- Email: [email protected]
Surface labeling of platelet membrane glycoproteins
412
PLATELET
RECEPTORS: ASSAYS AND PURIFICATION
[35]
platelet surfaces have resulted in the intriguing observation that these reagents specifically inhibit collagen-induced platelet aggregation but not adhesion to collagen or thrombin-induced platelet aggregation. The mechanism by which this specific inhibition occurs is not known. Control experiments with a monofunctional sulfosuccinimidyl ester have discounted the possibility that it is simply acylation of specific residues that gives rise to the observed inhibition. Perhaps a tertiary or quaternary structural change in a specific surface protein is a required step in platelet activation by collagen, and cross-linking by BS 3 or DTSSP locks this protein in the unactivated state. The selective reduction in intensity of bands corresponding to several major glycoproteins in an SDS gel profile of platelets treated with one of these reagents suggests that candidates for surface proteins involved in collagen-induced platelet activation might be explored by this approach. However, much additional work needs to be done before this question is clarified. Radioisotopically labeled BS 3 or DTSSP may be useful in this endeavor. Acknowledgment Work in this laboratory was supported by grants from the National Institutes of Health, DK25489 and DK31880.
[35] S u r f a c e L a b e l i n g o f P l a t e l e t M e m b r a n e G l y c o p r o t e i n s By DAVID R. PHILLIPS Introduction
Many reactions related to the hemostatic effectiveness of the platelet (e.g., binding of platelet agonists, platelet adhesion, platelet aggregation, and platelet procoagulant activity) occur on specific glycoproteins on the outer surface of the platelet plasma membrane. J Identification of the membrane glycoproteins involved in these reactions has been facilitated by procedures that specifically label platelet surface proteins. These procedures attempt to introduce specifically and exclusively a radioactive label only into macromolecules on the outer surface of the membrane. The basic premise of these procedures is that the labeling agent does not penetrate N. Kieffer and D. R. Phillips, Annu. Rev. Cell Biol. 6, 329 (1990).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[35]
413
SURFACE LABELING OF PLATELETS TABLE I RADIOLABELING PLATELET MEMBRANE GLYCOPROTEINS
Labeling method Lactoperoxidase-catalyzed iodination (125I or 131I) Periodate/sodium boro[3H]hydride labeling
Functional groups labeled
Major glycoproteins labele&
Tyrosine (histidine)
GPIIb (Cqlb), GPIIIa (f13), GPIV, IGPIIa (/30] GPIb, [GPIIb, GPIIIa, GPIV, GPV, GPIX]
Sialic acid
" Glycoproteins shown in brackets are labeled less intensely.
the plasma membrane, and hence only membrane surface components are radiolabeled. Two methods of labeling membrane surface glycoproteins will be described in this report: lactoperoxidase-catalyzed iodination 2 and periodate/ sodium boro[3H]hydride labeling. 3 These procedures were selected for presentation because they are sensitive, introduce minimal alterations into platelet glycoproteins, together label all known surface glycoproteins, and are widely used. The sites of labeling and the major glycoproteins identified by these techniques are extensive and up to 40 glycoproteins can be identified. The major ones are summarized in Table I. Additional agents have been identified that also label cell surface proteins on platelets, and the reader is referred to the original publications for a description of the procedures using them: neuraminidase/galactose oxidase/sodium boro[3H]hydride 4, transglutaminase 5, diazotized diiodosulfanylic acid 6, and Iodogen. 7 Protein labeling procedures are characteristically more facile with proteins in solution than with proteins in membranes. Consequently, platelets must be washed to remove plasma proteins prior to labeling, which results in two limitations of these techniques. First, buffers that include protein to "stabilize" platelets during isolation cannot be used for washing. Second, because radiolabeling of platelets involves numerous washing steps (first to eliminate plasma proteins and second to reduce the concentration of unincorporated isotope), the labeled platelets are usually less reactive than 2 D. R. Phillips, Biochemistry 11, 4582 (1972). 3 T. L. Steck and G. Dawson, J. Biol. Chem. 249, 2135 (1974). 4 D. R. Phillips and P. P. Agin, J. Clin. Invest. 60, 535 (1977). 5 T. Okumura and G. A. Jamieson, J. Biol. Chem. 251, 5944 (1976). 6 j. N. George, R. D. Potterf, D. C. Lewis, and D. A. Sears, J. Lab. Clin. Med. 88, 232 (1976). 7 G. P. Tuszynski, L. C. Knight, E. Kornecki, and S. Srivastava, Anal. Biochem. 130, 166 (1983).
414
iiiiii::i s, iiii!iiiiiiiiii
PLATELET RECEPTORS; ASSAYS AND PURIFICATION
o.+
12Sl. + I/2 H202
LP ~ (, Protein
[35]
)-~_~)--OH + H20
FIG. 1. The lactoperoxidase-catalyzed iodination reaction.
unlabeled platelets and have lost their characteristic discoid morphology. These limitations can be minimized by judicious selection ofplatelet isolation conditions. 8 Lactoperoxidase-Catalyzed Iodination Lactoperoxidase is used to oxidize 125I so that it rapidly iodinates tyrosine (and to a lesser extent histidine). The reaction catalyzed is diagrammed in Fig. 1. Because the platelet membrane is impermeable to lactoperoxidase (Mr 78,000), the iodination reaction occurs primarily with proteins on the platelet surface. 2'4 The reader is referred to Ref. 9 for a complete discussion of the iodination reaction and a description of the products produced. Platelets from freshly drawn blood are washed and suspended at ambient temperature in normal Tyrode's buffer (138 mM sodium chloride, 2.7 m M potassium chloride, 12 mM sodium bicarbonate, 0.36 mM sodium phosphate, 1.8 mM calcium chloride, 0.49 mM magnesium chloride, and 5.5 mM glucose, pH 7.4) to a platelet count of 109/ml. Other platelet washing buffers have been used, and all have proven suitable, providing they are free of protein, do not contain inhibitors of lactoperoxidase, and do not disproportionate hydrogen peroxide. To I ml of the platelet suspension, 1 mCi of carrier-flee NaJ25I is added, with gentle stirring at ambient temperature, followed by 0.25 nmol of lactoperoxidase (Sigma, St. Louis, MO) and five 10-/A aliquots of freshly prepared 3 mM hydrogen peroxide, added at 10-sec intervals. The use of flesh isotope (within 1 month of preparation) is desirable to minimize the presence ofI 2 and other oxidized species of iodide. Lactoperoxidase is stable when stored frozen in solution. The 3 m M hydrogen peroxide solution should be freshly prepared at 4° in 1 m M EDTA to avoid any disproportionation reaction. 8 j. E. B. Fox, C. C. Reynolds, and J. K. Boyles, this volume [6]. 9 M. Morrison, this series, Vol. 70, p. 214.
[35]
SURFACE LABELING OF PLATELETS
415
The labeled platelets are diluted 10-fold with Tyrode's buffer, and are sedimented by centrifugation at 2000 g for 15 min at 4 °. The labeled platelets are washed twice by resuspension in 10 ml of the Tyrode's buffer followed by centrifugation. Less than I% of the isotope is covalently bound to protein under these conditions. Higher yields can be achieved through more additions of hydrogen peroxide but should be avoided because platelets are activated by high concentrations of this oxidizing agent. Most of the 125I in the washed platelets (>90%) is intracellular, not bound to protein, and difficult to remove by these procedures. Platelets labeled by lactoperoxidase-catalyzed iodination are functional in that they secrete serotonin and aggregate when treated with thrombin. A modification of the lactoperoxidase-catalyzed iodination procedure can be used to monitor changes of the platelet surface that occur during physiological responses of the platelet. ~0In this modification, the iodination reaction is initiated by hydrogen peroxide and is terminated 15 sec later by the addition of catalase, which disproportionates all remaining hydrogen peroxide. An example of this reaction is illustrated in Fig. 2, in which platelet surface proteins were iodinated during aggregation. In this instance, platelet aggregation is monitored by light scattering in an aggregometer. Na~25I and lactoperoxidase are added before the agonist (thrombin). The iodination reaction is initiated 1 min later by the single addition of hydrogen peroxide. After 15 sec, the iodination reaction is terminated by adding catalase. To separate platelets and associated proteins from the nonassociated secreted proteins that are also labeled, the samples are carefully layered on 15% sucrose in Tyrode's solution and centrifuged for 3 min at 8500 g (microfuge; Beckman Instruments, Palo Alto, CA). The resulting pellet is processed for electrophoresis. The proteins that have become labeled during platelet stimulation are identified by comparing the proteins labeled in stimulated platelets to those labeled in unstimulated platelets. Iaeriodate/Sodium
Borohydride
Labeling
The periodate/sodium boro[3H]hydride procedure labels sialic acid residues on membrane glycoproteins and glycolipids3 (see Fig. 3). Periodate is used to cleave oxidatively carbon-carbon bonds between the 7-8 or 8-9 positions of sialic acid residues. The resulting aldehyde is then reduced with sodium boro[3H]hydride, producing the stable, radiolabeled alcohol derivative. Labeling can be restricted to the outer membrane surface by equilibrating platelets to 4° (which renders the membrane impermeable to the oxidizing agent) before exposing them to periodate. Io D. R. Phillips, L. K. Jennings, and H. R. Prasanna, J. Biol. Chem. 255, 11629 (1980).
416
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[35]
H202 Cat
c
.o Cat
l
r-
H202A /
I,-
Si,
Time I 1 rain
FIG. 2. lodination of platelets during thrombin-induced aggregation. Washed platelets (5 × 108) were suspended in 0.5 ml Tyrode's solution and iodinated either 10 or 60 sec after the induction of aggregation. The reaction was performed with stirring: aggregation was monitored by the decrease in light transmittance. Additions at the arrows were as follows: I, 0.25 mCi carrier-free 1251; LP, 1.5 × 10 -I° mol lactoperoxidase; Th, 0.05 unit thrombin; H202, 3.5 × 10 -9 mol hydrogen peroxide; Cat, 3.5 × 10 -9 mol catalase.
Platelets from freshly drawn blood are washed and suspended in 1 ml of a buffer containing 150 mM sodium chloride and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.6. Tris buffers are to be avoided as they quench the periodate. The suspension is equilibrated to 4 ° and, after the addition of sodium periodate (1 mM final concentration, freshly prepared), is incubated an additional 10 min at 4° in the
_~~ , - Acooc ~3.~-NoB.~,°,--k~. _ii_iiiii~iiA¸i¸ic~i !' i! I0~
Fit3. 3. The periodate/sodium borohydride labeling procedure.
[35]
SURFACE LABELING OF PLATELETS
417
dark. All remaining steps are performed at ambient temperature. The periodate-treated platelets are removed from solution by centrifugation at 800 g for 10 rain at 4°, washed once with the HEPES buffer, and resuspended in 1 ml of this buffer. Sodium boro[3H]hydride (0.5 mCi) is added, and the suspension is incubated for 5 rain. The sodium boro[3H]hydride should be stored in one-use-size aliquots at - 80° in 0.1 M sodium hydroxide and thawed immediately before use. The volume added should not affect the pH of the platelet suspension. The labeled platelets are centrifuged, washed once with the HEPES buffer, and resuspended in the original volume. Higher specific activities of labeled platelets can be achieved by (1) using more sodium boro[3H]hydride, (2) decreasing the pH of the periodate oxidation to 6, and (3) increasing to 8 the pH of the borohydride reduction, u Identification of Labeled Membrane Glycoproteins Membrane glycoproteins labeled by either the lactoperoxidase or periodate procedures are readily identifiable by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, as is illustrated in Fig. 4. In platelets labeled by lactoperoxidate-catalyzed iodination, the labeled proteins are detected by autoradiography of the dried gel. 4 As shown in lanes 2 and 4 of Fig. 4, glycoprotein (GP) IIb and GPIIIa are the most prominently labeled bands, reflecting their abundance in platelets (-50,000 copies per platelet). Glycoprotein IIIa (Mr 114,000, reduced) labels approximately three times more intensely by this method than does GPIIb (Mr 132,000, reduced), even though the two glycoproteins are present in equal concentrations; both of the disulfide-linked subunits of GPIIb, GPIIb~, and GPIIb~ are labeled. The identity of the GPIIb and GPIIIa bands can be confirmed by analyzing various parameters of these glycoproteins: (1) characteristic shifts in molecular weight on disulfide reduction (Mr 95,000 for nonreduced GPIIIa and Mr 142,000 for nonreduced GPIIb~2); (2) their absence in platelets from patients with Glanzmann's thrombasthenia% (3) staining by the periodic acid-Schiff reagent 2, (4) binding to Lens culinaris lectin 4, and (5) coimmunoprecipitation from Ca2+-con taining buffers by monoclonal antibodies ~3 or antibodies specific for one of the two glycoproteins in the complex. Electrophoresis of nonreduced samples of SDS-solubilized platelets should be performed immediately after solubilization to avoid protein polymerization. Alternatively, the u B. Steiner, K. J. Clemetson, and E. F. Liischer, Thromb. Res. 29, 43 (1983). t2 D. R. Phillips and P. P. Agin, J. Biol. Chem. 252, 2121 (1977). 13 R. P. McEver, E. M. Bennett, and M. N. Martin, J. Biol. Chem. 258, 5269 (1983).
418
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
Coomassie Blue
1251
12s I
aH
[35]
3H
;~i;~i:i!!i
200K G P lib,,, Gp11Ta -
lOOK
-GPIba -GPma -GPmb - GP I b a GPllb<~
5OK
-
-GPma i
- GPITIb
25K GP/Ib/3- m I
10% Acrylamide ~ J
6% A c r y l a m i d e
FIG. 4. Detection of cell surface glycoproteins by lactoperoxidase-catalyzed iodination and periodate/sodium boro[3H]hydride labeling. Proteins in lanes 1-3 were separated by electrophoresis in SDS through 10% polyacrylamide gels; 6% polyacrylamide gels were used in lanes 4 and 5. Lane 1 shows the Coomassie Brilliant blue-stained proteins in whole platelets; lanes 2 and 4 are autoradiograms of SDS gels showing the cell surface glycoproteins labeled by lactoperoxidase-catalyzed iodination; lanes 3 and 5 are fluorograms of SDS gels showing the cell surface glycoproteins labeled by the periodate/sodium boro[3H]hydride technique. The molecular weight scale is indicated.
solubilized samples can be stored in an anaerobic solution or treated with N-ethylmaleimide. ~2 The GPIIIb band (M r 97,000, also termed GPIV) is the primary labeled band just below reduced GPIIIa, and is recognizable because, unlike GPIIIa, it does not change electrophoretic mobility on disulfide bond reduction 4 and is resistant to chymotrypsin hydrolysis on intact platelets. 5 There are several labeled bands above GPIIb~ that can be visualized by nonreduced-reduced two-dimensional electrophoresis.22 Identity of these and other bands is also confirmed by electrophoresis according to O'Farrell (see Ref. 14) and immunoprecipitation with monospecific antibodies. 15 In platelets labeled by the periodate/sodium boro[3H]hydride procedure, the labeled glycoproteins are detected by autofluorography of the 14 K. J. Clemetson, A. Capitanio, and E. F. Liischer, Biochim. Biophys. Acta 553, 11 (1979). ~5 R. P. McEver and M. N. Martin, J. Biol. Chem. 259, 9799 (1984).
[35]
SURFACE LABELING OF PLATELETS
419
dried SDS-polyacrylamide gel. 16 Lanes 3 and 5 of Fig. 4 illustrate the glycoproteins labeled by this procedure; increased exposure of the film to the gel will permit detection of more than 30 labeled glycoproteins. Glycoprotein Ib is the most prominent of the labeled glycoproteins, reflecting its abundance in platelets (-30,000 copies per platelet) and its high sialic acid content. Both of the disulfide-linked subunits of GPIb are labeled by this procedure; GPIb~, Mr 128,000-141,000; and GPIb B, M r 22,000. The heterogeneity in the molecular weight of GPIb~ is due to the presence of allelic variants, which are usually quite rare. ~7 The identity of GPIb can be confirmed by several of its properties: (1) characteristic shift in molecular weight on reduction of disulfide bonds (Mr 170,000, nonreduced~2), (2) absence in platelets from patients with Bernard-Soulier syndrome ~8, (3) intense staining by the periodic acid-Schiff reagent 2, (4) binding to wheat germ agglutinin 19, (5) immunoprecipitation with monoclonal antibodies 2°, and (6) selective hydrolysis by Ca2+-dependent protease treatment of intact platelets. 21 Glycoprotein IX (Mr 17,000), also labeled by the periodate procedure, exists as a complex with GPIb 22 and therefore coimmunoprecipitates with GPIb. Fibrinogen, thrombospondin, and other a granule proteins and glycoproteins are not normally labeled by either of the surface-labeling methods described here. They do become prominently labeled components, however, if platelets have undergone the release reaction prior to labeling.l° Accordingly, care must be taken to maintain platelets in an unactivated state during isolation and to complete the labeling procedure without delay after isolation. Acknowledgments The work was supported by Grants HL28947 and HL 32254 from the National Institutes of Health. The author wishes to thank James X. Warger and Norma Jean Gargasz for graphics, Barbara Allen and Sally Gullatt Seehafer for editorial assistance, and Michele Prator and Linda Harris Odumade for manuscript preparation.
t6 W. A. Bonner and R. A. Laskey, Eur. J. Biochem. 46, 83 (1974). 17 M. Moroi, S. M. Jung, and N. Yoshida, Blood 64, 622 (1984). 18 A. T. Nurden and J. P. Caen, Nature (London) 255, 720 (1975). I9 K. J. Clemetson, S. L. Pfueller, E. F. Li~scher, and C. S. P. Jenkins, Biochim. Biophys. Acta 464, 493 (1977). 2o A. J. McMichael, N. A. Rust, J. R. Pilch, R. Sochyinsky, J. Morton, D. Y. Mason, C. Ruan, G. Tobelem, and J. Caen, Br. J. Haematol. 49, 501 (1981). 21 N. Yoshida, B. Weksler, and R. Nachman, J. Biol. Chem. 258, 7168 (1983). 2: M. C. Berndt, C. Gregory, A. Kabral, H. Zola, D. Fournier, and P. A. Castaldi, Eur. J. Biochem. 151, 637 (1985).
PLATELET
RECEPTORS: ASSAYS AND PURIFICATION
[35]
platelet surfaces have resulted in the intriguing observation that these reagents specifically inhibit collagen-induced platelet aggregation but not adhesion to collagen or thrombin-induced platelet aggregation. The mechanism by which this specific inhibition occurs is not known. Control experiments with a monofunctional sulfosuccinimidyl ester have discounted the possibility that it is simply acylation of specific residues that gives rise to the observed inhibition. Perhaps a tertiary or quaternary structural change in a specific surface protein is a required step in platelet activation by collagen, and cross-linking by BS 3 or DTSSP locks this protein in the unactivated state. The selective reduction in intensity of bands corresponding to several major glycoproteins in an SDS gel profile of platelets treated with one of these reagents suggests that candidates for surface proteins involved in collagen-induced platelet activation might be explored by this approach. However, much additional work needs to be done before this question is clarified. Radioisotopically labeled BS 3 or DTSSP may be useful in this endeavor. Acknowledgment Work in this laboratory was supported by grants from the National Institutes of Health, DK25489 and DK31880.
[35] S u r f a c e L a b e l i n g o f P l a t e l e t M e m b r a n e G l y c o p r o t e i n s By DAVID R. PHILLIPS Introduction
Many reactions related to the hemostatic effectiveness of the platelet (e.g., binding of platelet agonists, platelet adhesion, platelet aggregation, and platelet procoagulant activity) occur on specific glycoproteins on the outer surface of the platelet plasma membrane. J Identification of the membrane glycoproteins involved in these reactions has been facilitated by procedures that specifically label platelet surface proteins. These procedures attempt to introduce specifically and exclusively a radioactive label only into macromolecules on the outer surface of the membrane. The basic premise of these procedures is that the labeling agent does not penetrate N. Kieffer and D. R. Phillips, Annu. Rev. Cell Biol. 6, 329 (1990).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[35]
413
SURFACE LABELING OF PLATELETS TABLE I RADIOLABELING PLATELET MEMBRANE GLYCOPROTEINS
Labeling method Lactoperoxidase-catalyzed iodination (125I or 131I) Periodate/sodium boro[3H]hydride labeling
Functional groups labeled
Major glycoproteins labele&
Tyrosine (histidine)
GPIIb (Cqlb), GPIIIa (f13), GPIV, IGPIIa (/30] GPIb, [GPIIb, GPIIIa, GPIV, GPV, GPIX]
Sialic acid
" Glycoproteins shown in brackets are labeled less intensely.
the plasma membrane, and hence only membrane surface components are radiolabeled. Two methods of labeling membrane surface glycoproteins will be described in this report: lactoperoxidase-catalyzed iodination 2 and periodate/ sodium boro[3H]hydride labeling. 3 These procedures were selected for presentation because they are sensitive, introduce minimal alterations into platelet glycoproteins, together label all known surface glycoproteins, and are widely used. The sites of labeling and the major glycoproteins identified by these techniques are extensive and up to 40 glycoproteins can be identified. The major ones are summarized in Table I. Additional agents have been identified that also label cell surface proteins on platelets, and the reader is referred to the original publications for a description of the procedures using them: neuraminidase/galactose oxidase/sodium boro[3H]hydride 4, transglutaminase 5, diazotized diiodosulfanylic acid 6, and Iodogen. 7 Protein labeling procedures are characteristically more facile with proteins in solution than with proteins in membranes. Consequently, platelets must be washed to remove plasma proteins prior to labeling, which results in two limitations of these techniques. First, buffers that include protein to "stabilize" platelets during isolation cannot be used for washing. Second, because radiolabeling of platelets involves numerous washing steps (first to eliminate plasma proteins and second to reduce the concentration of unincorporated isotope), the labeled platelets are usually less reactive than 2 D. R. Phillips, Biochemistry 11, 4582 (1972). 3 T. L. Steck and G. Dawson, J. Biol. Chem. 249, 2135 (1974). 4 D. R. Phillips and P. P. Agin, J. Clin. Invest. 60, 535 (1977). 5 T. Okumura and G. A. Jamieson, J. Biol. Chem. 251, 5944 (1976). 6 j. N. George, R. D. Potterf, D. C. Lewis, and D. A. Sears, J. Lab. Clin. Med. 88, 232 (1976). 7 G. P. Tuszynski, L. C. Knight, E. Kornecki, and S. Srivastava, Anal. Biochem. 130, 166 (1983).
414
iiiiii::i s, iiii!iiiiiiiiii
PLATELET RECEPTORS; ASSAYS AND PURIFICATION
o.+
12Sl. + I/2 H202
LP ~ (, Protein
[35]
)-~_~)--OH + H20
FIG. 1. The lactoperoxidase-catalyzed iodination reaction.
unlabeled platelets and have lost their characteristic discoid morphology. These limitations can be minimized by judicious selection ofplatelet isolation conditions. 8 Lactoperoxidase-Catalyzed Iodination Lactoperoxidase is used to oxidize 125I so that it rapidly iodinates tyrosine (and to a lesser extent histidine). The reaction catalyzed is diagrammed in Fig. 1. Because the platelet membrane is impermeable to lactoperoxidase (Mr 78,000), the iodination reaction occurs primarily with proteins on the platelet surface. 2'4 The reader is referred to Ref. 9 for a complete discussion of the iodination reaction and a description of the products produced. Platelets from freshly drawn blood are washed and suspended at ambient temperature in normal Tyrode's buffer (138 mM sodium chloride, 2.7 m M potassium chloride, 12 mM sodium bicarbonate, 0.36 mM sodium phosphate, 1.8 mM calcium chloride, 0.49 mM magnesium chloride, and 5.5 mM glucose, pH 7.4) to a platelet count of 109/ml. Other platelet washing buffers have been used, and all have proven suitable, providing they are free of protein, do not contain inhibitors of lactoperoxidase, and do not disproportionate hydrogen peroxide. To I ml of the platelet suspension, 1 mCi of carrier-flee NaJ25I is added, with gentle stirring at ambient temperature, followed by 0.25 nmol of lactoperoxidase (Sigma, St. Louis, MO) and five 10-/A aliquots of freshly prepared 3 mM hydrogen peroxide, added at 10-sec intervals. The use of flesh isotope (within 1 month of preparation) is desirable to minimize the presence ofI 2 and other oxidized species of iodide. Lactoperoxidase is stable when stored frozen in solution. The 3 m M hydrogen peroxide solution should be freshly prepared at 4° in 1 m M EDTA to avoid any disproportionation reaction. 8 j. E. B. Fox, C. C. Reynolds, and J. K. Boyles, this volume [6]. 9 M. Morrison, this series, Vol. 70, p. 214.
[35]
SURFACE LABELING OF PLATELETS
415
The labeled platelets are diluted 10-fold with Tyrode's buffer, and are sedimented by centrifugation at 2000 g for 15 min at 4 °. The labeled platelets are washed twice by resuspension in 10 ml of the Tyrode's buffer followed by centrifugation. Less than I% of the isotope is covalently bound to protein under these conditions. Higher yields can be achieved through more additions of hydrogen peroxide but should be avoided because platelets are activated by high concentrations of this oxidizing agent. Most of the 125I in the washed platelets (>90%) is intracellular, not bound to protein, and difficult to remove by these procedures. Platelets labeled by lactoperoxidase-catalyzed iodination are functional in that they secrete serotonin and aggregate when treated with thrombin. A modification of the lactoperoxidase-catalyzed iodination procedure can be used to monitor changes of the platelet surface that occur during physiological responses of the platelet. ~0In this modification, the iodination reaction is initiated by hydrogen peroxide and is terminated 15 sec later by the addition of catalase, which disproportionates all remaining hydrogen peroxide. An example of this reaction is illustrated in Fig. 2, in which platelet surface proteins were iodinated during aggregation. In this instance, platelet aggregation is monitored by light scattering in an aggregometer. Na~25I and lactoperoxidase are added before the agonist (thrombin). The iodination reaction is initiated 1 min later by the single addition of hydrogen peroxide. After 15 sec, the iodination reaction is terminated by adding catalase. To separate platelets and associated proteins from the nonassociated secreted proteins that are also labeled, the samples are carefully layered on 15% sucrose in Tyrode's solution and centrifuged for 3 min at 8500 g (microfuge; Beckman Instruments, Palo Alto, CA). The resulting pellet is processed for electrophoresis. The proteins that have become labeled during platelet stimulation are identified by comparing the proteins labeled in stimulated platelets to those labeled in unstimulated platelets. Iaeriodate/Sodium
Borohydride
Labeling
The periodate/sodium boro[3H]hydride procedure labels sialic acid residues on membrane glycoproteins and glycolipids3 (see Fig. 3). Periodate is used to cleave oxidatively carbon-carbon bonds between the 7-8 or 8-9 positions of sialic acid residues. The resulting aldehyde is then reduced with sodium boro[3H]hydride, producing the stable, radiolabeled alcohol derivative. Labeling can be restricted to the outer membrane surface by equilibrating platelets to 4° (which renders the membrane impermeable to the oxidizing agent) before exposing them to periodate. Io D. R. Phillips, L. K. Jennings, and H. R. Prasanna, J. Biol. Chem. 255, 11629 (1980).
416
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[35]
H202 Cat
c
.o Cat
l
r-
H202A /
I,-
Si,
Time I 1 rain
FIG. 2. lodination of platelets during thrombin-induced aggregation. Washed platelets (5 × 108) were suspended in 0.5 ml Tyrode's solution and iodinated either 10 or 60 sec after the induction of aggregation. The reaction was performed with stirring: aggregation was monitored by the decrease in light transmittance. Additions at the arrows were as follows: I, 0.25 mCi carrier-free 1251; LP, 1.5 × 10 -I° mol lactoperoxidase; Th, 0.05 unit thrombin; H202, 3.5 × 10 -9 mol hydrogen peroxide; Cat, 3.5 × 10 -9 mol catalase.
Platelets from freshly drawn blood are washed and suspended in 1 ml of a buffer containing 150 mM sodium chloride and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.6. Tris buffers are to be avoided as they quench the periodate. The suspension is equilibrated to 4 ° and, after the addition of sodium periodate (1 mM final concentration, freshly prepared), is incubated an additional 10 min at 4° in the
_~~ , - Acooc ~3.~-NoB.~,°,--k~. _ii_iiiii~iiA¸i¸ic~i !' i! I0~
Fit3. 3. The periodate/sodium borohydride labeling procedure.
[35]
SURFACE LABELING OF PLATELETS
417
dark. All remaining steps are performed at ambient temperature. The periodate-treated platelets are removed from solution by centrifugation at 800 g for 10 rain at 4°, washed once with the HEPES buffer, and resuspended in 1 ml of this buffer. Sodium boro[3H]hydride (0.5 mCi) is added, and the suspension is incubated for 5 rain. The sodium boro[3H]hydride should be stored in one-use-size aliquots at - 80° in 0.1 M sodium hydroxide and thawed immediately before use. The volume added should not affect the pH of the platelet suspension. The labeled platelets are centrifuged, washed once with the HEPES buffer, and resuspended in the original volume. Higher specific activities of labeled platelets can be achieved by (1) using more sodium boro[3H]hydride, (2) decreasing the pH of the periodate oxidation to 6, and (3) increasing to 8 the pH of the borohydride reduction, u Identification of Labeled Membrane Glycoproteins Membrane glycoproteins labeled by either the lactoperoxidase or periodate procedures are readily identifiable by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, as is illustrated in Fig. 4. In platelets labeled by lactoperoxidate-catalyzed iodination, the labeled proteins are detected by autoradiography of the dried gel. 4 As shown in lanes 2 and 4 of Fig. 4, glycoprotein (GP) IIb and GPIIIa are the most prominently labeled bands, reflecting their abundance in platelets (-50,000 copies per platelet). Glycoprotein IIIa (Mr 114,000, reduced) labels approximately three times more intensely by this method than does GPIIb (Mr 132,000, reduced), even though the two glycoproteins are present in equal concentrations; both of the disulfide-linked subunits of GPIIb, GPIIb~, and GPIIb~ are labeled. The identity of the GPIIb and GPIIIa bands can be confirmed by analyzing various parameters of these glycoproteins: (1) characteristic shifts in molecular weight on disulfide reduction (Mr 95,000 for nonreduced GPIIIa and Mr 142,000 for nonreduced GPIIb~2); (2) their absence in platelets from patients with Glanzmann's thrombasthenia% (3) staining by the periodic acid-Schiff reagent 2, (4) binding to Lens culinaris lectin 4, and (5) coimmunoprecipitation from Ca2+-con taining buffers by monoclonal antibodies ~3 or antibodies specific for one of the two glycoproteins in the complex. Electrophoresis of nonreduced samples of SDS-solubilized platelets should be performed immediately after solubilization to avoid protein polymerization. Alternatively, the u B. Steiner, K. J. Clemetson, and E. F. Liischer, Thromb. Res. 29, 43 (1983). t2 D. R. Phillips and P. P. Agin, J. Biol. Chem. 252, 2121 (1977). 13 R. P. McEver, E. M. Bennett, and M. N. Martin, J. Biol. Chem. 258, 5269 (1983).
418
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
Coomassie Blue
1251
12s I
aH
[35]
3H
;~i;~i:i!!i
200K G P lib,,, Gp11Ta -
lOOK
-GPIba -GPma -GPmb - GP I b a GPllb<~
5OK
-
-GPma i
- GPITIb
25K GP/Ib/3- m I
10% Acrylamide ~ J
6% A c r y l a m i d e
FIG. 4. Detection of cell surface glycoproteins by lactoperoxidase-catalyzed iodination and periodate/sodium boro[3H]hydride labeling. Proteins in lanes 1-3 were separated by electrophoresis in SDS through 10% polyacrylamide gels; 6% polyacrylamide gels were used in lanes 4 and 5. Lane 1 shows the Coomassie Brilliant blue-stained proteins in whole platelets; lanes 2 and 4 are autoradiograms of SDS gels showing the cell surface glycoproteins labeled by lactoperoxidase-catalyzed iodination; lanes 3 and 5 are fluorograms of SDS gels showing the cell surface glycoproteins labeled by the periodate/sodium boro[3H]hydride technique. The molecular weight scale is indicated.
solubilized samples can be stored in an anaerobic solution or treated with N-ethylmaleimide. ~2 The GPIIIb band (M r 97,000, also termed GPIV) is the primary labeled band just below reduced GPIIIa, and is recognizable because, unlike GPIIIa, it does not change electrophoretic mobility on disulfide bond reduction 4 and is resistant to chymotrypsin hydrolysis on intact platelets. 5 There are several labeled bands above GPIIb~ that can be visualized by nonreduced-reduced two-dimensional electrophoresis.22 Identity of these and other bands is also confirmed by electrophoresis according to O'Farrell (see Ref. 14) and immunoprecipitation with monospecific antibodies. 15 In platelets labeled by the periodate/sodium boro[3H]hydride procedure, the labeled glycoproteins are detected by autofluorography of the 14 K. J. Clemetson, A. Capitanio, and E. F. Liischer, Biochim. Biophys. Acta 553, 11 (1979). ~5 R. P. McEver and M. N. Martin, J. Biol. Chem. 259, 9799 (1984).
[35]
SURFACE LABELING OF PLATELETS
419
dried SDS-polyacrylamide gel. 16 Lanes 3 and 5 of Fig. 4 illustrate the glycoproteins labeled by this procedure; increased exposure of the film to the gel will permit detection of more than 30 labeled glycoproteins. Glycoprotein Ib is the most prominent of the labeled glycoproteins, reflecting its abundance in platelets (-30,000 copies per platelet) and its high sialic acid content. Both of the disulfide-linked subunits of GPIb are labeled by this procedure; GPIb~, Mr 128,000-141,000; and GPIb B, M r 22,000. The heterogeneity in the molecular weight of GPIb~ is due to the presence of allelic variants, which are usually quite rare. ~7 The identity of GPIb can be confirmed by several of its properties: (1) characteristic shift in molecular weight on reduction of disulfide bonds (Mr 170,000, nonreduced~2), (2) absence in platelets from patients with Bernard-Soulier syndrome ~8, (3) intense staining by the periodic acid-Schiff reagent 2, (4) binding to wheat germ agglutinin 19, (5) immunoprecipitation with monoclonal antibodies 2°, and (6) selective hydrolysis by Ca2+-dependent protease treatment of intact platelets. 21 Glycoprotein IX (Mr 17,000), also labeled by the periodate procedure, exists as a complex with GPIb 22 and therefore coimmunoprecipitates with GPIb. Fibrinogen, thrombospondin, and other a granule proteins and glycoproteins are not normally labeled by either of the surface-labeling methods described here. They do become prominently labeled components, however, if platelets have undergone the release reaction prior to labeling.l° Accordingly, care must be taken to maintain platelets in an unactivated state during isolation and to complete the labeling procedure without delay after isolation. Acknowledgments The work was supported by Grants HL28947 and HL 32254 from the National Institutes of Health. The author wishes to thank James X. Warger and Norma Jean Gargasz for graphics, Barbara Allen and Sally Gullatt Seehafer for editorial assistance, and Michele Prator and Linda Harris Odumade for manuscript preparation.
t6 W. A. Bonner and R. A. Laskey, Eur. J. Biochem. 46, 83 (1974). 17 M. Moroi, S. M. Jung, and N. Yoshida, Blood 64, 622 (1984). 18 A. T. Nurden and J. P. Caen, Nature (London) 255, 720 (1975). I9 K. J. Clemetson, S. L. Pfueller, E. F. Li~scher, and C. S. P. Jenkins, Biochim. Biophys. Acta 464, 493 (1977). 2o A. J. McMichael, N. A. Rust, J. R. Pilch, R. Sochyinsky, J. Morton, D. Y. Mason, C. Ruan, G. Tobelem, and J. Caen, Br. J. Haematol. 49, 501 (1981). 21 N. Yoshida, B. Weksler, and R. Nachman, J. Biol. Chem. 258, 7168 (1983). 2: M. C. Berndt, C. Gregory, A. Kabral, H. Zola, D. Fournier, and P. A. Castaldi, Eur. J. Biochem. 151, 637 (1985).