[34]
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
403
by different agonists. Others reported a lack of correlation between ADPinduced platelet aggregation and serum insulin levels in patients with type I and type II diabetes 19 and a lack of effect of continuous subcutaneous insulin infusion on platelet aggregation to ADP and epinephrine or thromboxane B 2 generation. 2° t9 D. B. Jones, T. M. Davis, E. Brown, R. D. Carter, J. I. Mann, and R. J. Prescott, Diabetologia 29, 291 (1986). 2o L. H. Monnier, M. Rodier, A. Gancel, P. Crastes De Paulet, C. Colette, M. Piperno, and J. Crastes DePaulet, Diabete Metab. 13, 210 (1987).
[34] M e m b r a n e - I m p e r m e a n t Cross-Linking Reagents for S t r u c t u r a l a n d F u n c t i o n a l A n a l y s e s o f P l a t e l e t Membrane Glycoproteins
By JAMES V.
STAROS,
NICOLAS J. KOT1TE, and
LEON W. CUNNINGHAM
The interactions among platelet membrane glycoproteins and between these molecules and macromolecular components of the subendothelial matrix and of the plasma have been areas of great and growing interest. Chemical cross-linking is a technique that has proven useful in many studies of protein-protein interactions. 1-4 Indeed, cross-linking has provided useful information concerning platelet supramolecular structure 5-s and the interactions of specific macromolecules 7,9-J4 or synthetic pepF. Wold, this series, Vol. 25, p. 623. 2 K. Peters and F. M. Richards, Annu. Rev. Biochem. 46, 523 (1977). 3 T. H. Ji, this series, Vol. 91, p. 580. 4 j. V. Staros and P. S. R. Anjaneyulu, this series, Vol. 172, p. 609. 5 G. E. Davies and J. Palek, Blood 59, 502 (1982). 6 S. M. Jung and M. Moroi, Biochim. Biophys. Acta 761, 152 (1983). v N. J. Kotite, J. V. Staros, and L. W. Cunningham, Biochemistry 23, 3099 (1984). s A. Sonnenberg, H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers, J. Biol. Chem. 262, 10376 (1987). 9 N. E. Larsen and E. R. Simons, Biochemistry 20, 4141 (1981). l0 j. Lahav, M. A. Schwartz, and R. O. Hynes, Cell (Cambridge, Mass.) 31, 253 (1982). II j. Takamatsu, M. K. Horne, Ill, and H. R. Gralnick, J. Clin. Invest. 77, 362 (1986). t2 F. C. Molinas, J. Wietzerbin, and E. Falcoff, J. lmmunol. 138, 802 (1987). t~ M. Jandrot-Perrus, D. Didry, M.-C. Guillin, and A. T. Nurden, Eur. J. Biochem. 174, 359 (1988). ~4 R. K. Andrews, J. J. Gorman, W. J. Booth, G. L. Corino, P. A. Castaldi, and M. C. Berndt, Biochemistt3' 28, 8326 (1989).
METHODS IN ENZYMOLOGY.VOL. 215
Copyright © 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.
404
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[34]
o -o3sT-~J(\ o o L SO3II II / "IF L~oN-O-C-CHa-CH2-S-S-CH2-CH2-C-O-N0~ DTSSP
½N ,o
o,
-O-C-CHz -CH2-cHz-cHz-CH z -CHz--C-0-N .'
FIG. 1. The structures of 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) and bis (sulfosuccinimidyl) suberate (BS3). (Reprinted from Ref. 20 with permission. Copyright 1982 American Chemical Society.)
tides related to such macromolecules ]5-~8 with platelet surface components. One complication encountered in cross-linking studies of cell surfaces has been that the reagents available prior to the last decade were all membrane permeant, so that internal cellular components as well as surface components reacted with them. For example, when intact platelets were treated with the membrane-permeant reagent 3,3'-dithiobis(succinimidyl propionate), cytoskeletal components were the most prominent cross-linked complexes detected. 5 For many studies that focus on macromolecular interactions at the membrane surface, it would be desirable to restrict reactions to the extracytoplasmic surface of the cell. The introduction of membrane-impermeant cross-linkers 4']9-21 provides a class of reagents that meets this criterion. This chapter will focus on the preparation of bifunctional N-hydroxysulfosuccinimide active esters of dicarboxylic acids (Fig. 1) as high-yield, membrane-impermeant cross-linking reagents, 2° and their application to human platelet structure and function. 7 J5 S. A. Santoro and W. J. Lawing, Jr., Cell (Cambridge, Mass.) 48, 867 (1987). J6 S. E. D'Souza, M. H. Ginsberg, S. C.-T. Lam, and E. F. Plow, J. Biol. Chem. 263, 3943 (1988). 17 S. E. D'Souza, M. H. Ginsberg, T. A. Burke, S. C.-T. Lam, and E. F. Plow, Science 242, 91 (1988). ~8 S. E. D'Souza, M. H. Ginsberg, T. A. Burke, and E. F. Plow, J. Biol. Chem. 265, 3440 (1990). 19 j. V. Staros, D. G. Morgan, and D. R. Appling, J. Biol. Chem. 256, 5890 (1981). 2o j. V. Staros, Biochemistry 21, 3950 (1982). 2I j. V. Staros, Acc. Chem. Res. 21, 435 (1988).
[34]
405
C R O S S - L I N K I N G OF 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
0
,o r/'L"rS°; R-C-O-NN, A + R2-NH2 0
.o,, ~ R'-C-NH-R
0
+
N SO; HO0
FIG. 2. The reaction of a sulfosuccinimidyl active ester with a primary amino group to form an amide linkage with release of N-hydroxysulfosuccinimide. Sulfosuccinimidyl active esters react with amino groups to form stable amide bonds, with loss of N-hydroxysulfosuccinimide (Fig. 2). The sulfonate groups on the sulfosuccinimide rings render these reagents highly w a t e r soluble and m e m b r a n e impermeant. 2°a~ In the absence of nucleophiles, sulfosuccinimidyl active esters hydrolyze very slowly as c o m p a r e d with their rate of reaction with amino groups, 22 resulting in a very high yield of covalent linkage. Beside the chemical properties of a cross-linking reagent, another important criterion in reagent design is span, i.e., the distance between the reacted groups on the protein spanned by the cross-linker. For the alltrans configurations of the two reagents shown in Fig. 1, this distance is 11-12 A, as m e a s u r e d with C P K models. What does this distance m e a n in molecular terms? Within myoglobin, which is essentially a bundle of closely packed a helices, the center-to-center distance of a helices in pairwise contact averages 9 ,~.23 Thus, the two cross-linkers shown in Fig. 1 span a distance c o m p a r a b l e to the center-to-center distance between two closely p a c k e d a helices. It is important to r e m e m b e r when interpreting e x p e r i m e n t s employing these reagents that the cross-links formed are, on the scale of proteins, intramolecular in terms of distance. When intermoiecular cross-links are formed, it is implied that the two participating proteins were in very close proximity at the time of cross-linking. E x p e r i m e n t a l Procedures R e a g e n t s 24 S y n t h e s i s o f N - H y d r o x y s u l f o s u c c i n i m i d e Sodium Salt. 2° It is very important to keep N - h y d r o x y m a l e i m i d e , the immediate precursor of N - h y d r o x y s u l f o s u c c i n i m i d e , in an inert atmosphere. Therefore, solutions
22p. s. R. Anjaneyulu and J. V. Staros, Int. J. Pept. Protein Res. 30, ll7 (1987). 23T. J. Richmond and F. M. Richards, J. Mol. Biol. 119, 537 (1978). 24Commercial sources are now available for N-hydroxysulfosuccinimide sodium salt (Fluka Chemical Corp., Ronkonkoma, NY, Pierce Chemical Co., Rockford, II, Aldrich Chemical Co., Milwaukee, WI) and for cross-linking reagents incorporating this compound (Pierce Chemical Co.).
406
P L A T E L E T R E C E P T O R S : ASSAYS A N D P U R I F I C A T I O N
[34]
of this compound are prepared in a glove bag filled with N 2 and are transferred to a purged and Nz-filled reaction vessel in a syringe. In this manner, N-hydroxymaleimide (Fluka Chemical Corp.) (1.45 g, 12.8 mmol) is dissolved in absolute ethanol (15 ml) and the resulting solution is transferred to the reaction vessel under N 2. An aqueous solution (10 ml) of NazS205 (1.22 g, 6.4 mmol) is then added with stirring. The reaction mixture is stirred at room temperature for 2 hr under N2, and the vessel is then opened to the atmosphere. After the solvent is removed by rotary evaporation (bath temperature -40°), the product, a thick yellow oil, is dissolved in 50 ml of H20, filtered, and lyophilized. The resulting light yellow solid is triturated overnight with anhydrous ether. An off-white powder is then recovered by filtration [yield, 2.66 g (96%)]. This product forms a single spot when subjected to thin-layer chromatography on silica gel plates (0.20 mm with fluorescent indicator on aluminum backing from EM Industries, Hawthorn, NY) developed in 5 : 2 : 3 1-butanol-acetic acid-water. It gives a single peak when subjected to ion-pair reversedphase high-performance liquid chromatography (HPLC) using a C j8 column (Alltech, Deerfield, IL; Cat. No. 600RP) and a mobile phase of aqueous l0 mM tetrabutylammonium formate, pH 4.0 and methanol (60 : 40). If necessary, the product may be recrystallized from 95% ethanol.
Synthesis of 3,3'-Dithiobis(sulfosuccinimidyl propionate) Disodium Salt (DTSSP). 2° N-Hydroxysulfosuccinimide sodium salt (0.44 g, 2.0 mmol), 3,3'-dithiodipropionic acid (Aldrich Chemical Co., Milwaukee, WI) (0.21 g, 1.0 mmol), and N,N'-dicyclohexylcarbodiimide (Aldrich) (0.46 g, 2.2 mmol) are dissolved in 5.0 ml of anhydrous dimethylformamide. The reaction vessel is capped and the reaction mixture stirred overnight at room temperature. The reaction mixture is then cooled to 3° and stirred for 2-3 hr. The precipitated dicyclohexylurea is removed by filtration and washed with a small quantity of dry dimethylformamide. The product is then precipitated from the pooled filtrate by addition of - 2 0 vol of ethyl acetate, recovered by filtration, and stored in a vacuum dessicator [yield, 0.40 g (65%)], assuming a pure anhydrous product. The noncleavable cross-linker bis(sulfosuccinimidyl) suberate (BS 3) is synthesized by the same method by substituting suberic acid (0.175 g, 1.0 mmol) for 3,3'dithiodipropionic acid. DTSSP, BS 3, and other homologous cross-linkers are routinely assayed in our laboratory by testing their ability to crosslink rabbit muscle aldolase, as described below.
Methods Cross-Linking of Rabbit Muscle Aldolase.2° A suspension of crystalline rabbit muscle aldolase in 2.5 M (NH4)2SO4 (type IV, Sigma Chemical Co., St. Louis, MO) is exhaustively dialyzed against 50 mM sodium phosphate,
[34]
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
407
pH 7.4. The final concentration of aldolase is determined by absorbance l~ = 9.38).25 Equal aliquots are diluted with 50 mM sodium at 280 nm tr, ~,-,280 phosphate, pH 7.4, and are treated with various concentrations of crosslinker that have been prepared immediately before use as a I0 mM stock solution in the same buffer. After incubation for 30 min at room temperature, the reactions are quenched by addition of one-sixth volume of 50 mM ethanolamine, 20 mM N-ethylmaleimide, 50 mM sodium phosphate, pH 7.4. To each sample is then added sodium dodecyl sulfate (SDS) gelsolubilizing solution, with or without reductant, as required. Samples are incubated at 50° for 3 min, and then stored at - 6 5 °. Treatment o f Platelets with DTSSP. 7 Platelets are isolated 26'27 from freshly drawn blood and resuspended in platelet buffer (137 mM NaC1, 2.7 mM KC1, 4.25 mM Na2HPO4, 1.5 mM KH2PO4, 5 mM glucose 2 mM EDTA, pH 7.4) at a concentration of 0.5-1 x 109/ml. For some experiments, platelets are radiolabeled by the periodate-boro[3H]hydride method. 7 A 10 mM stock of DTSSP is prepared in the same buffer immediately before use. Appropriate aliquots of the DTSSP stock are added to samples of the platelet suspension (5 × 108/ml), and the reaction is allowed to proceed for 20 min at room temperature. The reaction is quenched by addition of Tris-HC1, pH 7.4, to a final concentration of 0.2 M, and by incubation for 5 rain. (Subsequent studies have suggested that other primary amines are better quenching agents than Tris. In other studies, such as the cross-linking of rabbit muscle aldolase described above, we have employed ethanolamine. We prepare a quench buffer by adding ethanolamine, typically to a final concentration of 20-50 mM, to a sample of the same buffer used for the reaction, and adjusting the pH with HCI back to its previous value.) Six volumes of platelet buffer are then added to the samples, which are then pelleted by centrifugation at 1200 g for 10 rain at 4 °. For aggregation assays, the platelets are washed once in aggregation buffer (platelet buffer without EDTA and with the addition of bovine serum albumin to 0.35%) by resuspension and pelleting as above, and are finally resuspended in aggregation buffer at a concentration of 2.5 x 108/ml. Immediately prior to assay, samples are adjusted to 2 mM CaClz, 1 mM MgC12, and 0.05% fibrinogen and are subjected to an aggregation assay 28'29 in a Chronolog aggregometer (Chronolog Corp., Haverstown, PA). 25 j. W. D o n o v a n , Biochemistry 3, 67 (1964). 26 S. A. Santoro and L. W. C u n n i n g h a m , Proc. Natl. Acad. Sei. U.S.A. 76, 2644 (1979). -,7 See also J. F. Mustard, R. L. K i n l o u g h - R a t h b o n e , and M. A. Packham, this series, Vol. 169, p. 3; S. T i m m o n s and J. Hawiger, ibid. p. 11. 28 S. A. Santoro and L. W. C u n n i n g h a m , J. Clin. Invest. 60, 1054 (1977). 29 See also M. Zucker, this series, Vol. 169, p. 117.
408
PLATELET RECEPTORS" ASSAYS AND PURIFICATION
[34]
For analysis of the effects of cross-linking on the profile of platelet surface glycoproteins, the platelets are washed once as above, but in platelet buffer containing 20 mM N-ethylmaleimide. This treatment alkylates free thiols to prevent thiol-disulfide exchange from scrambling the cross-linked products when the cells are disrupted. 19,20,30Finally, the platelets are washed once more in platelet buffer without N-ethylmaleimide, and the resulting pellets are stored at - 70° until dissolved for analysis by SDS-polyacrylamide gel electrophoresis. Results Cross-Linking o f Rabbit Muscle AIdolase. Once a cross-linking reagent has been prepared or purchased, it is important to assay its cross-linking activity in a well-defined system. We routinely assay all new reagents as well as all new batches of our well-characterized reagents by testing their ability to cross-link rabbit muscle aldolase, a tetrameric protein, under standard conditions. An example of a quality control assay testing a new batch of DTSSP against the current stock of the same reagent is shown in Fig. 3. In the assay the two batches of DTSSP were reacted with aldolase under identical conditions. This assay is also very useful for testing whether a new reagent is as efficient a cross-linker as a given standard or whether a proposed reaction buffer will support efficient cross-linking. Thus it is often useful to react aldolase with the cross-linker of choice in a new buffer and to compare the results to the reaction of identical concentrations of aldolase in the standard buffer with identical concentrations of the same reagent. Control samples, reacted with DTSSP but dissolved in SDS gel-solubilizing solution containing dithiothreitol and subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions, ran essentially quantitatively as monomers, i.e., like the sample in Fig. 3, lane 1.2° Effect o f D T S S P or BS 3 on Platelet Aggregation. 7 Figure 4 shows the results of treating platelets with various concentrations of DTSSP or BS 3 and subjecting them to an assay for aggregation induced by collagen. The response to collagen of platelets treated with either cross-linker was sharply reduced. Under the conditions of the experiment depicted in Fig. 3, the ECs0 of DTSSP for inhibition of collagen-induced aggregation was - 2 / z M and that of BS 3 was - 8 / z M . To distinguish whether the inhibition of collagen-induced platelet aggregation was the result of cross-linking of surface components or simply of the acylation of surface groups, platelets were treated with sulfosuccinimidyl propionate, a monofunctional analog of DTSSP, and were then 30 K. W a n g and F. M. Richards, J. Biol. Chem. 249, 8005 (1974).
[34]
409
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
Tetra~ Tri Di~
Mono--
1
2 A
3 B
A
4 B
A
B
FIG. 3. Reaction of rabbit muscle aldolase with two different preparations of DTSSP. Rabbit muscle aldolase, 1 mg/ml, was reacted with the following concentrations of DTSSP, as described in the test: lane 1,0; lanes 2, 0.05 mM; lanes 3, 0.20 raM; lanes 4, 1.0 mM. After quenching the reaction, samples were taken up in SDS gel-solubilizing solution without reductant and were subjected to SDS-polyacrylamide gel electrophoresis under nonreducing conditions. A and B denote two different synthetic preparations of DTSSP. Mono, Di, Tri, and Tetra refer to the positions of monomers, dimers, trimers, and tetramers of aldolase subunits, respectively.
subjected to the aggregation assay. No inhibition of collagen-induced platelet aggregation was observed up to a concentration of 200/xM sulfosuccinimidyl propionate, suggesting that it is cross-linking and not simply acylation of surface components that results in the observed inhibition. The inhibition of aggregation appears to be specific to the fibrillar collagen induction pathway. Treatment of platelets with DTSSP was found to have no effect on aggregation in response to thrombin or on plateletcollagen adhesion. 7 Effect ofBS 3 on Sodium Dodecyl Sulfate-Polyacrylamide Gel Profile of Platelet Surface Glycoproteins. 7 To assess whether intermolecular crosslinks formed between platelet surface glycoproteins might correlate with the inhibition of collagen-induced aggregation, radiolabeled platelets were treated with BS 3, dissolved in SDS gel buffer, and subjected to SDS-polya-
410
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[34]
IOO' z
o
8o
I-~9 UJ
60
20
0
/~
0
I
I
I
I
III
~1
I
i
I
I
I tJtll
~M
i
i
t
Itllll
I0
I00
FIG. 4. Effects of DTSSP ((3), BS 3 ([:]), and a monofunctional analog, sulfosuccinimidyl propionate (SSP) (A), on collagen-induced platelet aggregation. The concentration of SSP used was twice that indicated on the abscissa, so that the concentration of reactive groups would be the same as in the samples with the cross-linkers. Percentage aggregation was calculated from the slope of the aggregation profiles. The values shown represent the average of three determination -+ SD. (Reprinted from Ref, 7 with permission. Copyright 1984 American Chemical Society.)
crylamide gel electrophoresis. The resulting gel was subjected to fluorography, 3t and the fluorograph was scanned with a fiber optic densitometer (model 800; Kontes). The results of this analysis are shown in Fig. 5. At low concentrations of BS 3, which effectively inhibit collagen-induced platelet aggregation, radiolabeled bands corresponding to glycoproteins IIb, Ilia, and IV were significantly reduced in the SDS gel profile. In addition, the bands corresponding to glycoproteins IIb and Ilia disappeared in parallel, suggesting that they were cross-linked to one another. This observation is consistent with independent evidence that these proteins exist as a 1 : 1 noncovalent complex 32 that has been recognized as an important platelet integrin. 33
3[ W. M. Bonner and R. A. Laskey, Eur. J. Biochem. 46, 83 (1974). 32 L. K. Jennings and D. R. Phillips, J. Biol. Chem. 257, 10458 0982). 33 R. O. Hynes, Cell (Cambridge, Mass.) 48, 549 (1987).
[34]
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
411
20C
Z
z_ 180 / on~ O: hi t-LL Z
0
w
II. 0 (J >-
"J
(.9
IO(:~
8O
60
\\ 40
20
Ot¢ 0
I
I0
I00
I000
BS3(p.M) FIG. 5. Effect of BS 3 on the SDS gel profile of radiolabeled platelet surface glycoproteins. Radiolabeled proteins were treated with varying concentrations of BS 3. The reactions were quenched, and the samples were dissolved in SDS gel-solubilizing solution and were subjected to SDS-polyacrylamide gel electrophoresis. The resulting gel was subjected to fluorography, and the resulting fluorograph was analyzed densitometrically. The percentage of each glycoprotein at various concentrationsof BS 3 was calculated with reference to the uncross-linked control. (O), 150K; (A), GPIb; ((3), GPIIb; (12), GPIIIa; (A), GPIV. (Reprinted from Ref. 7 with permission. Copyright 1984 American Chemical Society.)
Discussion The advent of high-yield, membrane-impermeant cross-linking reagents has allowed the experimenter to probe protein-protein interactions at one face of a membrane. Platelets appear to present an especially rich variety of questions that can be addressed with these reagents. These include the identification of platelet surface proteins that specifically interact with protein components of the subendothelial matrix and of the plasma, as well as possible changes in tertiary or quaternary structure of surface proteins that accompany platelet activation. Initial studies in which these reagents have been used to cross-link
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.
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
403
by different agonists. Others reported a lack of correlation between ADPinduced platelet aggregation and serum insulin levels in patients with type I and type II diabetes 19 and a lack of effect of continuous subcutaneous insulin infusion on platelet aggregation to ADP and epinephrine or thromboxane B 2 generation. 2° t9 D. B. Jones, T. M. Davis, E. Brown, R. D. Carter, J. I. Mann, and R. J. Prescott, Diabetologia 29, 291 (1986). 2o L. H. Monnier, M. Rodier, A. Gancel, P. Crastes De Paulet, C. Colette, M. Piperno, and J. Crastes DePaulet, Diabete Metab. 13, 210 (1987).
[34] M e m b r a n e - I m p e r m e a n t Cross-Linking Reagents for S t r u c t u r a l a n d F u n c t i o n a l A n a l y s e s o f P l a t e l e t Membrane Glycoproteins
By JAMES V.
STAROS,
NICOLAS J. KOT1TE, and
LEON W. CUNNINGHAM
The interactions among platelet membrane glycoproteins and between these molecules and macromolecular components of the subendothelial matrix and of the plasma have been areas of great and growing interest. Chemical cross-linking is a technique that has proven useful in many studies of protein-protein interactions. 1-4 Indeed, cross-linking has provided useful information concerning platelet supramolecular structure 5-s and the interactions of specific macromolecules 7,9-J4 or synthetic pepF. Wold, this series, Vol. 25, p. 623. 2 K. Peters and F. M. Richards, Annu. Rev. Biochem. 46, 523 (1977). 3 T. H. Ji, this series, Vol. 91, p. 580. 4 j. V. Staros and P. S. R. Anjaneyulu, this series, Vol. 172, p. 609. 5 G. E. Davies and J. Palek, Blood 59, 502 (1982). 6 S. M. Jung and M. Moroi, Biochim. Biophys. Acta 761, 152 (1983). v N. J. Kotite, J. V. Staros, and L. W. Cunningham, Biochemistry 23, 3099 (1984). s A. Sonnenberg, H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers, J. Biol. Chem. 262, 10376 (1987). 9 N. E. Larsen and E. R. Simons, Biochemistry 20, 4141 (1981). l0 j. Lahav, M. A. Schwartz, and R. O. Hynes, Cell (Cambridge, Mass.) 31, 253 (1982). II j. Takamatsu, M. K. Horne, Ill, and H. R. Gralnick, J. Clin. Invest. 77, 362 (1986). t2 F. C. Molinas, J. Wietzerbin, and E. Falcoff, J. lmmunol. 138, 802 (1987). t~ M. Jandrot-Perrus, D. Didry, M.-C. Guillin, and A. T. Nurden, Eur. J. Biochem. 174, 359 (1988). ~4 R. K. Andrews, J. J. Gorman, W. J. Booth, G. L. Corino, P. A. Castaldi, and M. C. Berndt, Biochemistt3' 28, 8326 (1989).
METHODS IN ENZYMOLOGY.VOL. 215
Copyright © 1992by AcademicPress, Inc. All rights of reproduction in any form reserved.
404
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[34]
o -o3sT-~J(\ o o L SO3II II / "IF L~oN-O-C-CHa-CH2-S-S-CH2-CH2-C-O-N0~ DTSSP
½N ,o
o,
-O-C-CHz -CH2-cHz-cHz-CH z -CHz--C-0-N .'
FIG. 1. The structures of 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) and bis (sulfosuccinimidyl) suberate (BS3). (Reprinted from Ref. 20 with permission. Copyright 1982 American Chemical Society.)
tides related to such macromolecules ]5-~8 with platelet surface components. One complication encountered in cross-linking studies of cell surfaces has been that the reagents available prior to the last decade were all membrane permeant, so that internal cellular components as well as surface components reacted with them. For example, when intact platelets were treated with the membrane-permeant reagent 3,3'-dithiobis(succinimidyl propionate), cytoskeletal components were the most prominent cross-linked complexes detected. 5 For many studies that focus on macromolecular interactions at the membrane surface, it would be desirable to restrict reactions to the extracytoplasmic surface of the cell. The introduction of membrane-impermeant cross-linkers 4']9-21 provides a class of reagents that meets this criterion. This chapter will focus on the preparation of bifunctional N-hydroxysulfosuccinimide active esters of dicarboxylic acids (Fig. 1) as high-yield, membrane-impermeant cross-linking reagents, 2° and their application to human platelet structure and function. 7 J5 S. A. Santoro and W. J. Lawing, Jr., Cell (Cambridge, Mass.) 48, 867 (1987). J6 S. E. D'Souza, M. H. Ginsberg, S. C.-T. Lam, and E. F. Plow, J. Biol. Chem. 263, 3943 (1988). 17 S. E. D'Souza, M. H. Ginsberg, T. A. Burke, S. C.-T. Lam, and E. F. Plow, Science 242, 91 (1988). ~8 S. E. D'Souza, M. H. Ginsberg, T. A. Burke, and E. F. Plow, J. Biol. Chem. 265, 3440 (1990). 19 j. V. Staros, D. G. Morgan, and D. R. Appling, J. Biol. Chem. 256, 5890 (1981). 2o j. V. Staros, Biochemistry 21, 3950 (1982). 2I j. V. Staros, Acc. Chem. Res. 21, 435 (1988).
[34]
405
C R O S S - L I N K I N G OF 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
0
,o r/'L"rS°; R-C-O-NN, A + R2-NH2 0
.o,, ~ R'-C-NH-R
0
+
N SO; HO0
FIG. 2. The reaction of a sulfosuccinimidyl active ester with a primary amino group to form an amide linkage with release of N-hydroxysulfosuccinimide. Sulfosuccinimidyl active esters react with amino groups to form stable amide bonds, with loss of N-hydroxysulfosuccinimide (Fig. 2). The sulfonate groups on the sulfosuccinimide rings render these reagents highly w a t e r soluble and m e m b r a n e impermeant. 2°a~ In the absence of nucleophiles, sulfosuccinimidyl active esters hydrolyze very slowly as c o m p a r e d with their rate of reaction with amino groups, 22 resulting in a very high yield of covalent linkage. Beside the chemical properties of a cross-linking reagent, another important criterion in reagent design is span, i.e., the distance between the reacted groups on the protein spanned by the cross-linker. For the alltrans configurations of the two reagents shown in Fig. 1, this distance is 11-12 A, as m e a s u r e d with C P K models. What does this distance m e a n in molecular terms? Within myoglobin, which is essentially a bundle of closely packed a helices, the center-to-center distance of a helices in pairwise contact averages 9 ,~.23 Thus, the two cross-linkers shown in Fig. 1 span a distance c o m p a r a b l e to the center-to-center distance between two closely p a c k e d a helices. It is important to r e m e m b e r when interpreting e x p e r i m e n t s employing these reagents that the cross-links formed are, on the scale of proteins, intramolecular in terms of distance. When intermoiecular cross-links are formed, it is implied that the two participating proteins were in very close proximity at the time of cross-linking. E x p e r i m e n t a l Procedures R e a g e n t s 24 S y n t h e s i s o f N - H y d r o x y s u l f o s u c c i n i m i d e Sodium Salt. 2° It is very important to keep N - h y d r o x y m a l e i m i d e , the immediate precursor of N - h y d r o x y s u l f o s u c c i n i m i d e , in an inert atmosphere. Therefore, solutions
22p. s. R. Anjaneyulu and J. V. Staros, Int. J. Pept. Protein Res. 30, ll7 (1987). 23T. J. Richmond and F. M. Richards, J. Mol. Biol. 119, 537 (1978). 24Commercial sources are now available for N-hydroxysulfosuccinimide sodium salt (Fluka Chemical Corp., Ronkonkoma, NY, Pierce Chemical Co., Rockford, II, Aldrich Chemical Co., Milwaukee, WI) and for cross-linking reagents incorporating this compound (Pierce Chemical Co.).
406
P L A T E L E T R E C E P T O R S : ASSAYS A N D P U R I F I C A T I O N
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of this compound are prepared in a glove bag filled with N 2 and are transferred to a purged and Nz-filled reaction vessel in a syringe. In this manner, N-hydroxymaleimide (Fluka Chemical Corp.) (1.45 g, 12.8 mmol) is dissolved in absolute ethanol (15 ml) and the resulting solution is transferred to the reaction vessel under N 2. An aqueous solution (10 ml) of NazS205 (1.22 g, 6.4 mmol) is then added with stirring. The reaction mixture is stirred at room temperature for 2 hr under N2, and the vessel is then opened to the atmosphere. After the solvent is removed by rotary evaporation (bath temperature -40°), the product, a thick yellow oil, is dissolved in 50 ml of H20, filtered, and lyophilized. The resulting light yellow solid is triturated overnight with anhydrous ether. An off-white powder is then recovered by filtration [yield, 2.66 g (96%)]. This product forms a single spot when subjected to thin-layer chromatography on silica gel plates (0.20 mm with fluorescent indicator on aluminum backing from EM Industries, Hawthorn, NY) developed in 5 : 2 : 3 1-butanol-acetic acid-water. It gives a single peak when subjected to ion-pair reversedphase high-performance liquid chromatography (HPLC) using a C j8 column (Alltech, Deerfield, IL; Cat. No. 600RP) and a mobile phase of aqueous l0 mM tetrabutylammonium formate, pH 4.0 and methanol (60 : 40). If necessary, the product may be recrystallized from 95% ethanol.
Synthesis of 3,3'-Dithiobis(sulfosuccinimidyl propionate) Disodium Salt (DTSSP). 2° N-Hydroxysulfosuccinimide sodium salt (0.44 g, 2.0 mmol), 3,3'-dithiodipropionic acid (Aldrich Chemical Co., Milwaukee, WI) (0.21 g, 1.0 mmol), and N,N'-dicyclohexylcarbodiimide (Aldrich) (0.46 g, 2.2 mmol) are dissolved in 5.0 ml of anhydrous dimethylformamide. The reaction vessel is capped and the reaction mixture stirred overnight at room temperature. The reaction mixture is then cooled to 3° and stirred for 2-3 hr. The precipitated dicyclohexylurea is removed by filtration and washed with a small quantity of dry dimethylformamide. The product is then precipitated from the pooled filtrate by addition of - 2 0 vol of ethyl acetate, recovered by filtration, and stored in a vacuum dessicator [yield, 0.40 g (65%)], assuming a pure anhydrous product. The noncleavable cross-linker bis(sulfosuccinimidyl) suberate (BS 3) is synthesized by the same method by substituting suberic acid (0.175 g, 1.0 mmol) for 3,3'dithiodipropionic acid. DTSSP, BS 3, and other homologous cross-linkers are routinely assayed in our laboratory by testing their ability to crosslink rabbit muscle aldolase, as described below.
Methods Cross-Linking of Rabbit Muscle Aldolase.2° A suspension of crystalline rabbit muscle aldolase in 2.5 M (NH4)2SO4 (type IV, Sigma Chemical Co., St. Louis, MO) is exhaustively dialyzed against 50 mM sodium phosphate,
[34]
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
407
pH 7.4. The final concentration of aldolase is determined by absorbance l~ = 9.38).25 Equal aliquots are diluted with 50 mM sodium at 280 nm tr, ~,-,280 phosphate, pH 7.4, and are treated with various concentrations of crosslinker that have been prepared immediately before use as a I0 mM stock solution in the same buffer. After incubation for 30 min at room temperature, the reactions are quenched by addition of one-sixth volume of 50 mM ethanolamine, 20 mM N-ethylmaleimide, 50 mM sodium phosphate, pH 7.4. To each sample is then added sodium dodecyl sulfate (SDS) gelsolubilizing solution, with or without reductant, as required. Samples are incubated at 50° for 3 min, and then stored at - 6 5 °. Treatment o f Platelets with DTSSP. 7 Platelets are isolated 26'27 from freshly drawn blood and resuspended in platelet buffer (137 mM NaC1, 2.7 mM KC1, 4.25 mM Na2HPO4, 1.5 mM KH2PO4, 5 mM glucose 2 mM EDTA, pH 7.4) at a concentration of 0.5-1 x 109/ml. For some experiments, platelets are radiolabeled by the periodate-boro[3H]hydride method. 7 A 10 mM stock of DTSSP is prepared in the same buffer immediately before use. Appropriate aliquots of the DTSSP stock are added to samples of the platelet suspension (5 × 108/ml), and the reaction is allowed to proceed for 20 min at room temperature. The reaction is quenched by addition of Tris-HC1, pH 7.4, to a final concentration of 0.2 M, and by incubation for 5 rain. (Subsequent studies have suggested that other primary amines are better quenching agents than Tris. In other studies, such as the cross-linking of rabbit muscle aldolase described above, we have employed ethanolamine. We prepare a quench buffer by adding ethanolamine, typically to a final concentration of 20-50 mM, to a sample of the same buffer used for the reaction, and adjusting the pH with HCI back to its previous value.) Six volumes of platelet buffer are then added to the samples, which are then pelleted by centrifugation at 1200 g for 10 rain at 4 °. For aggregation assays, the platelets are washed once in aggregation buffer (platelet buffer without EDTA and with the addition of bovine serum albumin to 0.35%) by resuspension and pelleting as above, and are finally resuspended in aggregation buffer at a concentration of 2.5 x 108/ml. Immediately prior to assay, samples are adjusted to 2 mM CaClz, 1 mM MgC12, and 0.05% fibrinogen and are subjected to an aggregation assay 28'29 in a Chronolog aggregometer (Chronolog Corp., Haverstown, PA). 25 j. W. D o n o v a n , Biochemistry 3, 67 (1964). 26 S. A. Santoro and L. W. C u n n i n g h a m , Proc. Natl. Acad. Sei. U.S.A. 76, 2644 (1979). -,7 See also J. F. Mustard, R. L. K i n l o u g h - R a t h b o n e , and M. A. Packham, this series, Vol. 169, p. 3; S. T i m m o n s and J. Hawiger, ibid. p. 11. 28 S. A. Santoro and L. W. C u n n i n g h a m , J. Clin. Invest. 60, 1054 (1977). 29 See also M. Zucker, this series, Vol. 169, p. 117.
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PLATELET RECEPTORS" ASSAYS AND PURIFICATION
[34]
For analysis of the effects of cross-linking on the profile of platelet surface glycoproteins, the platelets are washed once as above, but in platelet buffer containing 20 mM N-ethylmaleimide. This treatment alkylates free thiols to prevent thiol-disulfide exchange from scrambling the cross-linked products when the cells are disrupted. 19,20,30Finally, the platelets are washed once more in platelet buffer without N-ethylmaleimide, and the resulting pellets are stored at - 70° until dissolved for analysis by SDS-polyacrylamide gel electrophoresis. Results Cross-Linking o f Rabbit Muscle AIdolase. Once a cross-linking reagent has been prepared or purchased, it is important to assay its cross-linking activity in a well-defined system. We routinely assay all new reagents as well as all new batches of our well-characterized reagents by testing their ability to cross-link rabbit muscle aldolase, a tetrameric protein, under standard conditions. An example of a quality control assay testing a new batch of DTSSP against the current stock of the same reagent is shown in Fig. 3. In the assay the two batches of DTSSP were reacted with aldolase under identical conditions. This assay is also very useful for testing whether a new reagent is as efficient a cross-linker as a given standard or whether a proposed reaction buffer will support efficient cross-linking. Thus it is often useful to react aldolase with the cross-linker of choice in a new buffer and to compare the results to the reaction of identical concentrations of aldolase in the standard buffer with identical concentrations of the same reagent. Control samples, reacted with DTSSP but dissolved in SDS gel-solubilizing solution containing dithiothreitol and subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions, ran essentially quantitatively as monomers, i.e., like the sample in Fig. 3, lane 1.2° Effect o f D T S S P or BS 3 on Platelet Aggregation. 7 Figure 4 shows the results of treating platelets with various concentrations of DTSSP or BS 3 and subjecting them to an assay for aggregation induced by collagen. The response to collagen of platelets treated with either cross-linker was sharply reduced. Under the conditions of the experiment depicted in Fig. 3, the ECs0 of DTSSP for inhibition of collagen-induced aggregation was - 2 / z M and that of BS 3 was - 8 / z M . To distinguish whether the inhibition of collagen-induced platelet aggregation was the result of cross-linking of surface components or simply of the acylation of surface groups, platelets were treated with sulfosuccinimidyl propionate, a monofunctional analog of DTSSP, and were then 30 K. W a n g and F. M. Richards, J. Biol. Chem. 249, 8005 (1974).
[34]
409
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
Tetra~ Tri Di~
Mono--
1
2 A
3 B
A
4 B
A
B
FIG. 3. Reaction of rabbit muscle aldolase with two different preparations of DTSSP. Rabbit muscle aldolase, 1 mg/ml, was reacted with the following concentrations of DTSSP, as described in the test: lane 1,0; lanes 2, 0.05 mM; lanes 3, 0.20 raM; lanes 4, 1.0 mM. After quenching the reaction, samples were taken up in SDS gel-solubilizing solution without reductant and were subjected to SDS-polyacrylamide gel electrophoresis under nonreducing conditions. A and B denote two different synthetic preparations of DTSSP. Mono, Di, Tri, and Tetra refer to the positions of monomers, dimers, trimers, and tetramers of aldolase subunits, respectively.
subjected to the aggregation assay. No inhibition of collagen-induced platelet aggregation was observed up to a concentration of 200/xM sulfosuccinimidyl propionate, suggesting that it is cross-linking and not simply acylation of surface components that results in the observed inhibition. The inhibition of aggregation appears to be specific to the fibrillar collagen induction pathway. Treatment of platelets with DTSSP was found to have no effect on aggregation in response to thrombin or on plateletcollagen adhesion. 7 Effect ofBS 3 on Sodium Dodecyl Sulfate-Polyacrylamide Gel Profile of Platelet Surface Glycoproteins. 7 To assess whether intermolecular crosslinks formed between platelet surface glycoproteins might correlate with the inhibition of collagen-induced aggregation, radiolabeled platelets were treated with BS 3, dissolved in SDS gel buffer, and subjected to SDS-polya-
410
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[34]
IOO' z
o
8o
I-~9 UJ
60
20
0
/~
0
I
I
I
I
III
~1
I
i
I
I
I tJtll
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i
i
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FIG. 4. Effects of DTSSP ((3), BS 3 ([:]), and a monofunctional analog, sulfosuccinimidyl propionate (SSP) (A), on collagen-induced platelet aggregation. The concentration of SSP used was twice that indicated on the abscissa, so that the concentration of reactive groups would be the same as in the samples with the cross-linkers. Percentage aggregation was calculated from the slope of the aggregation profiles. The values shown represent the average of three determination -+ SD. (Reprinted from Ref, 7 with permission. Copyright 1984 American Chemical Society.)
crylamide gel electrophoresis. The resulting gel was subjected to fluorography, 3t and the fluorograph was scanned with a fiber optic densitometer (model 800; Kontes). The results of this analysis are shown in Fig. 5. At low concentrations of BS 3, which effectively inhibit collagen-induced platelet aggregation, radiolabeled bands corresponding to glycoproteins IIb, Ilia, and IV were significantly reduced in the SDS gel profile. In addition, the bands corresponding to glycoproteins IIb and Ilia disappeared in parallel, suggesting that they were cross-linked to one another. This observation is consistent with independent evidence that these proteins exist as a 1 : 1 noncovalent complex 32 that has been recognized as an important platelet integrin. 33
3[ W. M. Bonner and R. A. Laskey, Eur. J. Biochem. 46, 83 (1974). 32 L. K. Jennings and D. R. Phillips, J. Biol. Chem. 257, 10458 0982). 33 R. O. Hynes, Cell (Cambridge, Mass.) 48, 549 (1987).
[34]
CROSS-LINKING OF PLATELET MEMBRANE GLYCOPROTEINS
411
20C
Z
z_ 180 / on~ O: hi t-LL Z
0
w
II. 0 (J >-
"J
(.9
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8O
60
\\ 40
20
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BS3(p.M) FIG. 5. Effect of BS 3 on the SDS gel profile of radiolabeled platelet surface glycoproteins. Radiolabeled proteins were treated with varying concentrations of BS 3. The reactions were quenched, and the samples were dissolved in SDS gel-solubilizing solution and were subjected to SDS-polyacrylamide gel electrophoresis. The resulting gel was subjected to fluorography, and the resulting fluorograph was analyzed densitometrically. The percentage of each glycoprotein at various concentrationsof BS 3 was calculated with reference to the uncross-linked control. (O), 150K; (A), GPIb; ((3), GPIIb; (12), GPIIIa; (A), GPIV. (Reprinted from Ref. 7 with permission. Copyright 1984 American Chemical Society.)
Discussion The advent of high-yield, membrane-impermeant cross-linking reagents has allowed the experimenter to probe protein-protein interactions at one face of a membrane. Platelets appear to present an especially rich variety of questions that can be addressed with these reagents. These include the identification of platelet surface proteins that specifically interact with protein components of the subendothelial matrix and of the plasma, as well as possible changes in tertiary or quaternary structure of surface proteins that accompany platelet activation. Initial studies in which these reagents have been used to cross-link
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.
