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Isolation of the lipoprotein associated coagulation inhibitor produced by HepG2 (human hepatoma) cells using bovine factor Xa affinity chromatography
THROMBOSIS RESEARCH 48; 253-259, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright
(c) 1987 Pergamon Journals
Ltd.
All rights reserved.
BRIEF COMMUNICATION _---
ISOLATION OF THE LIPOPROTEIN ASSOCIATED COAGULAlION INHIBITOR PRODUCED BY HepGZ (HUMAN HEPATOMA) CELLS USING BOVINE FAClOR Xa AFFINIlY CHROMAlOGRAPHY
Broze, Jr.*, Louise A. Warren*, James J. Girard*, and Joseph P. Miletlch** *Dlvlsion of Hematology/Oncology and **Laboratory Medicine Washington University School of Medicine Jewish Hospital, St. Louis, Missouri
George J.
USA
(Received 28.7.1987; Accepted in revised form 17.8 1987 by Editor C.T. Esmon) INTRODUCTION
Human
plasma
contains a 1lpOprOtein associated coagulation inhibitor the presence of activated factor X (Xa), LACI(P) rapidly inhibits the enzymatic activity of the factor VIIa-Tissue Factor (VIIa/TF) complex which is responslble for the initiation of coagulation through the classical extrlnslc pathway (l-5). An inhibitor with the same characteristics as LACJ(P) In plasma has recently been isolated from HepG2 cell (human hepatoma cell line) serum free conditioned media, and one step used in the purification procedure was affinity chromatography upon DIP-Xa-agarose (6). Although a critical step In the ultimate Isolation of the LACI produced by HepG2 cells [LACI(HG2)], the capacity of the DIP-Xa-agarose column was so low as to suggest that trace quantities of active Xa remaining on the matrix could Subsequent studles have have been responsible for the blndlng of LACI(HG2). confirmed this by showing that purified LACI(HG2) binds to Xa at or near Xa's active site and does not Interact wlth Xa which has been Inactivated with dilsopropylfluorophosphate (DFP) or dansyl-glutamyl-glycyl-arglnlne chloromethyl ketone (dansyl-GGACK) (7,8). Here a slmpllfled, three step purification procedure for LACI(HG2) Is presented utlllzlng bovine Xa-agarose affinity chromatography.
[LACI(P)I (1.2).
Key Words:
In
coagulation
inhibitor, Tissue Factor, Factor Xa
253
LACI(HG2) PURIFICATION
254
Vol. 48, No. 2
MATERIALS AND METHODS ~_______ 1251-iodide, carrier Sodium free, was purchased from New England Nuclear (Boston, MA) and Iodo-Gen was obtained from Pierce Chemical Co. (Rockford, IL). Dansyl-L-glutamyl-L-glycyl-L-arginine chloromethyl ketone (dansyl-GGACK) was from Calbiochem (La Jolla, CA). Affi-Gel 15 and low molecular weight for polyarylamide standards electrophoresis were gel purchased from Bio-Rad (Richmond, CA). Aprotinin, bovine serum albumin, acrylamide, methylenebis(acrylamide), rabbit brain cephalin, MOPS, and lrizma base were from Sigma (St. Louis, MO). All other chemicals were of reagent grade or better and came from Fisher or from Sigma. Factor X deficient human plasma was obtained from George King Biomedical (Overland Park, KS). Serum samples from healthy blood donors were provided by the American Red Cross (St. Louis, MO).
Cell Culture. described (6).
HepG2
cells
were
cultured
in serum-free
media
as
previously
Proteins. A crude preparation of 1F was prepared and washed extensively with EOTA (1). The X coagulant protein from Russell's viper venom (9). factor VIIa (1). and human factor X (10) were purified as described. Bovine factor X was isolated from the barium sulfate eluate (Sigma) from bovine plasma using DEAEagarose anion exchange chromatography (11). Bovine factor Xa was produced from purified factor X by incubation with insolubilized X coagulant protein (11). and linked to Affi-Gel 15 at a final concentration of 1 mg/ml of packed gel using the manufacturer's instructions (MOPS buffer, pH 7.5).
s-g*
In the first A three stage assay for TF inhibition (TFI) was used. factor VIIa (1 mg/ml). 10 111 of bovine factor Xa rll of (1 ys;ml;P 10 ~1 of CaCl, (40 mM), 10 ,J of TBSA (0.1 M NaCl, 0.05 M 50 ~,IL sample to iris-HCl, pH 7.5, with 1 mg/ml bovine serum albumin), tested, and 10 PL of crude, EDTA-washed TF (10% v/v) were incubated at room temperature. After 30 minutes, a 10 pL sample was diluted lOO-fold into Fifty PL of this diluted sample, 50 PL of VIIa TBSA with 5 mM CaCl,. (1 pg/ml), 50 ~JL of CaCl, (25 mM), and 50 PL of human factor X (10 ug/ml) were then incubated at 37°C. After 1 minute, 50 ~1 of a mixture containing 10 parts factor X-deficient plasma and 1 part rabbit brain cephalin stock reagent (prepared as described by Sigma) was added, and the time to clot formation was determined with a fibrometer (BBL, Cockeysville, MD). Mixtures in which TBSA rather than sample was incubated in the first stage The concentration of crude TF in the assay was chosen to served as controls. produce control clotting times of 35-40 seconds. Relative TFI activity was calculated from a standard curve constructed by plotting (on log-log paper) the prolongation in seconds of the clotting time beyond the control value vs. the final concentration of normal pooled serum (50 donors) in the first stage of This standard curve produced a linear response from l-10% (vol/vol) the assay. One unit of TFI activity was defined as that contained serum concentrations. in 1 ml of normal pooled serum. Results of assays of chromatography fractions are expressed as clotting times and have not been converted to units/ml.
LACI(HG2)
Vol. 48, No. 2
PURIFICATION
255
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laennnli (15% separatlng gel and 4% stacking gel) Reduced samples were heated to 100°C for 5 min in the presence of 10% (12). 2-mercaptoethanol prior to electrophoresis. LACI was extracted following SDS-PAGE by cutting the lane from the gel, slicing it into 2 mm sections, and incubating each slice overnight In 100 JJL of 0.1 M NaCl, 0.05 M Tris HCl, A 1:50 dllutlon in TBSA of the extracted material was then pH 7.5, 0.5% SDS. assayed as described above for TF inhibition. Western blotting was performed as described using 12sI-labeled bovine factor Xa as the probe (13). Protein was assayed with the bicinchoninic acid protein assay (Pierce Chemical Co.) using bovine serum albumln as standard.
RESULTS
The overall scheme for the purification of LACI(HG2) is shown in Table 1. HepG2 cell (human hepatoma cell line) serum free conditioned media was used as Four liters of media were centrifuged at 2500 x g x 30 min starting material. to remove particulate debris.
TABLE 1 Purification
Step
Protein mg
Serum free media 14,800 CdCl, preclpltatlon and elution 290 Bovine Xa afflgel 0.16 Superose 12 0.11
Activity units
of LACI(HG2) Specific Activity units/mg -
-
2,310
0.156
2,125
1.33
1,410 1,160
8,810 10,500
Yield % _ _._
Purification -fold__
100 92
47
61 50
56,500 67,300
(1.0 M) was added to the clarified serum CdCl, free media to a flnal concentration of 10 mM and the mixture stirred for 30 min. The precipitate was collected by centrifugation (10,000 x g x 20 min), The pellet was resuspended with 80 ml of 0.5 M and the supernatant discarded. EDTA containing aprotinin (100 KIU/ml) and insoluble material removed by centrlfugation (10,000 x g x 20 mln).
CdC1,_Precipitation.
The sample was diluted Bovln_e Factor Xa-Affi-Gel Affinity Chromatography. with an eaual volume of 0.1 M NaCl. 0.05 M Iris-HCl. pH 7.5. 0.2% Lubrol PX and applied at a flow rate of 4 ml/hr to a 2 ml column o-f Sepharose 4 B agarose connected in series with a 3 ml column of bovine factor Xa-Affi-Gel which had been equilibrated in 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.5, 0.1% Lubrol PX. The columns were washed with 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.5, 0.1% Lubrol (20 ml), and then 0.1 M NaCl, 0.05 M 'Iris-HCl, pH 7.5, 2% lubrol
256
LACI(HG2) PURIFICATION
Vol. 48, No. 2
PX (20 ml). The Sepharose 4 B pre-column was then removed from the circuit and the bovine factor Xa-Affi-Gel column was further washed with 0.1 M NaCl, (20 ml) and eluted with 0.5 M 0.05 M Iris-HCl, pH 7.5, 2% R-Octylglucoside benzamidine, 0.05 M Iris-HCl, pH 1.5, 7% B-octylglucoside. Fractions containing LACI(HG2) as determined by the 7FI assay were pooled (Fig. 1, #62-66) and concentrated to '- 0.4 ml using a YM5 (Amicon) membrane. lhe large quantity of A,,, in the eluted fractions is due to benzamidinc.
20
15
'0
60.
I 0 *w
FIG. 1 Bovine Xa Affi-Gel Affinity Chro matography. Fractions 1 .60 wore 4 ml in volume, and 60.70, 1 ml in A,,,, 0 volume. -0; 111 activity of lOOO-fold diluted samples, 0 -II
,35
40.
FRACTION
NUMBER
lhe concentrated sample was applied at d flow Sgerose 12 Gel Filtration. rate of 0.3 ml/min to two 25 ml Superose 12 columns connected in series and equilibrated in 1 M NaSCN, 0.02 M MdPS. pH 7.0, 2% O-octylglucosidc (fig. 1). Fractions 27-31 containing IF1 activity were pooled and stored at 4°C.
FIG,_2
Superose 12 Gel Filtration. Fraction size 0.9 ml. AzeO, -. --; lfl activity of lOOO-fold diluted samples, 0.- .-0.
FRACTION
NUMBER
LACI(HG2)
Vol. 48, No. 2
257
PURIFICATION
Characterization of Purified LACI(HG2). SDS-PAGE of reduced LACI(HG2) showed _~______ a diffuse band wlth an apparent MW of 39,000 (Fig. 3). In Fig. 4, unreduced samples of LACI(HG7) were also subjected to SDS-PAGE. The results show the co-migration of silver stained protein, l*sI-Xa binding activity following western blotting, and functional TFI activity.
21.5
Unreduced ‘25 I-X0
FIG A
3
SDS-Polyacrylamlde Gel Electrophoresis of Purified LACI(HG2). Silver stain of 2 pg of reduced LACI(HG2).
FIG d
4
SDS-Polyacrylamide Gel Electrophoresis of Purified LACI(HG2). Unreduced = Silver stain of 2pg of unreduced LACI(HG2). 1251-Xa Blot = 1 ;g of unreduced LACITHGP),lectrophoretically transferred to nitrocellulose, "stained" with 1251-Xa and autoradiographed. Top Figure: TFI activity of extracted gel slices following SDS-PAGE of 1 ug of LACI(HG2).
LACI(HG2)
PURIFICATION
Vol. 48, No. 2
pISCUSSION Although LACI activity is produced by several cultured cell lines, including U937 (human monocytic) and endothelial cells (umbilical vein) (14), HepG2 cells remain an excellent source for starting material since they can be maintained under serum free conditions for prolonged periods of time. The media used in the Isolation procedure presented here was harvested from cells which have been cultured > 6 months in the absence of serum. lhe realization that the binding of LACI(HG7) to Xa required Xa's active site explained the very low apparent capacity of the DIP-Xa-Affi-Gel affinity column which was used in our previous purification procedure (7). Bovine rather than human Xa was used to construct the affinity column employed in the present isolation procedure because in initial experiments LACI(HG2) appeared to be a more potent inhibitor of bovine Xa than human Xa (unpublished Additional considerations favoring the use of bovine X were its ease data). of purification, and the avoidance of the need to handle large volumes of human plasma with its potential infectious risks. Since previous studies suggested that LACI(HG2) bound in a non-covalent fashion at or near Xa's active catalytic site (7, 8). benzamidine, a competitive inhibitor of Xa and many other serine proteases, was used to elute LACI(HG2) from the Xa-Affi-Gel column. No additional 1FI activity was eluted from the affinity column by 0.5% SOS, 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.5. On subsequent Superose 12 gel filtration, LACI(HG2) was well separated Benzamidine, present in the concentrated sample from other peaks of A,,,. which was applied to the column, eluted anomalously late in the chromatography (fraction 90, not shown in Fig. 7). No protein could be detected by silver staining in the peak of A,,, which eluted after LACI(HG7). lhe isolation procedure presented here is faster, simpler, and produces a greater yield of LACI(HG2) than our previously published procedure (6). Both are, however, limited by the fact that conditioned media from a cultured The LACI derived from neoplastic cell line is used as starting material. HepG2 cells and that present in human plasma, nevertheless, appear to be very (factor Xa slmilar since they possess the same functional characteristics dependent inhibition of the VIIa-TF complex but not TF alone) (6) and since antisera directed against a synthetic peptide based on the NH,-terminal sequence of LACI(HG2) inhibits the LACI(P) activity present in plasma (8).
REFERENCES
Characterization 1. BROZE, G.J., JR. and MILETICH, J.P. tissue factor in serum. Blood 69, 150-155, 1987.
of the inhibition
of
Inhlbltion of the tissue factor-factor 2. HUBBARD, A.R. and JENNINGS, C.A. VII complex: Involvement of factor Xa and lipoproteins. Thromb. Res. 46, 527-537, 1987.
Inhibition 3. SANDERS, N.L., BAJAJ, S.P., ZIVELIN, A. and RAPAPORT S.I. tissue factor/factor VIIa activity In plasma requires factor X and Blood 66, 204-212, 1985. additional plasma component.
of an
Vol.
4.
48,
LACI(HG2)
No. 2
PURIFICATION
259
and JENNINGS, C.A. Inhlbltion of tissue thromboplastinblood coagulation. mromb. Res, 42, 489-498, 1986.
HUBBARD, A.R.
mediated
5.
RAO, L.V.M. and RAPAPORl, S.I. Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation. Blood _-_. 69 645-651, ____ 1987.
6.
and MILETICH, J.P. Isolation of the tissue factor BROZE, G.J., JR. inhibitor produced by HepG2 hepatoma cells. Proc. Natl. Acad. Sci .-...... _- : USA 84, 1886-1890, 1987.
7.
The tlssue factor JR. BROZE, G.J., Clin. Res_:s, 597A. 1987. inhibitor.
8.
BROZE, G.J., JR. and MILflICH, J.P. The lipoprotein factor inhibitor also inhibits factor Xa. Submitted.
9.
ESMON, C. Washington
10.
inhibitor
Function of factor V in prothrombin University, St. Louis, MO, 1974.
is
also
a
factor
associated
activation.
Ph.D.
Xa
tissue
thesis,
BROZE, G.J., JR. and MAJFRUS, P.W. Purification of human MILETICH, J.P., coagulation factors II, IX, and X using sulfated dextran beads. Methods &Enzymology #O, 221-228, 1981.
Chymotrypsin treated factor Xa 11. BROZE, G.J., JR. and MILETICH, J.P. interacts wlth the lipoprotein associated coagulation inhibitor but does not support the Inhibition of the factor VIIa/tissue factor complex. Submitted.
Cleavage of structural proteins during the assembly 12. LAEMMLI, M.K. 227, 680-685, 1970. head of bacteriophage T4. Nature (L0nd.l -13. BROZE, G.J., VII factor antlgenically
3,
937-946,
HICKMAN, S. and MILETICH, J.P. Detection in plasma antibodies: and genetically related to factor 1985.
JR.,
of the
Monoclonal anti-human second protein of a Invest J Clin VII. -z---.-'.----__._1.
Inhibitor of 14. BAJAJ, M.S., RANA, S.V., WYSOLMERSKI, R.B. and BAJAJ, S. P. the factor VIIa-tissue factor complex is reduced in patients with dlssemlnated intravascular coagulation but not In patients with severe J. Clln. Invest. 79, 1874-1878, 1987. hepatocellular disease.
(c) 1987 Pergamon Journals
Ltd.
All rights reserved.
BRIEF COMMUNICATION _---
ISOLATION OF THE LIPOPROTEIN ASSOCIATED COAGULAlION INHIBITOR PRODUCED BY HepGZ (HUMAN HEPATOMA) CELLS USING BOVINE FAClOR Xa AFFINIlY CHROMAlOGRAPHY
Broze, Jr.*, Louise A. Warren*, James J. Girard*, and Joseph P. Miletlch** *Dlvlsion of Hematology/Oncology and **Laboratory Medicine Washington University School of Medicine Jewish Hospital, St. Louis, Missouri
George J.
USA
(Received 28.7.1987; Accepted in revised form 17.8 1987 by Editor C.T. Esmon) INTRODUCTION
Human
plasma
contains a 1lpOprOtein associated coagulation inhibitor the presence of activated factor X (Xa), LACI(P) rapidly inhibits the enzymatic activity of the factor VIIa-Tissue Factor (VIIa/TF) complex which is responslble for the initiation of coagulation through the classical extrlnslc pathway (l-5). An inhibitor with the same characteristics as LACJ(P) In plasma has recently been isolated from HepG2 cell (human hepatoma cell line) serum free conditioned media, and one step used in the purification procedure was affinity chromatography upon DIP-Xa-agarose (6). Although a critical step In the ultimate Isolation of the LACI produced by HepG2 cells [LACI(HG2)], the capacity of the DIP-Xa-agarose column was so low as to suggest that trace quantities of active Xa remaining on the matrix could Subsequent studles have have been responsible for the blndlng of LACI(HG2). confirmed this by showing that purified LACI(HG2) binds to Xa at or near Xa's active site and does not Interact wlth Xa which has been Inactivated with dilsopropylfluorophosphate (DFP) or dansyl-glutamyl-glycyl-arglnlne chloromethyl ketone (dansyl-GGACK) (7,8). Here a slmpllfled, three step purification procedure for LACI(HG2) Is presented utlllzlng bovine Xa-agarose affinity chromatography.
[LACI(P)I (1.2).
Key Words:
In
coagulation
inhibitor, Tissue Factor, Factor Xa
253
LACI(HG2) PURIFICATION
254
Vol. 48, No. 2
MATERIALS AND METHODS ~_______ 1251-iodide, carrier Sodium free, was purchased from New England Nuclear (Boston, MA) and Iodo-Gen was obtained from Pierce Chemical Co. (Rockford, IL). Dansyl-L-glutamyl-L-glycyl-L-arginine chloromethyl ketone (dansyl-GGACK) was from Calbiochem (La Jolla, CA). Affi-Gel 15 and low molecular weight for polyarylamide standards electrophoresis were gel purchased from Bio-Rad (Richmond, CA). Aprotinin, bovine serum albumin, acrylamide, methylenebis(acrylamide), rabbit brain cephalin, MOPS, and lrizma base were from Sigma (St. Louis, MO). All other chemicals were of reagent grade or better and came from Fisher or from Sigma. Factor X deficient human plasma was obtained from George King Biomedical (Overland Park, KS). Serum samples from healthy blood donors were provided by the American Red Cross (St. Louis, MO).
Cell Culture. described (6).
HepG2
cells
were
cultured
in serum-free
media
as
previously
Proteins. A crude preparation of 1F was prepared and washed extensively with EOTA (1). The X coagulant protein from Russell's viper venom (9). factor VIIa (1). and human factor X (10) were purified as described. Bovine factor X was isolated from the barium sulfate eluate (Sigma) from bovine plasma using DEAEagarose anion exchange chromatography (11). Bovine factor Xa was produced from purified factor X by incubation with insolubilized X coagulant protein (11). and linked to Affi-Gel 15 at a final concentration of 1 mg/ml of packed gel using the manufacturer's instructions (MOPS buffer, pH 7.5).
s-g*
In the first A three stage assay for TF inhibition (TFI) was used. factor VIIa (1 mg/ml). 10 111 of bovine factor Xa rll of (1 ys;ml;P 10 ~1 of CaCl, (40 mM), 10 ,J of TBSA (0.1 M NaCl, 0.05 M 50 ~,IL sample to iris-HCl, pH 7.5, with 1 mg/ml bovine serum albumin), tested, and 10 PL of crude, EDTA-washed TF (10% v/v) were incubated at room temperature. After 30 minutes, a 10 pL sample was diluted lOO-fold into Fifty PL of this diluted sample, 50 PL of VIIa TBSA with 5 mM CaCl,. (1 pg/ml), 50 ~JL of CaCl, (25 mM), and 50 PL of human factor X (10 ug/ml) were then incubated at 37°C. After 1 minute, 50 ~1 of a mixture containing 10 parts factor X-deficient plasma and 1 part rabbit brain cephalin stock reagent (prepared as described by Sigma) was added, and the time to clot formation was determined with a fibrometer (BBL, Cockeysville, MD). Mixtures in which TBSA rather than sample was incubated in the first stage The concentration of crude TF in the assay was chosen to served as controls. produce control clotting times of 35-40 seconds. Relative TFI activity was calculated from a standard curve constructed by plotting (on log-log paper) the prolongation in seconds of the clotting time beyond the control value vs. the final concentration of normal pooled serum (50 donors) in the first stage of This standard curve produced a linear response from l-10% (vol/vol) the assay. One unit of TFI activity was defined as that contained serum concentrations. in 1 ml of normal pooled serum. Results of assays of chromatography fractions are expressed as clotting times and have not been converted to units/ml.
LACI(HG2)
Vol. 48, No. 2
PURIFICATION
255
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laennnli (15% separatlng gel and 4% stacking gel) Reduced samples were heated to 100°C for 5 min in the presence of 10% (12). 2-mercaptoethanol prior to electrophoresis. LACI was extracted following SDS-PAGE by cutting the lane from the gel, slicing it into 2 mm sections, and incubating each slice overnight In 100 JJL of 0.1 M NaCl, 0.05 M Tris HCl, A 1:50 dllutlon in TBSA of the extracted material was then pH 7.5, 0.5% SDS. assayed as described above for TF inhibition. Western blotting was performed as described using 12sI-labeled bovine factor Xa as the probe (13). Protein was assayed with the bicinchoninic acid protein assay (Pierce Chemical Co.) using bovine serum albumln as standard.
RESULTS
The overall scheme for the purification of LACI(HG2) is shown in Table 1. HepG2 cell (human hepatoma cell line) serum free conditioned media was used as Four liters of media were centrifuged at 2500 x g x 30 min starting material. to remove particulate debris.
TABLE 1 Purification
Step
Protein mg
Serum free media 14,800 CdCl, preclpltatlon and elution 290 Bovine Xa afflgel 0.16 Superose 12 0.11
Activity units
of LACI(HG2) Specific Activity units/mg -
-
2,310
0.156
2,125
1.33
1,410 1,160
8,810 10,500
Yield % _ _._
Purification -fold__
100 92
47
61 50
56,500 67,300
(1.0 M) was added to the clarified serum CdCl, free media to a flnal concentration of 10 mM and the mixture stirred for 30 min. The precipitate was collected by centrifugation (10,000 x g x 20 min), The pellet was resuspended with 80 ml of 0.5 M and the supernatant discarded. EDTA containing aprotinin (100 KIU/ml) and insoluble material removed by centrlfugation (10,000 x g x 20 mln).
CdC1,_Precipitation.
The sample was diluted Bovln_e Factor Xa-Affi-Gel Affinity Chromatography. with an eaual volume of 0.1 M NaCl. 0.05 M Iris-HCl. pH 7.5. 0.2% Lubrol PX and applied at a flow rate of 4 ml/hr to a 2 ml column o-f Sepharose 4 B agarose connected in series with a 3 ml column of bovine factor Xa-Affi-Gel which had been equilibrated in 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.5, 0.1% Lubrol PX. The columns were washed with 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.5, 0.1% Lubrol (20 ml), and then 0.1 M NaCl, 0.05 M 'Iris-HCl, pH 7.5, 2% lubrol
256
LACI(HG2) PURIFICATION
Vol. 48, No. 2
PX (20 ml). The Sepharose 4 B pre-column was then removed from the circuit and the bovine factor Xa-Affi-Gel column was further washed with 0.1 M NaCl, (20 ml) and eluted with 0.5 M 0.05 M Iris-HCl, pH 7.5, 2% R-Octylglucoside benzamidine, 0.05 M Iris-HCl, pH 1.5, 7% B-octylglucoside. Fractions containing LACI(HG2) as determined by the 7FI assay were pooled (Fig. 1, #62-66) and concentrated to '- 0.4 ml using a YM5 (Amicon) membrane. lhe large quantity of A,,, in the eluted fractions is due to benzamidinc.
20
15
'0
60.
I 0 *w
FIG. 1 Bovine Xa Affi-Gel Affinity Chro matography. Fractions 1 .60 wore 4 ml in volume, and 60.70, 1 ml in A,,,, 0 volume. -0; 111 activity of lOOO-fold diluted samples, 0 -II
,35
40.
FRACTION
NUMBER
lhe concentrated sample was applied at d flow Sgerose 12 Gel Filtration. rate of 0.3 ml/min to two 25 ml Superose 12 columns connected in series and equilibrated in 1 M NaSCN, 0.02 M MdPS. pH 7.0, 2% O-octylglucosidc (fig. 1). Fractions 27-31 containing IF1 activity were pooled and stored at 4°C.
FIG,_2
Superose 12 Gel Filtration. Fraction size 0.9 ml. AzeO, -. --; lfl activity of lOOO-fold diluted samples, 0.- .-0.
FRACTION
NUMBER
LACI(HG2)
Vol. 48, No. 2
257
PURIFICATION
Characterization of Purified LACI(HG2). SDS-PAGE of reduced LACI(HG2) showed _~______ a diffuse band wlth an apparent MW of 39,000 (Fig. 3). In Fig. 4, unreduced samples of LACI(HG7) were also subjected to SDS-PAGE. The results show the co-migration of silver stained protein, l*sI-Xa binding activity following western blotting, and functional TFI activity.
21.5
Unreduced ‘25 I-X0
FIG A
3
SDS-Polyacrylamlde Gel Electrophoresis of Purified LACI(HG2). Silver stain of 2 pg of reduced LACI(HG2).
FIG d
4
SDS-Polyacrylamide Gel Electrophoresis of Purified LACI(HG2). Unreduced = Silver stain of 2pg of unreduced LACI(HG2). 1251-Xa Blot = 1 ;g of unreduced LACITHGP),lectrophoretically transferred to nitrocellulose, "stained" with 1251-Xa and autoradiographed. Top Figure: TFI activity of extracted gel slices following SDS-PAGE of 1 ug of LACI(HG2).
LACI(HG2)
PURIFICATION
Vol. 48, No. 2
pISCUSSION Although LACI activity is produced by several cultured cell lines, including U937 (human monocytic) and endothelial cells (umbilical vein) (14), HepG2 cells remain an excellent source for starting material since they can be maintained under serum free conditions for prolonged periods of time. The media used in the Isolation procedure presented here was harvested from cells which have been cultured > 6 months in the absence of serum. lhe realization that the binding of LACI(HG7) to Xa required Xa's active site explained the very low apparent capacity of the DIP-Xa-Affi-Gel affinity column which was used in our previous purification procedure (7). Bovine rather than human Xa was used to construct the affinity column employed in the present isolation procedure because in initial experiments LACI(HG2) appeared to be a more potent inhibitor of bovine Xa than human Xa (unpublished Additional considerations favoring the use of bovine X were its ease data). of purification, and the avoidance of the need to handle large volumes of human plasma with its potential infectious risks. Since previous studies suggested that LACI(HG2) bound in a non-covalent fashion at or near Xa's active catalytic site (7, 8). benzamidine, a competitive inhibitor of Xa and many other serine proteases, was used to elute LACI(HG2) from the Xa-Affi-Gel column. No additional 1FI activity was eluted from the affinity column by 0.5% SOS, 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.5. On subsequent Superose 12 gel filtration, LACI(HG2) was well separated Benzamidine, present in the concentrated sample from other peaks of A,,,. which was applied to the column, eluted anomalously late in the chromatography (fraction 90, not shown in Fig. 7). No protein could be detected by silver staining in the peak of A,,, which eluted after LACI(HG7). lhe isolation procedure presented here is faster, simpler, and produces a greater yield of LACI(HG2) than our previously published procedure (6). Both are, however, limited by the fact that conditioned media from a cultured The LACI derived from neoplastic cell line is used as starting material. HepG2 cells and that present in human plasma, nevertheless, appear to be very (factor Xa slmilar since they possess the same functional characteristics dependent inhibition of the VIIa-TF complex but not TF alone) (6) and since antisera directed against a synthetic peptide based on the NH,-terminal sequence of LACI(HG2) inhibits the LACI(P) activity present in plasma (8).
REFERENCES
Characterization 1. BROZE, G.J., JR. and MILETICH, J.P. tissue factor in serum. Blood 69, 150-155, 1987.
of the inhibition
of
Inhlbltion of the tissue factor-factor 2. HUBBARD, A.R. and JENNINGS, C.A. VII complex: Involvement of factor Xa and lipoproteins. Thromb. Res. 46, 527-537, 1987.
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