Vox Sang. 35: 3 6 4 0 (1978)

Variable Degradation of Factor VIII-Related Protein in Lyophilised Concentrates of Antihaemophilic Factor (AHF) ’ T . Jukub, H. Pflugshaupt, M . Furlan and E. A . Beck Central Haernatology Laboratory, lnselspital, and School of Medicine, University of Rerne, and Central Laboratory, Blood Transfusion Service of the Swiss Red Cross, Bernc

Abstract. Factor VIII-related properties (coagulant = V l l l : C, ‘Willebrand’ factor = V l l I R : WF, antigen = V l l l R : AG) are measured in a constant proportion in normal plasma and certain preparations of highly purified factor V l l I (relative ratios: 0.5-1.5). We tested these activities in some commercial, lyophilised concentrates of factor V l l l and found a variable increase of the ratio V l l l R : AGjVllI R : WF. The relative increase of V l l l R : AG, and/or loss of V l l l R : WF, was attributed to variable degradation of factor VIII-related protein(s) which was directly visualised by electrophoresis on 2.75% polyacrylamide gels in the presence of sodium dodecyl sulphate.

The widespread availability of lyophilised factor V l l l concentrates has improved the mobility of patients with haemophilia. However, alarmingly high proportions of haemophiliacs with inhibitors have been reported, possibly in relation with more intensive treatment [2]. Furthermore, minor qualitative differences of factor VIII concentrates have become clinically important, since they arc not all equally suitable for

Supported by the Stanley Thomas Johnson Foundation. 2 The authors are obliged to Miss A . Z’gruggen, Miss A . Clunzniann, Mr. A . Aluney, Mrs. M. Gierisch and Mr. D.Morris for skilful technical assistance. 1

treatment of von Willebrand disease and related disorders [ 6 ] . We still know very little about the native structure of factor VIII. A relatively constant proportion of several properties of factor VIII is, however, found in fresh normal plasma, cryoprecipitate from fresh normal plasma and in highly purified factor VIlI which has been prepared with special precautions [3]. Assuming a ratio of 1 . 0 for quantitatively measurable properties of factor VIII in a normal plasma pool, we compared the following parameters in several commercially available factor VIII concentrates: coagulant (VIIl:C), factor VIII-related antigen (VIII R:AG) and ‘Willebrand activity’ (VIII R:WF). Our results suggest

Factor VIII-Kelated Protein in Lyophilised AHF

that an increased ratio of VIII R:AG/VIII R:WF characteristically accompanies irreversible degradation of high molecular weight factor VIII, irrespective of residual coagulant activity.

Materials and Methods Highly purified fiicfor V111 was prepared from 500 ml lipid-poor single donor plasma by cryoprecipitation and subsequent agarose gel filtration as previously described 141. Coniriierciiil f ( i i ~ o V r l l l ~~oncentriites. Several batches of commercially available AHF concentrates were purchased. Following dissolution acco:ding to the manufacturer’s specification, aliquots were either immediately tested or stored at -SO ’C until further analysis. Samples were numbered and distributed for ‘blind’ testing. We report here o n l y on some relevant results and indicate the corresponding methods used for analysis: T o t a l protein was estimated in commercial factor Vlll concentrates by the biuret method, in highly purified factor Vlll using Fohn’s reagent. Bovine serum albumin served as standard. VIII:C was measured by the partial thromboplastin time using plasma from patients with severe haemophilia as substrate 191. V l l l R:AC was determined according to Zit>)nieriniin c’t ul. 1141. 100% or 1 U correspond to Vlll R:AG in I ml normal plasma pool or a rocket height of about 1 cm. Factor VIlI concentrates were adjusted with barbital buffer to concentrations of 0.5-2.0 U/niI. V l l l R : W F was assayed by ristocetin aggregation of washed and formaldehyde-treated platelets 111. For calibration, the same normal plasma pool was used as for measurement of VIlI R:AG. P o l y iicry It: I iiide ge I electrophoresis ( P A C E ). Factor VIIl concentrate ( I S ; r l ) was added to 200 ! t l sodium dodecyl sulphate (SDS, I % ) in 8 M urea and applied on 6 x 70 mm gels containing 2,7S% polyacrylamide cross-linked with 2.75% bisacrylamide. The time of electrophoresis was 3.5 h at a constant current of 4 mA per gel. This minor modification of our recently described procedure [4] improved separation of factor VIII-related protein(s) in the absence of reducing agents.

31

Highly purified factorVIl1 (- 10,ug or 1.2 antigen unit per gel) served as reference material. Crosslinked fibrinogen oligomers were used as molecular weight standard [4]. Following staining with Coomassie brilliant blue, the gels were photographed as suggested by Oliver cind Chnlkley [lo]. The resulting pictures were then cut and the photographs of the individual gels (the upper third only) mounted on white drawing paper.

Results

The specific increase of VI1I:C is the principal yardstick by which fractionation of plasma for preparation of AHF concentrate has been guided thus far. Following purification of factor VIII from lipid-poor, fresh plasma we were able to obtain V1II:C with a purification factor of at least 10,000 (table I). By qualitative analysis of such preparations we reproducibly find a relatively homogeneous species of non-reduced protein on 2.75% gels (fig. 1). Its estimated molecular weight is in excess of several millions [7]. Using our normal plasma pool as a reference, the ratio of VIII R:AGNIII R:WF was close to 1.0 (1.2 in the example shown in fig. 1). This ratio remained stable upon storage for several weeks at 4 ^ C of highly purified material, in contrast to VII1:C which disappeared gradually. In some of our factor VIII preparations, as well as in commercial factor VIII concentrates, the VIII R:AG/VIII R:WF ratio was greater than 2.0 and increased upon prolonged storage, dialysis at room temperature [3] or following deliberate enzymatic degradation [5]. The ratio of VIII R:AG/VIII R:WF is variable in different commercial factor VIII preparations, although in the freshly dissolved material VII1:C was found in the concentration (or activity) as indicated by

Jakab/Pflugshaupt/Furlan/Beck

38

Table 1. Comparison of four commercial factor V l l l concentrates and highly purified factor V l l l Preparation

A B C D Pure F V l l l

VIII: C, U/ml

VllI R : A C U/ml

declared

found

20 25 23 31

34 34

100

21

100 100

41 20

Vlll R : WF U/ml

40 46

I30

18

4 20

25

Ratio Vlll R:AG/ Vlll R : WF 2.5 2.8 5.5 25.0 1.2

Protein mg/ml

14 36 20 19

-0.15

Table 11. Comparison of four batches of factor VIll concentrate (same producer and method of preparations) Preparation

A1 A2 A3 A4

VIII: C, U/ml declared

found

20 20 20 20

34 24 29 22

Vlll R:AG U/ml

VllI R : F U/ml

Ratio V l l l R: AG/ V l l l H: W F

Protein mg/ml

100 100 I50

40 31 40 14

2.5 3.2 3.1 6.2

14 13

87

the manufacturer (table I). When several batches of a product prepared by an identical procedure from large pools of freshfrozen plasma were compared (table 11), the variability was less pronounced but at least one batch (A4) had a significantly increased VIII R:AGNIII R:WF ratio. A correlation between the increase of VIII R:AG over VIII R:WF and molecular alteration of factor VIII-related protein is indicated by the electrophoretic patterns in polyacrylamide gels. Highly purified material hardly enters 2.75% gels (fig. 1 ) suggesting that the protein material is a high molecular weight complex. A similar stained band of an extremely large size could also be regularly demonstrated in cryoprecipitates prepared from fresh human plasma (not shown in fig. 1). The amounts of fac-

19 18

torVIII in concentrates A, B, C and D would have been sufficient, in comparison with highly purified material, to produce a strong protein band of very low mobility. Instead, our gels showed a number of stained bands which were normally absent in fresh cryoprecipitates. A similar pattern of multiple bands and an increase in antigen/WF ratio was recently shown to result following degradation of purified factor VIII by endogeneous contaminating enzymes [3]. The apparent molecular weight of these degradation products was still in excess of one million, when compared with the mobilities of chemically cross-linked fibrinogen oligomers (fig. 1). Sample D, the concentrate with the highest VIII R:AG/VIII R:WF ratio, was turbid in solution and contained some material which remained on top of polyacryl-

Factor VIII-Related Protein in Lyophilised AHF

39

WF ratio was accompanied by a gradual disappearance of slowly moving proteins indicating a progressive degradation of factor VIII.

Discussion

Fig. 1. SDS-polyacrylamide gel electrophoresis of factor Vlll concentrates. Letters A, €3, C, D and ‘pure VIII’ designate the same preparations as in table 1. Non-reduced protein was electrophoresed in 2.75% polyacrylamide gels. AG/WF = ratio of VllI K:AG/VIII K:WF; Fbg polymer = fibrinogen treated with glutaraldehyde serving as marker for molecular weight estimates (0.68 = dimer, etc.).

Fig. 2. SDS-polyacrylamidc gel electrophoresis of different batches of factor VIlI concentrate from the same producer. Arabic numerals designate the same batches as in table 11. Method: cf. legend to figure I .

amide gels. This band probably represents insoluble aggregates or particles (chylomicrons?). Electrophoretic patterns in figure 2 show that the different batches (1-4) from the same manufacturer produced similar patterns; again, an increase in the antigen/

By convention, haemophiliacs have been treated with a biological entity lacking in their plasma and bearing the neutral designations antihaemophilic factor, factor VIIl or V1II:C. Since we now know that V1II:C is associated with other related subunits (or activities) in a macromolecular complex, one should not overlook the fate of this complex during plasma fractionation. Our approach to quality assessment of factor VIII concentrates was deduced from experiments indicating that factor VIII complex may be reversibly or irreversibly degraded by several enzymes [5, 81. Irreversible degradation, e.g. by plasmin, is accompanied by a rapid loss of VIII:C, a slower inactivation of VIII R:WF and an apparent increase of VIII R:AG measured by crossed immunoelectrophoresis [5]. Analysis of reduced factor VIII indicated a reduction of the molecular size of the typical subunit chain following proteolytic degradation [5, 121. Interpretation of events occurring during purification of VIII:C becomes confused by the still unresolved issue concerning dissociation of VII1:C from ‘carrier’ protein [ I l l . If one would accept the hypothesis that dissociated, low molecular weight and highly purified VIII:C is the ideal material to treat haemophiliacs, our present investigation would be rather superfluous. However, such evidence is lacking. To the contrary, recent investigations suggest that VI1I:C obtained from out-dated plasma ex-

Jakab/Pflugshaupt/Furlan/Beck

40

hibits different in vivo activity from that commonly seen for cryoprecipitate [13]. Furthermore, low molecular weight VIII:C, i.e. separated from the bulk of related protein(s), is certainly not suitable for treatment of von Willebrand disease or von Willebrand syndromes [6]. We conclude from our observations that a certain degree of degradation (and/or dissociation) of factor VIll complex during collection and fractionation of plasma is probably unavoidable. The heterogeneity of high molecular weight material as shown by SDS-PAGE (2.75% gels) may be due to alterations of factor VIII-related protein(s), but direct evidence that this material is derived from factorVIII in every instance cannot be obtained by this method. However, the striking variability of various commercial preparations with respect to VIII R:WF activity and VIII R:AG/VIII R:WF ratios indicates at least that the problem is worthy of further studies. The measurement of VIlI R:AGNIII R:WF ratios during purification of factor VIlI on an industrial scale offers the advantage of a simple, rapid quality assessment. References I Allain, J. P.; Cooper, H. A.; Wagner, R. H., and Brinkhous, K. M.: Platelets fixed with paraformaldehyde; a new reagent for assay of von Willebrand factor and platelet aggregating factor. 1. Lab. clin. Med. 85: 318-328 (1975). 2 Allain, J. P.: Gaillandre, A.; Meunier, L. ct Frommel, D.: Anticorps anti-facteur VIII: rkactivitk immunologique dcs hkmophiles A a p r h injection d e facteur V111. Nouv. Revue fr. H k mat. 14: 784-785 (1975). 3 Furlan, M. and Beck, E. A.: Degradation of purified factor VIll by endogenous contaminating enzymes. Thromb. Res. 10: 153-158 (1977).

4 Furlan, M.; Jakab, T., and Beck, E. A,: Dissociation of human factor V l l l by rhizopus lipase. Thromb. Kes. 10: 431-443 (1977). 5 Furlan, M.; Jakab, T., and Beck, E. A.: Lipolytic versus proteolytic degradation o f human factor VIII. Thromb. Res. 10: 445-456 (1977). 6 Green, D. and Potter, E.V.: Failure of A H F concentrate to control blceding in von Willebrand’s diseasc. Am. J. Med. 60: 357-360 (1976). 7 Hershgold, E. J.: Properties of factor V l l l (antihemophilic factor). Prog. Hemostasis Thromb. 2: 99- I39 ( 1 974). 8 McKee, P. A,; Andersen, J.C.. and Switzer, M. E.: Molecular structural studies of human factor V111. Ann. N.Y. Acad. Sci. 240: 8-33 (1974). 9 Moser, S. und Pflugshaupt, K.: Vergleichende Untersuchungen iiber die Bestimmung der Faktor-VIII-Aktivitat. Schweiz. med. Wschr. 104: 1372-1375 (1974). 10 Oliver, D. and Chalkley, K.: An improved system for polyacrylaniidc gels. Analyt. Biochem. 44: 540-542 (1971). 11 Sussman, 1. 1.; Kosner, W., and Weiss, H. 1.: Concentration dependent dissociation of factor Vlll i n I M NaCI. Am. J . Physiol. 230: 434440 ( 1976). 12 Switzer, M. E. and McKee, P. A.: Studies o n human antihemophilic factor. J . clin. Invest. 57: 925-937 (1976). 13 Whitman, D. E.; Switzer, M. E., and McKcc, P. A.: The recovery of factor V l l l from freshfrozen, indatcd and outdated human plasma. Thrombos. Haemostas. 36: 71-77 (1976). 14 Zimmcrman, T. S.; Ratnoff. 0.D., and Powell, A. E.: Immunologic differentiation of classic hemophilia (factor VIIl deficiency) and von Willebrand’s disease. 1. clin. Invest. 50: 224254 (1971).

Keceived: April 18, 1977 Accepted: Septernbcr 7, 1977 Prof. E. A . Beck, Hamatologisches Zentrallabor, Inselspital, CH-3010 Bern (Switzerland)