British Journal of Haematology, 1990, 76, 80-8 7
Factor VIII heavy chain polypeptides in plasma and concentrates G. KEMBALL-COOK,s. A . BEVAN A N D T. W. BARROWCLIFFE National Institute for Biological Standards and Control, Potters Bur, Herts.
Received 10 October 1989; accepted for publication 9 May 7 990
Summary. Factor VIII polypeptides in plasma and FVIII concentrates have been analysed by an electrophoretic technique based on that of Weinstein et aI (1981). Samples were complexed with radiolabelled anti-FVIII Fab’, and the immunocomplexes visualized by SDS-polyacrylamide electrophoresis. The technique visualized FVIII heavy chain polypeptides in all types of samples, including plasma, without further purification. Fresh or frozen normal plasma (collected into protease inhibitors) contained a range of polypeptides with the largest dominant band at an apparent M, of 250-300 kDa, and the smallest at 80-90 kDa: no bands were produced from samples of severe haemophilic plasma. Cryoprecipitate had a similar polypeptide distribution to normal plasma, but intermediate purity FVIII concentrates
showed more degraded patterns which varied between products: the 2 50-300 kDa bands were reduced or absent, the 80-90 kDa bands were more pronounced than in plasma, and in one product a polypeptide was seen at approximately 4050 kDa. In some products heat treatment for viral inactivation increased the proportion of smaller FVIII polypeptides. Highly-purified FVIII concentrate derived from plasma was also degraded relative to plasma FVIII, and two products obtained by recombinant DNA technology both showed degraded, though slightly different.profiles. The native structure of FVIII in fresh plasma appears heterogeneous with a predominance of higher M, forms: these are degraded to a greater or lesser extent during concentrate production, dependent on the manufacturing processes used.
The biological activity of factor VIII (FVIII)is related closely to its polypeptide structure: the single-chain form of relative molecular mass (M,) approximately 267 kDa deduced from sequencing of the cDNA for human FVIII (Toole et al, 1984; Vehar et al, 1984)is thought to possess little activity. Limited proteolysisto a metal ion-linked complex of FVIII heavy chain (HC)-derived and light chain (LC)-derived polypeptides is correlated with the generation of activated FVIII (FVIIIa), while further proteolysis or dissociation inactivates the cofactor (Eaton et al, 1986; Fay et al, 1986). The structure of human FVIIIa has been reported to be a heterodimer of HCderived 51 kDa and LC-derived 73 kDa polypeptides (Fay, 1987 ) .although a stable form ofporcine FVIIIa consisting of a heterotrimer has also been reported (Lollar & Parker, 1989). From estimates of the specific activity of purified FVIII preparations, FVIII is present in normal plasma at a concentration of approximately 100-200 ng/ml, and in most conventional FVIII concentrates (specificactivity approximately 2-5 iu/mg) is present as a trace constituent making up
only about 0.1% of total protein. Traditional structural analysis of FVIII polypeptides in plasma or concentrates by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with monoclonal antibodies to FVIII antigen (FVIII:Ag) is difficult or impossible without further purification. Since FVIII has been shown to be highly susceptible to degradation by trace amounts of a variety of coagulation and fibrinolytic enzymes including (see above) thrombin, activated protein C (APC), activated FX (FXa) and plasmin (Guisasola et al, 1 978; Holmberg et al, 1983)it is preferable to perform analyses on samples without additional processing, in order to avoid the possibility of alterations in FVIII structure due to these or other contaminating proteases. In 1981 Weinstein et a1 described a technique which could be used to investigate FVIII structure in highly impure materials such as plasmas: samples were incubated with lZ5Ilabelled specific anti-FVIII Fab’ fragments (derived from a haemophilic patient with anti-FVIII:C antibodies), then immune complexes formed were separated by SDS-PAGE followed by autoradiography. This technique has been modified in our laboratory and used to investigate FVIII structure in a variety of samples without additional purification: these include plasma, cryopreciptate, intermediate
Correspondence: Dr G . Kemball-Cook. Division of Haematology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms. Potters Bar. Herts. EN6 3QG.
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Analysis of FVZZZ by SDS-PAGE purity FVIII concentrates and high purity products derived from both plasma and recombinant DNA sources. MATERIALS A N D METHODS Preparation of 1251-labelledspecific anti-FVIII Fab’. This was prepared from inhibitor plasma CC8000 essentially as previously described (Lazarchick & Hoyer, 1978). Briefly, IgG was purified from inhibitor plasma by ammonium sulphate fractionation and precipitation of impurities with caprylic acid (Fine& Steinbuch, 1970) then pepsin-treated to produce Fab’ fragments (Reisner et al, 1979). After iodination of Fab’ by the Chloramine T method (Greenwood et al, 1963) antiFVIII Fab’ was purified by fluid-phase immunoisolation and acid dissociation (Lazarchick & Hoyer, 1978). using a Sepharose CL-4B void volume fraction of an intermediate purity FVIII concentrate as antigen. CC8000 inhibitor plasma was a kind gift of Dr E. G . D. Tuddenham. Fresh plasmas were obtained by clean venepuncture from healthy donors followed by immediate anticoagulation of the blood by mixing 9: 1 with 0.109 M trisodium citrate and centrifugation at 50000 g for 5 min. Where an inhibitor cocktail was used in the anticoagulant, the final concentrations of protease inhibitors were: Trasylol (Bayer, Newbury), 10’ kIU/ml: leech hirudin (a gift of Professor F. Markwardt. Erfurt Medical Academy, G.D.R.), 2.5 ATU/ml; 1-2581 (Kabi. Uxbridge), 2 5 p ~ PMSF : (Sigma, Poole), 10 mM. Severe haemophilic plasmas. Frozen plasmas were kind gifts from Dr P. B. A. Kernoff (Royal Free Hospital, London) and Dr G. Savidge (St Thomas’ Hospital, London). Plasma-derivedfactor Vlll concentrates were Hemofil HT and Hemofil M (Baxter Hyland, Thetford): Koate HT (Bayer, Slough): Profilate HT (Alpha, Thetford): Factorate HT and Monoclate (Rorer, Eastbourne); Kryobulin HT (Immuno, Sevenoaks); Octonativ (KabiVitrum, Uxbridge): HT Factor VIII 8 Y (Blood Products Laboratory, Elstree); and HT Factor VIII Z8 (Protein Fractionation Centre, Edinburgh). Hemofil M and Monoclate are products in which the FVIII has been purified using immobilized monoclonal antibodies to FVIII or von Willebrand factor. Recombinant DNA-derived FVIII products were kindly provided by both Baxter Hyland and Bayer U.K. In both cases the full-length wild-type human FVIII gene was used. Cryoprecipitate and crgosupernatant were prepared as follows: frozen normal pool plasma was allowed to thaw in melting ice for several hours, then centrifuged at 50 000 g for 10 min at 4°C. After removing the cryosupernatant. the precipitate was redissolved in one-fifth the original volume of buffered saline. Molecular weight markers for use in non-reduced Laemmli SDS-PAGE were HMW kit (Pharmacia, Milton Keynes) and SDS-6H (Sigma, Poole). Preparation of FVlll sarnplesfor SDS-PAGE was by diluting generally to between 0.5 and 3 iu/ml FVII1:C: then 10 pl were mixed with 10 pl of IzSI-Fab’(3000-10 000 cpm) and 4 pl of 9% (w/v) PEG 4000 (Sigma, Poole). and incubated in a closed plastic tube at 37OC for 2 h to allow formation of immunocomplexes. 20 pl of sample buffer (62 mM Tris-HC110%glycerol-2% SDS-O.OOl% bromophenol blue, pH 6.8)
81
were added and incubation continued for a further 30 min, after which samples (30-35 pl) were loaded onto the gel. Electrophoresis was carried out in a Hoefer Mighty Small system at 20 mA per gel, using 4-10% gradient slab gels, until the tracking dye reached the bottom of the gel. A discontinuous buffer system was used (Laemmli, 1970). Autoradiography of the fixed dried gels was carried out at - 70°C for between 2 and 7 d i n Protex cassettes fitted with a Philips Ultra S intensifying screen, using Kodak X-Omat XS-1 film. Assignment of apparent relative molecular masses ( M r ) . Calculation of approximate M, was by measurement of polypeptide migration distance on the autoradiograph and comparison with molecular weight markers on the dried Coomassie blue-stained gels, with subtraction of 50 kDa ( = M, of one Fab’ molecule) to give the apparent net mass of the polypeptide.
RESULTS Visualization of FVIII in plasma 1L51-labelledanti-FVIII Fab’ (A/FVIII Fab’) was incubated with both frozen pooled normal plasma and a frozen aliquot of severe haemophilic plasma, then electrophoresed in an SDS-polyacrylamide slab gel. Following autoradiography, the pattern seen in Fig l(a) was obtained. In the absence of plasma sample, the labelled Fab’ ran as a single band of relative molecular mass (M,) 45-50 kDa. with a small amount of diffuse staining at M , > 300 kDa due to limited self-association of Fab’ monomers. Normal plasma (NP)shows a pattern of several bands with apparent M, between approximately 2 50-300 kDa and approximately 80-90 kDa (M, estimated after subtraction of 45-50 kDa for one Fab’ molecule). Each band corresponds to a species of labelled immunocomplex consisting of one polypeptide chain and one Fab molecule: one to one binding of Fab’ fragments of polypeptides has been confirmed elsewhere (Weinstein et al, 1981) and in our laboratory by varying widely the Fab’:FVIII ratio, in which case the FVIII patterns remained the same. Severe haemophilic plasma (SHP) showed no bands at all, indicating that the bands visualized in normal plasma were due to FVIII polypeptides alone. Eflect of protease inhibitors In order to determine whether the multiple band pattern seen with frozen pooled plasma (Fig l a ) was due to proteolysis of larger FVIII polypeptides during either freeze/thawing or the incubations with labelled Fab’ and sample buffer, whole blood from each of two healthy donors was collected into trisodium citrate in the presence or absence of a cocktail of protease inhibitors (seeMethods), centrifuged and the plasma used immediately. Following incubation with labelled antibody, SDS-PAGE and autoradiography the patterns seen in Fig l(b) were obtained. In both plasmas the bands visualized were identical regardless of the presence of inhibitors and were very similar to Fig l(a):this indicates that the multiple
82
G. Kemball-Cook, S. A. Bevan and T . W. Barrowclifle
Fig 1. (a) Autoradiograph of immunocomplexes after SDS-PAGE in 410%slab gel. Fab’. sample contained lZ51labelled Fab alone: SHP. severe haemophilic plasma incubated with 1L51-labelledFab’: NP. normal plasma plus 1251-Fab’.Arrows indicate approximate M, range by comparison with SDS-PAGE markers after subtraction of 50 kDa (=M, of one Fab’ molecule), with Fab the position of uncomplexed Fab’ fragments. (b) Effect of protease inhibitors: Fab’. sample was 1251Fab’ alone: P1 and P2. two fresh plasmas collected into anticoagulant in the absence ( -I) and presence ( + I ) of a cocktail of protease inhibitors. Arrows indicate approximate M, range of bands seen after autoradiography .
bands represent FVIII structure in freshly anticoagulated plasma rather than proteolytic breakdown products produced during freeze/thawing or incubation of the samples. Effect of plasma storage on FVlll polypeptides Fresh citrated plasmas from four normal donors were each divided into three portions: the first were incubated with labelled antibody containing protease inhibitor cocktail and electrophoresed immediately, the second were subjected to three cycles of freezing and thawing from -40°C before inhibitor addition and electrophoresis, and the third were held in a water bath at 37OC for 24 h before analysis. Fig 2 shows the results obtained from fresh plasma and plasma held at 37°C for 24 h. In all four plasmas the lowest M, band at approximately 80-90 kDa was intensified after 24 h storage at 3 7°C. with no other consistent alterations visible: as seen above, the patterns obtained from freeze-thawed
plasma (not shown) were indistinguishable from fresh plasma. Thus prolonged storage at 3 7°C but not freezing and thawing alone, generates increased quantities of a smaller polypeptide(s). The ‘doublet’ band seen in lane 4 (24 h. 37°C) and sometimes in other Figures superficially resembles that often seen when FVIII light chains (78 and 80 kDa) are visualized by SDS-PAGE however, this antibody preparation does not recognize FVIII light chains, nor does the method have sufficient resolution to visualize polypeptides so close in size. FV111 polypeptides in cryoprecipitate and cryosupernatant Fig 3 shows a comparison between the FVIII visualized in pooled normal plasma (NP), cryosupernatant (CS) and cryoprecipitate (CP),prepared as described in Methods. There was no significant difference in band pattern between the three samples, indicating that all polypeptides were fractio-
Analysis of FVIII by SDS-PAGE
83
Fig 2. Effect of plasma storage at 3 7 T for 24 h on four normal plasmas: Arrows indicate approximate M, range of bands seen after autoradiography.
Pig 3. FVIII poIypeptides visualized in normal plasma (NP). cryosupernatant (CS) and cryoprecipitate diluted x 3 (CP). Arrows indicate approximate M, range of bands seen after autoradiography.
nated equally during the precipitation. Similar results were obtained from two further preparations of cyroprecipitate (results not shown). F V l l l polypeptides in intermediate purity FVHI concentrates Fig 4 shows samples of six different products (five commercial
materials plus one NHS product, all heat-treated and of specific activity between 0.5 and 3 iu/mg). together with severe haemophilic plasma (HP) and normal plasma pool (NP). All the polypeptides present in normal plasma are also present in each concentrate, but with relatively larger amounts of the smaller polypeptides: in addition, in product E an almost complete lack of the larger polypeptides is accom-
panied by the appearance of a band of further degraded FVIII at M, 40-50 kDa. This comparison has been performed five times with different batches of each product each time: the results were very similar in each case indicating both that the technique itself is reproducible and that there is little batch to batch variation in any one product. The fainter appearance of all FVIII polypeptides in the NHS sample was due to a lower antigen loading: in other gels with higher loading (see Fig 5a) a clearer pattern was seen. In addition, these results are very similar to those we presented in a previous report (Barrowclife et al, 1986), with each product including E showing similar patterns to those in Fig 4.
84
G. Kernball-Cook, S. A. Bevan and T. W. Barrowclifle
Fig 4. FVIII polypeptides visualized in haemophilic plasma (HP), normal plasma (NP), five brands of commercial FVIII concentrate (A-E) and one brand of NHS-produced FViII concentrate. Arrows indicate approximate M, range of polypeptides after subtraction of 50 kDa (=M, of one Fab' fragment).
Fig 5. Effect of dry heat treatment ( 7 2 h. 80°C) on FVIII polypeptides in FVIII IP concentrates. (a) NHS-produced IP concentrate: NP. normal plasma: -HT. beforeheat treatment: +HT, after heat treatment. (b) Commercial IP concentrate: pre and post heat treatment samples from two batches are shown. Arrows indicate approximate M, range of FVIII polypeptides.
Effect of heat treatment on FVlIl polypeptides The effects of virucidal heat treatment on some FVIII concentrates have been investigated by this technique. Fig S(a) shows results obtained with dry heating of an intermediate purity (IP) product manufactured by an NHS fractionation centre and currently distributed in the U.K.: an unheated ( - HT) sample was compared with a sample heated in the freeze-dried state (+HT) for 72 h at 80°C. No major differenceswere seen in FVIII polypeptide distribution following heat treatment, although in each case the FVIII was more degraded than that of a frozen normal plasma pool (NP) as
+
judged by increased amounts of the lowest M, band at 80-90 kDa. Fig S(b) shows that in another commercial IP concentrate dry-heated under the same conditions a little reduction of the largest polypeptide was seen in batch 2, but there were no major changes. However, in a third IP product two methods of dry heating did produce differingpatterns: Fig 6 shows that heating of the concentrate powder suspended in heptane (6O0C, 20 h) led to more FVIII degradation than dry heating without heptane (60°C 36 h). Finally, a fourth IP concentrate was subjected either to dry heating (68°C. 72 h) or to pasteurization (60°C.
Analysis of FVllZ by SDS-PAGE
85
including those prepared by elution from anti-FVIII or antivon Willebrand factor (vWf) monoclonal antibody columns. Initially the FVIII was plasma-derived, but recently the methodology has been applied to recombinant DNA-derived FVIII expressed in mammalian cell culture. Fig 8 presents results obtained from samples of two plasma-derived and two recombinant DNA-derived monoclonal antibody-purified products. Compared to plasma the two plasma-derived VHP products are almost completely depleted in the larger FVIII polypeptides relative to the smaller ones and one of the products shows a faint degraded band at an M, or 40-50 kDa (this band did not reproduce well on photography of the original autoradiograph). The two recombinant DNA-derived VHP products, while also possessing relatively more lower M, polypeptides than plasma, differ in their patterns: product 1 has a faint band at about 130 kDa (not present in product 2 ) in addition to the strong band at 80-90 kDa, while product 2 has a stronger band at 2 50-300 kDa. DISCUSSION Fig 6. FVIII polypeptide patterns seen in a commercial IP concentrate heat treated either as powder in the dry state (Dry)or as a suspension in heptane (Hept): two batches of each type are shown.
Fig 7 . Analysis of a commercial IP FVIII concentrate heat treated by either pasteurization (Wet) or by dry heating (Dry). Arrows indicate
approximate M, range of FVIII polypeptides.
10 h), Fig 7 shows that FVIII was clearly depleted in high M, polypeptides in the pasteurized material compared to the dry heated concentrate, indicating limited proteolysis during the procedure.
Very highly-pur$ed FVlll concentrates Recent advances in fractionation technology have led to the development of very highly purified (VHP) FVIII products,
FVIII activity in vitro has been shown to be dependent on its polypeptide structure, which in turn is determined by limited proteolysis. The main enzymes involved in this modulation are probably thrombin, factor Xa and activated protein C (Cockburnet al, 1981;Fulcher & Zimmerman, 1982: Fulcher et al. 1983, 1984; Toole et al, 1984: Vehar et al, 1984: Rotblat et al, 1985; Eaton et nl, 1986: Andersson et al. 1986; Fay et al, 1986). It is possible to assay FVIII cofactor activity in many different types of sample, including plasmas with FVIII levels as low as 1 ng/ml: however, analysis of FVIII polypeptides by conventional structural analysis (for example by immunoblotting with monoclonal antibodies) requires samples to be both considerably more concentrated (approximately 1 pg/ ml), and also processed to remove the large majority of contaminating proteins. The electrophoretic technique described here is based on that of Weinstein et a1 (1981): the main modifications have been the substitution of polyacrylamide gradient slab gels for acrylamide/agarose gels, and the use of a human antibody with reactivity to a greater range of FVIII polypeptides. As performed, the method can visualize FVIII polypeptides in many types of FVIII sample including plasma, with a detection limit as low as 30 ng/ml and without any form of FVIII purification. However, unlike conventional SDS-PAGE, it is not possible to assign precise relative molecular masses (M,) to the bands seen after autoradiography, since samples are run unreduced in non-denaturing buffer (to avoid dissociation of immunocomplexes) and it is necessary to subtract the approximate M, of one molecule of Fab’ to obtain the net M, of the FVIII polypeptide. The usefulness of the technique lies in the ability to compare directly samples of widely differing purity, including plasma, without any additional processing. Conventional SDS-PAGE analysis of reduced highly-purified plasma-derived FVIII normally reveals the presence of one or more bands of the heavy chain (HC) at M, between 90
86
G. Kernball-Cook, S. A. Bevan and T. W. Barrowcliffe
Fig 8. FVIII polypeptides visualized in VHP
FVIII concentrates produced using immobilized monoclonal antibodies. NP: normal plasma; p d plasma-derived;rDNA:
recombinant DNA-derived. Arrows indicate approximate M, range of polypeptides.
kDa and 2 10 kDa, and a roughly equivalent amount of the light chain doublet (LC) at M, 79/80 kDa: on treatment with thrombin these are degraded first of all to M, 50 40 kDa and 69/70 kDa respectively, and further to smaller polypeptides (Fulcher et al. 1983, 1985: Rotblat et al. 1985; Eaton et al, 1986; Hamer et al, 1986; Anderson et al, 1986). Of these species, the polypeptides detected by the electrophoretic method described here will be dependent on the epitope specificities of the homologous anti-FVIII:C antibody used: studies of such inhibitors arising in polytransfused haemophiliacs have shown that they may be directed against either or both HC-derived and LC-derived polypeptides, although antibodies binding to the Mr 50 kDa thrombin fragment of HC are rare (Fulcher et al, 1987; Scandella et al, 1988). In our experiments the largest polypeptide (apparent M, 2 50-300 kDa) seen on the autoradiographs corresponded to the M,2 10 kDa HC species reported for purified FVIII, and the smaller band at M, 80-90 kDa corresponded to the Mr 90 kDa HC species. Where a band at M, 40-50 kDa was detected this was clearly an HC-derived cleavage product, either one of the M, kDa or 50 kDa degradation products seen on thrombin treatment of purified FVIII, or the reported M, 45 kDa polypeptidereleased by FXa or activated protein C. Deliberate thrombin treatment of plasma led to the disappearance of the bands at 250-300 kDa and 80-90 kDa, and appearance of a band in the 40-50 kDa position (data not shown). Although this inhibitor plasma has been shown to contain antibodies directed against both HC and LC determinants (OBrien, 1985; Kemball-Cook et al, 1988; Scandella et al, 1988). in most experiments the labelled antibody did not visualize the M, 79/80 kDa LC or its 69/70 kDa thrombin degradation product, indicating that anti-HC rather than anti-LC antibodies predominate in the affinity-purified preparation used. FVIII in fresh normal plasma appeared least degraded of all the samples tested. with one strong band at M, 250-300 kDa and several fainter bands at smaller M, as previously
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described by Weinstein et a1 (1981): even when blood was collected in the presence of powerful protease inhibitors these smaller polypeptides were present, indicating that FVIII may circulate as a mixture of different HC sizes complexed with LC, rather than as the full-length single chain molecule. Intermediate purity factor VIII concentrates displayed a variable pattern of HC bands, some products being obviously more degraded than others: these variations were generally reproducible from batch to batch and also over different preparations of labelled antibody. Different FVIII concentrate manufacturing protocols clearly lead to different degrees of FVIII degradation, but it is not known whether these differences lead to alterations in the products' bioavailability or immunogenicity. An essential stage in the production of current plasmaderived FVIII concentrates is virucidal treatment to reduce the risk of transmission of infectious viruses, such as HIV or the hepatitis viruses. For many products heat-treatment, in stabilized aqueous solution (pasteurization) or in the freezedried state either under vacuum, in steam, or as a suspension in organic solvent has been the method of choice: however, some factor VIII activity is always lost through degradation in these processes. Our results show that the method of heating can affect the FVIII polypeptide structure: while some IP concentrates were not affected by dry heat, even at 8OoCfor 72 h, pasteurization (6OoC, 10 h) led to increased FVIII breakdown. In one product heating of lyophilized powder in organic solvent suspension (6OoC, 20 h) yielded a more degraded product than ordinary dry heating (60"C, 36 h). It is likely that highly purified FVIII products, both plasmaand recombinant DNA-derived, will be increasingly used for patient treatment in the future. We found two such plasmaderived products to be heavily depleted in larger FVIII polypeptides, although whether this is due to the purification steps or viral inactivation procedures is not known. The recombinant products are also degraded relative to plasma
Analysis of FVIII by SDS-PAGE FVIII, but their patterns differ: again, whether this is due to differences in the structure of the expressed protein or to stages in processing is unknown. Since all these products are ultimately efficacious in patients. it is clear that the larger FVIII heavy chain polypeptides are not essential for its activity following infusion: however, at least in the plasma-derived products they are a marker for the potential loss of FVIII activity due to proteolytic degradation during manufacture. ACKNOWLEDGMENT The authors wish to acknowledge the excellent technical assistance of Mr S. J, Edwards in performing some of the gel analyses. KEFEKENCES Andersson. L.-O., Forsman. N.. Huang, K., Lundin, A., Pavlu, B.. Sandberg. H., Sewerin. K. & Smart, J. (1986) Isolation and characterisation of human factor VIII: molecular forms in commercial factor VIII concentrate, cryoprecipitate, and plasma. Proceedings ofthe National Academy of Scierices of the United States q/ America. 83, 2979-2983. Barrowcliffe, T.W.. Edwards, S.]., Kemball-Cook. G. & Thomas, D.P. (1986) Factor VIII degradation products in heated concentrates. Lancet. i, 1448-1449. Cockburn, C.C., De Beaufre-Apps, R.J., Wilson, 1. & Hardisty, R.M. { 198 1) Parallel destruction of factor VIII procoagulant activity and an 85.000 dalton protein in highly purified factor VIII/vWF. Tlirumbosis Research, 21, 29 5- 309. Eaton. D.. Rodriguez, H. & Vehar, G.A. (1986) Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin. factor Xa. and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry. 25, 505-51 2. Fay. P.J.. Anderson, M.T.. Chavin. S.I. &Marder, V.J. (1986) The size of human factor VIII heterodimers and the effects produced by thrombin. Biochiniica et Biophysica Acta, 871, 268-278. Fay. P.1. (1987) Subunit structure of thrombin-activated human factor VIIIa. Biochirnica et Biophysica Acta. 952, 181-190. Fine. J.M. & Steinbuch, M. (1970) A simple technique for the isolation of monoclonal IgG and IgA. Kevire Europeene Etirdes Cliniqires et Biologiques, 1 5 , 1 1 15-1 121. Fulcher, C.A.. Gardiner, J.E., Griffin, J.H. & Zimmerman. T.S. (1984) Proteolytic inactivation of human factor VIII procoagulant protein by activated protein C and its analogy with factor V. Blood. 63, 486-489. Fulcher. C.A.. Mahoney, S. De G. & Zimmerman. T.S. (1987) FVIII inhibitor subclass and FVIII polypeptide specificity determined by immunoblotting. Blood, 69, 197551480. Fu1cher.C.A.. Roberts.J.R..Holland.L.Z.&Zimmerman,T.S.(19851 Human factor VIII procoagulant protein: monoclonal antibodies define precursor-product relationships and functional epitopes. lournal of'Cliriica1 Znvestigation, 76, 1 17-124. Fulcher. C.A.. Roberts, J.R. & Zimmerman, T.S. ( 1 98 3) Thrombin proteolysis of purified factor VIII procoagulant protein: correlation
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of activation with generation of a specific polypeptide. Blood, 61, 807-811. Fulcher. C.A. & Zimmerman. T.S. (1982) Characterisation of the human factor VIII procoagulant protein with a heterologous precipitating antibody. Proceedings of the National Academy of Sciences ofthe United States of America, 79, 1648-1 652. Greenwood. F.C., Hunter, W.M. & Glover, J.S. (1963) The preparation of 13'I-labelled human growth hormone of high specific radioactivity. Biochemical Journal. 89, 114-1 2 3. Guisasola. J.A., Cockburn, C.G. & Hardisty, R.M. (1978) Plasmin digestion of factor VIII: characterisation of the breakdown products with respect to antigenicity and von Willebrand activity. Thrombosis arid Haernostasis, 40, 302-3 1 5. Hamer. H.J., Koedam. J.A.. Beeser-Visser, N.H. & Sixma. J.J. (1986) Human factor VIII: purification from commercial factor VIII concentrate. characterisation, identification and radiolabelling. Biocfiiniicu et Biophysica Acta, 873, 356-366. Holmberg, L., Ljung, R. & Nilsson, I.M. (1983) The effects of plasmin and protein C, on factor VIII :C and VIII :CAg. Thrombosis Research, 31,41-50. Kernball-Cook, G.. Edwards. S.J., Sewerin. K.. Anderson. L.-0. & Barrowcliffe, T.W. (1988) Factor VIII procoagulant protein interacts with phospholipid vesicles via its 8 0 kDa light chain. Thrombosis and Haemustasis. 60, 442-446. Laemmli. 1J.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-685. Lazarchick. J. & Hoyer. L.W. (1978) Immunoradiometric measurement of the factor VIII procoagulant antigen. ]ourrial of Clinical Investigation. 62, 1048-1052, Lollar. P. & Parker. C.G. (1989) Subunit structure of thrombinactivated porcine factor VIII. Biochemistry, 28, 666-674. O'Brien, D.P. (1985) Purification and characterisation of human factor VIII. Ph.D. thesis. University of London. Reisner. H.M., Barrow, E.S. & Graham, J.B. (1979) Radioimmunoassay for coagulant factor VIII-related antigen (VIII :CAB). Thronibosis Research. 14, 235-239. Rotblat, F.. O'Brien. D.P.. O'Brien, F.J.. Goodall. A.H. & Tuddenham. E.G.D. ( 1 98 5) Purification of human factor VIII: C and its characterisation by Western blotting using monoclonal antibodies. Biochemistry, 24, 4294-4300. Scandella. D., Mahoney, S. De G., Mattingley. M., Roeder. D.. Timmons, L. & Fulcher, C.A. (1988) Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Eseherischia coli. Proceedings of the National AcadenryofSciencesofthe UnitedStatesofAmerica. 85, 6152-61 56. Toole. J.J., Knopf. J.L.. Wozney. J.M., Sultzman, LA., Buecker. J.L.. Pittman. D.D.. Kaufman, R.J.. Brown, E.. Shoemaker. C.. Orr. E.C.. Amphlett, G.W.. Foster, W.B., Coe. M.L.. Knutson. G.L., Fass. D.N. & Hewick. R.M. (1984) Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature. 312, 342-347. Vehar, G.A., Keyt. B.. Eaton, D.. Rodriguez, H., O'Brien. D.P.. Rotbtat. F., Oppermann, H.. Keck. R.. Wood, W.I., Harkins. R.N., Tuddenham, E.G.D., Lawn. R.M. & Capon, D.J. (1984) Structure of human factor VIII. Nature, 312, 337-342. Weinstein. M., Chute, L. & Deykin. D. (1981)Analysis of factor VIII coagulant antigen in normal, thrombin-treated and hemophilic plasma. Proceedirigs of the Nationd Acaderng of Sciences of the United States ofAmerica, 78, 5137-5141.
Factor VIII heavy chain polypeptides in plasma and concentrates G. KEMBALL-COOK,s. A . BEVAN A N D T. W. BARROWCLIFFE National Institute for Biological Standards and Control, Potters Bur, Herts.
Received 10 October 1989; accepted for publication 9 May 7 990
Summary. Factor VIII polypeptides in plasma and FVIII concentrates have been analysed by an electrophoretic technique based on that of Weinstein et aI (1981). Samples were complexed with radiolabelled anti-FVIII Fab’, and the immunocomplexes visualized by SDS-polyacrylamide electrophoresis. The technique visualized FVIII heavy chain polypeptides in all types of samples, including plasma, without further purification. Fresh or frozen normal plasma (collected into protease inhibitors) contained a range of polypeptides with the largest dominant band at an apparent M, of 250-300 kDa, and the smallest at 80-90 kDa: no bands were produced from samples of severe haemophilic plasma. Cryoprecipitate had a similar polypeptide distribution to normal plasma, but intermediate purity FVIII concentrates
showed more degraded patterns which varied between products: the 2 50-300 kDa bands were reduced or absent, the 80-90 kDa bands were more pronounced than in plasma, and in one product a polypeptide was seen at approximately 4050 kDa. In some products heat treatment for viral inactivation increased the proportion of smaller FVIII polypeptides. Highly-purified FVIII concentrate derived from plasma was also degraded relative to plasma FVIII, and two products obtained by recombinant DNA technology both showed degraded, though slightly different.profiles. The native structure of FVIII in fresh plasma appears heterogeneous with a predominance of higher M, forms: these are degraded to a greater or lesser extent during concentrate production, dependent on the manufacturing processes used.
The biological activity of factor VIII (FVIII)is related closely to its polypeptide structure: the single-chain form of relative molecular mass (M,) approximately 267 kDa deduced from sequencing of the cDNA for human FVIII (Toole et al, 1984; Vehar et al, 1984)is thought to possess little activity. Limited proteolysisto a metal ion-linked complex of FVIII heavy chain (HC)-derived and light chain (LC)-derived polypeptides is correlated with the generation of activated FVIII (FVIIIa), while further proteolysis or dissociation inactivates the cofactor (Eaton et al, 1986; Fay et al, 1986). The structure of human FVIIIa has been reported to be a heterodimer of HCderived 51 kDa and LC-derived 73 kDa polypeptides (Fay, 1987 ) .although a stable form ofporcine FVIIIa consisting of a heterotrimer has also been reported (Lollar & Parker, 1989). From estimates of the specific activity of purified FVIII preparations, FVIII is present in normal plasma at a concentration of approximately 100-200 ng/ml, and in most conventional FVIII concentrates (specificactivity approximately 2-5 iu/mg) is present as a trace constituent making up
only about 0.1% of total protein. Traditional structural analysis of FVIII polypeptides in plasma or concentrates by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with monoclonal antibodies to FVIII antigen (FVIII:Ag) is difficult or impossible without further purification. Since FVIII has been shown to be highly susceptible to degradation by trace amounts of a variety of coagulation and fibrinolytic enzymes including (see above) thrombin, activated protein C (APC), activated FX (FXa) and plasmin (Guisasola et al, 1 978; Holmberg et al, 1983)it is preferable to perform analyses on samples without additional processing, in order to avoid the possibility of alterations in FVIII structure due to these or other contaminating proteases. In 1981 Weinstein et a1 described a technique which could be used to investigate FVIII structure in highly impure materials such as plasmas: samples were incubated with lZ5Ilabelled specific anti-FVIII Fab’ fragments (derived from a haemophilic patient with anti-FVIII:C antibodies), then immune complexes formed were separated by SDS-PAGE followed by autoradiography. This technique has been modified in our laboratory and used to investigate FVIII structure in a variety of samples without additional purification: these include plasma, cryopreciptate, intermediate
Correspondence: Dr G . Kemball-Cook. Division of Haematology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms. Potters Bar. Herts. EN6 3QG.
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Analysis of FVZZZ by SDS-PAGE purity FVIII concentrates and high purity products derived from both plasma and recombinant DNA sources. MATERIALS A N D METHODS Preparation of 1251-labelledspecific anti-FVIII Fab’. This was prepared from inhibitor plasma CC8000 essentially as previously described (Lazarchick & Hoyer, 1978). Briefly, IgG was purified from inhibitor plasma by ammonium sulphate fractionation and precipitation of impurities with caprylic acid (Fine& Steinbuch, 1970) then pepsin-treated to produce Fab’ fragments (Reisner et al, 1979). After iodination of Fab’ by the Chloramine T method (Greenwood et al, 1963) antiFVIII Fab’ was purified by fluid-phase immunoisolation and acid dissociation (Lazarchick & Hoyer, 1978). using a Sepharose CL-4B void volume fraction of an intermediate purity FVIII concentrate as antigen. CC8000 inhibitor plasma was a kind gift of Dr E. G . D. Tuddenham. Fresh plasmas were obtained by clean venepuncture from healthy donors followed by immediate anticoagulation of the blood by mixing 9: 1 with 0.109 M trisodium citrate and centrifugation at 50000 g for 5 min. Where an inhibitor cocktail was used in the anticoagulant, the final concentrations of protease inhibitors were: Trasylol (Bayer, Newbury), 10’ kIU/ml: leech hirudin (a gift of Professor F. Markwardt. Erfurt Medical Academy, G.D.R.), 2.5 ATU/ml; 1-2581 (Kabi. Uxbridge), 2 5 p ~ PMSF : (Sigma, Poole), 10 mM. Severe haemophilic plasmas. Frozen plasmas were kind gifts from Dr P. B. A. Kernoff (Royal Free Hospital, London) and Dr G. Savidge (St Thomas’ Hospital, London). Plasma-derivedfactor Vlll concentrates were Hemofil HT and Hemofil M (Baxter Hyland, Thetford): Koate HT (Bayer, Slough): Profilate HT (Alpha, Thetford): Factorate HT and Monoclate (Rorer, Eastbourne); Kryobulin HT (Immuno, Sevenoaks); Octonativ (KabiVitrum, Uxbridge): HT Factor VIII 8 Y (Blood Products Laboratory, Elstree); and HT Factor VIII Z8 (Protein Fractionation Centre, Edinburgh). Hemofil M and Monoclate are products in which the FVIII has been purified using immobilized monoclonal antibodies to FVIII or von Willebrand factor. Recombinant DNA-derived FVIII products were kindly provided by both Baxter Hyland and Bayer U.K. In both cases the full-length wild-type human FVIII gene was used. Cryoprecipitate and crgosupernatant were prepared as follows: frozen normal pool plasma was allowed to thaw in melting ice for several hours, then centrifuged at 50 000 g for 10 min at 4°C. After removing the cryosupernatant. the precipitate was redissolved in one-fifth the original volume of buffered saline. Molecular weight markers for use in non-reduced Laemmli SDS-PAGE were HMW kit (Pharmacia, Milton Keynes) and SDS-6H (Sigma, Poole). Preparation of FVlll sarnplesfor SDS-PAGE was by diluting generally to between 0.5 and 3 iu/ml FVII1:C: then 10 pl were mixed with 10 pl of IzSI-Fab’(3000-10 000 cpm) and 4 pl of 9% (w/v) PEG 4000 (Sigma, Poole). and incubated in a closed plastic tube at 37OC for 2 h to allow formation of immunocomplexes. 20 pl of sample buffer (62 mM Tris-HC110%glycerol-2% SDS-O.OOl% bromophenol blue, pH 6.8)
81
were added and incubation continued for a further 30 min, after which samples (30-35 pl) were loaded onto the gel. Electrophoresis was carried out in a Hoefer Mighty Small system at 20 mA per gel, using 4-10% gradient slab gels, until the tracking dye reached the bottom of the gel. A discontinuous buffer system was used (Laemmli, 1970). Autoradiography of the fixed dried gels was carried out at - 70°C for between 2 and 7 d i n Protex cassettes fitted with a Philips Ultra S intensifying screen, using Kodak X-Omat XS-1 film. Assignment of apparent relative molecular masses ( M r ) . Calculation of approximate M, was by measurement of polypeptide migration distance on the autoradiograph and comparison with molecular weight markers on the dried Coomassie blue-stained gels, with subtraction of 50 kDa ( = M, of one Fab’ molecule) to give the apparent net mass of the polypeptide.
RESULTS Visualization of FVIII in plasma 1L51-labelledanti-FVIII Fab’ (A/FVIII Fab’) was incubated with both frozen pooled normal plasma and a frozen aliquot of severe haemophilic plasma, then electrophoresed in an SDS-polyacrylamide slab gel. Following autoradiography, the pattern seen in Fig l(a) was obtained. In the absence of plasma sample, the labelled Fab’ ran as a single band of relative molecular mass (M,) 45-50 kDa. with a small amount of diffuse staining at M , > 300 kDa due to limited self-association of Fab’ monomers. Normal plasma (NP)shows a pattern of several bands with apparent M, between approximately 2 50-300 kDa and approximately 80-90 kDa (M, estimated after subtraction of 45-50 kDa for one Fab’ molecule). Each band corresponds to a species of labelled immunocomplex consisting of one polypeptide chain and one Fab molecule: one to one binding of Fab’ fragments of polypeptides has been confirmed elsewhere (Weinstein et al, 1981) and in our laboratory by varying widely the Fab’:FVIII ratio, in which case the FVIII patterns remained the same. Severe haemophilic plasma (SHP) showed no bands at all, indicating that the bands visualized in normal plasma were due to FVIII polypeptides alone. Eflect of protease inhibitors In order to determine whether the multiple band pattern seen with frozen pooled plasma (Fig l a ) was due to proteolysis of larger FVIII polypeptides during either freeze/thawing or the incubations with labelled Fab’ and sample buffer, whole blood from each of two healthy donors was collected into trisodium citrate in the presence or absence of a cocktail of protease inhibitors (seeMethods), centrifuged and the plasma used immediately. Following incubation with labelled antibody, SDS-PAGE and autoradiography the patterns seen in Fig l(b) were obtained. In both plasmas the bands visualized were identical regardless of the presence of inhibitors and were very similar to Fig l(a):this indicates that the multiple
82
G. Kemball-Cook, S. A. Bevan and T . W. Barrowclifle
Fig 1. (a) Autoradiograph of immunocomplexes after SDS-PAGE in 410%slab gel. Fab’. sample contained lZ51labelled Fab alone: SHP. severe haemophilic plasma incubated with 1L51-labelledFab’: NP. normal plasma plus 1251-Fab’.Arrows indicate approximate M, range by comparison with SDS-PAGE markers after subtraction of 50 kDa (=M, of one Fab’ molecule), with Fab the position of uncomplexed Fab’ fragments. (b) Effect of protease inhibitors: Fab’. sample was 1251Fab’ alone: P1 and P2. two fresh plasmas collected into anticoagulant in the absence ( -I) and presence ( + I ) of a cocktail of protease inhibitors. Arrows indicate approximate M, range of bands seen after autoradiography .
bands represent FVIII structure in freshly anticoagulated plasma rather than proteolytic breakdown products produced during freeze/thawing or incubation of the samples. Effect of plasma storage on FVlll polypeptides Fresh citrated plasmas from four normal donors were each divided into three portions: the first were incubated with labelled antibody containing protease inhibitor cocktail and electrophoresed immediately, the second were subjected to three cycles of freezing and thawing from -40°C before inhibitor addition and electrophoresis, and the third were held in a water bath at 37OC for 24 h before analysis. Fig 2 shows the results obtained from fresh plasma and plasma held at 37°C for 24 h. In all four plasmas the lowest M, band at approximately 80-90 kDa was intensified after 24 h storage at 3 7°C. with no other consistent alterations visible: as seen above, the patterns obtained from freeze-thawed
plasma (not shown) were indistinguishable from fresh plasma. Thus prolonged storage at 3 7°C but not freezing and thawing alone, generates increased quantities of a smaller polypeptide(s). The ‘doublet’ band seen in lane 4 (24 h. 37°C) and sometimes in other Figures superficially resembles that often seen when FVIII light chains (78 and 80 kDa) are visualized by SDS-PAGE however, this antibody preparation does not recognize FVIII light chains, nor does the method have sufficient resolution to visualize polypeptides so close in size. FV111 polypeptides in cryoprecipitate and cryosupernatant Fig 3 shows a comparison between the FVIII visualized in pooled normal plasma (NP), cryosupernatant (CS) and cryoprecipitate (CP),prepared as described in Methods. There was no significant difference in band pattern between the three samples, indicating that all polypeptides were fractio-
Analysis of FVIII by SDS-PAGE
83
Fig 2. Effect of plasma storage at 3 7 T for 24 h on four normal plasmas: Arrows indicate approximate M, range of bands seen after autoradiography.
Pig 3. FVIII poIypeptides visualized in normal plasma (NP). cryosupernatant (CS) and cryoprecipitate diluted x 3 (CP). Arrows indicate approximate M, range of bands seen after autoradiography.
nated equally during the precipitation. Similar results were obtained from two further preparations of cyroprecipitate (results not shown). F V l l l polypeptides in intermediate purity FVHI concentrates Fig 4 shows samples of six different products (five commercial
materials plus one NHS product, all heat-treated and of specific activity between 0.5 and 3 iu/mg). together with severe haemophilic plasma (HP) and normal plasma pool (NP). All the polypeptides present in normal plasma are also present in each concentrate, but with relatively larger amounts of the smaller polypeptides: in addition, in product E an almost complete lack of the larger polypeptides is accom-
panied by the appearance of a band of further degraded FVIII at M, 40-50 kDa. This comparison has been performed five times with different batches of each product each time: the results were very similar in each case indicating both that the technique itself is reproducible and that there is little batch to batch variation in any one product. The fainter appearance of all FVIII polypeptides in the NHS sample was due to a lower antigen loading: in other gels with higher loading (see Fig 5a) a clearer pattern was seen. In addition, these results are very similar to those we presented in a previous report (Barrowclife et al, 1986), with each product including E showing similar patterns to those in Fig 4.
84
G. Kernball-Cook, S. A. Bevan and T. W. Barrowclifle
Fig 4. FVIII polypeptides visualized in haemophilic plasma (HP), normal plasma (NP), five brands of commercial FVIII concentrate (A-E) and one brand of NHS-produced FViII concentrate. Arrows indicate approximate M, range of polypeptides after subtraction of 50 kDa (=M, of one Fab' fragment).
Fig 5. Effect of dry heat treatment ( 7 2 h. 80°C) on FVIII polypeptides in FVIII IP concentrates. (a) NHS-produced IP concentrate: NP. normal plasma: -HT. beforeheat treatment: +HT, after heat treatment. (b) Commercial IP concentrate: pre and post heat treatment samples from two batches are shown. Arrows indicate approximate M, range of FVIII polypeptides.
Effect of heat treatment on FVlIl polypeptides The effects of virucidal heat treatment on some FVIII concentrates have been investigated by this technique. Fig S(a) shows results obtained with dry heating of an intermediate purity (IP) product manufactured by an NHS fractionation centre and currently distributed in the U.K.: an unheated ( - HT) sample was compared with a sample heated in the freeze-dried state (+HT) for 72 h at 80°C. No major differenceswere seen in FVIII polypeptide distribution following heat treatment, although in each case the FVIII was more degraded than that of a frozen normal plasma pool (NP) as
+
judged by increased amounts of the lowest M, band at 80-90 kDa. Fig S(b) shows that in another commercial IP concentrate dry-heated under the same conditions a little reduction of the largest polypeptide was seen in batch 2, but there were no major changes. However, in a third IP product two methods of dry heating did produce differingpatterns: Fig 6 shows that heating of the concentrate powder suspended in heptane (6O0C, 20 h) led to more FVIII degradation than dry heating without heptane (60°C 36 h). Finally, a fourth IP concentrate was subjected either to dry heating (68°C. 72 h) or to pasteurization (60°C.
Analysis of FVllZ by SDS-PAGE
85
including those prepared by elution from anti-FVIII or antivon Willebrand factor (vWf) monoclonal antibody columns. Initially the FVIII was plasma-derived, but recently the methodology has been applied to recombinant DNA-derived FVIII expressed in mammalian cell culture. Fig 8 presents results obtained from samples of two plasma-derived and two recombinant DNA-derived monoclonal antibody-purified products. Compared to plasma the two plasma-derived VHP products are almost completely depleted in the larger FVIII polypeptides relative to the smaller ones and one of the products shows a faint degraded band at an M, or 40-50 kDa (this band did not reproduce well on photography of the original autoradiograph). The two recombinant DNA-derived VHP products, while also possessing relatively more lower M, polypeptides than plasma, differ in their patterns: product 1 has a faint band at about 130 kDa (not present in product 2 ) in addition to the strong band at 80-90 kDa, while product 2 has a stronger band at 2 50-300 kDa. DISCUSSION Fig 6. FVIII polypeptide patterns seen in a commercial IP concentrate heat treated either as powder in the dry state (Dry)or as a suspension in heptane (Hept): two batches of each type are shown.
Fig 7 . Analysis of a commercial IP FVIII concentrate heat treated by either pasteurization (Wet) or by dry heating (Dry). Arrows indicate
approximate M, range of FVIII polypeptides.
10 h), Fig 7 shows that FVIII was clearly depleted in high M, polypeptides in the pasteurized material compared to the dry heated concentrate, indicating limited proteolysis during the procedure.
Very highly-pur$ed FVlll concentrates Recent advances in fractionation technology have led to the development of very highly purified (VHP) FVIII products,
FVIII activity in vitro has been shown to be dependent on its polypeptide structure, which in turn is determined by limited proteolysis. The main enzymes involved in this modulation are probably thrombin, factor Xa and activated protein C (Cockburnet al, 1981;Fulcher & Zimmerman, 1982: Fulcher et al. 1983, 1984; Toole et al, 1984: Vehar et al, 1984: Rotblat et al, 1985; Eaton et nl, 1986: Andersson et al. 1986; Fay et al, 1986). It is possible to assay FVIII cofactor activity in many different types of sample, including plasmas with FVIII levels as low as 1 ng/ml: however, analysis of FVIII polypeptides by conventional structural analysis (for example by immunoblotting with monoclonal antibodies) requires samples to be both considerably more concentrated (approximately 1 pg/ ml), and also processed to remove the large majority of contaminating proteins. The electrophoretic technique described here is based on that of Weinstein et a1 (1981): the main modifications have been the substitution of polyacrylamide gradient slab gels for acrylamide/agarose gels, and the use of a human antibody with reactivity to a greater range of FVIII polypeptides. As performed, the method can visualize FVIII polypeptides in many types of FVIII sample including plasma, with a detection limit as low as 30 ng/ml and without any form of FVIII purification. However, unlike conventional SDS-PAGE, it is not possible to assign precise relative molecular masses (M,) to the bands seen after autoradiography, since samples are run unreduced in non-denaturing buffer (to avoid dissociation of immunocomplexes) and it is necessary to subtract the approximate M, of one molecule of Fab’ to obtain the net M, of the FVIII polypeptide. The usefulness of the technique lies in the ability to compare directly samples of widely differing purity, including plasma, without any additional processing. Conventional SDS-PAGE analysis of reduced highly-purified plasma-derived FVIII normally reveals the presence of one or more bands of the heavy chain (HC) at M, between 90
86
G. Kernball-Cook, S. A. Bevan and T. W. Barrowcliffe
Fig 8. FVIII polypeptides visualized in VHP
FVIII concentrates produced using immobilized monoclonal antibodies. NP: normal plasma; p d plasma-derived;rDNA:
recombinant DNA-derived. Arrows indicate approximate M, range of polypeptides.
kDa and 2 10 kDa, and a roughly equivalent amount of the light chain doublet (LC) at M, 79/80 kDa: on treatment with thrombin these are degraded first of all to M, 50 40 kDa and 69/70 kDa respectively, and further to smaller polypeptides (Fulcher et al. 1983, 1985: Rotblat et al. 1985; Eaton et al, 1986; Hamer et al, 1986; Anderson et al, 1986). Of these species, the polypeptides detected by the electrophoretic method described here will be dependent on the epitope specificities of the homologous anti-FVIII:C antibody used: studies of such inhibitors arising in polytransfused haemophiliacs have shown that they may be directed against either or both HC-derived and LC-derived polypeptides, although antibodies binding to the Mr 50 kDa thrombin fragment of HC are rare (Fulcher et al, 1987; Scandella et al, 1988). In our experiments the largest polypeptide (apparent M, 2 50-300 kDa) seen on the autoradiographs corresponded to the M,2 10 kDa HC species reported for purified FVIII, and the smaller band at M, 80-90 kDa corresponded to the Mr 90 kDa HC species. Where a band at M, 40-50 kDa was detected this was clearly an HC-derived cleavage product, either one of the M, kDa or 50 kDa degradation products seen on thrombin treatment of purified FVIII, or the reported M, 45 kDa polypeptidereleased by FXa or activated protein C. Deliberate thrombin treatment of plasma led to the disappearance of the bands at 250-300 kDa and 80-90 kDa, and appearance of a band in the 40-50 kDa position (data not shown). Although this inhibitor plasma has been shown to contain antibodies directed against both HC and LC determinants (OBrien, 1985; Kemball-Cook et al, 1988; Scandella et al, 1988). in most experiments the labelled antibody did not visualize the M, 79/80 kDa LC or its 69/70 kDa thrombin degradation product, indicating that anti-HC rather than anti-LC antibodies predominate in the affinity-purified preparation used. FVIII in fresh normal plasma appeared least degraded of all the samples tested. with one strong band at M, 250-300 kDa and several fainter bands at smaller M, as previously
+
described by Weinstein et a1 (1981): even when blood was collected in the presence of powerful protease inhibitors these smaller polypeptides were present, indicating that FVIII may circulate as a mixture of different HC sizes complexed with LC, rather than as the full-length single chain molecule. Intermediate purity factor VIII concentrates displayed a variable pattern of HC bands, some products being obviously more degraded than others: these variations were generally reproducible from batch to batch and also over different preparations of labelled antibody. Different FVIII concentrate manufacturing protocols clearly lead to different degrees of FVIII degradation, but it is not known whether these differences lead to alterations in the products' bioavailability or immunogenicity. An essential stage in the production of current plasmaderived FVIII concentrates is virucidal treatment to reduce the risk of transmission of infectious viruses, such as HIV or the hepatitis viruses. For many products heat-treatment, in stabilized aqueous solution (pasteurization) or in the freezedried state either under vacuum, in steam, or as a suspension in organic solvent has been the method of choice: however, some factor VIII activity is always lost through degradation in these processes. Our results show that the method of heating can affect the FVIII polypeptide structure: while some IP concentrates were not affected by dry heat, even at 8OoCfor 72 h, pasteurization (6OoC, 10 h) led to increased FVIII breakdown. In one product heating of lyophilized powder in organic solvent suspension (6OoC, 20 h) yielded a more degraded product than ordinary dry heating (60"C, 36 h). It is likely that highly purified FVIII products, both plasmaand recombinant DNA-derived, will be increasingly used for patient treatment in the future. We found two such plasmaderived products to be heavily depleted in larger FVIII polypeptides, although whether this is due to the purification steps or viral inactivation procedures is not known. The recombinant products are also degraded relative to plasma
Analysis of FVIII by SDS-PAGE FVIII, but their patterns differ: again, whether this is due to differences in the structure of the expressed protein or to stages in processing is unknown. Since all these products are ultimately efficacious in patients. it is clear that the larger FVIII heavy chain polypeptides are not essential for its activity following infusion: however, at least in the plasma-derived products they are a marker for the potential loss of FVIII activity due to proteolytic degradation during manufacture. ACKNOWLEDGMENT The authors wish to acknowledge the excellent technical assistance of Mr S. J, Edwards in performing some of the gel analyses. KEFEKENCES Andersson. L.-O., Forsman. N.. Huang, K., Lundin, A., Pavlu, B.. Sandberg. H., Sewerin. K. & Smart, J. (1986) Isolation and characterisation of human factor VIII: molecular forms in commercial factor VIII concentrate, cryoprecipitate, and plasma. Proceedings ofthe National Academy of Scierices of the United States q/ America. 83, 2979-2983. Barrowcliffe, T.W.. Edwards, S.]., Kemball-Cook. G. & Thomas, D.P. (1986) Factor VIII degradation products in heated concentrates. Lancet. i, 1448-1449. Cockburn, C.C., De Beaufre-Apps, R.J., Wilson, 1. & Hardisty, R.M. { 198 1) Parallel destruction of factor VIII procoagulant activity and an 85.000 dalton protein in highly purified factor VIII/vWF. Tlirumbosis Research, 21, 29 5- 309. Eaton. D.. Rodriguez, H. & Vehar, G.A. (1986) Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin. factor Xa. and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry. 25, 505-51 2. Fay. P.J.. Anderson, M.T.. Chavin. S.I. &Marder, V.J. (1986) The size of human factor VIII heterodimers and the effects produced by thrombin. Biochiniica et Biophysica Acta, 871, 268-278. Fay. P.1. (1987) Subunit structure of thrombin-activated human factor VIIIa. Biochirnica et Biophysica Acta. 952, 181-190. Fine. J.M. & Steinbuch, M. (1970) A simple technique for the isolation of monoclonal IgG and IgA. Kevire Europeene Etirdes Cliniqires et Biologiques, 1 5 , 1 1 15-1 121. Fulcher, C.A.. Gardiner, J.E., Griffin, J.H. & Zimmerman. T.S. (1984) Proteolytic inactivation of human factor VIII procoagulant protein by activated protein C and its analogy with factor V. Blood. 63, 486-489. Fulcher. C.A.. Mahoney, S. De G. & Zimmerman. T.S. (1987) FVIII inhibitor subclass and FVIII polypeptide specificity determined by immunoblotting. Blood, 69, 197551480. Fu1cher.C.A.. Roberts.J.R..Holland.L.Z.&Zimmerman,T.S.(19851 Human factor VIII procoagulant protein: monoclonal antibodies define precursor-product relationships and functional epitopes. lournal of'Cliriica1 Znvestigation, 76, 1 17-124. Fulcher. C.A.. Roberts, J.R. & Zimmerman, T.S. ( 1 98 3) Thrombin proteolysis of purified factor VIII procoagulant protein: correlation
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of activation with generation of a specific polypeptide. Blood, 61, 807-811. Fulcher. C.A. & Zimmerman. T.S. (1982) Characterisation of the human factor VIII procoagulant protein with a heterologous precipitating antibody. Proceedings of the National Academy of Sciences ofthe United States of America, 79, 1648-1 652. Greenwood. F.C., Hunter, W.M. & Glover, J.S. (1963) The preparation of 13'I-labelled human growth hormone of high specific radioactivity. Biochemical Journal. 89, 114-1 2 3. Guisasola. J.A., Cockburn, C.G. & Hardisty, R.M. (1978) Plasmin digestion of factor VIII: characterisation of the breakdown products with respect to antigenicity and von Willebrand activity. Thrombosis arid Haernostasis, 40, 302-3 1 5. Hamer. H.J., Koedam. J.A.. Beeser-Visser, N.H. & Sixma. J.J. (1986) Human factor VIII: purification from commercial factor VIII concentrate. characterisation, identification and radiolabelling. Biocfiiniicu et Biophysica Acta, 873, 356-366. Holmberg, L., Ljung, R. & Nilsson, I.M. (1983) The effects of plasmin and protein C, on factor VIII :C and VIII :CAg. Thrombosis Research, 31,41-50. Kernball-Cook, G.. Edwards. S.J., Sewerin. K.. Anderson. L.-0. & Barrowcliffe, T.W. (1988) Factor VIII procoagulant protein interacts with phospholipid vesicles via its 8 0 kDa light chain. Thrombosis and Haemustasis. 60, 442-446. Laemmli. 1J.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-685. Lazarchick. J. & Hoyer. L.W. (1978) Immunoradiometric measurement of the factor VIII procoagulant antigen. ]ourrial of Clinical Investigation. 62, 1048-1052, Lollar. P. & Parker. C.G. (1989) Subunit structure of thrombinactivated porcine factor VIII. Biochemistry, 28, 666-674. O'Brien, D.P. (1985) Purification and characterisation of human factor VIII. Ph.D. thesis. University of London. Reisner. H.M., Barrow, E.S. & Graham, J.B. (1979) Radioimmunoassay for coagulant factor VIII-related antigen (VIII :CAB). Thronibosis Research. 14, 235-239. Rotblat, F.. O'Brien. D.P.. O'Brien, F.J.. Goodall. A.H. & Tuddenham. E.G.D. ( 1 98 5) Purification of human factor VIII: C and its characterisation by Western blotting using monoclonal antibodies. Biochemistry, 24, 4294-4300. Scandella. D., Mahoney, S. De G., Mattingley. M., Roeder. D.. Timmons, L. & Fulcher, C.A. (1988) Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Eseherischia coli. Proceedings of the National AcadenryofSciencesofthe UnitedStatesofAmerica. 85, 6152-61 56. Toole. J.J., Knopf. J.L.. Wozney. J.M., Sultzman, LA., Buecker. J.L.. Pittman. D.D.. Kaufman, R.J.. Brown, E.. Shoemaker. C.. Orr. E.C.. Amphlett, G.W.. Foster, W.B., Coe. M.L.. Knutson. G.L., Fass. D.N. & Hewick. R.M. (1984) Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature. 312, 342-347. Vehar, G.A., Keyt. B.. Eaton, D.. Rodriguez, H., O'Brien. D.P.. Rotbtat. F., Oppermann, H.. Keck. R.. Wood, W.I., Harkins. R.N., Tuddenham, E.G.D., Lawn. R.M. & Capon, D.J. (1984) Structure of human factor VIII. Nature, 312, 337-342. Weinstein. M., Chute, L. & Deykin. D. (1981)Analysis of factor VIII coagulant antigen in normal, thrombin-treated and hemophilic plasma. Proceedirigs of the Nationd Acaderng of Sciences of the United States ofAmerica, 78, 5137-5141.
