THROMBOSIS RESEARCH Vol. 13. pp. 15-24. 0 Pngamon Press Ltd. 19%. Printed in Great Britain.
DISPERSITY QF !iUE1A?I FACTOR VIII - !,QvWILLFBRAND FACTOR Jan A. van Mourik* and Piet A. Bolhuis+ *Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9190, Amsterdam, The Netherlands and t Pediatric Clinic, Laboratory of Biochemistry, Binnen Gasthuis, GrimburCwal IO, Amsterdam, The Metherlands.
(,Received
PSSTRACT
17.4.1973.
Accepted
by
Editor
P.J.
Gaffney)
Human factor VIII - Von Wille'brand factor Furified hy gel chromatography in the presence of dextran has been studied with large pore polyacrylamide gel electrophoresis designed for analysis of highmolecular-weight proteins. It was demonstrate.1 that purified factor VIII-Von Willebrand factor is not a single homogeneous entity, but consists of a series of immunologically related polymers. The polydispersity of factor VIII-Von Willebrand factor was confirmed by an imnunochemical variant of the reversal-electrophoresis spreading test. With this technique it could be visualized that also factor VIII in plasma and cryoprecipitate is polydisperse in nature. As a consequence,changes in the polymer distribution of factor VIII-Von Willebrand factor can be an alternative explanation for results of dissociation experiments and for abnormalities in various diseases as demonstrated by cross-immunoelectrophoresis.
INTRODUCTION Attempts to purify antihaemophilic factor A (factor VIII) have led to the discovery of a high-molecular-weight glycoprotein with both factor VIII
and
Von Willebrand factor activities (l-7). Dissociation into smaller fragments has been observed under a variety of conditions, e.g. high ionic strength (814), low ionic strength (15,16), reducino
conditions (4,6,17,18) and incuba-
tion with thrombin (19). It has been proposed that both non-covalent bonds and disulphide bonds are involved in the stabilization of the macromolecular structure (4,E,15-18).
16
POLYDISPERSITY
OF FYI11
- vWF
v01.13,x0.1
Only one protein band appears upon polyacrylamide gel electrophoresis of samples reduced with dithiothreitol or Z-mercaptoethanol under denaturing conditions (4,6,17,18). These results have led to the assumption that the factor
VIII
and Von Willebrand factor activities are located on a homogeneous mole-
cular entity (3,6,18,20,21) composed of identical or similar constituent units (4,6,17,18).
In this
paper
we show that
complex of factor VIII
the
and the Von Wille-
brand factor in purified form as well as in cryoprecipitate and in plasma is polydisperse and is not a homogeneous molecular entity. For these studies special electrophoretic techniques were developed: (i) large-pore polyacrylamide gel electrophoresis suited for analysis of high-molecular-weight proteins, and (ii) a new variant of the reversal-electroohoresis spreading test (22,23), which can be applied to unpurified samples.
MATERIALS AND METHODS Sepharose 68, dextran (Rheomacrodex, average molecular weight 4O.OOn) and columns were purchased from Pharmacia Fine Chemicals,lJppsala, Sweden. The columns were siliconized prior to use. Factor VIII
activity was determined with a one-stage assay (24). Von
Willebrand factor activity was evaluated on the basis of the ability to support the ristocetin (Lundbeck, Copenhagen) aggregation in washed platelet suspensions (25). Fibrinopeptide A was determined by a radioimmunoassay as described previously (26) omitting the dialysis step. Protein concentrations were determined by the method of Lowry (27) using bovine serum albumin (Sigma Chemical Co.j as standard. Heparin (Trombo:ikine) was purchased from Organon, The Netherlands. Trasylol was obtained from Bayer, Germany, benzamidine from Aldrich, Belgium and soybean trypsin inhibitor from Sigma Chemical Co. Chemicals used for polyacrylamide crelelectrophoresis were purchased from Bio-rad. Agarose
was
obtained from Hoechst, W. Germany. Anti-cold insoluble globuiin serum was kindly supplied by Or. Mossesson (Down State Med. Ctr., State Univ. of New York). All other antisera used for identification of plasma proteins were supplied by the Netherlands Red Cross Blood transfusion Service, Amsterdam. Factor VIII-VHF was purified
as described previously (2,15). Concentra-
ted factor VIII-VGIFwas ohtained by
dialysis against 35% saturated amnonium-
sulfate during 18 h at 4'C. The cloudy precipitate was collected by centrifu-
Vol.l3,No.l
POLYDISPERSITY
OF FYI11
- vk-F
17
gation and dissolved in phosphate buffered saline (pH 7.4)
to a concentration
of approximately 0.5 mg/ml. Undissolved particles were removed by centrifugation. The final product was purified approximately 5000 to 75r)O-foldover the starting plasma. Analytical large-pore polyacrylamide gel electrophoresis was performed as described previously (15) using 0.37 M Tris-glycine (pH 9.5). The total gel concentration (T) was 4.35% and the degree of cross-linkage (C) was 0.67% (notation according to Hjerten (28)). On this large-pore gel system consisting of a network of long fibers (29) relatively large samples can be applied. Since the retardation coefficient of factor VIII-VWF
(see
below) is high, con-
siderable concentration is obtained. Factor VIII-VWF related antigen was detected by incubating the acrylamide gels for 1 h at 37'C and 18 h at 4'C with monospecific rabbit anti-factor VIII-VWF serum. In this way precipitation of the antigen-antibody complexes could be visualized directly. Factor VIII-VWF was reduced by incubation during 2 h at 37'C in sodium dodecylsulfate, urea and dithiothreitol to a concentration of 1%,8Mand
1% respectively.Prior to polyacrylamide gel electrophoresis,
the above reaction mixture was dialyzed at room temperature overnight against a solution containing 0.01 M sodium phosphate, 0.1% sodium dodecylsulfate, 0.125 M NaCl (pH 7.0).
0.5 M urea and 0.1% 2-mercaptoethanol.
A new variant of the reversal electrophoresis spreading test was used for inspection of the homogeneity of factor VIII-VWF.
The basis of the test is
the determination of the dependence of the band width on the electrophoresis time and the effect of reversal of the field polarity (22,23). The original procedure was carried out in a free-ele:trophoresis system. In the present case the migration of the protein is visualized by cross-immunoelectrophoresis using rabbit anti-factor VIII-VWF serum.Electrophoresis at 5 V/cm was carried out simultaneously on six identical plates. With intervals of one hour, one of the plates was placed perpendicular to the original direction, where upon the electrophoresis was continued into the agarose layer containing 0.2% antifactor VIII-VWF serum. The polarity of the electric field was reversed after three hours. Electrophoresis was carried out in 0.67% (w/v) agarose gels with Tris-barbital buffer (pH 8.8, ionic strength 0.933) on glass plates (8 x 9 cm) in LKB Multiphors at a temperature of 15'C. The field strength was measured with pin-pointed electrodes connected with a Philips Multimeter. The gels were washed with 0.15 M NaCl and stained with Coomassie Brilliant Blue.
‘8
POLYDISPERSITY
OF FVIIT
- VW
VOf.13,SO.l
RESULTS AND DISCUSSION Factor VIII-Von Willebrand factor (factor VIII-VWF) did not enter polyacrylamide gels of the type commonly used (Fig. lA), and, after reduction, migrated as one band (Fig. IS). However, when large-pore acrylamide gels with extremely low cross-linkage were used, (unreduced) factor VIII-VWF was seen to comprise a series of components (Fig. 1C). All bands reacted with a monospecific rabbit anti-factor VIII-VWF serum (7,30) (Fig. 1D). Thus, the product of purification was not a homogeneous entity but apparently consisted of a series
FIG. 1 Polyacrylamide gel electrophoresis of factor VIII-VWF. Large-pore gels (T=4.65%, &0.67%) were used, except gel A (T=5%, C=3%). Factor VIII-VWF was applied to gel B after reduction and to other gels in unreduced state. Samples of 0.5 ml (25 ug) were used for gels A,B and C, while concentrated factor VIII-VWF (0.24 ml, 0.1 mg) was applied to gels D and E. Gels D and E were incubated with rabbit anti-factor VIII-VWF serum (see Materials and Methods). Gels were stained with Coomassie Brilliant Blue, except gel D which is shown unstained.
Vol.‘3.xo.l
P0LYDISPERSTTY
OF TV111
- vk'F
19
The observed dispersity of unreduced factor VIII-VWF on large-pore polyacrylamide gel electrophoresis is not due to contaminations as (i) no precipitation line is seen in imnunodiffusion against anti-N2
macroglobulin, anti-fibrino-
gen and anti-cold insoluble globulin. The absence of substantial amounts of fibrinogen was confirmed by the radioimmunoassay of fibrinopeptide A (26). The top of the void volume peak contains approx. 1 ng fibrinopeotide
A per ml,
which corresponds to 2 mg fibrinogen per g protein. (ii) When cryoprecipitate of a patient with Von Willebrand disease is used as starting material for purification, no 275 nm- abscrbingmaterial
is eluted at the void volume.
(iii) When reduced factor VIII-VWF is analyzed by sodium dodecyl sulfate electrophoresis one band is observed (Fig. 1B). Whether differences in mobility of the various components are due to differences either in size or in charge can be derived from gel filtration experiments (Fig. 2). Fractions corresponding to the ascending part, top and descending part of the void volume peak were submitted to large-pore acrylamide gel electrophoresis. The concentration of the fast migrating components increased with elution volume, and the concentration of more slowly migrating components decreased. Thus, differences in electrophoretic mobility reflect differences in size rather than differences in charge. Although successive void
volume fractions differed quantitatively in
polymer distribution, reduction of these samples resulted in similar polyacrylamide electrophoresis patterns, indicating that all polymers are constructed of similar or identical constituent units. Similar elution and electrophoresis patterns were observed when the blood was collected in citrate that contained proteolytic inhibitors of broad specificity. Cryoprecipitation and gel chromatography (2) were performed in the presence of the same inhibitors (benzamidine In mM, soybean trypsin inhi-
.
bitor 10 mg/l, trasylol 10 U/ml, heparin 1.25 U/ml). It therefore seems unlikely that the polydispersity is the result of partial proteolytic breakdown, at least in vitro, of a single homogeneous macromolecular complex. The polydispersity of factor VIII-VWF was confirmed by means of a new variant of the reversal-electrophoresis spreading test (22,23). By this test, originally described by Tiselius in 1930 (see ref. 22), small differences in electric mobility can be detected. Classically, the test is carried out in a free-electrophoresis system. In this study, cross-immunoelectrophoresis was used for visualization of the protein band (Fig. 3).
20
.POLYDISPERSITY
OF FVIII
Vol.l3,No.l
- VW
t
1
A. 500 A
-
-1~ fL_x.
62
_*
C”’ 6;
1000 1500
2000
elutionvolume (ml)
“0
.: I
66
2
0
0
I
70
65
,,
75 fraction nu 600
500
cr
elution volume (ml)
FIG. 2 Elution pattern (inset, upper right) of cryoprecipitate from 400 ml fresh plasma that had been subjected to gel chromatography. The sample, 15 ml, was chromatographed on a column (5 cm x 80 cm) of Sepharose 68 developed with downward flow at room temperature with a veronal-NaCl buffer (pH 7.0) containing 3.3% dextran (2.15). The flow rate was 75 ml/h. The void volume part of the elution pattern, indicated by the vertical arrow, is shown in detail in the lower portion of the figure. Polyacrylamide gel of selected column fractions, containing both pro-coagulant activity and Von Willebrand factor activity (indicated by tube number), are shown inset adjacent to the Sepharose 68 chromatogram. Before electrophoresis, samples (unreduced) were diluted to the same protein concentration (about 25 ug/ml) with column buffer. For further experimental details, see refs. 2, 14 and Fig. 1. No heterogeneity was observed from a single cross-immunoelectrophoresis pattern of concentrated factor
VIII-WF.
However, the band width was conside-
rably larger than expected from spreading by diffusion. Furthermore, upon reversal of the polarity of the electric field, the band width decreased, which is indicative of heterogeneity. It seems likely that the increase and decrease of the band width are caused by the presence of components with different mobilities and not by phenomena unaffected by field reversal (e.g.
Vol.13,No.f
POLkDISPERSITY
OF FVITI
-
VW
21
termal gradients, electro-endosmosis or adsorption to the agarose).
FIG.
3
Reversal-electrophoresis spreading test of purified factor VIII-VWF. The dependence of the band width on the electrophoresis time and the effect of field reversal on the band width are shown in Plates l-6. Samples of 8 ul (5 ug of protein) were apolied'to elites as used for crossed-imnunoelectrophoresis. The migration in the layer free of antiserum is indicated by the arrows.
A paramount question is whether the polydispersity of the purified protein reflects the state in vivo. The use of the variant of the reversalelectrophoresis spreading test also makes it possihle to determine the heterogeneity of impure samples. Patterns similar to those shown in
Fig. 3 were
observed when cryoprecipitate, fresh titrated or fresh heparinized plasma were submitted to the reversal spreading test. The plasma samples were obtained from single donors and the test was started within 15 min after the
22
POLYDISPERSITY
OF FVIII
- vWF
v01.13,~0.1
venipuncture. Therefore, the polydispersity cannot be ascribed to the purification procedure, freezing of the plasma, an effect of citrate or to the mixing of donor-specific homogeneous species. Since the term heterogeneity is usually restricted to molecular species that differ in chemical composition , we prefer the term polydiserpsity, which indicates that differences in size between chemically identical sub-species are the main cause of differences in physico-chemical behaviour. As a consequence of these studies, data about variants of the factor VIII-VWF in Von Willebrand disease or liver disease (31-33) can be interpreted in terms of the polydispersity,that is, the changes in electrophoretic mobility may be a reflection of changes in the molecular weight distribution. Whether these differences are due to structural modifications of the constituent units remains to be established. The same argument applies to dissociation (and recombination) experiments on factor VIII-VWF. Shifts in polymer distribution may be induced under various conditions, which complicates the interpretation of these experiments considerably.
ACKNOWLEDGE?lENTS We thank Dr. B.N. Bouma for helpful discussions and E.M. Hoorweg and A.J. Seinen for technical assistance. The investigations were supported in part by the Foundation for Medical Research FUNGO which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
REFERENCES 1. RATNOFF, O.D., KASS, L. and LANG, P.D. Studies on the purification of anti hemophilic factor (factor VIII), II. Separation of partially purified antihemophilic factor by gel filtration of plasma. J. Clin. Invest. 48, 957, 1969. 2. MOURIK, J.A. van and MOCHTAR, I.A. Purification of human antihemophilic factor (factor VIII) by gel chromatography). Biochim. Biophys. Acta 221, 667, 1970. 3. HERSGOLD, E.J., DAVISON, A.M. and JANSZEN, M.E. Isolation and some chemical properties of human factor VIII (antihemophilic factor). J. Lab. Clin. Med. 77, 185, 1971. 4. SCHMER, G., KIRBY, E.P., TELLER, D.C. and DAVIE, E.W. The isolation and characterization of bovine factor VIII (antihemophilic factor). J. Biol. Chem. 247, 2412, 1972. 5. OWEN, W.G. and WAGNER, R.H. Antihemophilic factor. A new method for puri-
~ol.l~.Yo.l
POLYDISPERSITY
OF FVIII
- vWF
23
6. LEGAZ, M.E., SCHMER, G., COUNTS, R.8. and OAVIE, E.W. Isolation and characterization of human factor VIII (antihemophilic factor). J. Biol. Chem. 248, 3946, 1973. 7. BOUMA, B.N., WIEGERINCK, Y., SIXMA, J.J., MOURIK, J.A. van and MOCHTAR, I.A. Immunological characterization of purified antihaemophilic factor A which corrects abnormal platelet retention in Von Wille(factor VIII) brand's disease. Nature New Biol. 236, 104, 1972. 8. THELIN, G.M. and WAGNER, R.H. Sedimentation of plasma antihemophilic factor. Arch. Biochem. Biophys. 95, 70, 1961. 9. WEISS, H.J. and KOCHWA, S. Molecular forms of anti-hemophilic globulin in plasma, cryoprecipitate and after thrombin activation. Brit. J. Haematol. 18, 89. 1970. 10. OWEN, W.G. and WAGNER, R.H. Antihemophilic factor: separation of an active fragment following dissociation by salts or detergents. Thromb. Diathes. Haemorrh. 27, 502, 1972. 11. WEISS, H.J., PHILLIPS, L.L. and ROSMER, W. Separation of subunits of antihemophilic factor (AHF) by agarose gel chromatography. Thromb.Diathes. Haemorrh. 27, 212, 1972. 12. WEISS, H.J. and HOYER, L.W. Dissociation of antihemophilic factor procoagulant activity from the Von Willebrand factor. Science 132, 1149, 1973 13. RICK, M.E. and HOYER, L.W. Immunologic studies of antihemophilic factor (AHF ; factor VIII), V. Immunologic properties of AHF subunits produced by salt dissociation. Blood 42, 737, 1973. 14. RICK, M.E. and HOYER, L.W. Molecular weight of human factor VIII procoagulant activity. Thrombosis Research 7, 909, 1975. 15. MOURIK, J.A. van, BOUMA, B.N., BRUYERE, W.T.la, GRAPF, S. de and MOCHTAR, a series of homologous oligomers and a complex of two I.A. Factor VIII, proteins. Thrombosis Research 4, 155, 1974. 16. BOUMA, B.N., MOURIK, J.A. van, GRAAF, S. de, HORDIJK-HOS, J.M. and SIXMA, J.J. Immunologic studies on human factor VIII (antihemophilic factor A; AHF) components produced by low ionic strength dialysis. Blood 47, 253, 1976. 17. MARCHESI, S.L., SHULMAN, N.R. and GRALNICK, H.R. Studies on the purification and characterization of human factor VIII. J. Clin Invest. 51, 2151, 1972. 18. SHAPIRO, G.A., ANDERSEN, J.C., PIZZO, S.V. and MCKEE, P.A. The subunit J. Clin Invest. 52, 2193, structure of normal and hemophilic factor VIII. 1973. 19. VEHAR, G.A. and DAVIE, E.W. Formation of a serine enzyme in the presence of bovine factor VIII (antihemophilic factor)and thrombin. Science 197, 374, 1977. 20. HERSHGOLD, E.J. Properties of factor VIII (antihemophilic factor). In: Progress in Hemostasis and Thrombosis, Vol. 2, T.H. Spaet (Ed.), New York, Grune and Stratton, 1974, p 99. 21. SWITZER, M.E. and MCKEE, P.A. Studies on human antihemophilic factor. Evidence for a covalently linked subunit structure. J.Clin. Invest. 57, 925, 1976. 22. ALBERTY, R.A. A quantitative study of reversible boundary spreading in the electrophoresis of proteins. J. Amer. Chem. Sot. 70, 1675, 1948. 23. ALBERTY, R.A. Electrochemical properties. In: The Proteins, Vol 1, H. Neurath and K. Bailey (Ed.), New York, Academic Press, 1953, p. 537. 24. VELTKAMP, J.J.. DRION, E.F. and LOELIGER, E.A. Detectionofthe carrier state in hereditary coagulation disorders. Thromb. Diathes. tiaemorrh 19, 279, 1968. 25. WEISS, H.J., HOYER, L.W., RICKLES, R.F., VARMA, A. and ROGERS, J. Quantitative assay of a plasma factor deficient in Von Willebrand's disease that is necessary for platelet aggregation. J. Clin. Invest. 52,
24
POLYDISPERSITY
OF FVIII
-
vWF
Vol.
13,No.l
2708, 1973. 26. GERRITS, W.B.J., FLIER, 0.Th.N. and MEER, J. van der.Fibrinopeptide A immunoreactivity in human plasma. Thrombosis Research 5, 197, 1974. 27. LOWRY, O.,H., ROSENBOURGH, N.J., FARR, A.L. and RANDALL, R.J. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265, 1951 28. HJERTEN, 5. Molecular-sieve electrophoresis in cross-linked polyacrylamide gels. J. Chromatogr. 11, 66, 1963. 29. RODBARD, D. and CHRAMBAC , A. Unified theory for gel electrophoresis and gel filtration. Proc. Nat. Acad. Sci. USA 65, 970, 1970. 30. BOUMA, B.N., MOURIK, J.A. van, WIEGERINCK, Y., SIXMA, J.J. and MOCHTAR, I.A. Immunological characterization of anti-haemophilic factor A related antigen in haemophili A. Stand. J. Haematol. 11, 184, 1973. 31. ZIMMERMAN, T.S., ROBERTS, J. and EDGINGTON, T.S. Factor VIII related anti. gen: multiple molecular forms in human plasma. Proc. Natl. Acad. Sci. USA 72, 5121, 1975. 32. MAISONNEUVE, P. and SULTAN, Y. Modification of factor VIII-complex properties in patients with liver disease. J. Clin. Pathol. 30, 221, 1977. 33. WEISS, H.J., SUSSMAN, 1.1. and HOYER, L.W. Stabilization of factor VIII in plasma by the Von Willebrand factor. J. Clin. Invest. 60, 390, 1977.
DISPERSITY QF !iUE1A?I FACTOR VIII - !,QvWILLFBRAND FACTOR Jan A. van Mourik* and Piet A. Bolhuis+ *Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9190, Amsterdam, The Netherlands and t Pediatric Clinic, Laboratory of Biochemistry, Binnen Gasthuis, GrimburCwal IO, Amsterdam, The Metherlands.
(,Received
PSSTRACT
17.4.1973.
Accepted
by
Editor
P.J.
Gaffney)
Human factor VIII - Von Wille'brand factor Furified hy gel chromatography in the presence of dextran has been studied with large pore polyacrylamide gel electrophoresis designed for analysis of highmolecular-weight proteins. It was demonstrate.1 that purified factor VIII-Von Willebrand factor is not a single homogeneous entity, but consists of a series of immunologically related polymers. The polydispersity of factor VIII-Von Willebrand factor was confirmed by an imnunochemical variant of the reversal-electrophoresis spreading test. With this technique it could be visualized that also factor VIII in plasma and cryoprecipitate is polydisperse in nature. As a consequence,changes in the polymer distribution of factor VIII-Von Willebrand factor can be an alternative explanation for results of dissociation experiments and for abnormalities in various diseases as demonstrated by cross-immunoelectrophoresis.
INTRODUCTION Attempts to purify antihaemophilic factor A (factor VIII) have led to the discovery of a high-molecular-weight glycoprotein with both factor VIII
and
Von Willebrand factor activities (l-7). Dissociation into smaller fragments has been observed under a variety of conditions, e.g. high ionic strength (814), low ionic strength (15,16), reducino
conditions (4,6,17,18) and incuba-
tion with thrombin (19). It has been proposed that both non-covalent bonds and disulphide bonds are involved in the stabilization of the macromolecular structure (4,E,15-18).
16
POLYDISPERSITY
OF FYI11
- vWF
v01.13,x0.1
Only one protein band appears upon polyacrylamide gel electrophoresis of samples reduced with dithiothreitol or Z-mercaptoethanol under denaturing conditions (4,6,17,18). These results have led to the assumption that the factor
VIII
and Von Willebrand factor activities are located on a homogeneous mole-
cular entity (3,6,18,20,21) composed of identical or similar constituent units (4,6,17,18).
In this
paper
we show that
complex of factor VIII
the
and the Von Wille-
brand factor in purified form as well as in cryoprecipitate and in plasma is polydisperse and is not a homogeneous molecular entity. For these studies special electrophoretic techniques were developed: (i) large-pore polyacrylamide gel electrophoresis suited for analysis of high-molecular-weight proteins, and (ii) a new variant of the reversal-electroohoresis spreading test (22,23), which can be applied to unpurified samples.
MATERIALS AND METHODS Sepharose 68, dextran (Rheomacrodex, average molecular weight 4O.OOn) and columns were purchased from Pharmacia Fine Chemicals,lJppsala, Sweden. The columns were siliconized prior to use. Factor VIII
activity was determined with a one-stage assay (24). Von
Willebrand factor activity was evaluated on the basis of the ability to support the ristocetin (Lundbeck, Copenhagen) aggregation in washed platelet suspensions (25). Fibrinopeptide A was determined by a radioimmunoassay as described previously (26) omitting the dialysis step. Protein concentrations were determined by the method of Lowry (27) using bovine serum albumin (Sigma Chemical Co.j as standard. Heparin (Trombo:ikine) was purchased from Organon, The Netherlands. Trasylol was obtained from Bayer, Germany, benzamidine from Aldrich, Belgium and soybean trypsin inhibitor from Sigma Chemical Co. Chemicals used for polyacrylamide crelelectrophoresis were purchased from Bio-rad. Agarose
was
obtained from Hoechst, W. Germany. Anti-cold insoluble globuiin serum was kindly supplied by Or. Mossesson (Down State Med. Ctr., State Univ. of New York). All other antisera used for identification of plasma proteins were supplied by the Netherlands Red Cross Blood transfusion Service, Amsterdam. Factor VIII-VHF was purified
as described previously (2,15). Concentra-
ted factor VIII-VGIFwas ohtained by
dialysis against 35% saturated amnonium-
sulfate during 18 h at 4'C. The cloudy precipitate was collected by centrifu-
Vol.l3,No.l
POLYDISPERSITY
OF FYI11
- vk-F
17
gation and dissolved in phosphate buffered saline (pH 7.4)
to a concentration
of approximately 0.5 mg/ml. Undissolved particles were removed by centrifugation. The final product was purified approximately 5000 to 75r)O-foldover the starting plasma. Analytical large-pore polyacrylamide gel electrophoresis was performed as described previously (15) using 0.37 M Tris-glycine (pH 9.5). The total gel concentration (T) was 4.35% and the degree of cross-linkage (C) was 0.67% (notation according to Hjerten (28)). On this large-pore gel system consisting of a network of long fibers (29) relatively large samples can be applied. Since the retardation coefficient of factor VIII-VWF
(see
below) is high, con-
siderable concentration is obtained. Factor VIII-VWF related antigen was detected by incubating the acrylamide gels for 1 h at 37'C and 18 h at 4'C with monospecific rabbit anti-factor VIII-VWF serum. In this way precipitation of the antigen-antibody complexes could be visualized directly. Factor VIII-VWF was reduced by incubation during 2 h at 37'C in sodium dodecylsulfate, urea and dithiothreitol to a concentration of 1%,8Mand
1% respectively.Prior to polyacrylamide gel electrophoresis,
the above reaction mixture was dialyzed at room temperature overnight against a solution containing 0.01 M sodium phosphate, 0.1% sodium dodecylsulfate, 0.125 M NaCl (pH 7.0).
0.5 M urea and 0.1% 2-mercaptoethanol.
A new variant of the reversal electrophoresis spreading test was used for inspection of the homogeneity of factor VIII-VWF.
The basis of the test is
the determination of the dependence of the band width on the electrophoresis time and the effect of reversal of the field polarity (22,23). The original procedure was carried out in a free-ele:trophoresis system. In the present case the migration of the protein is visualized by cross-immunoelectrophoresis using rabbit anti-factor VIII-VWF serum.Electrophoresis at 5 V/cm was carried out simultaneously on six identical plates. With intervals of one hour, one of the plates was placed perpendicular to the original direction, where upon the electrophoresis was continued into the agarose layer containing 0.2% antifactor VIII-VWF serum. The polarity of the electric field was reversed after three hours. Electrophoresis was carried out in 0.67% (w/v) agarose gels with Tris-barbital buffer (pH 8.8, ionic strength 0.933) on glass plates (8 x 9 cm) in LKB Multiphors at a temperature of 15'C. The field strength was measured with pin-pointed electrodes connected with a Philips Multimeter. The gels were washed with 0.15 M NaCl and stained with Coomassie Brilliant Blue.
‘8
POLYDISPERSITY
OF FVIIT
- VW
VOf.13,SO.l
RESULTS AND DISCUSSION Factor VIII-Von Willebrand factor (factor VIII-VWF) did not enter polyacrylamide gels of the type commonly used (Fig. lA), and, after reduction, migrated as one band (Fig. IS). However, when large-pore acrylamide gels with extremely low cross-linkage were used, (unreduced) factor VIII-VWF was seen to comprise a series of components (Fig. 1C). All bands reacted with a monospecific rabbit anti-factor VIII-VWF serum (7,30) (Fig. 1D). Thus, the product of purification was not a homogeneous entity but apparently consisted of a series
FIG. 1 Polyacrylamide gel electrophoresis of factor VIII-VWF. Large-pore gels (T=4.65%, &0.67%) were used, except gel A (T=5%, C=3%). Factor VIII-VWF was applied to gel B after reduction and to other gels in unreduced state. Samples of 0.5 ml (25 ug) were used for gels A,B and C, while concentrated factor VIII-VWF (0.24 ml, 0.1 mg) was applied to gels D and E. Gels D and E were incubated with rabbit anti-factor VIII-VWF serum (see Materials and Methods). Gels were stained with Coomassie Brilliant Blue, except gel D which is shown unstained.
Vol.‘3.xo.l
P0LYDISPERSTTY
OF TV111
- vk'F
19
The observed dispersity of unreduced factor VIII-VWF on large-pore polyacrylamide gel electrophoresis is not due to contaminations as (i) no precipitation line is seen in imnunodiffusion against anti-N2
macroglobulin, anti-fibrino-
gen and anti-cold insoluble globulin. The absence of substantial amounts of fibrinogen was confirmed by the radioimmunoassay of fibrinopeptide A (26). The top of the void volume peak contains approx. 1 ng fibrinopeotide
A per ml,
which corresponds to 2 mg fibrinogen per g protein. (ii) When cryoprecipitate of a patient with Von Willebrand disease is used as starting material for purification, no 275 nm- abscrbingmaterial
is eluted at the void volume.
(iii) When reduced factor VIII-VWF is analyzed by sodium dodecyl sulfate electrophoresis one band is observed (Fig. 1B). Whether differences in mobility of the various components are due to differences either in size or in charge can be derived from gel filtration experiments (Fig. 2). Fractions corresponding to the ascending part, top and descending part of the void volume peak were submitted to large-pore acrylamide gel electrophoresis. The concentration of the fast migrating components increased with elution volume, and the concentration of more slowly migrating components decreased. Thus, differences in electrophoretic mobility reflect differences in size rather than differences in charge. Although successive void
volume fractions differed quantitatively in
polymer distribution, reduction of these samples resulted in similar polyacrylamide electrophoresis patterns, indicating that all polymers are constructed of similar or identical constituent units. Similar elution and electrophoresis patterns were observed when the blood was collected in citrate that contained proteolytic inhibitors of broad specificity. Cryoprecipitation and gel chromatography (2) were performed in the presence of the same inhibitors (benzamidine In mM, soybean trypsin inhi-
.
bitor 10 mg/l, trasylol 10 U/ml, heparin 1.25 U/ml). It therefore seems unlikely that the polydispersity is the result of partial proteolytic breakdown, at least in vitro, of a single homogeneous macromolecular complex. The polydispersity of factor VIII-VWF was confirmed by means of a new variant of the reversal-electrophoresis spreading test (22,23). By this test, originally described by Tiselius in 1930 (see ref. 22), small differences in electric mobility can be detected. Classically, the test is carried out in a free-electrophoresis system. In this study, cross-immunoelectrophoresis was used for visualization of the protein band (Fig. 3).
20
.POLYDISPERSITY
OF FVIII
Vol.l3,No.l
- VW
t
1
A. 500 A
-
-1~ fL_x.
62
_*
C”’ 6;
1000 1500
2000
elutionvolume (ml)
“0
.: I
66
2
0
0
I
70
65
,,
75 fraction nu 600
500
cr
elution volume (ml)
FIG. 2 Elution pattern (inset, upper right) of cryoprecipitate from 400 ml fresh plasma that had been subjected to gel chromatography. The sample, 15 ml, was chromatographed on a column (5 cm x 80 cm) of Sepharose 68 developed with downward flow at room temperature with a veronal-NaCl buffer (pH 7.0) containing 3.3% dextran (2.15). The flow rate was 75 ml/h. The void volume part of the elution pattern, indicated by the vertical arrow, is shown in detail in the lower portion of the figure. Polyacrylamide gel of selected column fractions, containing both pro-coagulant activity and Von Willebrand factor activity (indicated by tube number), are shown inset adjacent to the Sepharose 68 chromatogram. Before electrophoresis, samples (unreduced) were diluted to the same protein concentration (about 25 ug/ml) with column buffer. For further experimental details, see refs. 2, 14 and Fig. 1. No heterogeneity was observed from a single cross-immunoelectrophoresis pattern of concentrated factor
VIII-WF.
However, the band width was conside-
rably larger than expected from spreading by diffusion. Furthermore, upon reversal of the polarity of the electric field, the band width decreased, which is indicative of heterogeneity. It seems likely that the increase and decrease of the band width are caused by the presence of components with different mobilities and not by phenomena unaffected by field reversal (e.g.
Vol.13,No.f
POLkDISPERSITY
OF FVITI
-
VW
21
termal gradients, electro-endosmosis or adsorption to the agarose).
FIG.
3
Reversal-electrophoresis spreading test of purified factor VIII-VWF. The dependence of the band width on the electrophoresis time and the effect of field reversal on the band width are shown in Plates l-6. Samples of 8 ul (5 ug of protein) were apolied'to elites as used for crossed-imnunoelectrophoresis. The migration in the layer free of antiserum is indicated by the arrows.
A paramount question is whether the polydispersity of the purified protein reflects the state in vivo. The use of the variant of the reversalelectrophoresis spreading test also makes it possihle to determine the heterogeneity of impure samples. Patterns similar to those shown in
Fig. 3 were
observed when cryoprecipitate, fresh titrated or fresh heparinized plasma were submitted to the reversal spreading test. The plasma samples were obtained from single donors and the test was started within 15 min after the
22
POLYDISPERSITY
OF FVIII
- vWF
v01.13,~0.1
venipuncture. Therefore, the polydispersity cannot be ascribed to the purification procedure, freezing of the plasma, an effect of citrate or to the mixing of donor-specific homogeneous species. Since the term heterogeneity is usually restricted to molecular species that differ in chemical composition , we prefer the term polydiserpsity, which indicates that differences in size between chemically identical sub-species are the main cause of differences in physico-chemical behaviour. As a consequence of these studies, data about variants of the factor VIII-VWF in Von Willebrand disease or liver disease (31-33) can be interpreted in terms of the polydispersity,that is, the changes in electrophoretic mobility may be a reflection of changes in the molecular weight distribution. Whether these differences are due to structural modifications of the constituent units remains to be established. The same argument applies to dissociation (and recombination) experiments on factor VIII-VWF. Shifts in polymer distribution may be induced under various conditions, which complicates the interpretation of these experiments considerably.
ACKNOWLEDGE?lENTS We thank Dr. B.N. Bouma for helpful discussions and E.M. Hoorweg and A.J. Seinen for technical assistance. The investigations were supported in part by the Foundation for Medical Research FUNGO which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
REFERENCES 1. RATNOFF, O.D., KASS, L. and LANG, P.D. Studies on the purification of anti hemophilic factor (factor VIII), II. Separation of partially purified antihemophilic factor by gel filtration of plasma. J. Clin. Invest. 48, 957, 1969. 2. MOURIK, J.A. van and MOCHTAR, I.A. Purification of human antihemophilic factor (factor VIII) by gel chromatography). Biochim. Biophys. Acta 221, 667, 1970. 3. HERSGOLD, E.J., DAVISON, A.M. and JANSZEN, M.E. Isolation and some chemical properties of human factor VIII (antihemophilic factor). J. Lab. Clin. Med. 77, 185, 1971. 4. SCHMER, G., KIRBY, E.P., TELLER, D.C. and DAVIE, E.W. The isolation and characterization of bovine factor VIII (antihemophilic factor). J. Biol. Chem. 247, 2412, 1972. 5. OWEN, W.G. and WAGNER, R.H. Antihemophilic factor. A new method for puri-
~ol.l~.Yo.l
POLYDISPERSITY
OF FVIII
- vWF
23
6. LEGAZ, M.E., SCHMER, G., COUNTS, R.8. and OAVIE, E.W. Isolation and characterization of human factor VIII (antihemophilic factor). J. Biol. Chem. 248, 3946, 1973. 7. BOUMA, B.N., WIEGERINCK, Y., SIXMA, J.J., MOURIK, J.A. van and MOCHTAR, I.A. Immunological characterization of purified antihaemophilic factor A which corrects abnormal platelet retention in Von Wille(factor VIII) brand's disease. Nature New Biol. 236, 104, 1972. 8. THELIN, G.M. and WAGNER, R.H. Sedimentation of plasma antihemophilic factor. Arch. Biochem. Biophys. 95, 70, 1961. 9. WEISS, H.J. and KOCHWA, S. Molecular forms of anti-hemophilic globulin in plasma, cryoprecipitate and after thrombin activation. Brit. J. Haematol. 18, 89. 1970. 10. OWEN, W.G. and WAGNER, R.H. Antihemophilic factor: separation of an active fragment following dissociation by salts or detergents. Thromb. Diathes. Haemorrh. 27, 502, 1972. 11. WEISS, H.J., PHILLIPS, L.L. and ROSMER, W. Separation of subunits of antihemophilic factor (AHF) by agarose gel chromatography. Thromb.Diathes. Haemorrh. 27, 212, 1972. 12. WEISS, H.J. and HOYER, L.W. Dissociation of antihemophilic factor procoagulant activity from the Von Willebrand factor. Science 132, 1149, 1973 13. RICK, M.E. and HOYER, L.W. Immunologic studies of antihemophilic factor (AHF ; factor VIII), V. Immunologic properties of AHF subunits produced by salt dissociation. Blood 42, 737, 1973. 14. RICK, M.E. and HOYER, L.W. Molecular weight of human factor VIII procoagulant activity. Thrombosis Research 7, 909, 1975. 15. MOURIK, J.A. van, BOUMA, B.N., BRUYERE, W.T.la, GRAPF, S. de and MOCHTAR, a series of homologous oligomers and a complex of two I.A. Factor VIII, proteins. Thrombosis Research 4, 155, 1974. 16. BOUMA, B.N., MOURIK, J.A. van, GRAAF, S. de, HORDIJK-HOS, J.M. and SIXMA, J.J. Immunologic studies on human factor VIII (antihemophilic factor A; AHF) components produced by low ionic strength dialysis. Blood 47, 253, 1976. 17. MARCHESI, S.L., SHULMAN, N.R. and GRALNICK, H.R. Studies on the purification and characterization of human factor VIII. J. Clin Invest. 51, 2151, 1972. 18. SHAPIRO, G.A., ANDERSEN, J.C., PIZZO, S.V. and MCKEE, P.A. The subunit J. Clin Invest. 52, 2193, structure of normal and hemophilic factor VIII. 1973. 19. VEHAR, G.A. and DAVIE, E.W. Formation of a serine enzyme in the presence of bovine factor VIII (antihemophilic factor)and thrombin. Science 197, 374, 1977. 20. HERSHGOLD, E.J. Properties of factor VIII (antihemophilic factor). In: Progress in Hemostasis and Thrombosis, Vol. 2, T.H. Spaet (Ed.), New York, Grune and Stratton, 1974, p 99. 21. SWITZER, M.E. and MCKEE, P.A. Studies on human antihemophilic factor. Evidence for a covalently linked subunit structure. J.Clin. Invest. 57, 925, 1976. 22. ALBERTY, R.A. A quantitative study of reversible boundary spreading in the electrophoresis of proteins. J. Amer. Chem. Sot. 70, 1675, 1948. 23. ALBERTY, R.A. Electrochemical properties. In: The Proteins, Vol 1, H. Neurath and K. Bailey (Ed.), New York, Academic Press, 1953, p. 537. 24. VELTKAMP, J.J.. DRION, E.F. and LOELIGER, E.A. Detectionofthe carrier state in hereditary coagulation disorders. Thromb. Diathes. tiaemorrh 19, 279, 1968. 25. WEISS, H.J., HOYER, L.W., RICKLES, R.F., VARMA, A. and ROGERS, J. Quantitative assay of a plasma factor deficient in Von Willebrand's disease that is necessary for platelet aggregation. J. Clin. Invest. 52,
24
POLYDISPERSITY
OF FVIII
-
vWF
Vol.
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2708, 1973. 26. GERRITS, W.B.J., FLIER, 0.Th.N. and MEER, J. van der.Fibrinopeptide A immunoreactivity in human plasma. Thrombosis Research 5, 197, 1974. 27. LOWRY, O.,H., ROSENBOURGH, N.J., FARR, A.L. and RANDALL, R.J. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265, 1951 28. HJERTEN, 5. Molecular-sieve electrophoresis in cross-linked polyacrylamide gels. J. Chromatogr. 11, 66, 1963. 29. RODBARD, D. and CHRAMBAC , A. Unified theory for gel electrophoresis and gel filtration. Proc. Nat. Acad. Sci. USA 65, 970, 1970. 30. BOUMA, B.N., MOURIK, J.A. van, WIEGERINCK, Y., SIXMA, J.J. and MOCHTAR, I.A. Immunological characterization of anti-haemophilic factor A related antigen in haemophili A. Stand. J. Haematol. 11, 184, 1973. 31. ZIMMERMAN, T.S., ROBERTS, J. and EDGINGTON, T.S. Factor VIII related anti. gen: multiple molecular forms in human plasma. Proc. Natl. Acad. Sci. USA 72, 5121, 1975. 32. MAISONNEUVE, P. and SULTAN, Y. Modification of factor VIII-complex properties in patients with liver disease. J. Clin. Pathol. 30, 221, 1977. 33. WEISS, H.J., SUSSMAN, 1.1. and HOYER, L.W. Stabilization of factor VIII in plasma by the Von Willebrand factor. J. Clin. Invest. 60, 390, 1977.
