276
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[24]
[24] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f G l y c o p r o t e i n Ib
By
A N D R E A S N . W I C K I , J E A N N I N E M . CLEMETSON, B E A T STEINER, W O L F G A N G SCHNIPPERING,
and K E N N E T H J. CLEMETSON
Glycoprotein Ib is one of the major components of the outer surface of the platelet plasma membrane and contains most of the sialic acid of the platelet membrane contributing to the surface charge.1 Because of its absence in platelets in the inherited bleeding disorder Bernard-Soulier syndrome, it was one of the first platelet glycoproteins to which a functional role was ascribed. 2'3 It is thus clear that the GPIb-IX complex plays an essential role in normal platelet adhesion and activation, particularly at high shear rates. The first structural information came with the isolation and partial characterization of the proteolytic degradation products "macroglycopeptide ''4 and glycocalicin, 5 which were later shown to be derived from GPIb. Glycocalicin is described in [25] in this volume. Methods have been established for the isolation of amounts of intact, purified GPIb-IX adequate for biochemical characterization. All three of the constituent chains of the GPIb-IX complex, GPIb~, GPIb¢, and GPIX have now been cloned and s e q u e n c e d 6-9 so that a great deal of structural information is now available. Glycoprotein Ib-IX, in common with most other membrane glycoproteins, requires the presence of a detergent for its solubilization from the membrane. Several different detergents have been used. 10When nondenaturing detergents are employed to maintain the molecule in a biologically I N. O. Solum, I. Hagen, C. Filion-Myklebust, and T. Stabaek, Biochim. Biophys. Acta 597, 235 (1980). 2 C. S. P. Jenkins, D. R. Phillips, K. J. Clemetson, D. Meyer, M.-J. Larrieu, and E. F. Lfischer, J. Clin. Invest. 57, 112 (1976). 3 A. T. Nurden and J. P. Caen, N a t u r e (London) 255, 720 (1975). 4 D. S. Pepper and G. A. Jamieson, Biochemistry 9, 3706 (1970). 5 T. Okamura, C. Lombart, and G. A. Jamieson, J. Biol. Chem. 251, 5950 (1976). 6 j. A. Lopez, D. W. Chung, K. Fujikawa, F. S. Hagen, T. Papayannopoulou, and G. J. Roth, Proc. Natl. Acad. Sci. U.S.A. 84, 5615 (1987). 7 K. Titani, K. Takio, M. Handa, and Z. M. Ruggeri, Proc. Natl. Acad. Sci. U.S.A. 84, 5610 (1987). 8 j. A. Lopez, D. W. Chung, K. Fujikawa, F. S. Hagen, E. W. Davie, and G. J. Roth, Proc. Natl. Acad. Sci. U.S.A. 85, 2135 (1988). 9 M. J. Hickey, S. A. Williams, and G. J. Roth, Proc. Natl. Acad. Sci. U.S.A. 86, 6773 (1989). l0 H. A. Cooper, K. J. Clemetson, and E. F. LiJscher, Proc. Natl. Acad. Sci. U.S.A. 76, 1069 (1979).
METHODS IN ENZYMOLOGY.VOL. 215
Copyright © 1992by AcademicPress, Inc. All rightsof reproductionin any form reserved.
[24]
ISOLATION AND CHARACTERIZATION OF G P I b
277
active state, a second problem is encountered, because GPIb-IX remains specifically associated with a group of cytoskeletal components. In contrast to GPIIb-IIIa, which is not generally linked to the cytoskeleton in resting platelets, 11 GPIb-IX seems to be largely associated with the cytoskeleton, which influences the amount of GPIb-IX that can be solubilized. This linkage to actin-binding protein can be disrupted by treating the platelets with N-ethylmaleimide lz before preparation of membranes or Triton X-114 phase separation. Although N-ethylmaleimide is clearly a cysteine-blocking reagent, the mechanism of action in this case is not yet understood. The linkage of GPIb to the cytoskeleton is via actin-binding protein, 13 which is rapidly degraded by calpain. It is retained in the presence of the inhibitors of this enzyme. 14The optimal conditions for this first solubilization stage to avoid loss of GPIb-IX in the cytoskeleton fraction are still poorly defined but affect the yield of GPIb-IX. Although it is always possible to have platelets in a defined state by working with fresh blood from individual donors, the large amount of blood bank platelets required for preparative work often means that storage and handling conditions cannot be defined before isolation starts. Use of N-ethylmaleimide to uncouple GPIb-IX from the cytoskeleton plus efficient calpain inhibitors to prevent degradation to glycocalicin may solve these problems. Although platelets contain several proteases, the principal activity is due to calcium-activated neutral thiol protease (calpain). 15When released by lysed platelets calpain cleaves GPIb, forming glycocalicin. Proteases from other cells may also be a problem. In preparative-scale isolations it is difficult to remove all contaminating leukocytes that are rich in enzymes that degrade platelet glycoproteins. 16Thus, it is essential to add a cocktail of protease inhibitors to the platelets before solubilization to avoid the degradation of GPIb and the production of cleavage products that might interfere at later purification stages. Lectin affinity chromatography on wheat germ agglutinin was an early method that was shown to be of value in purifying GPIb-IX. 17Wheat germ agglutinin interacts predominantly with glycoproteins rich in sialic acid I1 D. R. Phillips, L. K. Jennings, and H. H. Edwards, J. Cell Biol. 86, 77 (1980). 12j. E. B. FOX, L. P. Aggerbeck, and M. C. Berndt, J. Biol. Chem. 263, 4882 (1988). 13 j. R. Okita, D. Pidard, P. J. Newman, R. R. Montgomery, and T. J. Kunicki, J. Cell Biol. 100, 317 (1985). i4 N. O. Solum, T. M. Olsen, G. O. Gogstad, I. Hagen, and F. Brosstad, Biochim. Biophys. Acta 729, 53 (1983). 15 D. R. Phillips and M. Jakfibovgt, J. Biol. Chem. 252, 5602 (1977). 16 K. Bykowska, J. Kaczanowska, M. Karpowicz, J. Strachurska, and M. Kopec, Thromb. Haemostasis 50, 768 (1983). 17 K. J. Clemetson, S. L. Pfueller, E. F. Lilscher, and C. S. P. Jenkins, Biochim. Biophys. Acta 464, 493 (1977).
278
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[24]
present on O-linked oligosaccharides. On platelets these are relatively few and include GPIb, GPIIIb (GPIV), and G P V . 17'j8 A more selective lectin is peanut agglutinin, which binds fairly specifically to GPIb, although only after sialic acid residues are removed with neuraminidase.19 The problems of specificity are overcome by using the Triton X-114 phase separation method. Glycoprotein Ib displays exotic behavior by partitioning into the aqueous phase, rather than in the Triton phase, as would be expected for an integral hydrophobic glycoprotein. 6 Most of the other glycoproteins, including GPIIIb and GPV, partition into the detergent phase. Thus, together with wheat germ agglutinin affinity chromatography, the Triton X-114 phase separation method provides the basis for an isolation procedure for GPIb-IX that is relatively simple and effective. Still further purification can be effected by ion-exchange chromatography on Q-Sepharose or by taking advantage of the fact that GPIb contains a thrombin-binding site by affinity chromatography on thrombin-Sepharose. Monoclonal antibodies to GPIb-IX also provide a method for isolating the GPIb-IX complex. 2°'21 Questions of availability and expense, however, necessarily restrict the generalization of this approach. Experimental Procedures
Isolation and Washing of Platelets Platelets are isolated from citrate-treated blood within 20 hr of collection. The buffy coats from I00 units are transferred into one-quarter of their volume of 9.6 mM glucose, 3 mM KC1, 100 mM NaCI, 10 mM EDTA, 30 mM sodium citrate, pH 6.5, to a final concentration of about 3 × 109 platelets/ml. The platelets are collected by centrifugation at 1500 g for 10 min and washed twice in 30 mM glucose, 120 mM NaC1, I0 mM EDTA, 30 mM sodium citrate, pH 6.5, and once in 134 mM NaCI, 10 mM EDTA, 10 mM Tris-HCl buffer, pH 7.4, giving about 75 ml of platelet pellet.
Solubilization and Triton X-114 Separation The pellet is suspended in 100 ml of 124 mM NaCI, 20 mM EDTA, 2 mM N-ethylmaleimide (omit this if GPIb-IX linked to actin-binding protein is required), I0 mM Tris-HC1 buffer, pH 7.4. The mixture is cooled to 4 ° and
18 M. Moroi and S. M. Jung, Biochim. Biophys. Acta 798, 295 (1984). 19 p. S. Judland and D. J. Anstee, Protides Biol. Fluids 27, 871 (1979). 20 M. C. Berndt, C. Gregory, A. Kabral, H. Zola, D. Fournier, and P. A. Castaldi, Eur. J. Biochem. 151, 637 (1985). -*~V. Vicente, R. A. Houghten, and Z. M. Ruggeri, J. Biol. Chem. 265, 274 (1990).
[24]
ISOLATION AND CHARACTERIZATION OF G P I b
279
mixed with 100 mE of 2% (w/v) Triton X-114 at 4°. Phenylmethylsulfonyl fluoride (PMSF) in methanol is added to give a final concentration of 2 mM, and the mixture is stirred for 15 min and centrifuged at 10,000 g for 30 min at 4 °. The supernatant is further centrifuged at 100,000 g for I hr at 4°; the supernatant from this second centrifugation (220-230 ml) should be clear. The clear supernatant (20-25 ml) is carefully overlayered on 20ml cushions of 6% (w/v) sucrose, 154 mM NaC1, 1 mM EDTA, 0.06% (w/v) Triton X-114, 10 mM Tris-HC1, pH 7.4, in 50-ml centrifuge tubes. The tubes are heated to 37 ° for 5 min in a water bath. The upper phase becomes opaque as clouding of the Triton X-114 occurs. The tubes are then centrifuged at 1000 g for 10 min in an uncooled centrifuge. To avoid mixing the phases, it is important that the centrifuge is well balanced and that no braking is used. The upper phase is carefully removed, and Triton X-114 is added to give a final concentration of 1% (w/v). The mixture is stirred at 4 ° until a clear solution is obtained, again layered above the sucrose solution in the centrifuge tubes, heated to 37° for 5 min, and again centrifuged at 1000 g for 10 min. The upper phases are pooled and Brij 99 (Sigma, St. Louis, MO) is added to a final concentration of 0.5% (w/v). The pH is checked, adjusted (if necessary) to 7.4 with 1 N HCI, and the solution is left standing at 4 ° for 2-3 hr. Generally a precipitate forms at this stage, which is removed by centrifuging at 10,000 g for 10 min. If the preparation must be interrupted and the material stored frozen, then the best point is just before this centrifugation, which is then carried out on thawing.
Wheat Germ Agglutinin-Sepharose 4B Affinity Chromatography The clear supernatant is applied to a column of wheat germ agglutininSepharose 4B [15 x 3 cm; 1 mg wheat germ agglutinin (WGA)/ml Sepharose], which is equilibrated in 0.5% (w/v) Brij 99, 154 mM NaCI, 20 mM Tris-HCl, pH 7.4, and washed through with the same buffer until the optical density (OD) of the eluate returns to baseline. The bound material is then eluted using 0.5% Brij 99, 30 mM NaC1, 20 mM Tris-HC1, pH 7.4, plus 2.5% (w/v) N-acetylglucosamine. A silver-stained, two-dimensional polyacrylamide gel electrophoresis separation of the bound material is shown in Fig. 1.
Q-Sepharose Ion-Exchange Chromatography The fractions containing material that bound to the wheat germ agglutinin-Sepharose 4B are pooled, adjusted to pH 7, and applied to a 10 x 2.5cm Q-Sepharose (Pharmacia, Piscataway, N J) column equilibrated with 0.5% Brij 99, 30 mM NaC1, 20 mM Tris-HCl, pH 7.0, and washed through with the same buffer until the flow-through has eluted. A 200-ml gradient
280
[24]
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
8
7
I
I
pl
6
5.5
4.5
I
I
I
kDa
--93 ---67
O
.......~
-
O
--43
--20 FlG. 1. Two-dimensional isoelectric focusing/5-17% gradient polyacrylamide gel, under reducing conditions, of the platelet components present in the aqueous phase of a Triton X-114 phase separation that bind to wheat germ agglutinin-Sepharose. Glycoprotein Ibt~and GPIX stain relatively weakly with the silver method. ABP, Actin-binding protein.
[24]
ISOLATION AND CHARACTERIZATION OF G P I b
a
281
b kDa 170 140
93
ABC FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6% gel), under nonreducing conditions, of GPIb purifications. (a) Glycoprotein Ib purified by Triton X-114 phase separation, wheat germ agglutinin chromatography, and Q-Sepharose ion-exchange chromatography. Note the presence of two, barely separated bands due to size polymorphism. (b) Lane A, glycoproteins from Triton X-100-solubilized platelets that bind to wheat germ agglutinin; lane B, flow-through on thrombin-Sepharose chromatography of the material shown in lane A; lane C, thrombin-Sepharose-bound material from lane A eluted with 0.5 M NaC1.
from 0.25 to 0.5 M NaC1 in 0.5% Brij 99, 20 mM Tris-HC1, pH 7.0, is then applied to the column to elute the bound material. The fractions containing protein (OD280) are examined by SDS-polyacrylamide gel electrophoresis 22 with silver staining 23 (Fig. 2a) and those containing GPIb complex (a low, broad peak eluting between 0.38 and 0.48 M NaCI) are pooled, dialyzed against water, and lyophilized. '-2 U. K. Laemmli, Nature (London) 227, 680 (1970). 23 j. H. Morrissey, Anal. Biochern. 117, 307 (1981).
282
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[24]
Preparation of Thrombin-Sepharose 4B Bovine thrombin is purified by the method of Fenton et a l . 24 and stored at - 8 0 ° in phosphate-buffered saline containing l M NaC1. The affinity matrix is prepared by reacting 45 mg thrombin with I0 ml CNBr-activated Sepharose 4B (Pharmacia) according to standard procedures. To block the enzymatic activity of thrombin, the thrombin-Sepharose is then incubated with 0.32 mM o-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK; Calbiochem, La Jolla, CA) in 150 mM NaC1, 20 mM Tris-HCl buffer, pH 7.0, for 1 hr at room temperature. The column is then washed and stored at 4 ° in the same buffer containing 0.05% (w/v) NAN3.
Thrombin-Sepharose Affinity Chromatography Washed, outdated platelets are lysed at 4 ° for 15 hr in a buffer containing 1% (w/v) Triton X-100, 150 mM NaC1, 1 mM CaCIE, 1 mM MgCI z, 0.02% (w/v) NAN3, 10 /xM leupeptin, 0.5 mM PMSF, 1 mM N-ethylmaleimide, 20 mM Tris-HC1, pH 7.3. The lysate is centrifuged for 30 min at 30,000 g to remove insoluble material. The supernatant is stored at - 8 0 ° until use or applied directly at room temperature to a wheat germ agglutinin-Sepharose column preincubated in 0.1% (w/v) Triton X-100, 150 mM NaCI, 1 mM CaC12, 1 mM MgCI 2, 0.02% (w/v) NaN 3, 20 mM Tris-HC1, pH 7.0. The column is washed with the same buffer until the optical density of the flow-through is
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[24]
[24] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f G l y c o p r o t e i n Ib
By
A N D R E A S N . W I C K I , J E A N N I N E M . CLEMETSON, B E A T STEINER, W O L F G A N G SCHNIPPERING,
and K E N N E T H J. CLEMETSON
Glycoprotein Ib is one of the major components of the outer surface of the platelet plasma membrane and contains most of the sialic acid of the platelet membrane contributing to the surface charge.1 Because of its absence in platelets in the inherited bleeding disorder Bernard-Soulier syndrome, it was one of the first platelet glycoproteins to which a functional role was ascribed. 2'3 It is thus clear that the GPIb-IX complex plays an essential role in normal platelet adhesion and activation, particularly at high shear rates. The first structural information came with the isolation and partial characterization of the proteolytic degradation products "macroglycopeptide ''4 and glycocalicin, 5 which were later shown to be derived from GPIb. Glycocalicin is described in [25] in this volume. Methods have been established for the isolation of amounts of intact, purified GPIb-IX adequate for biochemical characterization. All three of the constituent chains of the GPIb-IX complex, GPIb~, GPIb¢, and GPIX have now been cloned and s e q u e n c e d 6-9 so that a great deal of structural information is now available. Glycoprotein Ib-IX, in common with most other membrane glycoproteins, requires the presence of a detergent for its solubilization from the membrane. Several different detergents have been used. 10When nondenaturing detergents are employed to maintain the molecule in a biologically I N. O. Solum, I. Hagen, C. Filion-Myklebust, and T. Stabaek, Biochim. Biophys. Acta 597, 235 (1980). 2 C. S. P. Jenkins, D. R. Phillips, K. J. Clemetson, D. Meyer, M.-J. Larrieu, and E. F. Lfischer, J. Clin. Invest. 57, 112 (1976). 3 A. T. Nurden and J. P. Caen, N a t u r e (London) 255, 720 (1975). 4 D. S. Pepper and G. A. Jamieson, Biochemistry 9, 3706 (1970). 5 T. Okamura, C. Lombart, and G. A. Jamieson, J. Biol. Chem. 251, 5950 (1976). 6 j. A. Lopez, D. W. Chung, K. Fujikawa, F. S. Hagen, T. Papayannopoulou, and G. J. Roth, Proc. Natl. Acad. Sci. U.S.A. 84, 5615 (1987). 7 K. Titani, K. Takio, M. Handa, and Z. M. Ruggeri, Proc. Natl. Acad. Sci. U.S.A. 84, 5610 (1987). 8 j. A. Lopez, D. W. Chung, K. Fujikawa, F. S. Hagen, E. W. Davie, and G. J. Roth, Proc. Natl. Acad. Sci. U.S.A. 85, 2135 (1988). 9 M. J. Hickey, S. A. Williams, and G. J. Roth, Proc. Natl. Acad. Sci. U.S.A. 86, 6773 (1989). l0 H. A. Cooper, K. J. Clemetson, and E. F. LiJscher, Proc. Natl. Acad. Sci. U.S.A. 76, 1069 (1979).
METHODS IN ENZYMOLOGY.VOL. 215
Copyright © 1992by AcademicPress, Inc. All rightsof reproductionin any form reserved.
[24]
ISOLATION AND CHARACTERIZATION OF G P I b
277
active state, a second problem is encountered, because GPIb-IX remains specifically associated with a group of cytoskeletal components. In contrast to GPIIb-IIIa, which is not generally linked to the cytoskeleton in resting platelets, 11 GPIb-IX seems to be largely associated with the cytoskeleton, which influences the amount of GPIb-IX that can be solubilized. This linkage to actin-binding protein can be disrupted by treating the platelets with N-ethylmaleimide lz before preparation of membranes or Triton X-114 phase separation. Although N-ethylmaleimide is clearly a cysteine-blocking reagent, the mechanism of action in this case is not yet understood. The linkage of GPIb to the cytoskeleton is via actin-binding protein, 13 which is rapidly degraded by calpain. It is retained in the presence of the inhibitors of this enzyme. 14The optimal conditions for this first solubilization stage to avoid loss of GPIb-IX in the cytoskeleton fraction are still poorly defined but affect the yield of GPIb-IX. Although it is always possible to have platelets in a defined state by working with fresh blood from individual donors, the large amount of blood bank platelets required for preparative work often means that storage and handling conditions cannot be defined before isolation starts. Use of N-ethylmaleimide to uncouple GPIb-IX from the cytoskeleton plus efficient calpain inhibitors to prevent degradation to glycocalicin may solve these problems. Although platelets contain several proteases, the principal activity is due to calcium-activated neutral thiol protease (calpain). 15When released by lysed platelets calpain cleaves GPIb, forming glycocalicin. Proteases from other cells may also be a problem. In preparative-scale isolations it is difficult to remove all contaminating leukocytes that are rich in enzymes that degrade platelet glycoproteins. 16Thus, it is essential to add a cocktail of protease inhibitors to the platelets before solubilization to avoid the degradation of GPIb and the production of cleavage products that might interfere at later purification stages. Lectin affinity chromatography on wheat germ agglutinin was an early method that was shown to be of value in purifying GPIb-IX. 17Wheat germ agglutinin interacts predominantly with glycoproteins rich in sialic acid I1 D. R. Phillips, L. K. Jennings, and H. H. Edwards, J. Cell Biol. 86, 77 (1980). 12j. E. B. FOX, L. P. Aggerbeck, and M. C. Berndt, J. Biol. Chem. 263, 4882 (1988). 13 j. R. Okita, D. Pidard, P. J. Newman, R. R. Montgomery, and T. J. Kunicki, J. Cell Biol. 100, 317 (1985). i4 N. O. Solum, T. M. Olsen, G. O. Gogstad, I. Hagen, and F. Brosstad, Biochim. Biophys. Acta 729, 53 (1983). 15 D. R. Phillips and M. Jakfibovgt, J. Biol. Chem. 252, 5602 (1977). 16 K. Bykowska, J. Kaczanowska, M. Karpowicz, J. Strachurska, and M. Kopec, Thromb. Haemostasis 50, 768 (1983). 17 K. J. Clemetson, S. L. Pfueller, E. F. Lilscher, and C. S. P. Jenkins, Biochim. Biophys. Acta 464, 493 (1977).
278
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[24]
present on O-linked oligosaccharides. On platelets these are relatively few and include GPIb, GPIIIb (GPIV), and G P V . 17'j8 A more selective lectin is peanut agglutinin, which binds fairly specifically to GPIb, although only after sialic acid residues are removed with neuraminidase.19 The problems of specificity are overcome by using the Triton X-114 phase separation method. Glycoprotein Ib displays exotic behavior by partitioning into the aqueous phase, rather than in the Triton phase, as would be expected for an integral hydrophobic glycoprotein. 6 Most of the other glycoproteins, including GPIIIb and GPV, partition into the detergent phase. Thus, together with wheat germ agglutinin affinity chromatography, the Triton X-114 phase separation method provides the basis for an isolation procedure for GPIb-IX that is relatively simple and effective. Still further purification can be effected by ion-exchange chromatography on Q-Sepharose or by taking advantage of the fact that GPIb contains a thrombin-binding site by affinity chromatography on thrombin-Sepharose. Monoclonal antibodies to GPIb-IX also provide a method for isolating the GPIb-IX complex. 2°'21 Questions of availability and expense, however, necessarily restrict the generalization of this approach. Experimental Procedures
Isolation and Washing of Platelets Platelets are isolated from citrate-treated blood within 20 hr of collection. The buffy coats from I00 units are transferred into one-quarter of their volume of 9.6 mM glucose, 3 mM KC1, 100 mM NaCI, 10 mM EDTA, 30 mM sodium citrate, pH 6.5, to a final concentration of about 3 × 109 platelets/ml. The platelets are collected by centrifugation at 1500 g for 10 min and washed twice in 30 mM glucose, 120 mM NaC1, I0 mM EDTA, 30 mM sodium citrate, pH 6.5, and once in 134 mM NaCI, 10 mM EDTA, 10 mM Tris-HCl buffer, pH 7.4, giving about 75 ml of platelet pellet.
Solubilization and Triton X-114 Separation The pellet is suspended in 100 ml of 124 mM NaCI, 20 mM EDTA, 2 mM N-ethylmaleimide (omit this if GPIb-IX linked to actin-binding protein is required), I0 mM Tris-HC1 buffer, pH 7.4. The mixture is cooled to 4 ° and
18 M. Moroi and S. M. Jung, Biochim. Biophys. Acta 798, 295 (1984). 19 p. S. Judland and D. J. Anstee, Protides Biol. Fluids 27, 871 (1979). 20 M. C. Berndt, C. Gregory, A. Kabral, H. Zola, D. Fournier, and P. A. Castaldi, Eur. J. Biochem. 151, 637 (1985). -*~V. Vicente, R. A. Houghten, and Z. M. Ruggeri, J. Biol. Chem. 265, 274 (1990).
[24]
ISOLATION AND CHARACTERIZATION OF G P I b
279
mixed with 100 mE of 2% (w/v) Triton X-114 at 4°. Phenylmethylsulfonyl fluoride (PMSF) in methanol is added to give a final concentration of 2 mM, and the mixture is stirred for 15 min and centrifuged at 10,000 g for 30 min at 4 °. The supernatant is further centrifuged at 100,000 g for I hr at 4°; the supernatant from this second centrifugation (220-230 ml) should be clear. The clear supernatant (20-25 ml) is carefully overlayered on 20ml cushions of 6% (w/v) sucrose, 154 mM NaC1, 1 mM EDTA, 0.06% (w/v) Triton X-114, 10 mM Tris-HC1, pH 7.4, in 50-ml centrifuge tubes. The tubes are heated to 37 ° for 5 min in a water bath. The upper phase becomes opaque as clouding of the Triton X-114 occurs. The tubes are then centrifuged at 1000 g for 10 min in an uncooled centrifuge. To avoid mixing the phases, it is important that the centrifuge is well balanced and that no braking is used. The upper phase is carefully removed, and Triton X-114 is added to give a final concentration of 1% (w/v). The mixture is stirred at 4 ° until a clear solution is obtained, again layered above the sucrose solution in the centrifuge tubes, heated to 37° for 5 min, and again centrifuged at 1000 g for 10 min. The upper phases are pooled and Brij 99 (Sigma, St. Louis, MO) is added to a final concentration of 0.5% (w/v). The pH is checked, adjusted (if necessary) to 7.4 with 1 N HCI, and the solution is left standing at 4 ° for 2-3 hr. Generally a precipitate forms at this stage, which is removed by centrifuging at 10,000 g for 10 min. If the preparation must be interrupted and the material stored frozen, then the best point is just before this centrifugation, which is then carried out on thawing.
Wheat Germ Agglutinin-Sepharose 4B Affinity Chromatography The clear supernatant is applied to a column of wheat germ agglutininSepharose 4B [15 x 3 cm; 1 mg wheat germ agglutinin (WGA)/ml Sepharose], which is equilibrated in 0.5% (w/v) Brij 99, 154 mM NaCI, 20 mM Tris-HCl, pH 7.4, and washed through with the same buffer until the optical density (OD) of the eluate returns to baseline. The bound material is then eluted using 0.5% Brij 99, 30 mM NaC1, 20 mM Tris-HC1, pH 7.4, plus 2.5% (w/v) N-acetylglucosamine. A silver-stained, two-dimensional polyacrylamide gel electrophoresis separation of the bound material is shown in Fig. 1.
Q-Sepharose Ion-Exchange Chromatography The fractions containing material that bound to the wheat germ agglutinin-Sepharose 4B are pooled, adjusted to pH 7, and applied to a 10 x 2.5cm Q-Sepharose (Pharmacia, Piscataway, N J) column equilibrated with 0.5% Brij 99, 30 mM NaC1, 20 mM Tris-HCl, pH 7.0, and washed through with the same buffer until the flow-through has eluted. A 200-ml gradient
280
[24]
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
8
7
I
I
pl
6
5.5
4.5
I
I
I
kDa
--93 ---67
O
.......~
-
O
--43
--20 FlG. 1. Two-dimensional isoelectric focusing/5-17% gradient polyacrylamide gel, under reducing conditions, of the platelet components present in the aqueous phase of a Triton X-114 phase separation that bind to wheat germ agglutinin-Sepharose. Glycoprotein Ibt~and GPIX stain relatively weakly with the silver method. ABP, Actin-binding protein.
[24]
ISOLATION AND CHARACTERIZATION OF G P I b
a
281
b kDa 170 140
93
ABC FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6% gel), under nonreducing conditions, of GPIb purifications. (a) Glycoprotein Ib purified by Triton X-114 phase separation, wheat germ agglutinin chromatography, and Q-Sepharose ion-exchange chromatography. Note the presence of two, barely separated bands due to size polymorphism. (b) Lane A, glycoproteins from Triton X-100-solubilized platelets that bind to wheat germ agglutinin; lane B, flow-through on thrombin-Sepharose chromatography of the material shown in lane A; lane C, thrombin-Sepharose-bound material from lane A eluted with 0.5 M NaC1.
from 0.25 to 0.5 M NaC1 in 0.5% Brij 99, 20 mM Tris-HC1, pH 7.0, is then applied to the column to elute the bound material. The fractions containing protein (OD280) are examined by SDS-polyacrylamide gel electrophoresis 22 with silver staining 23 (Fig. 2a) and those containing GPIb complex (a low, broad peak eluting between 0.38 and 0.48 M NaCI) are pooled, dialyzed against water, and lyophilized. '-2 U. K. Laemmli, Nature (London) 227, 680 (1970). 23 j. H. Morrissey, Anal. Biochern. 117, 307 (1981).
282
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[24]
Preparation of Thrombin-Sepharose 4B Bovine thrombin is purified by the method of Fenton et a l . 24 and stored at - 8 0 ° in phosphate-buffered saline containing l M NaC1. The affinity matrix is prepared by reacting 45 mg thrombin with I0 ml CNBr-activated Sepharose 4B (Pharmacia) according to standard procedures. To block the enzymatic activity of thrombin, the thrombin-Sepharose is then incubated with 0.32 mM o-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK; Calbiochem, La Jolla, CA) in 150 mM NaC1, 20 mM Tris-HCl buffer, pH 7.0, for 1 hr at room temperature. The column is then washed and stored at 4 ° in the same buffer containing 0.05% (w/v) NAN3.
Thrombin-Sepharose Affinity Chromatography Washed, outdated platelets are lysed at 4 ° for 15 hr in a buffer containing 1% (w/v) Triton X-100, 150 mM NaC1, 1 mM CaCIE, 1 mM MgCI z, 0.02% (w/v) NAN3, 10 /xM leupeptin, 0.5 mM PMSF, 1 mM N-ethylmaleimide, 20 mM Tris-HC1, pH 7.3. The lysate is centrifuged for 30 min at 30,000 g to remove insoluble material. The supernatant is stored at - 8 0 ° until use or applied directly at room temperature to a wheat germ agglutinin-Sepharose column preincubated in 0.1% (w/v) Triton X-100, 150 mM NaCI, 1 mM CaC12, 1 mM MgCI 2, 0.02% (w/v) NaN 3, 20 mM Tris-HC1, pH 7.0. The column is washed with the same buffer until the optical density of the flow-through is
