Visualization of VIII:C-like material in a CRM negative hemophiliac

THROMBOSIS

Pergamon

RESEARCH 16; 7!+7-;j7 Printed Press Ltd.1979.

in C+rear Srit.nin

VISUXLIZATIOX OF VIII:C-LIKE XATERIAL I?;b CRM XEGATIVE HEMOPHILIAC

G.A.

Rock*,

D.S. Palmer and X.X. Cruickshank

Tine Canadian Red Cross Blood Transfusion Service Ottawa, RlS 3E?, Canada, and *Faculty of ?1edicine, University of Ottawa Ottawa, RlX 6x5, Canada. in revised form 27.8.19Tc). (Received 5.6.1979; Accepted by Editor J.X. Jamieson) ABSTRACT A low molecular weight protein which is immunologically and structurally analogous to the VIII:C in normal individuals has been demonstrated in a CRX negative hemophiliac following column chromatography using two different techniques, both of which affect the ionic environment. Elution patterns suggest a close similarity between normal and hemophilic proteins. Data from isoelectric focusing also indicates similarity with both proteins showing two acidic and one neutral band. Identity is further confirmed by visual demonstration on SDS polyacrylamide gel electrophoresis. Both LNW proteins show a dark band at approximately 150,000 with faint traces of protein at 71,000 and 43,000. This material does not react with anti-albumin, anti-whole human serum, anti-fibrinogen or rabbit antibody to Factor VIII suggesting that no contaminating protein material is present. However, upon elution of these bands from the gels they react with human antibody in the inhibition assay, a test which is said to be an accurate indicator of the presence of VIII:C-like material. Upon reduction with DTT, both samples again show identical patterns with a single band at 43,000. The data demonstrates the first visualization, on gels, of a VIII:C-like material from a CIWnegative individual and indicates that, like the normal, it has a ver\ low molecular weight.

Contribution number 15 from the Ottawa Centre of the Canadian Red Cross Blood Transfusion Service. Presented in part at the Royal College of Canada, Montreal, Canada (1979). liey

Words:

Visualization,VIII:C

Material,CR"I

747

Segatir-e

Hemophiliac

defect &hi&h acc0l~nts for tji-.rlinisal2res.+ntatiS3 2! Tne a012cu13.r : Fun 5 th at hemophilia X has not been specifically determined. Ea r 1:;n <, --2rmLit? Ii), :;na refuted 'by Zi..".. hemophiliacs completely lack Factor T.YIIw.ercz showed that immunologically the plasma of a hemophiliac can cross-react :Gith heterologous rabbit antisera. As well, obs2rvations that some or 311 htmomaterial capable of cross-reacting with human Factor :'I11 philiacs possess inhibitor (?,3) suggest the presence of a VIII:C-like material which carries an intact antibody binding site but lacks biological activity. In a previous report from this laboratory (4) we demonstrated that following salt dissociation and column chromatography of henophilic plasma, a low molecular weight component can be produced which is analogous to the VIII:C found with normal individuals. This finding iJas confirmed by a subsequent study (5) in which it was found :hat a species analogous to c'III:C could be isolated from both CPA positive and CRX negative hemophilic plasma. The present study demonstrates visualization of this LXW \JIII:C-likemat2rial from a CRM negative hemophiliac and presents a comparison with the LX material from normal individuals. XATERIALS ;tUD XETHODS Preparation of Low Xolecular Weight Factor VIII The procedure employed for the isolation of low molecular weight Factor VIII was similar to that used in previous investigations (6). Cryoprecipitate prepared from 500 ml of titrated plasma by the rapid thaw procedure (7) was made Itifin benzamidine and centrifuged at 3000 x g for 10 minutes. The supernatant (20 ml containing 1000 - 2000 mg of protein) was warmed at 37oC for 5 minutes and then applied to a 2.h x 95 cm column of Bio-Gel A-15X equilibrated with ammonium bicarbonate buffer (0.05 ?fammonium bicarbonate, 0.014 PI sodium citrate, 1 ti benzamidine, pH 8.6). The column was eluted at room temperature by upward flow at a rate of 15 ml/hr. Fractions of 5 ml were collected and their optical density at 280 nm determined. Each of the fractions eluting at the V region of the column was assayed for fibrinogen using the Thrombo-Wellcotes? latex bead agglutination procedure (Wellcome Reagents Limited) and those comprising the fibrinogen-free area were pooled. This sample was concentrated to 10 ml by pressure dialysis using a PM-10 membrane (Amicon Corporation), made 1 Sl in sodium chloride and allowed to stir for 30 minutes at room temperature. This material (9 - 20 mg protein) was applied to a 2.6 x 46 cm column of Bio-Gel A-15?lwhich had been equilibrated with tris-sodium chloride buffer (0.025 >l tris, 1.0 >I sodium chloride, 1 mM benzamidine, pH 7.4). The column was eluted at room temperature by upward flow at a rate of 18 ml/hr. Fractions of 5 ml were collected and their optical density at 280 nm was measured. Those fractions eluting in the 2.3 V region of the column were pooled, concentrated by pressure dialysis to a f?nal volume of 300 - 1000 ~1 containing 100 - 1000 ng protein and tested for activity in the human antibody neutralization assay (8). Recovery of protein from the concentration stage ranged from 30 - 60% provided the membrane was washed with a small volume of buffer. Disc Gel Electrophoresis Samples of the 2.3 V. material from both normal and hemophilic plasma were subjected to sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis (PAGE) according to the procedure of Fairbanks et al (9). All gels were 5% acrylamide and the ratio of N,N'-methylene bisacrylamide to acrylamide was 1:150. Samples containing 5 to 30 ug of protein in 30 i.ll of buffer were applied and the electrophoresis carried out at room temperature with a current

The gels w2r2 stained with Coonassie 31~2 R?jQ and th2 Rf of 5 mligel. each band 5;as meas*ured r2lati3:e to bronphenol blue. ?I? localization of faintly staining bands was facilitated by scanning the gels at 5% nm using a Beckman model R-112 Gel Scanner. A mixture of standard proteins (Bio-Rad High >IolecularWeight Karkers) was run in conjunction with all samples and the molecular weights of th2 sample bands wer2 d2tetmined according to standard procedures. Samples which were to be reduced w2re made 0.1 X in ditiiothrietol (DTT) and incubated at 60°C for 30 minutes prior to their application to th2 gal. . 1n the Son-reduced 2.3 V material was also subjected to e:~ctrop:horesls absence of SDS in whit R case SDS was omitted from the gel preparation and ths running buffer. Samples of 2.3 V material containing 5 - 15 _g of protein in 30 ;L of buffer were appli2d tg tit2gels and folio-zingelectrophorosis t'h2 321s were sliced a: 1 mm intervals, the individual siizes mashed and allowed to stand with 0.15 >I sodium chloride at 4'C overnight. At an eq-ivafent warprotein concentration of 15 '+,/30 31 volume load, t-hehenophilic L?f,< J only l/3 as effective in neutralizing 1 unit of human anti-L%' compared to its normal LXX counterpart. A blank gel to which Jnl:; sample buffer had beer, applied was treated in an identical fashion and th2 supernatants from the slices of the blank and sample gels were tested for asti./ity in the human antibody neutralization assay (8). 35

Frsparation of Ilz5 Low Molecular Weight Factor VIII Radiolabellin of both normal and hemophilic low molecular weight Factor VIII with IHvs - was performed according to th2 method of Hunter (10). The reaction mixture was passed over a small column (3.5 x 10 cm) of Sephadex C-175, and the fractions containing the labelled protein which eluted at the Y of the column were pooled. This material was exhaustively dialyzed against OoX ammonium bicarbonate and the retentate then lyopiilized. Isoelectric Focusing Analytical isoelectric focusing of normal and henophilic radiolabelled low molecular weight Factor VIII was performed on an LRB Multiphor system according to the manufacturer's instructions. The position of the sample bands was then determined by autoradiography. Chromatography of Heparinized Plasma Normal and hemophilic blood was collected into heparin to a final concentration of 3 - 4 U.S.P. units/ml whole blood. The plasma was separated and column chromatography carried out according to a previously described procedure (11). In the case of normal plasma, each fraction was assayed for Factor VIII procoagulant activity usinga modification of the partial thromboplastin time method (12) with Pacific Hemostasis Reference Plasma as standard. When hemophilic plasma was used, human antibody neutralization assays were performed on each fraction by the method of Poon and Ratnoff (8) using citrated human serum containing an antibody directed against Factor VIII procoagulant activity. Molecular weight determinations on this low molecular weight material were made using chromatography on Sephadex G-100 (11). RESULTS Column Chromatography of Normal and Hemophilic Factor VIII Both normal and hemophilic plasma show similar elution profiles following column chromatography, with small amounts of protein eluting at the column V and the bulk of the protein emerging later (Figures la and lb). The normal plasma shows a typical pattern of Factor VIII distribution (13) in which the

NORMAL AHF:COL.I

I,;.-J.

3

11

4

%

*

#

(

I

I

120 130 140 150 160 170 180 190 200 210 220 230 ELUTION VOLUME (ml)

HEMOPHILIAC AHF:COL.IV,

L--J.

’

’

’

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(

2

*

1

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170 180 190 200 210 220 230 240 250 260 270 280 ELUTION VOLUME(mlf FIG. 1 Gel filtration on Bio-Gel A-15 >i of (a) normal and (b) hemophilic cryoprecipitate from CPD plasma. Fractions of 5 ml per tube were collected and the O.D. of each fraction measured. 200

V fraction contains all the procoagulant activity as well as all the material w R. ich is cross-reactive with the rabbit and human antibody. The V material from the hemophilic plasma does not possess any procoagulant activyty; however, it is cross-reactive with both human and rabbit antibody. Following dissociation in the presence of 1 M sodium chloride, both preparations demonstrate the presence of low molecular weight material at approximately 2.3 - 2.5 V as shown in Figures 2a and 2b. As expected, the low molecular weight frac?ion obtained from normal plasma contains procoagulant activity and is active in the inhibition assay with human antibody. It does not cross-react with rabbit antibody. The corresponding fraction from hemophilic plasma does not contain procoagulant activity but is cross-reactive with human anti-factor VIII and not with rabbit antibody. Both the normal and hemophilic low molecular weight material was assessed for purity by immunoelectrophoresis using heterologous anti-whole human serum and anti-albumin. Samples of both preparations were also tested for fibrinogen content by the latex bead agglutination procedure. The results were negative in all cases. Polyacrylamide Gel Electrophoresis In order to further compare these low molecular weight components, samples were concentrated and run on 5% polyacrylamide gels. When 5 ug

CRY

SEGXTIVE

HEXOPHILIAC

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0.2 -

40

60

80

100

ELUTION

120

160

180

200

t

f

220

240

220

240

VOLUME (ml)

HEMOPHILIAC 1.2- b 1.0 -

140

AHF:COL.Il

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&a-Gel *-lSM (2J.38cm)

2.5’f.

40

60

80

100

120

140

MO

180 200

ELUTION VOLUME (ml) FIG.

2

Gel filtration on Bio-Gel A-15H of (a) normal and (b) hemophilic high Fractions of 5 ml per molecular weight Factor VIII in 1 M sodium chloride. of each fraction measured. tube were collected and the O.D. 280

samples were run, a single band with an R indicative of a molecular weight of f approximately 150,000 was seen for both normal and hemophilic LNW. In both cases if larger amounts (30 up) of protein were used, two additional faint bands with apparent molecular weights of 71,000 and 43,000 would be detected by scanning the gel at 550 nm. These could be clearly demonstrated when the gels were scanned at 550 nm. AEter reduction with dithiothreitol, both the normal and hemophilic samples had only 1 band with an R value corresponding to a molecular weight of 43,000. The results of the SDi gel electrophoresis are summarized in Figures 3a, 3b, 3~ and 3d. In order to confirm that these bands did contain VIII:C material, 10 ~g samples of both the normal and hemophilic material were subjected to non-SDS gel electrophoresis. The gels were then sliced and the material eluted fro:: each slice and assayed for cross-reactivity with human Factor VIII inhibitor. A gel to which no sample was applied was run at the same time to serve as a control. 30th samples showed two major areas of cross-reactivity as illustrated in Figures 4a and 4b. The mobility of the peaks, relative to bromphenol blue, from normal low molecular weight material was 0.20 and 0.74; that of the hemophilic material was 0.47 and 0.90. Isoelectric Focusing Both normal and hemophilic low molecular weight Factor VIII were labellez with 1"' and then subjected to isoelectric focusing. Autoradiography showed

NORMAL

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ziy SO

HEMOPHILIC

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TRACKER

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Anode FIG. 3

SDS polyacrylamide gel electrophoresis of (a) non-reduced and (b) DTT reduced normal (N) and hemophilic (H) low molecular weight Factor VIII. The 550 nm scan of the non-reduced gels is shown in (c) and (d). A Bio-Rad high molecular weight standards kit was used to calibrate the gels.

NORMAL

30-

C

BLANK

20 10 f-~-~~~%~,

,c 4 L-W\.

0.1

0.3

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RELATIVE

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+

FIG. 4 Antigenic activity of (a) normal and (b) hemophilic low molecular weight Factor VIII eluted from gel slices following non-SDS gel electrophoresis. Xobilities are expressed relative to bromphenol blue. The results for a blank gel are shown in (c).

bands with isoelectric points of 7.4, 4.4 and 3.9 for both the normal and hemophilic samples as shown in Figure 5. There was insufficient material in any of these bands to permit their detection by normal protein stainins procedures. Chromatography of Heparinized Plasma In a recent report from this laboratory (ll), it was shown that, in heparinized plasma where calcium ions are not chelated, a large proportion (approximately 50%) of the procoagulant activity exists by itself in a form which is not associated with either VIII:XAg or VIII:VWF. Consequently, it

Isoelectric focusing of normal (N) and hemo?hilic (H) I"' low molecular weight Factor VIII. Two separate runs with di'fferent origins were performed in order to facilitate resolution of the acidic bands.

30 25 20 15 10 5

10

20

30

40

ELUTION

50

60

VOLUME

70

80

90

(ml1

FIG. 6 Gel filtration on Sepharose CL-6B of (a) normal and (b) hemophilic heparinized plasma. Fractions of 2.5 ml idere collected and their O.D.,a, measured. The fractions from both samples were assayed for activity in the human antibody inhibition assay and against rabbit antibody to AHF; those from the normal sample were also assayed for procoagulant activity.

CPM

SEGATIVE

HEMOPHILIXC

___ i 33

bias decided to assess heparinized hemophilic plasma by the same proacedure. iis shown in Figure 6, following chromatography of heparinized hemophilic plasma the elution profile is essentially the same as that of normal plasma. That is, there are two peaks which both exhibit activity in the human antibody neutralization assay. The first of these elutes at the V of the column and contains 43% of the activity while the remaining 57X of thz antibody neutralization activity is associated with material eluting at 2.3 V . Upon rechromatography on a calibrated column of Sephadex GlOO the materizl eluting at 2.3 vo was determined to have a molecular weight of 22,000. DISCUSSION Tnne Factor VIII molecule in the normal individual contains at least three separately identifiable functional capabilities: it precipitates with rabbit antibody, and has bothvonWillebrand's andprocoagulant activity. The henophilic material also reacts with rabbit antibody and has von Willebrand's activity but it does not have procoagulant activity. Nonetheless, the presence of some material which is antigenically similar to VIII:C is suggested by immunological studies which define CRM positive and negative individuals based on their reactivity with human Factor VIII antibody (2). Indeed, this classification may be too limiting since Zimmerman (3) has reported that if a specific protocol is followed, virtually all hemophiliacs can be demonstrated to be CRX positive, therefore suggesting that at least the antibody binding site of VIII:C is present in hemophiliacs. In the present study, we have been able to demonstrate the presence of VIII:C-like material in a CR&inegative hemophiliac following column chromatography using two different techniques, both of which affect the ionic environment of the molecule. In each case the elution profiles are identical io those obtained for the normal individual. While the material which is obtained from the hemophiliac is not functional in the clotting assay, it is cross-reactive with the human antibody in an assay systen which has been shown (5) to be a valid indicator for the presence of low molecular weightLike material. Following rechromatography of the heparin-derived material a molecular weight of 22,000 is obtained concurring with our previous report for the VLJlW procoagulant material found in the normal individual when physiological levels of calcium are constantly maintained (11). The isoelectric focusing data also indicates a marked similarity in the electrical charge of the proteins obtained from the normal and hemophiliac with two acidic and one neutral band in each preparation. As well, the VIII:C-like material from the hemophiliac migrates in the same way as does the normal on polyacrylamide gel electrophoresis. The dark band has a molecular weight of 150,000 which is in close agreement with previous reports of 180,000 (on column chromatography) (14) and 150,000 (on PAGE) (6) for the procoagulant material. As well, two other very faint bands can be shown on both of these gels when higher concentrations of protein are applied. Since the 2.3 V material did not react with anti-albumin or antihuman serum and did not a&ay for fibrinogen or VIII:RAg, it is unlikely that these bands are contaminants of the more common plasma proteins. A more likely possibility is that these bands represent smaller molecular weight forms of VIII:C since it has been demonstrated (15,16) that LKW VIII:C has a propensity to aggregate with itself to form intermediate and very high (106) molecular weight complexes. Upon reduction with DTT, both the normal and hemophilic samples again show identical patterns with a single band corresponding to a molecular weight oE 43,000. Therefore, it may be that the 22,000 molecular weight indicated by Sephadex G-100 chromatography of both the normal (11) and the

a minimum molefular hemophilic material collected into heparin r53resent3 1 . . DTT repies2n:s a weignt and that the 43,000 species demonstrated on PAGE vith diner aggregate of this basic unit which is relatively resistant to reduction. That is, VIII:C may, in various associated forms, present a pictiireof marked heterogeneity in the same way as does albumin (17,18). By using the inhibitor assay on the material efuted from the gzla, it was possible to confirm that on PAGE both samples did contain VIII:C-like material or at least that portion of the protein which contains the antibody binding site. While only the two bands with activity in the human antibody inhibitor assay were detectable following non-SDS gel electrophoresis, the materiai corresponding to the third band observed on SDS gel electrophoresis ma:; have been present at too low a concentration to permit detection. An alternate explanation is that the third band represents the material with a pI of 7.4 which would not enter the gel when electrophoresis was carried out at pH 7.4. The difference in mobility of the two peaks may indicatti critical differences in basic structure between the two proteins. It is also possible that some degree of aggregation occurred in one or the other of the samples during processing since even the mildest of manipulations will result in aggregation of LMW material (15). As well, since an automated device was used to slice and grind the gels to extract the protein for antibody reaction there may have been considerable distortion oE the gels which could account for these apparent differences. Indeed, if differences in molecular weight do exist they must be relatively small since they are not exhibited on Sephadex G-100 or on SDS gel electrophoresis. In any case, the results of the non-SDS gels conclusively demonstrate that the material which is shown on the discs following polyacrylamide gel electrophoresis represents a visualization of VIII:C in the normal individual and in the CR&lnegative hemophiliacs and that, as shown by PAGE, both these proteins are of a relatively small molecular weignt (150.000). X detailed analysis of the difference between the normal and hemophilic low molecular weight component is a topic of continuing investigation in our laboratory. REFERENCES 1.

ZIMMERMAN, T.S., RATNOFF, O.D. and POWELL, A.E. Immunologic differentiation of classic hemophilia (Factor VIII Deficiency) and von Willebrand's disease. J. Clin. Invest. 50,244-254, 1971.

2.

HOYER, L.W. and BPECKENRIDGE, R.T. Immunologic studies on antihemophilic factor (AHF, Factor VIII): Cross-reacting material in a genetic variant of hemophilia A. Blood. 32,962-971, 1968.

3.

ZIMMERMAN, T.S., DE LA POINTE, L. and EDGINGTON, T.S. Interaction of Factor VIII antigen in hemophilic plasma with human antibodies in Factor VIII. J. Clin. Invest. 59,984-989, 1977.

4.

ROCK, G.A. and CRUICKSHANK, W.H. Low molecular weight Factor VIII: comparison of the electrophoretic maps of the normal and hemophilic molecule. Thrombosis and Haemostasis. 38,52, 1977.

5.

POON, M.C. and R4TNOFF, O.D. Immunologic evidence that the antihemophilic factor (Factor VIII)-like material in hemophilic plasma possesses a non-functional low molecular weight subcomponent. Blood. 50,367-376, 1977.

A

6.

G.A. "isuali-ation. of low CRUICi(SFlXK,‘n’.H. and ROC:::, :KA_XG, E.P., of henzanidin2. molecular weight Factor VIII purified in t:h?~jT132?.i? Thromb. Res. Submitted for publication.

7.

ROCK, G.A. and TITTLEY, P. Variations in yryoprzeipitate production. Transfusion. 17,50-53, 1977.

8.

POOX, X.C. and RATSOFF, O.D. Evidence t-hat functional subunits of antihemophilis factor (Factor VIII) are lin'kedbv consc:*alent bonds. 91oo.d. 48,37-94, 1976.

9.

ChIRBX'XS, G. , STACK, T.L. and WALLXCH, D.F.Y. Electrophortisis analysis of the major polypeptides of the human erythrocy:e -.?mbrane. Biochemistry. 10,2606-2617, 1971.

10.

HLXIER, W.X. In: Sandbook of Experimental Immunol3$:;. D.L!.WEIR (Ed.j Oxford-London-Edinburgh-Melbourne: Blackwell Scientific Publications, 1978, c‘n. 14.1.

11.

ROCK, G.A., PALMER, D.S., TXCMBERRY, E.S. and CRZICKSHAXK, !;.H. The presence of high and low molecular weight forms of Factor VIII in heparinized plasma. Thromb. Res. 13,85-96, 1978.

12.

BRECKENRIDGE, R.T. and R\T&OFF, O.D. Studies on the nature of the circulating anticoagulant directed against antihemophilic factor: with no:es on an assay for antihemophilic factor. Blood. '0,137-147, 1962.

13.

WEISS, H.J., PHILLIPS, L.L. and ROSNER, W. Separation of subunits of antihemophilic factor (AHF) by agarose gel chromatography. Thromb. Diath. Haemorrh. 27,212-219, 1972.

14.

SHLZMAN, S., LUDABLRU, R.H. and SEEGERS, W.H. Biophysical studies on platelet cofactor I preparations. Thromb. Diath. Haeaorrh. 4,336-341, 1960.

15.

ROCK, G.A., TACKABERRY, E.S. and PALMER, D.S. Factors affecting the relative distribution of high and low molecular weight forms of Factor VIII. Thromb. Res. 14,573-587, 1979.

16.

MJSTEN, D.E.G. Factor VIII of small molecular weight and its aggregation. Brit. J. Haem. 27,89-100, 1974.

17.

SAIFER, A. and PALO, J. Amino acid composition of monomeric and polymeric human serum albumin. Anal. Biochem. 27.1-14, 1969.

18.

SAIFER, A., ROBIN, M. and VENTRICE, X. Starch-gel electrophoresis of "purified" albumins, Arch. Biochem. Biophys. 92,409-419, 1961.