Action of insolubilized homologous anti-factor VIII antibodies on factor VIII-related antigen

THROHBOSIS RESEARCH Prinred in Great

ACTION

OF

INSOLUBILIZED ON FACTOR

C.

MAZURIER,

M.

13, pp. 661-667, Pergamon Press,

l-01.

Britain

KACEM,

HOMOLOGOUS

ANTI-FACTOR

VIII-RELATED

A.

VIII

1977

Lsd.

ANTIBODIES

ANTIGEN

PARQUET-GERNEZ,

M.

GOUDEMAND

Laboratoire d'Hemostase, Centre Regional de Transfusion Sanguine 21, rue C. Guerin - 59000 LILLE - FRANCE

1.8.1976; (Received Accepted by

in revised Editor M.J.

form

8.3.1977.

Larrieu)

ABSTRACT Immunoadsorption experiments were performed to study the action of homologous anti-factor VIII antibodies on factor VIII-related antigen (VIII R : Ag). Two factor VIII inhibitors, one from a hemophiliac patient, the second from an otherwise normal patient, were studied after coupling to CNBr-activated Sepharose. Both antibodies bound VIII R : Ag specifically. The presence of factor VIII procoagulant activity (VIII : C) did not appear necessary for this interaction since VIII R : Ag dissociated from VIII : C by salt dissociation and VIII R : Ag of a severe hemophiliac were also equally bound. These results suggest that the action of homologous anti-factor VIII antibodies is not limited to the neutralizing effect on VIII : C.

INTRODUCTION

Until recently, factor VIII had been characterized

only by its pro-

coagulant activity (VIII : C). Immunological studies have since helped to clarify some of its properties. Heterologous antibodies raised in rabbits against purified human factor VIII not only inhibit VIII : C but also precipitate a normal plasma protein which appears to be closely associated with it. This antigenic material (VIII R : Ag) is also present in hemophilia A plasma and in normal serum, but is decreased in the plasma of patients with von Willebrand disease. Homologous antibodies to VIII : C occur either in some polytransfused patients with hemophilia A or spontaneously in patients with no prior history of bleeding. None of these human inhibitors precipita661

662

ANTI

VIII

HOMOLOGOUS

ANTIBODIES

Vol.lO,No.~

tes VIII R : Ag. However by "inhibitor neutralization test" Goudemand (1) showed that homologous anti-VIII : C antibodies are neutralized not only by normal plasma but also by normal sera or plasmas from some patients with hemophilia A (2). Hemophilia A can thus be divided into two types based on the presence (A+) or absence (A-) of inhibitor neutralizing material. However a number of authors (3.4) have shown that by varying the antibody concentration and incubation times, the majority of hemophiliacs can be shown to have antigenic material capable of interacting with and neutralizing homologous inhibitors. The present study attempts to elucidate the action of human anti-VIII

: C antibodies on the VIII R : Ag of normal and hemophilia A plasma.

MATERIALS

AND

METHODS

Antibodies. The experiments were carried out using two human anti-VIII : C antisera. The first one had developed in a patient with hemophilia A (S.T.) who had been transfused exclusively with human fractions. In this patient's plasma, the inhibitor titer, expressed by the method of Rasper (5) was 1500 Bethesda units/ml. The second was a spontaneous inhibitor occuring in a JJ-year old woman (D.E.) with no definite evidence of an associated disease. The inhibitor titer was 500 Bethesda u/ml. Samples analyzed. Normal and hemophilia A plasmas were prepared by anticoagulation of freshly collected whole blood with 0.38 p.cent sodium citrate and centrifugation at 1200 g for 30 min. at 4°C. A high molecular weight fraction of factor VIII (HMW) was obtained by gel filtration of a factor VIIIrich fraction on Sepharose 6 B equilibrated in 0.02 M imidazole, I M NaCl, pH 6.5 buffer (6). The fraction collected at the void volume was devoid of procoagulant activity but contained

8 u/ml of VIII R : Ag.

Factor VIII coagulant activity and VIII-related antigen Assays.

VIII : c was

assayed by a one-stage method (J). The procoagulant activity could not be assayed in phosphate containing buffer.The factor VIII R : Ag was measured by the Laurel1 quantitative immunoelectrophoresis (8) using dilutions of plasma. The standard was a pool of normaL titrated plasmas prepared Erom 20 normal healthy individuals stored at -80°C for a maximum of one month.

Vol.

10,No. j

ANTI

\;I11 HOMOLOGOUS

XS'IIBODIES

663

Immunoadsorbents. One volune of saturated .Ammoniua sulphate was separately added to two volumes each of the anti-VIII : C antisera and of a serum from a hemophiliac without anti-VIII : C activity. The precipitate obtained after centrifugation was redissolved in two volumes of 0.05 M sodium carbonate,pH 9.0 buffer and dialysed against this buffer for 48 hrs. The anti-VIII : C activity in each was about 1500 u/ml (S.T.) and 500 u/ml (D.E.). 400 mg of the dialysed gamma-globulin fractionswere coupled to 40 ml of CNBr-activated Sepharose 43 (Pharmacia - Uppsala - Sweden) according to the method of Axen et al. (9) and separately packed into columns (2 x 28 cm), washed alternatively vith pH 4 and 8.5 buffers and then equilibrated in 0.1 M phosphate buffered saline, pH 7.5 (PBS). In consecutive experiments 0.5 ml of normal plasma, plasma from a patient with severe hemophilia A (without inhibitor) and HMW fraction were applied to the columns and the columns were washed with 250 ml PBS. The flow rate was 8 ml/hr. Samples were collected in two ml aliquots and the protein continuously monitored by absorbance at 280 nm. The filtrates obtained during washing were pooled and concentrated by pressure dialysis to the initial volume of the sample using a UH 100 membrane (Schleicher and Schiill- Dassel - Germany). Then the specifically bound proteins were eluted (18 ml/hr) with NaSCN pH 6.0,

3 M in 0.1 M acetate buffer

collected and concentrated as before. Each experiment was performed

in duplicate and each sample tested for VIII R : Ag.

RESULTS

Homologous anti-VIII : C antibodies insolubilized onto Sepharose appear to be capable of interacting with VIII R : Ag. Less than 10 % of the VIII R : Ag of plasma applied to such columns was recovered in the filtrate. However following application of 3 M NaSCN a protein peak was eluted which contained the residual 80 % VIII R : Ag (table I). Anti-VIII : C antibodies arising in a hemophiliac (S.T.) or occuring spontaneously in a non-hemophiliac patient (D.E.) appeared to have similar properties (table I and II). These factor VIII inhibitors also appeared to bind VIII R : Ag from severe hemophiliacs and from the HMW fraction obtained by salt dissociation (table II and III). Both these sources of VIII R : Ag are devoid of VIII : C. Over six experiments tith different sources of VIII R : Ag the yield of VIII R : Ag recovered from the column by NaSCN elution varied from 40-86 % with a mean of 69 %.

66ti

XSTI

VIII HOMOLOGOUS

XSTIBODIES

TABLE I 0.5 ml plasma were applied to two separate columns to which had been bound either IgG from a hemophiliac without circulating inhibitor (H.A.) or IgG from a hemophiliac with a circulating inhibitor (S.T.). The initial filtrate after washing with 250 ml PBS and the eluate after subsequent washing with NaSCX were collected and concentrated to 0.5 ml and assayed for VIII R : Ag.

Starting material

Filtrate

Control Column (H.A.)

Homologous Antibody Column (S.T.)

Normal plasma 1.10 u/ml

Normal plasma 1.00 u/ml

0.6

Eluate

< 0.10 u/ml

u/ml I

I < 0.10 u/ml

0.8 u/ml

TABLE II Recovery of VIII R : Ag in PBS filtrate and subsequent NaSCN eluate from column to which had been bound the IgG from a patient with a spontaneous inhibitor to VIII : C. Both normal plasma and the plasma of two patients with severe hemophilia A were tested.

Kormal plasma

Hemophilic A plasma (VIII : C < 0.01 u/ml)

Hemophilic A plasma (VIII : C < 0.01 u/ml)

This ability to bind VIII R : Ag appeared to be specific. The Sepharose column to which had been bound the control IgG (hemophiliac without inhibitor) did not appear to significantly adsorb VIII R : Ag. Sixty per

cent of the applied VIII R : Ag was recovered in t'nefiltrate during i;as'nin; and no significant VIII R : Ag was elgutedwi:h ?iaSCS (table I).

TABLE III Recovery of VIII R : hg in PBS filtrate and subsequent NaSC?i eluate from homologous antibody column (ST) after application of :XO different sources of VIII R : hg devoid of VIII : C

Hemophilic A plasma Starting material Filtrate Eluate

I

H?fWfraction I

1.10 u/ml

3.00 u/ml

< 0.10 u/ml

< 0.10 u/ml

0.90 u/ml

6.90 u/ml

DISCUSSION

Homologous antibodies, whether spontaneous or acquired in hemophilia A, neutralize the human factor VIII procoagulant activity (VIII : C). Contrary to heterologous rabbit antibodies, homologous antibodies do not precipitate with VIII R : Ag and it is uncertain if they have any action on VIII R : Ag. From the results presented here it appears that the VIII R : Ag of a normal plasma is adsorbed onto two human inhibitors which are insolubilized on Sepharose 4 B. This fixation is specific. It does not occur with similarly insolubilized gamma-globulin fraction of hemophilic serum with no VIII : C inhibitor activity. The two homologous anti-factor VIII also bind the VIII R : Ag of different patients with hemophilia A who had seemed to have no detectable antigenic material by the "inhibitor neutralization test" of Goudemand et al. (A-). However this immunoadsorption method does not allow any quantitative measurement of the amount of VIII R : hg in the different samples studied. It should also be considered possible that heterogeneity amongst homologous antibodies could exist with respect to their action on VIII R : Ag. Previously, other authors (IO, II) reported the absence of fixation of VIII R : Ag onto homologous antibodies. Zimmerman and Edgington (IO) showed that there was a 58 X loss of VIII : C without decrease of VIII R :

666

XXTI

VIII

HOMOLOGOUS

AMTIBODIES

Vol. 10,?;0.5

Ag in the supernatant of a normal plasma incubated vith hemophilia antibody beads in a batch systen. Moreover, Hougie and Sargeant (ll), demonstrated differential precipitation of the procoagulant activity but not of the antigen following complex formation between anti-VIII homologous antibodies and cryoprecipitate. Our experiments performed under different experimental conditions show that homologous antibodies, characterized in particular by their

neu-

tralizing action on VIII : C are able to bind VIII R : Xg even if it is devoid of procoagulant activity. These results suggest that factor VIII : C and VIII R : Ag have common antigenic determinants, a hypothesis which has already been made by others authors (2, 12, 14, 15).

REFERENCES

I . GOUDEMAND, M., FOUCAUT, M., HUTIN, A. and PARQUET-GERNEZ, A. Les anti-

coagulants circulants anti-facteur VIII au tours de l'hgmophilie A. Nouv. Rev. Franq. H&mat. 3, 703, 1963 2. GOUDEMAND, M., PARQUET-GERNEZ, A. and FECELLE, A. Hgmophilie. Les variants des facteurs VIII et IX - Discussion I. Nouv. Rev. Frang. Hemat. IO, 627, 1970 3. DENSON, K., BIGGS, R., HADDON, M., BORRETT, R. and COBB, K. Two types of haemophilia (A+ and A-) : a study of 48 cases. Br. J. Haemat. 17, 163, 1969 4. BIGGS, R. The absorption of human factor VIII neutralizing antibody by factor VIII. Br. J. Haemat. 26, 259, 1974 5. KASPER, C.K. A more uniform measurement of factor VIII inhibitors.Thromb. Diath. Haemorrh. (Stuttg.). 34, 869, 1975 6. MAZURIER, C., PARQUET-GERNEZ, A., GOUDEMAND, M. and MONTREUIL, J. Contribution 1 l'isolement et B l'etude de la masse molkulaire du facteur VIII humain. Path. Biol. 23 (Suppl.), 11, 1975 7. SOULIER, J.P. and LARRIEU, M.J. Nouvelle methode de diagnostic de l'hemophilie. Dosage des facteurs antihgmophiliques A et B. Sang 24, 205, 1953 8. LAURELL, C.B., Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15, 45, 1966 9. AXEN, R., PORATH, 3. and ERNBACK, S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature. 214, 1302, 1967

i'ol.lO,~o.~

10.

XNTI

VIII

,YO?~OLOGOUS XYTIBODIES

567

ZIX?fE~R.VXi, T. and EDGIXGTON, T. Factor VIII coagulant activity and factor VIII-like antigen : independant molecular entities. J. Exp. !-fed.IX, 1015, 1973

II. HOAGIE, C. and SARGEAXI, R. Antigen/ Biological activity ratio for iactor VIII. Lancet. 7814, 1247, 1973 12. BOUMA, B.N., de GRUF, S., BORDIJK-HOS, J.?1., Van XOtiRIK, J.A. and SLX?U, J.J. Investigations on the relationship of factor VIII related antigen, factor VIII procoagulant activity and von Willebrand factor activity using insolubilized rabbit antiserum. Thromb. Res. 7, 695, i975 13. BENNET, B., FOMAN, W. and RATSOFF, 0. Studies on the nature of antihemophilic factor. Further evidence relating the AHF-like antigens in normal and hemophilic plasmas. J. of Clin. Invest. 52, 2191, 1973 IS. PEAKE, I. and BLOOM, A.L. The dissociation of factor VIII by reducing agents, high salt concentration and affinity chromatography. Thrombos. Haemostas. (Stuttg.) 35, 191, 1976 15.

STRATTON, R.D., XAGNER, R.H., %?BSTER, W.P. and BRINKHOUS, K.X. Antibody nature of circulating inhibitor of plasma von Willebrand factor. Psoc. Nat. Acad. Sci. (USA) 72, 4167, 1975