5
Control and isotopic quantification of affinityof antithrombin III for heparin4ike surfaces J. Caix, V. Migonney’ , A. Baquey, C. Fougnot” , A. Perrot Minnot, A. Beziade, L. Vuillemin, C. Baquey and D. Ducassou CNRS-GRECO no48 Polymeres Hemocompatiblas, Laboratoire de Biophysiqoe et d’lmmunologie Cellulaire, Universite de Bordeaux IL and *Laboratoire de Recherche sur les Macromolecules Villetaneose, Universite Paris-Non-L Paris, France Presented at Biointeractions ‘8 7, Cambridge, UK in July 198 7 Heparin-like materials, characterized by a defined superficial density of functional groups which activate antithrombin Ill (AT Ill), when in contact with blood specifically inhibit thrombin as soon as it appears. This paper describes an isotopic method to estimate this density and to visualize the distribution of the affinity sites concerned, both directly with AT Ill labelled with ‘2510dine and indirectly with an anti AT Ill monoclonal antibody labelled with “‘lndium. Keywords: Haemocompatibility, haparin-like. antithrombin III, monoclonal antibody, radiotracers. scintigraphy Some people consider that a material to be put in contact with blood is haemocompatible if its surface can inhibit the effects of thrombin which may be generated at the interface. This hypothesis explains the conception of materials demonstrating a specific affinity for antithrombin III (AT III) and able to catalyse the inhibition of thrombin by AT III as does heparin. This has led us to conceive of two types of materials: heparinized materials to which heparin is fixed in a covalent way whilst conserving its biological properties; and heparin-like materials, characterized by a defined superficial density of functional groups, contributing to the specific adsorption and activation of AT III. The objective of this paper is to describe an isotopic method based on the use of AT III labelled with lz510dine and of a monoclonal antibody labelled with “‘lndium, in orderto evaluate the density of such affinity sites and to visualize their repartition towards heparin-like materials. MATERIALS Heparin-like tubes The experiments described here were performed with polyethylene tubes of 3 mm diameter, manufactured by Becton Dickinson, the inner surfaces of which had undergone a treatment, previously described’,’ giving it an affinity for AT III and an antithrombin activity. Briefly, this treatment consists of covering the inner surface with polystyrene Correspondence to Mme Josselyne Caix, Ceemasi, lnserm 11.306, Universitb de Bordeaux II, 146 rue LBo Saignat, 33076 Bordeaux CBdex, France. (2 mg/cm2) by means of a radiochemical grafting of styrene. The aromatic rings are then chlorosulphonated by chloro- sulphonic acid in the presence of nitromethane and dichloro- methane. (A) PI Two kinds of tube can then be prepared: the inner surface bears (i) S02CI-Asp coming from the coupling of S02Cl with aspatticacid and (ii) -SO, anionic sites coming from side hydrolysis reactions of SO,CI groups, and the S02Cl groups are directly hydrolysed into SO, anionic sites. The proteins and their labelling Antithrombin III (30 units/ml) was provided by the Centre Regional de Transfusion Sanguine de Lille (batch 82023) and divided immediately into 0.5 ml aliquots which were kept at - 80°C. A purity control of the protein was carried out using affinity chromatography on the heparin Ultrogel (A&IBF-LKB). The so-called IODO-GEN labelling technique used3, is particularly adapted for fragile substrata: 10 jrg of 1, 3,4,6- tetrachloro-3a6a-diphenylglycoluril is introduced in advance into the tube used for labelling and allowed to evaporate at 37°C. The substratum, made in a phosphate buffer (0.01 M, pH: 7.3), and the radioactive iodide solution (1251 IMS 30, Amersham, Bucks, UK) were then added. After incubation for 30 min. the radioactive mixture was passed through heparin Ultrogel for affinity chromatography (Figure 1). A first elution, performed with a phosphate buffer at low ionic 8 1988 Butterworth Et Co (Publishers) Ltd. 0142-96 12/88/010062-05$03.00 62 Biomaterials 1988, Vol9 January

Control and isotopic quantification of affinity of antithrombin III for heparin-like surfaces

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Page 1: Control and isotopic quantification of affinity of antithrombin III for heparin-like surfaces

Control and isotopic quantification of affinity of antithrombin III for heparin4ike surfaces J. Caix, V. Migonney’ , A. Baquey, C. Fougnot” , A. Perrot Minnot, A. Beziade, L. Vuillemin, C. Baquey and D. Ducassou CNRS-GRECO no48 Polymeres Hemocompatiblas, Laboratoire de Biophysiqoe et d’lmmunologie Cellulaire, Universite de Bordeaux IL and *Laboratoire de Recherche sur les Macromolecules Villetaneose, Universite Paris-Non-L Paris, France

Presented at Biointeractions ‘8 7, Cambridge, UK in July 198 7

Heparin-like materials, characterized by a defined superficial density of functional groups which activate antithrombin Ill (AT Ill), when in contact with blood specifically inhibit thrombin as soon as it appears. This paper describes an isotopic method to estimate this density and to visualize the distribution of the affinity sites concerned, both directly with AT Ill labelled with ‘2510dine and indirectly with an anti AT Ill monoclonal antibody labelled with “‘lndium.

Keywords: Haemocompatibility, haparin-like. antithrombin III, monoclonal antibody, radiotracers. scintigraphy

Some people consider that a material to be put in contact with blood is haemocompatible if its surface can inhibit the effects of thrombin which may be generated at the interface. This hypothesis explains the conception of materials demonstrating a specific affinity for antithrombin III (AT III) and able to catalyse the inhibition of thrombin by AT III as does heparin. This has led us to conceive of two types of materials: heparinized materials to which heparin is fixed in a covalent way whilst conserving its biological properties; and heparin-like materials, characterized by a defined superficial density of functional groups, contributing to the specific adsorption and activation of AT III.

The objective of this paper is to describe an isotopic method based on the use of AT III labelled with lz510dine and of a monoclonal antibody labelled with “‘lndium, in orderto evaluate the density of such affinity sites and to visualize their repartition towards heparin-like materials.

MATERIALS

Heparin-like tubes

The experiments described here were performed with polyethylene tubes of 3 mm diameter, manufactured by Becton Dickinson, the inner surfaces of which had undergone a treatment, previously described’,’ giving it an affinity for AT III and an antithrombin activity. Briefly, this treatment consists of covering the inner surface with polystyrene

Correspondence to Mme Josselyne Caix, Ceemasi, lnserm 11.306, Universitb de Bordeaux II, 146 rue LBo Saignat, 33076 Bordeaux CBdex, France.

(2 mg/cm2) by means of a radiochemical grafting of styrene. The aromatic rings are then chlorosulphonated by chloro- sulphonic acid in the presence of nitromethane and dichloro- methane.

(A)

PI

Two kinds of tube can then be prepared:

the inner surface bears (i) S02CI-Asp coming from the coupling of S02Cl with aspatticacid and (ii) -SO, anionic sites coming from side hydrolysis reactions of SO,CI groups, and the S02Cl groups are directly hydrolysed into SO, anionic sites.

The proteins and their labelling

Antithrombin III (30 units/ml) was provided by the Centre Regional de Transfusion Sanguine de Lille (batch 82023) and divided immediately into 0.5 ml aliquots which were kept at - 80°C. A purity control of the protein was carried out using affinity chromatography on the heparin Ultrogel (A&IBF-LKB).

The so-called IODO-GEN labelling technique used3, is particularly adapted for fragile substrata: 10 jrg of 1, 3,4,6- tetrachloro-3a6a-diphenylglycoluril is introduced in advance into the tube used for labelling and allowed to evaporate at 37°C.

The substratum, made in a phosphate buffer (0.01 M,

pH: 7.3), and the radioactive iodide solution (1251 IMS 30, Amersham, Bucks, UK) were then added. After incubation for 30 min. the radioactive mixture was passed through heparin Ultrogel for affinity chromatography (Figure 1). A first elution, performed with a phosphate buffer at low ionic

8 1988 Butterworth Et Co (Publishers) Ltd. 0142-96 12/88/010062-05$03.00

62 Biomaterials 1988, Vol9 January

Page 2: Control and isotopic quantification of affinity of antithrombin III for heparin-like surfaces

Affinity of AT IN for heparin-like surfaces: J, Caix et a!.

I I I I I I I f

0 IO 20 30 40 50 60 70 80

Time t (mln 1 I

0 IO 20 30 40 50 60 70 80

Time t (mln)

Figure 1 Affinity chromatography profile of labelled AT ill on Ultrogel heparin (A4R-IBF-LKB). Conditions: phosphate buffer 0.0 1 M, pli 7.3, flow rate 30 m//h, temperat~ra 4”C, dual path monitor UV 2, Pbarmacia-Fine chemicals.

strength (NaC10.4 M), released in the dead volume a protein fraction without affinity for heparin and, in the total absorption volume, the excess iodide ions. A second elution was then performed at high ionic strength (NaCI 1.5 M). The protein fraction, having a high affinity for heparin, was recovered in the dead volume; this corresponded to a recovering yield (RY), which was computed from the total amount of AT III involved. RY was generally found to be between 30 and 409/04.

We checked by means of PAG electrophoresis that this labelled protein fraction had the same migration pattern as native AT III. The radioactive purity of the labelled protein was confirmed by autoradiography.

The anti-AT HI monoclonal antibody

After cell fusion, supernatant liquor from growing cultures was screened for antibody production by ELISA (Enzyme Linked lmmunosorbent Assay) using AT III as an antigen. Two hybridoma, IEI and llC9, were selected and purified by chromatography on protein A Sepharose.

For labelling, the antibody was at first linked with DTPA (diethylenetriaminopenta-acetic anhydride) and the adduct was mixed with an ’ ” In chloridesolution (CEA-ORIS, Gif sur Yvette, France). Purification was carried out by gel filtration using Sephadex-R G 200 (Pharmacia, Uppsala, Sweden). The labelling yield was between 50 and 60%.

A control of affinity of labelled monoclonal antibodies towards AT III was performed (Figure 2).

About 2 pg (20 ~1) of AT III and human serum albumin (HSA) were spotted into nitrocellulose strips (MILLIPORE, HAWP 304 FO) and incubated with different concentrations of mon~lonal antibodies (I El ) labelled with “‘In DTPAfor one night at 4°C and then 1 h at 37°C. After further washings (PBS 0.1 M, pH: 7.4), each spot was counted in a gamma scintillation counter. No significant binding to the labelled antibody could be observed whatever human serum albumin concentration was used. On the contray, a significant binding was obtained with the related antigen AT III when its concentration was less than 7 00 ng.

RADIOACTIVITY ANALYSIS DEVICES

Two kinds of investigation were performed.

Quant~tat~e analyses. Radioactivity was measured from ‘*?odine (related to antigen AT Ill) and from “‘lndium (related to monoclonal antibody anti AT Ill) by means of two high-purity germanium diode-based detectors (Enertec, Schlumberger, Lingolsheim 67380, Strasbourg), to an acquisition and processing system connected an Apple II micr~omputer.

Qualitative analyses. A scintigraphic visualization of the spatial distribution of each of the labelled proteins involved in the experiment was obtained with a gamma camera (Acti Camera 3400~CGR lssy les Moulineaux, France) successively set on the energy windows related to the major gamma peak of each isotope used. This technique has been explained in a former paper5.

METHODS

A quantitative study of the adsorption of lz51 labelled AT Ill on the tubes was carried out. This first step was followed by the study of the behaviour of antibodies meeting the surfaces eventually bearing adsorbed AT I II. The use of these labels allowed scintigraphic imaging of their topographic distribution on the surfaces studied and the functional homogeneity of the latter could therefore be checked.

Tubes A, Band C (polyethylene control) were connected in series. Both ends of the series were fitted to each end of the motive tube of a peristaltic pump, using two three-way T valves, in order to make a closed loop (Figure 3).

Antibody concentration, pq

Figure 2 Affinity of monoclonai antibodies labelled with “‘in DTPA towards cold AT Ill. These curves show the specificity of anti-AT Ill towards its antigen. 0, AT //I, antithrombin II/; v. HSA human serum albumin.

Eiomaterials f988, Voi 9 January 63

Page 3: Control and isotopic quantification of affinity of antithrombin III for heparin-like surfaces

Affinity of AT Ill for heparin-like surfaces: J. Caix et al.

Figure 3 Circuit of tubes A B and C connected to a peristaltic pomp

The procedure comprised the following steps: the circuit was initially filled with Michaelis’ buffer; a dynamic incubation for 30 min with radiolabelled AT III (AT Ill*) (1 unit/ml) (flow rate was set at 30 ml/min); at the end of this period, the radioactivity of a given part of each tube is measured under two different conditions. First, tubes were kept filled with AT Ill* solution and the value, RIoo, of the radioactivity measure estimated the maximal amount of AT Ill* available for absorption onto the internal surface of the tubes. Second, the tubes were emptied and rinsed with 9% NaCl for 15 min. under the same conditions. The value, R,, corresponds to the amount of AT Ill* actually absorbed onto the inner surface of the tubes; for each tube, the quantity

RX ~ X 100 (retention percentage) R 100

characterizes its affinity for AT III, then the labelled anti AT III antibody was introduced into the circuit at the same molar concentration used for the antigen in step (b) and for an incubation of 15 minutes, using the same procedure, measures of radioactivity R’,oo and R! were carried out.

RESULTS

Measurements with semiconductor detectors (Table 1)

AT III seems to adsorb as well onto tube A as onto tube B.

The retention percentage was indeed around 30%. compared to less than 10% for control tubes (tube C). However, AT III can only be recognized by its related antibody, when it is adsorbed on tube A. In this case it can be noticed that the number of retained antibody molecules is equal to the number of adsorbed AT III molecules, which was consistant with mole-to-mole formation of the antigen antibody complex.

Measurement with gamma camera

lz51 scintigraphyshows that AT III is well fixed along tubes A and B, but the adsorption appears less homogeneous on the latter. Taking into account the technical conditions of imagery and the low radioactivity level of tube C, this cannot be visualized (Figure 4). ’ ’ ’ In scintigraphy shows the anti AT III antibody is only retained at a significant radioactivity level by tube A, bearing adsorbed AT III (Figure 5).

DISCUSSION

In this study, we have shown that polyethylene tubes can have their inner surface modified to allow AT III to adsorb on it. The specific fixation of an antibody on the related antigenic molecule AT III proves AT III is adsorbed according to different mechanisms depending on whether the material bears sulphonated groups or sulphonamide groups. In the former case, the antigenic site probably becomes hindered, or suffers a conformational change through the adsorption process (tube B). However, adsorption of AT III on tube A seems to preserve its ability to bind to its specific antibody. We can state with confidence that, the ability of non-specific interaction of immunoglobulins, IgG, with preadsorbed AT I II on the same tubes has been shown to be negligible.

CONCLUSION

The radioisotopic technique used here appears to be a very efficient tool with which to characterize biomaterials especially designed to feature affinity for AT III. We can distinguish different interaction modes of AT III with such materials. Moreover, the use of such methods can be generalized: every time a biomaterial is designed to demon- strate a specific affinity for a given protein, it is possible to apply the same methodology in so far as the protein and its antibody can be radiolabelled.

Table 1 Measurements with semiconductor detectors

Tubes tested

A B C

<

SO,_ PE ~ PS PE - PS so,- PE

SO*-Asp

Retained radioactivity 29 30 30 26 29 28 8 10 9

in percentage (lz51), RIO0

Retained concentration of 67.3 69.6 69.6 60.3 67.3 64.9 18.6 23.2 20.8

AT Ill* in p mol/cm* of inner surface, R,

Retained radioactivity 30 30 31 3 4 3 0

in percentage (“‘In). R;oo

Retained concentration of 69.6 69.6 71.9 6.9 9.2 6.9 0

anti AT Ill* antibody in p mole/cm2 of inner surface,

R;

64 Biomaterials 1988, Vol9 January

Page 4: Control and isotopic quantification of affinity of antithrombin III for heparin-like surfaces

Affinity of AT l/i for heparin-like surfaces: J. Ca/x et al

Figure anti-A7

4 ~~5~scintigrsp~ showing tubes R 8 and C after two incubation steps, firstly wifh a solution ofAT fit I&e/led with f25 ’ fff monoclonal antibody labelled with ’ ’ ‘ln

f and secondly with a solution of

Figure 5 1 “‘l/l scintigraphy showing tubes R 8 and C after the two incubation steps

Biomaterials f988, Vol 9 January i

Page 5: Control and isotopic quantification of affinity of antithrombin III for heparin-like surfaces

Affinity of AT Ill for heparin-like surfaces: J. Caix et al.

REFERENCES

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Artificial Organs, 1986, 10, 481-488 6 Migonney, V., Fo~ctionnal;satioR de la surface inteme de mat&iaux

tobulai~es. Etude de ~jflbibitioR de fa th~ombine par ~e~tithfombiffe 1118 /a surface de ces mat&iaux, Doctoral Thesis Paris, 1986

7 Fougnot, C., Jozefowicz, M., Bara. L. and Samama, M., Interactions of anticoagulant insoluble modified polystyrene resins with plasmatic proteins, Thromb. Res. 1982, 28, 37-46

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Biomaterials 1988, Vol9 January 65