = isoelectric focusing pI = isoelectric point PBS = phosphate-buffered saline TM = thrombomodulin

	Acknowledgments

 
This work was supported by grants from the Swedish Heart Lung Foundation, the Swedish Medical Research Council (project No. 09100), the Karolinska Institute, and the King Gustaf V Foundation.

Received December 21, 1995; revision received March 7, 1996;

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