Jun 13, 1986 - human plasmas were from George King Biomedical; factor. VI11 ..... Fulcher, C. A., Gardiner, J. E., Griffin, J. H., and Zimmerman,. 12. Lollar, P.
Vol . 262, No. 7, Issue of March 5, pp. 3285-3290,1987 Printed Ln U.S.A.
CHEMISTRV T H E .JOURNAL OF BIOLOGICAL M 1987 by The American Society of Biological Chemists, Inc
Characterizationof Recombinant Human FactorVIII* (Received for publication, June 13, 1986)
Dan L. Eaton, Philip E. Hass, LavoneRiddle$, Jennie Matherg, Mike Wiebes, Timothy Gregory$, and Gordon A. Vehar From the Departments of Molecular Biology, $Process Development, and §Research and Deuelopment, Genentech, Znc. South San Francisco, California94080
Recently, complete human factor VI11 DNA clones This single-chain formis readily proteolyzed both i n vivo and have been obtained and subsequently expressed in babyi n uitro. As isolated from plasmaor commercial concentrates, hamster kidney cells (Wood, W. I., Capon, D. J., Si- human factor VI11 consists of multiple polypeptides with M , monsen, c. c., Eaton, D. L., Gitschier, J., Keyt, B., -80,000-210,000 (6,8,9). Analysis of these proteinsby amino Seeburg, P. H., Smith, D. H., Hollingshead, P., Wion, acid sequencing shows that theM, 210,000 and 80,000 proteins K. L., Delwart, E., Tuddenham, E. G. D., Vehar, G. A., represent the amino and carboxyl-terminal regions of factor 330-337). The VIII,respectively (2, 9, 13). Further proteolytic processing and Lawn, R. M. (1984)Nature 312, recombinant factor VI11 (rVIII) protein secreted from within the carboxyl-terminal segmentof the M , 210,000 prothese cells has now been purified allowing its structein yields a series of proteins of M, 90,000-188,000 (9). Each tural analysis andcomparison to plasma-derived factor of the M , 90,000-210,000 proteins hasbeen suggested to form VI11 (pdVIII). Analysis of purified rVIII by sodium a complex with the M , 80,000 protein mediated by a metal dodecyl sulfate-polyacrylamide gel electrophoresis shows that it consists of multiple polypeptides with ion, perhaps calcium (2, 5, 9). The availabilityof the cDNAfor human factorVI11 allowed from 80,000relative mobilities (M,) ranging 210,000.The same patternof polypeptides is also ob- the construction of plasmids which would direct the expresserved for pdVIII resolved bysodium dodecyl sulfate- sion of factor VI11 protein in transfected mammaliancells (2, 3, 15). Factor VI11 secreted from these cells exhibits many of polyacrylamide gel electrophoresis. The proteins asthe functional characteristics associated with plasma-derived sociated withrVIII are recognized by pdVIII antibodies in a Westernblot. When rVIII and pdVIII are subjected factor VI11 (pdVIII)’ (2, 3). The development of mammalian to isoelectric focusing they are resolved into a similar cell lines producing recombinant factor VIII (rVIII) has perpattern of protein bands. Thrombin, factor Xa, and mitted the production of highly purified factor VIII. Use of of hemoactivated protein C, which modulate factor VI11 activ- such preparations as therapeutics in the treatment ity by proteolysis, process rVIII in the same manner philia offers a major advantage over current factorVI11 prepthey do pdVIII. As is the case for pdVIII, thrombin arations that are used for the treatment of hemophilia. These activation of rVIII coagulant activity correlates with latter preparations are derived from human plasma, highly the generation of subunits with M , of 73,000,50,000 impure, and inevitably contaminated with virus particles. and 43,000.These subunits appear to form a metalIn the present study we report the purification and char(perhaps Ca2+) linked complex. EDTA inactivates acterization of rVIIIobtainedfrom a mammlian cell line thrombin-activated rVIII and pdVIII, with the activity transfected with a factor VI11 expression vector (3). Results being regenerated after the addition of a molar excess presented in this paper show that rVIII is functionally and of MnC12. The resultssuggest that rVIII is structurally structurally similar to pdVIII. and functionally very similar topdVIII. EXPERIMENTALPROCEDURES ~
Factor Xa, thrombin, and activated protein C were all generous gifts of Dr. Walter Kisiel (The University of New Mexico). Factor Factor VI11 (antihemophilia factor) functions in the middle VI11 concentrates were gifts from Cutter Laboratories. Rabbit brain of the coagulation cascade, acting as a cofactor for factor X cephalin and phenylmethanesulfonyl fluoride were from Sigma; plaactivation by factor IXa in the presence of calcium ions and telin was from General Diagnostics; factor VIII-deficient and normal VI11 phospholipid (1). Because of the low levels of factor VI11 in humanplasmaswerefromGeorgeKingBiomedical;factor plasma (200 ng/ml), its apparent instability, and its associa- chromagenicCoatestassay was fromHelena;Dubecco’smodified Eagle’s Medium and Ham’s F-12medium were obtained from Gibco. tionwith von WillebrandFactor,attemptstopurifyand Purification ofpdVZZZ and rVZZZ-Plasma-derived factor VI11 was characterize the factor VI11 protein were hindered for many purifiedfromcommerciallyavailablefactor VI11 concentrates (9). years. However, the cloning of factor VI11 cDNA (2, 4, 15) The concentrates were diafiltered into 0.02 M Tris buffer, pH 7.4, and the purification and characterization of factor VI11 pro- containing 135 mM NaCI, 5 mM sodium citrate, 1 mM CaC12, 5% tein from several species (5-9) has now provided a detailed glycerol, and 1 mM phenylmethanesulfonyl fluoride. Subsequently,p mercaptoethanolwas addedto solubilized concentrates to 35 mM, and understanding of the structure of factor VIII. factor VI11was purified using DEAE-Sepharose and a factor VI11 The amino acid sequence deduced from human factor VI11 immunoaffinity column as describedpreviously (9). Recombinant cDNA clones predicts a mature single-chain protein (2,332 factor VI11 was purified from serum-free media (Dulbecco’s modified amino acids) that, after accounting for25 potential N-linked Eagle’s medium/F-12, 1:l)conditioned by baby hamster kidney cells glycosylation sites, would have a M , of -300,000 (2, 3, 15). transfected with a factor VI11 expression plasmid (3) for 48 h. The __ media werecollected and diluted with distilled H20 to reduce the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby The abbreviations used are: pdVIII, plasma-derived factor VIII; marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 rVIII, recombinantfactor VIII; SDS-PAGE, sodium dodecylsulfatesolely to indicatethis fact. polyacrylamide gel electrophoresis;APC, activated proteinC. ”
3285
3286
Factor
VIII
Recombinant
conductivity below 10 mmho. Purification of rVIII was accomplished using the.same chromatographic steps used to isolate pdVIII (9). We found that the recoveries across these columns for rVIII and pdVIII were very similar. Purified rVIII and pdVIII were stored at -80 "C in 0.05 M Tris buffer, pH 7.5, containing 150 mM NaCl, 2.5 mM CaCl,, 1 mM phenylmethanesulfonyl fluoride, and 5% glycerol. Factor VI11 activity was measured by coagulation analysis or by the factor VI11 Coatest assay as described previously (3). Protein concentrationwas determined by the method of Bradford (17). Isolation of Factor VIII Subunits-Separation of the M , 80,000 protein from the M , 90,000-210,000 proteins was accomplished by absorbing rVIII or pdVIII in 0.05 M Tris buffer, pH 7.5, 150 mM NaC1,2.5 mM CaC12, and 5% glycerol to a factor VI11 monoclonal antibody column that binds the M,80,000 protein (3, 9). The M, 90,000-210,000 proteins were eluted with the above buffer containing 50 mM EDTA. The M,80,000 protein was then eluted underthe same conditions used to elute intact factor VI11 (9). To obtain the M,73,000 subunit, the M,80,000 protein was treated with thrombin (1:50 ratio, w/w) for 1 h a t 37 "C a t which time hirudin was added to inhibit thrombin. Cleavage and NH2-terminal Sequence Analysis of rVIII-For NH2terminal amino acid sequencing, rVIII (0.2-0.5 mg) was incubated with either thrombin (1:lOO ratio, w/w), factor Xa (1:lOO ratio), or activated protein C (APC)(1:25 ratio) for 30-60 min a t 37 "C. In the case of factor Xa and APC, l/lOth sample volume of rabbit brain cephalin was included as a source of phospholipid. The reactions were terminated by adding SDS to afinalconcentration of 0.4% and immediately heating samples to 80 "C. Proteolyzed rVIII was resolved on a 6-12% polyacrylamide gradient gel in the presence of SDS (SDSPAGE). Electrophoresis was carried out by the method of Laemmli (18).Proteins were detected by staining with Coomassie Blue, excised, and electroeluted according to the method of Hunkapiller et al. (19). Peptides eluted from gels weresubjected to NH2-terminal aminoacid sequence analysis using an Applied Biosystems gas-phase sequenator (20) modified for on-line phenylthiohydantoin identification (21). Changes in subunit structure andcoagulant activity of rVIII during proteolysis by thrombin,factorXa, or APCwere determined as described previously (9). Briefly, rVIII (2.5 pg) was incubated with either thrombin (0.05 pg), factor Xa (0.05 pg), or APC (0.1 pg) over a 1.5- to 2-h time course. Rabbit brain cephalin was added to factor Xa andAPC reactions. At various time pointsduring the incubation, coagulant activity was determined and subunit structure was analyzed by SDS-PAGE. Proteins were visualized by silver staining (22).
Isoelectric Focusing of Factor VIII-Isoelectric focusing gels were carried out according to the method of O'Farrell (16) using 0.4-mm ultrathin horizontal gels in place of tube gels. The gels contained 2.5% pH 3.5-10.0 carrier Ampholines. Proteins to be focused were diafiltered into 20 mM Tris buffer, pH 7.5, containing 4 M urea, 2.5 mM CaC12,and 0.01% Tween 80; electrophoresis was carried out a t 8 watts (1000 V limit) a t 18 "C for 7 h. RESULTS
Structure of Purified rVIII-As in the case with pdVIII, analysis of purified rVIII by SDS-PAGE shows that it consists of several polypeptide chains withM , 80,000-210,000 (Fig. 1). The patternof polypeptides observed forrVIII isvery similar to thatof pdVIII. Analysis by Western blotshows that all the bands associated withrVIIIaredetected by a polyclonal antibody derived against pdVIII (Fig. 1).That the patternof polypeptides for rVIII and pdVIII are strikingly similarsuggests that rVIII and pdVIII are processed in the same manner. This indicates that the initial cleavage sites within thesinglechain form of factor VI11 are as accessible to proteolysis in rVIII as they arepdVIII. in It is notknown a t present whether thisinitial proteolysis isanintracellularor extracellular event, oroccurs during thepurification of rVIII. Proteolysis of rVIII by Thrombin, Factor Xu,and Activated Protein C-Factor VI11 coagulant activityis altered by thrombin, factor Xa, and activated protein C (7-12). These proteases, through specific cleavages, process factor VI11 into active and inactive forms (9). The proteins generated after cleavage of rVIII by thrombin, factor Xa, orAPC comigrated on SDS-PAGE with thosederived from pdVIII (Fig. 2). Furthermore, these proteins were found to have the same NH2terminal sequence as their counterparts derived from pdVIII (Fig. 3). This indicates that the cleavage sites (see "Discussion") of thrombin, factor Xa, and APC in rVIII andpdVIII are the same. Activation and Inactivation of rVIII Coagulant ActivityCorrelatingchangesinfactor VI11 subunitstructure with
.*.-. THROMBIN "-
MW x
C
D
Xa
APC
F-"
I
MW x
73-
67-
43 -
45-
50
PD
R
45-
PD
I
R
FIG. 1. SDS-PAGE andwestern blotting of rVIII and pdVIII.Approximately 15 pg of rVIII ( B and C ) or pdVIII ( A and D)were resolved on a 6-12% SDS-polyacrylamide gel. The proteins were either visualized by staining with Coomassie Blue ( A and B ) or transferred to nitrocellulose (C and D) for western blotting. For westerns a polyclonal antibody derived against pdVIII was used to detect factorVI11 bound to nitrocellulose. FIG. 2. Cleavage of rVIII and pdVIII by thrombin, factor Xa, and APC. Recombinant VI11 or pdVIII (-15 pg) were incubated with either thrombin (0.3 pg), factor Xa (0.3 pg), or APC (0.6 pg) for 1 h a t 37 "C. Rabbit brain cephalin (l/lOth volume) was included in factor Xa and APC reactions. The reactions were stopped by the addition of SDS to 0.5% and heating samples to 90"C for 5 min. Proteins wereresolved on a 6-12% SDSpolyacrylamide gel and visualized by staining with Coomassie Blue.
PD
R
r
Recombinant Factor VIII Thrombin Polypeptide
73,000
rvln pdVlll
67,000
rVll1 pdVlll
50.000
rvm pdVlll
APC
Xn
(SIFQKKTRHYFIAAV (SIFQKKTRHYFIAAVDERL
(SFQKKTRHYFIAAV T6ZKKTRHYRAAV
llpo
AQSGWFQFKK ?.SGSVFQFKK ATRRWLGAVEL A’TRRWLGAVELS
ATRRYYLGAVELSOD ATRRYYLGAVEL-W -yM
A?RRYYLGAVE ATRRYYLGAV 43.000
rvln pdVlll
ATRRYYLGAVEL ATRRYYLGAVEL I
SVAKKHPKT SVAKKHPK
(SIVAKKHPKTWV (SIVAKK--K- WVI ill
,n
~~
FIG.3. NHz-terminal sequence of rVIII proteins and their comparison to pdVIII proteins. Recombinant factor VI11 was treated with either thrombin, factor Xa, or APC and subsequently resolved by SDS-PAGE. Proteins to be sequenced were electroeluted as described under “Experimental Procedures.” T h e position of these proteins within factor VI11 were determined by alignment of the obtained sequence to the cDNA sequence of factor VI11 (2, 3). Sequence shown for DdVIII proteins are taken from Eaton et al. (9). r
210-
9080-
I
.
3287
210,000 proteins and theappearance of an M , 45,000 protein (Fig. 6). The M , 45,000 protein is derived from the NHzterminal portion of the M , 90,000-120,000 proteins (Ref. 9 and Fig. 3). The COOH-terminal portions of the M , 90,000210,000 proteins that would arise after cleavage by APC are not apparent by SDS-PAGE (Fig. 6), which has also been observed for pdVIII (9, 11). Factor Xa cleaves factor VI11 at thesame sites as thrombin or APC (Figs. 2 and 3; Ref. 9). Factor Xa has been observed to initially activate and thenultimately inactivate pdVIII (9). Incubation of rVIII with factor Xa over a 90-min time course resulted initially in a&fold activation followed by a decrease in coagulant activity to below control levels (Fig. 7). Analysis of rVIII subunit structureduring this time course shows that activation is best correlated with the appearance of the Mr 73,000, 50,000, and 43,000 subunits, while inactivation coincides with the degradation of the Mr 73,000 and 50,000 subunits to fragments of M , 67,000 and 45,000 (Fig. 7, inset). Similar results have been obtained in our laboratory with pdVIII (9). Subunit Dissociation and Reassociation-It has been suggested that each of the M , 90,000-210,000 proteins form a calcium-linked complex with the M , 80,000 protein of factor VI11 (5, 9). When rVIII or pdVIII are adsorbed to a factor VI11 monoclonal antibody column, which is specific for the Mr 80,000 protein (9, 13), the M , 90,000-210,000 proteins can beeluted with EDTA (Fig. 8). The M , 80,000 protein is subsequently eluted under conditions normally used to elute factor VI11 (9). Thissuggests that pdVIII and rVIII each exist as a metal-linked complex. Neither the isolated M , 80,000 protein nor the M , 90,000-210,000 proteins exhibited coagulant activity. It is likely that the M , 73,000, 50,000, and 43,000 subunits of thrombin-activated factor VI11 (VIIIa) also form a metallinked complex. Treatment of either pdVIIIa or rVIIIa with EDTA results in inactivation (Fig. 8). Coagulant activity can be subsequently regenerated by quenching the EDTA with excess MnClZ(Fig. 9). Activity was also regenerated, but to a lesser extent, by the addition of excess CaClZ.Incubation of EDTA-inactivated factor VIIIa with 50 mM MnC12 for 1 h resulted in restorationof -80% of the initial activity, whereas only -30% of the activity was regenerated when 50 mM CaClz was used. This is similar to what hasbeen previously observed for factor Va by Esmon (25). When factor VIIIa is subjected to gel permeation chromatography all three subunits comigrate, while they do not if factor VIIIa is pretreated with EDTA (data not shown). These results suggest that EDTA inactivates both pdVIIIa and rVIIIa by causing subunit dissociation, supporting the notion that factor VIIIa exists as a metal- (perhaps Ca2+)linked complex. Isoelectric Focusing of r VIII and pdVIII-When rVIII and pdVIII were subjected to isoelectric focusing only the MI 80,000 protein was resolved (Fig. 9). The M , 90,000-210,000 proteins of either rVIII or pdVIII could not be focused, even under denaturingconditions, using several isoelectric focusing gel systems (Fig. 10). The isolated Mr 80,000 protein of rVIII or pdVIII resolved into a clusterof four bands with isoelectric points of 6.9-7.2 and a separate bandwith an isoelectric point of 6.5 (Fig. 9). These bands corresponded to thefocused bands of intact rVIII and pdVIII. The M , 73,000 subunit of both rVIIIa and pdVIIIa resolved into a single broad band with an isoelectric point of 8.1 (Fig. 10). A t this time we do not have sufficient quantities of the MI 50,000 and 43,000 subunits of activated factor VI11 to perform isoelectric focusing.
2oo c 0 ,2
x)
40
60
5
10
15 20 3” 45 6 0 9 0 l 2 0
80
100
IM
IncubationTime (minutes) FIG.4. Thrombin activation of rVIII. Recombinant factor VI11 (2.5 pg) was incubated with (U or ) without (o”--o) thrombin (0.05 pg) for 0-120 min at 37 “C at the time points shown. Both coagulant activity and subunit structurewere determined as described under “Experimental Procedures.”
activation by thrombin has been the subject of several recent reports (6-9,12, 14). All of these reports show that thrombin activation is correlated with the proteolytic processing of the M , 80,000-210,000 proteins to lower MI subunits. We have observed that thrombin activation of pdVIII correlates with the generation of M , 73,000, 50,000, and 43,000 subunits (9). Similarly, a time course treatment of rVIII with thrombin resulted in approximately an 80-fold increase in rVIIIcoagulant activity (Fig. 4). Analysis of the changes insubunit structure of rVIII by SDS-PAGE and gel scanning shows that this activation coincided with the processing of the M , 80,000210,000 precursor species to theM , 73,000,50,000, and 43,000 active subunits (Figs. 4 and 5). As determined by aminoterminal sequencing, COOH-terminal proteolysis of the Mr 210,000 proteingenerates the Mr 90,000 protein which is subsequently cleaved at arginine 372 to yield the M , 50,000 and 43,000 proteins (Fig.3,Refs.2, 9, 13). Concomitantly, the M , 80,000 protein is cleaved at arginine 1689 to yield the M , 73,000 protein (Fig. 3). Thrombin-activatedrVIII remained stable for at least 1 h a t 37 “C after reaching the apparent maximum activity (Fig. 4). This stability has also been observed for thrombin-activated pdVIII (9). The kinetics of inactivation of rVIII by APC is shown in Fig. 6. Similar to pdVIII (9, 11) the inactivation of rVIII by APC is correlated with the proteolysis of the Mr 90,000-
3288
Factor
VIII
Recombinant
-
I . 80.
FIG.5. Quantitation of changes in .C rVIII subunit structure during actiea. vation. The various protein species & present a t each time point in the gel of 3 Fig. 4 were quantitated by scanning each 5 ' lane using anLKBlaserdensitometer $ gel scanner. P. I ..
0
o
. . . . m. . ea. . l. o .o .m
P
o
u)
zo
u)
ea
1m1a
04 ! 0
. P. . 40. . ea. . ea. . l.o o.1 2 l
r
Incubation time
c -80 c "45
9080-
1
I
x)
I
I
I
I
40
1
60
I
80
I
I 100
FIG.8. Separation of factor VI11 protein chains. Either pdVIII or rVIII were adsorbed to a factor VI11 monoclonal antibody FIG.6. Inactivation of rVIII by APC. rVIII (2.5 pg) was incu- column and the M,90,000-210,000 and M,80,000 proteins were eluted bated with APC (100 ng) and l/lOth volume of rabbit brain cephalin from the column as described under "Experimental Procedures." The for 0-90 min a t 37 "C. A t the time points indicated, coagulant activityisolated subunits were resolved on a ti-12% SDS-polyacrylamide gel, and proteins were visualized by silver staining (18). and subunit structure were determined as described under "Experimental Procedures." Incubation Time (minutes)
r .
" " "
- 1400 600
200 o
l
x)
40
60
80
I00
lncubat ion Time (minutes 1 FIG.7. Activation of rVIII by factor Xa. rVIII (2.5 pg) was or) without (W) factor Xa (50 ng) and incubated with (M l/lOth volume of rabbit brain cephalin for 0-120 min a t 37 "C. At the times indicated subunit structure and coagulant activity were determined as described under "Experimental Procedures." DISCUSSION
The recent cloning and protein characterization studiesof factor VI11 have elucidated the structure of factor VI11 (2, 3, 5-14). The DNA sequence of factor VI11 predicts a singlechain glycoprotein with an approximate M,of 300,000. The single-chain form is readily proteolyzed in uiuo (23) and in vitro (8) to yield a form consisting of multiple polypeptides with M,ranging from 80,000-210,000 (6,8,9). TheM,210,000 and 80,000 proteins represent the NH2-terminal andCOOHterminal regions of factor VIII, respectively (2, 13). Proteolysiswithin theCOOH-terminal region of the M , 210,000 protein generates a series of proteins with M, ranging from
pdVllla
I Control
EDTA EDTA MnC12
r
RVllla
EDTA EDTA MnC12
FIG.9. Regeneration of factor VIIIa coagulant activity after E D T A treatment. Both pdVIII and rVIII in 0.05 M Tris, pH 7.5, 0.15 M NaC1, 2.5 mM CaCI,, and 5% glycerol were activated by incubation with thrombin (1:50 ratio) for 1 h a t 37 "C a t which time hirudin was added. T o pdVIIIa or rVIIIa either no addition was made (control) or EDTA wasadded to 20 mM. The samples were then incubated for 1 h a t 3 7 ° C a t which timecoagulant activitywas determined. To the EDTA-treated samples, MnCI, was added to 50 mM and incubated an additional 1 h a t 37 "C. Subsequently, coagulant activity was determined. 110,000 to 180,000 (9). As discussed below the M, 80,000 protein forms a metal-linked complex with each of the M, 90,000-210,000 proteins (Fig. 11).Both the single-chain form and the above mentioned multiple polypeptide form of factor VI11 have been suggested to be inactive or less active precursors that can be activated by factorXa or thrombin and inactivated by APC through specific proteolytic processing (8-12, 14). The proteolysis of factor VI11 by thrombin, factor Xa, and APC is summarized in Fig. 11.
Factor
Recombinant
VIII
3289
Analysis of purified rVIIIshows that, like pdVIII, it consists of multiple polypeptides. By SDS-PAGE the patternof polypeptides observed for rVIII is strikingly similar to that of pdVIII (Fig. 1).Both are resolved into seven or eight distinct }Thrombin bands with M , ranging from 80,000 to 210,000 (Fig. 1).That the M , 210,000 proteins of rVIII and pdVIII comigrate on SDS-PAGE suggests that these two proteins may be glycosylated to a similar extent. The M , 210,000 protein contains 23 potential N-linked glycosylation sites (2, 3, 13). It is also interesting that the M , 80,000 proteins of either rVIIIor pdVIII resolve into a doublet on SDS-PAGE (Fig. 1).These observations suggest that the DNA-predictedsingle-chain form of rVIII and pdVIII are post-translationally processed in a similar manner toyield the multiple polypeptide form. Treatment of rVIII with thrombin, factor Xa, and APC shows that these proteasesprocess rVIII in the same manner they do pdVIII(Figs. 2 and 3).Activation of rVIII by thrombin or factor Xa correlatedwith the generation of M , 73,000, x FIG. 10. Isoelectric focusing of pdVIII and rVIII. pdVIII, 50,000, and 43,000 subunits (Figs. 4, 5, and 7) while inactirVIII, and the various isolated subunits were diafiltered into 20 m M vation of rVIIIa by factor Xa coincided with the degradation Tris, pH 7.2, 2.5 mM CaC12,0.01% Tween 80, and 4 M urea. Approx- of the M , 73,000 and 50,000 subunits (Fig. 7). Inactivation of imately 10 pg ofeach were resolved on pH3.5-10.0 gradient isoelectric rVIII by APC resulted in the proteolysis of the M , 90,000focusing gels as described under “Experimental Procedures.” Proteins 210,000 proteins (Fig. 6). Theseresults clearly show that rVIII were visualized by silver staining (22). is proteolytically processed by thrombin, factor Xa, andAPC to activated and inactivated forms in the same manner as Recently the cleavage sites of thrombin, factor Xa, and APC within factor VI11 have been identified (2,9,13). Throm- pdVIII. Initial results of Fass et al. ( 5 ) , using porcine factor VIII, bin and factor Xa initially cleave the M , 110,000-210,000 proteins at position 740 to yield the M , 90,000 protein (2, 9), suggested that, like factor V (24, 25), factor VI11 may exist as which is subsequently cleaved at position 372 to generate the a Caz+-linked complex. Similarly, results presented here inM , 50,000 and 43,000 subunits of factor VIIIa (Fig. 11, Ref. dicate that bothrVIII and pdVIII exist as metal-linked com9). Concomitantly,the M , 80,000 protein is cleaved at position plexes in that treatment of factor VI11 or VIIIa with EDTA 1689 to generate the M , 73,000 subunit of factor VIIIa (Fig. causes separation of the various protein chains. We found 11, Ref. 9). Further proteolysis of the M , 73,000 and 50,000 that when separated the M , 80,000 and M , 90,000-210,000 subunits by factor Xa generates fragmentsof M , 67,000 and proteins of factor VI11 and the individual subunits of factor 45,000 (Fig. 11, Ref. 9). One or both of these latter cleavages VIIIa did not exhibit coagulant activity even in the presence correlate with inactivation of factor VIIIa (9). APC hasbeen of excess MnClz or CaC12 (data not shown). However, the proposed to cleave the M , 90,000-210,000 proteins of factor coagulant activity of EDTA-treated rVIIIa or pdVIIIa could VI11 at position 336 to generate a M , 45,000 protein (Fig. 11, be restored by the addition of a molar excess of MnClZ (Fig. Ref. 9). This appears tobe the same site atwhich factor Xa 9). Since EDTA appears to dissociate factor VIIIa subunits, cleaves the M , 50,000 subunit of factor VIIIa (Fig. 10, Ref. 9). these results suggest that the subunits of factor VIIIa must It has been proposed that cleavage at position 336 by either be in a complex to exhibit coagulant activity. A t this time it factor Xa or APC correlates with inactivation (9). Cleavage is not known if all three subunits are required for activity or at position 372 bythrombin orfactor Xa is in partresponsible just two. For a more detailed comparison of rVIII and pdVIII both for activation (9).This suggests that the region between positions 336-372, which contains 15 aspartic and glutamic were analyzed by isoelectric focusing. When rVIII or pdVIII acid residues and only4lysineor arginine residues, is of were subjected to isoelectric focusing only the M , 80,000 functional importance. protein was resolved. The isolated M , 80,000 protein of either
1
1
740
90,000
N 1649
I
I*
ca
80,000
32 C
+
80,000 FIG. 11. Proteolytic processing of human factor VI11 by thrombin, factor Xa, and APC.
degradation peptides
3290
Factor
Recombinant
rVIII or pdVIII was resolved into a cluster of four distinct bands with isoelectric points between 6.9-7.2 and a separate band with an isoelectric point of 6.5 (Fig. 10). The M , 90,000210,000 proteins of either rVIII or pdVIII were not resolved by a numberof isoelectric-focusing gel systems. This probably reflects the heterogeneity of these proteins as well as the potentially high content of carbohydrate associated withthese proteins (13). The M , 73,000 subunit of either rVIIIaor pdVIIIa was resolved into a single broad band with an isoelectric point of 8.1 (Fig. 9). Cleavage of the M , 80,000 protein by thrombin to generate the M , 73,000 subunit, results in the possible removal of a 44-residue polypeptide that contains 15 asparticand glutamic acid residues and only 4 lysine or arginine residues (9, 13).Hence, the M , 73,000 subunit has a more basic isoelectric point than that of the Mr 80,000 protein from which it is derived. These results indicate that at least the charge density of the M , 80,000 and 73,000 proteins of rVIII and pdVIII are similar. The expression and secretion of recombinant factor VI11 from mammalian cells has enabled the purification of factor VI11 from a plasma-free system. This is in marked contrast to initial purifications of pdVIII accomplished only a few years ago that were laborious and required a large quantity of plasma (5-8). The initial characterization studies done here suggest that rVII1 is both structurally andfunctionally similar to pdVIII. Recentpharmacological data also suggest that, based on a number of pharmacological and physiological parameters, rVIII and pdVIII function similarly in vivo (26). The availability of highly purified factor VI11 will allow for both the development of a safe pharmaceutical for treating hemophilia and pure protein for characterization studies. This latter capability will hopefully better our understanding of the factor VI11 molecule. Acknowledgments-We thank RichardLazar of Genentech and Cutter Laboratories for supplying baby hamster kidney cell-conditioned media containing rVIII. We also thank Henry Rodriguez for amino acid sequencing of factor VI11 and Dr. Robert Hershberg for critically reviewing the manuscript. REFERENCES 1. Jackson, C.M., and Nemerson, Y. (1980) Annu. Reu. Biochem. 49,767-811 2. Toole, J. J., Knopf, J. L., Wozney, J. M., Sultzman, L. A., Buecker, J. L., Pittman, D.D., Kaufman, R. J., Brown, E.,
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