Sulfiydryl analogues of adenosine diphosphate: chemical synthesis and activity as platelet-aggregating agents
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Department ofBioclzemistiy, University ofAlberta, Edmonton, Alhertcr T6G 2H7 Received October 6, 1975
Stone, J. V . , Singh, R . KO, Horhk, H. & Barton, P. G. (1976)Sulfhydryl analogues of adenosine Biochrm. . 54, diphosphate: chemical synthesis and activity as platelet-aggregating agents. Can. .I 529-533 2-Thioadenosine 5'-diphosphrate (2-SH WDP), 2,2'-dithiobisadenosine 5'-diphosphate (2,2'(S-ADP),), 8-thioadenosine 5 'diphosphate (8-SH ADP),and6-mercaptopurine riboside 5 '-disphosphate (6-MPRDP) were synthesized as potential affinity labels for ADP receptors on the bloodplatelet membrane. The mean relative activities of these compounds in aggregating human platelets suspended in homologous plasma were 155% (2,2'-(S-ADP),), 74% (2-SH ADP), 0.65% (8-SH ADP), and 0.08% (dMPRDP). The mean relative activities against washed platelets were 249% (2,2'-(S-ADP),) and 115% (2-SH ADP), whereas no aggregation occurred with 8-SH ADP or 6-MBRDP. The last two compounds were found to be weak inhibitors of ADP-induced aggregation. Therefore, thio-substitution at position 2 followed by oxidation to a disulfide appears to be the most promising approach to further studies of affinity labelling of membrane ADP-receptors. Stone, J. V., Singh, R . K . , Horak, H. $ Barton, P. G . (1976) Sulfhydryl analoguesof adenosine diphosphate: chemical synthesis and activity as pHateIet aggregating agents. Can. J . Bitac-hem.54, 529-533 Nous avons synthetise le 2-thioadenosine 5'-diphosphate (2-SH ADPI, Be 2,2'-dithiobisadenosine 5'-diphosphate (2,2'-(S-ADP),), Be 8-thioadknosine 5'-diphosphate (8-SH ADP) et le Cmercaptopurine riboside 5'-diphssphate (6-MPRDP) et nous les avons utilises comme marqueurs possibles de B'affinite pour les ADP rkceptetars sur la membrane des plaquettes sanguines. L'activite relative msyenne de ces substances dans 19agr6gationdes plaquettes humaines suspendues dam le plasma homologue est de 155% (2.2'-(S-ADP),), 74% (2-SW ADP), 0.65% ((8-SH ADP) et 0.88% (6-MPRDPb. L'activite moyewne relative contre les plaquettes 1avCes est de 249% (2,2'-(S-ADP),) et 115% (2-%PI ADP). k e &SH ADP et le 6-MPRDP ne prduisent aucune agregation et ils inhibent faiblement l'agregation induite par I'ADP. La this-substitution en position 2 suivie Be l'oxydation en disulfure seraable donc l'approche la plus prometteuse pour les prochaines etudes du rnarquage de I'affinitC des recepteurs ADP membranaires. [Traduit gar le journal]
Introduction A D P causes aggregation of blood platelets. Binding o f A D P t o receptor sites on the platelet membrane is likely t o be a n important initial s t e p in this process ( 1-31. T h e nature of these receptors and the mechanism by which A D P causes the platelets t o aggregate are currently under investigation in many laboratories (4, 5). O n e possibility is that platelet A ~ s a ~ v r a ~ a 2-SH s ~ s : ADP, 2-thioadenosine 5'-diphosphate; 2,2'-(S-ADP),, 2,2'-dithiobisadenosine 5'-diphosphate; 8-SH ADP, tkthioadenosine 5'-diphosphate; 4-MPRDP, 6-mercaptopurine riboside 5'-diphosphate; ADB, adenosine 5'-diphosphate; 6-MPRMP, 6-thiopurine riboside 5'-monophosphate; 8-Br AMP, 8-bromoadenosine 5'-monophosphate; 2-SH AMP, Zthioadenosine 5'monsphosphate; 2-CI AMP, 2-chloroadenosine 5'-monophosphate; DMF, N,M-dimethylformamide; tlc, thinlayer chromatography. 'Present address: Department of Zoology and Applied Entomology, Inaperia! College of Science and Technology, London SW7, U.K.
actomyosin may be implicated in the early stages of ADP-induced aggregation ( 6 )and could furnish binding sites for ADB ('7). Since membrane-associated components of actomyc~slncould control t h e distribution of intrinsic membrane proteins in some cells (8, 91, w e have suggested that in platelets A D P may dissociate a network of filamentous protein, thereby permitting more rapid lateral and rotational diffusion o f intrinsic membrane proteins. This would allow specific protein-protein interactions t o occur in the membrane and could account for the development of prothrcsmbin-converting activity a t the cell surface during secondary aggregation (18). T o verify t h e nature of t h e A D P receptors o n the platelet membrane, one possible approach is t o label them covalently using reactive analogues of ADP. Sulfkydryl groups may be implicated in t h e ADP binding sites o n the platelet membrane since N-ethylmaleimide, p-chloromercuribenzoate. and pkaenyl mercuric acetate react with specific membrane components t o inhibit platelet aggregation ( B I ,
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CAN. 9. BHOCHEM. V8L. 54, 8976
12). Since sulfhydryl groups can undergo nucleophilic displacement or mixed disulfide formation with cysteine residues in proteins (13, 141, sulfiydry1 analogues sf ADB could be very useful for affinity Babelling (15) of A D P receptors. BPI this paper we report the synthesis of 2-SH ADP, 2,%'-(S-ADP),, 8-SH ADP, and 6-MPWDP, together with their effects on pIatelet aggregation in virro.
in V ~ C U Oto a yeilow oily residue, dissolved in isopropansl0.25 M N ~ H C O (2: , I), and chromatographed on a Whatman cellulose CF 1 1 column (2.5 x 4.0cm) in isopropanol0.25 M NI-$HCO, (2: 1). Appropriate fractions were collected, evaporated in vacuo, and the evaporation repeated with H,O to remove NH,HC03 and give a yellow oily residue sF2-C1AMP NH4+salt (155 mg). A single spot was obtained after tlc on silica gel (solvent system A) (A,,, (0.1 N HCI) 265 nm).
2-SH A MP (20,21) For tlc, silica gel plates (13181) and cellulose plates (6064) were obtained from Eastman K d a k Company, Rochester, N.Y. Whatman cellulose CF 11 and Whatman cellulose BE 32 were purchased from Reeve Angel, Clifton, N.J., BioRad AG50W-X8 resin (100-208 mesh, H9 form) was purchased from BioRad Laboratories (Canada) Ltd., Mississauga, Ont. GMPRMP and 8-Br AMP were purchased from PL Biochemicals, Milwaukee, Wisc,, 2-chloroadenosine was purchased from Cyclochemicals, Los Angeles, Calif. and 1,l'-carbonyldiimidazole was from Aldrich Chemical Co. Inc., Milwaukee, Wisc. Silica gel and cellulose tlc plates were developed in CH,CN : B M NH40H (7.3) (solvent system A) and in isobutyric acid : 1 M N&OH (5:3) (solvent system B), respectively. Spots were visualized under uv light. Fractions from the coiurnns were monitored by uv spectrophotometer at appropriate wavelength. Evaporations were carried out under reduced pressure at temperatures less than 35 C . Reaction products were dried under vacuum over P205.POCI,, DMF, and pyridine were all dried and freshly distilled. Samples were stored under N, at -20 "C. The preparation of platelet-rich plasma and washedplatelet suspensions and the assessment of the rate and extent of aggregation induced by nucleotides were as described before (16). Phosphate analyses were carried out by the method of Fiske and Subbarow (17) using the modification of Way and Parks (18). Synthesis of2-SH ADP sodium salt 2-C/ A MP (19) To an ice-cold solution of triethylphosphate (28 ml) and 2-chloroademosine (505 mg, 1.68 mmol), cold POCB, (1.65 ml, 17.2 mmol) and H 2 0 (30 ,al) were added with magnetic stirring. The reaction mixture was held at 4 T for 48 h. The solution was then poured over ice (66 g) with stirring and the pH was adjusted to 9 with concentrated aqueous LiOH. The solution was kept for 3 h and maintained at pH 9 by further addition of LiBH. The precipitate was removed and washed with H,O (five times) by centrifugation. The combined supernatants were extracted with chloroform (2 x BOO ml). The supernatant was diluted to 200 mP with H28, barium acetate (0.87 g, 3.4 mmol) was added with continuous stirring, and the solution was left overnight. The precipitate was removed by centrifugation and the supernatant was evaporated under vacuum to about 50 mmB. Ethanol (100 ml) was added and the precipitated Ba salt was centrifuged and washed with cold ethanol-H20 (2: l), ethanol, acetone, and ether. A slurry of the Ba salt was made with BioRad AG50W-X8 (PI9 form, 2 ml) and poured onto an ion-exchange column ( l x 40cm) of the same resin. The effluent was neutralized with N&OH and evaporated
The ammonium salt of 2-Cl AMP (150 n~g,0.32 mmol) was dissolved in 7 ml of dry DMF and then saturated with H,S. NaSM (179 mg, 3.2 rnmol) was added and the reaction IC for 24 h with continuous mixture was heated at %O stirring under anhydrous conditions. The soHution was cooled in ice and H 2 0 (10 ml) was added, neutralized with acetic acid, and evaporated under vacuum. The residue was dissolved in H 2 0and chromatographed on a Whatman cellulose DE 32 column (2.5 x 30 crn) with a linear gradient of 4 l of triethylammonium bicarbonate (0.024.4 M, pH 9.5). Appropriate fractions were collected, evaporated under vacuum, and the triethylamrnonium bicarbonate was removed by repeated addition and evaporation of ethanol. The residue was dissolved in methanol (6 mi, 0.05 M )and five volumes of an acetone solution of sodiuq perchlorate (15 equivalents) were added. The precipitated sodium salt was centrifuged and washed with acetone (2 x 3 ml) and then dried over P205under vacuum (I I6 mg). A single spot was obtained after tlc on cellulose (solvent system B) (h,,, (0. I N HCl) 293 and 238 nm, and La,(8.1 N NaOH) 283 and 240 nm (21)).
a - s ADB ~ 422) Pyridinium Salt o f 2 - S H AMP-An aqueous solution of the sodium salt of 2-SH AMP (71 mg, 175 pmol) was passed through a column of BioRad AGSOW-X8 (pyridinium) resin (50 ml). The effluent was evaporated to a small volume. Tribcdtylamrnoniusn Salt-Tributylamine (0.842 ml, 175 pmol) was added to the above solution and concentrated under vacuum. The residue was dried by repeated addition and evaporation of dry pyridine (3 x 7 ml) followed by dry DMF (3 x 5 ml). 2-SM ADP-The anhydrous tributylammonilarn salt of 2-SH AMP was dissolved in dry DM%;(2 ml) and 1,1 '-carbonyldiirnidazole (141 mg, 8.87 mmol) in dry DM%: (2 mu was added dropwise with continuous stirring. The mixture was stirred in a tightly stoppered container for 30 min, then kept overnight in vacuo over P205.Methanol (55 pl) was added and, after 30 min at room temperature, tributy8ammonium orthophosphate (8.87 mmol) in dry DMF ('7 ml) was added dropwise with vigorous stirring. The tributylammoniurn orthophosphate had been prepared by adding tributylarnine (209 pl, 0.87 mrnoi) to a solution of 85% o-phosphoric acid (56 pl, 6.87 mmol) in dry pyridine (3 mil), stirring for 15 min, and drying under vacuum (22). The o 1 day. The prestoppered mixture was kept in ~ ~ n c ufor cipitate was removed and washed with dry BMF (2 x 3 ml) by centrifugation. The supernatant was treated with an equal volume of methanol, and the solution was evaporated to dryness. The residue was chromatographed on a Whatman cellulose DE 32 column (2.5 x 30 cm) with a linear gradient of 4 1 of triethylammonium bicarbonate (0.04-0.4 M ,pH 7.5). Appropriate fractions were evaps-
53 1
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STONE ET AE.
rated under vacuum and triethylammonium bicarbonate was removed by addition and evaporation of ethanol. The salt was dissolved in methanol (2.5 ml, 0.05 M) and an acetone solution (12.5 ml) of sodium perchlorate (180 rng, 15 equivalents) was added. The precipitate of the sodium salt of 2-SH ADP was collected by centrifugation and washed with acetone (2 x 3 ml). It was dried over PaOg under vacuum. Yield was 53 mg as determined from E 18400 at 293 nm for 2-SH adenosine (21). A single spot was obtained after tlc on cellulose with solvent system B, Rf 0.46 (A,, (0.1 N HC1) 293 and 238 nm, and A,, (0.1 N NaOH) 284 and 242 wm). The ratio of labile phosphate total phosphate was 1.02:2.17.
2,2 '-(S- ADP)2 The sodium salt of 2-SH ADP (0.1 pmol in 50 pLi1 HzO) and iodine (0.05 pmol in 18 pl H,O) were mixed with 0. I M phosphate bufler (1 ml, pH 7.5) and the solution was stirred for 5 min at room temperature. The resulting solution (0.1 N HCl) of 270 nm, close to the reported showed ha, value of A,, of 273 (0.1 N HCI) for 2,2'-dithiobisadenosine (21). There was no absorption at 293 nm expected for 2-SH ADP. Synthesis of 8 S H A D P 8-SH A M P (20,21) Dry DMF (4 ml) was added to 8-Br AMP (51 mg, 0.1 mmol) and saturated with H,S. NaSH (56 mg, 1 mmol) was added and the reaction mixture was heated at 90 'T for 24 h under anhydrous conditions. It was cooled in ice and H,O (10 ml) was added, neutralized with acetic acid, and evaporated under vacuum. The residue was chromatographed on a Whatman cellulose DE 32 cs8rnmn (2.5 x 38 cm) with linear gradient of 3 1 of triethylammonium bicarbonate (0.844.4 M), pH 7.5. Appropriate fractions were evaporated under vacuum and triethylammonium bicarbonate was removed by addition and evaporation of ethanol to give the triethylammoniurn sa!t of 8-SH AMP, yield 45 mg. A single spot was obtained on tlc on cellulose with solvent system B (A,, (0.1 N HCI) 308,241, and 222 nm, Amax (0.1 N NaOH) 297 and 228 nm). 8-SHADP (22) Pyridiniunt Salt of &SHAMP-An aqueous solution of 8-SH AMP triethylammonium salt was passed through a The column of BioRad SOW-X8 (pyridinium) resin (40d). effluent was evaporated under vacuum to a small volume. Tributylamrnonium Salt-Tributylamine (19 p1, 80 pmol) was added to the pyridiniurn salt and the solution was concentrated under vacuum. The residue was dried by repeated addition and evaporation of dry pyridine (3 x 6 ml) followed by dry DMF (3 x 5 mf). 8-SH ADP-Anhydrous tributylammonium salt of 8-SH AMP was dissolved in dry DMF (1 ml) and l,lf-carbonyldiimidazole (65 mg, 0.4 mmol) in dry DMF (1 ml) was added dropwise with continuous stirring. The mixture was stoppered and stirred for SO wain and kept under vacuum overnight and then treated with methanol (34 pl). After 30 min at room temperature, tributylammonium orthophos; phate (0.5 mmol, prepared as described above) in dry DMF (3 ml) was added dropwise with vigorous stirring. The stoppered reaction mixture was kept under vacuum for 1 day. The precipitate was removed and washed with DMF (3 x 1ml) and collected by centrifugation. The supernatant
was treated with an equal volume of methanol and the solution was evaporated under vacuum to dryness. The residue was chromatographed on a Whatman cellulose DE 32 column (2 x 30 cm) with 4 1 of a linear gradient of triethylammonium bicarbonate (0.088.4 M, pH 7.5). Appropriate fractions were evaporated under vacuum and triethylammonium bicarbonate was removed by addition and evaporation of ethanol. The residue was dried under vacuum over P20g.The yield of triethylmmonium salt of 8-SH ADP was 31 mg (Bm). A single spot was obtained after tlc on cellulose (solvent system B), W f0.57 (Amax(0. I N HCI) 308,244, and 222 nm, A,, (0. I N NaBH) 297 and 227 nm). The ratio of labile phosphate - total phosphate was l.OQ:2.1. Synthesis of 6- MPWBP (23) Pyridiniurn Salt of 6-MPRMP-An aqueous solution of 6MPRMP sodium salt (100 mg, 224 pmol) was passed through a column of BioRad 5QW-X8(pyridinium) resin (50 ml). The effluent was evaporated under vacuum t o a small volume. Triburylarnmoraium Salt-Tributylamine (54 pA, 224 pmol) was added t o the pyridinium salt and the solution was concentrated under vacuum. The residue was dried by repeated addition and evaporation of dry pyridine (3 x 12 ml) followed by dry DMF (3 x 6 ml). 4-MPRDP-The residue of the anhydrous tributylammonium salt of 6-MPRMP was dissolved In dry DMF (2 ml), and I,I1-carbonyldiimidazole(143 mg, 0.88 mmol) in dry DMF (2 mi) was added dropwisc with continuous stirring. The mixture was stoppered and stirred for 30 min, kept under vacuum for 4 h, and then treated with methanol (75 pl). After 30 min at room temperature, tributylarnmonium orthophosphate (1.03 mmoB, prepared as above) in dry DMF (6 ml) was added dropwise with vigorous stirring. The stoppered reaction mixture was kept under vacuum for 1 day. The precipitate was removed and washed with dry DMF (3 x 2 ml) by centrifugation. The supernatant was treated with an equal volume of methanol and the solution was evaporated under vacuum to dryness. The residue was chromatographed on a Whatman cellulose DE 32 column (2.5 x 30 cm) with 4 1 of a Binear gradient of triethylammonium bicarbonate (0.005-0.4 M, p H 7.5). Appropriate fractions were evaporated under vacuum and triethylammonium bicarbonate was removed by addition and evaporation of ethanol. The salt was dissolved in methanol (4 ml, 0.85 M) and 20 ml of an acetone solution of sodium perchlorate (474 mg,15 equivalents) was added. The precipitate of sodium salt was collected by centrifugation and washed with acetone (3 x 2 ml). Finally, it was dried over P20, under vacuum. The yield was 94.2 mg (85.6%) as determined from E 2%100 a t 322 nm for 6mercaptopurine riboside 5'-triphosphate (23). A single spot was obtained after tlc om cellulose (with solvent system B), Rf 0.46 (A, (0.81 M acetate buffer, pH 4.4) 322 and 210 nm). The ratio of labile phosphate - total phosphate was 1.0~2.2.
Results and Dbcussion Synthesis 2-@1 AMP was synthesized from 2-cklsroadenosine by the method of Gough et aE. (19) and was
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CAN. J. BBOCHEM. VOL. 54, 1976
converted to 2-SH AMP by a modification of the method of Ikehara and Yamada (20) and Kikugawa L'P 618. (2%). In this modification, substitution of DMF for methanol as soivent increased the yield from 58% t 90%). In the second phosphorylation step (22), orthophosphate and the carbonyldiimidazolium derivative of ZSH AMP were used to yield 2-SH ADP. These sulfhydryl compounds dimerize very easily by aerial oxidation. The dimer can be reduced by passing it through - H,S - (24). - ~ - B ~ A M was P converted to 8-SH AMP by NaSH in DMF (20, 21). The second phosphate group was introduced as described for 2-SH ADP (22), which gave 8-SH ADP in 60% overall yield. The sodium salt of 8-SH ADP could not be prepared from acetone solution of sodium perchlorate, since the product was soluble in acetone. 8-SH ADP was used as triethylammonium salt, since triethylammonium ions do not affect the aggregation of platelets (Stone, J . V . . Singh, Ha. K., Horhk, PI., and Barton, P. G . : unpublished resuBt). 6-MPRMP was phosphoryBated as above (22) to give 6-MPWDP in $5% yield. A c-fii9iq%,To~i'ords P~LZPC~POS
The activities of these compounds were studied in human platelet-rich plasma and in suspensions of washed human platelets. The results are presented in Table I . The possibility of ernplc~yingsulfhydryl analogues of ADP as affinity labels was suggested by the work of Brox and Hampton (13) and Murphy and Morales (14), who used drnercaptopurine riboside 5 '-monophosphate and 6mercaptopurine riboside 5'-triphosphate to Babel guanosine 5'-phosphate reductase and myosin, respectively. The sulfhydryl groups in these analogues undergo slow nucleophilic displacement or disulfide exchange with cysteine side chains in proximity to the active sites of these enzymes. A more rapid reaction of 6-tkioinosinyHimidodiphosphate with myosin fragments was reported (26). In all cases, &substituted compounds were found to be suitable for the enzymes concerned. In contrast, Tables I and 2 show that 6-MP RDP was ineffective in causing platelet aggregation and was only a weak inhibitor of aggregation induced by ADP. This is consistent with an earliesobservation that removal of the $-amino group of ADP causes a loss of activity (27). Therefore, alternative analogues, retaining the 6-amino group were sought. The next compound synthesized, 2-SH ADP, was found to possess activity quantitatively similar to that of ADP. This is also consistent with the reported high activity of 2-Cl ADP (19, 28) and other 2-substituted analogues (29). High activity of 2-Cl
TABLE BA. Wdative activitiesa of ring-substituted analogues of ABP in causing aggregation sf human platelets suspended in hornslogous plasma (PRP)
Analogue 2,2'-(S-ADP)Z 2-SH ADP 8-SH ADP 6-MPRDP
ratee#" 1542 14 (6) 66+ 16 (18) 0 . 7 % + Q . W(5) 0.06 (2)
% extenteed
+
155 14 (5) 442 16 (18) 0.65+0.08 (5)
0.08 (2)
TABLE1B. Relative activitiesa sf ring-substituted analogues of ADP in causing aggregationb
of washed human platelets suspended in Tyrode's albumin bufFer (1 6) Analogue
% ratecod
2,2'-(S-ADP)2 2-C1 ADP 2-SH ADP
229 52 (4) 131 (2) 98221 (16)
+
% extentc** 249k 33 (4) 142 (2) 115+22 (16)
aCalculated assording to Ref. 25 as [([ADPI required to produse a given rate (extent) of aggregation)/([analogud required to produce the same rate (extent))] x BOO%',. bNucleotide added after 3-min preincubation of the platelet suapension with 0.3 mg fibrinogen per millilitre. cMeasurement of the rate and extent s f aggregation were as described earlier (16). dMean values + SD. The number of measurements is given in parentheses.
ADP was confirmed in this study ('Fable 1). Furthermore, the disulfide derivative, 2,2'-(S-ADB),, was even more active (Table 1). Yount et a/. (26) have reported that bis compounds of this type are extremely sensitive to disulfide cleavage, especially in the presence of Ca2+ and MgZ+and in the case of 6,6'-dithiobisinosinylimidodiphosphate this enhances the biologicai activity considerably. Therefore, 2.2'-(S-ADP), appears to be potentially the most useful reagent of those tested for specific labelHing of any reactive sulfiydryl group that may be present at the ADP binding site. In the presence of 4 rnM EDTA, 2-SH ADP (2 x IO-W)caused only the characteristic shape change without subsequent aggregation, as is the case for ADP (25). In washed platelets, both Ca2+ and fibrinogen are required for aggregation induced by 2-SH ABP (Table 1B). Somewhat surprisingly, 8-SH ADP was much less active thaw 2-SH ADP. It is possibie that the conformation of the ADP receptor is such that substitution at position 8 wc~uldproduce a steric hinderance to binding. In support of this speculation, 8-SH ABP was also found to be a poor inhibitor of ADPinduced aggregation (Table 2), implying that it failed to bind tightly at the ADB binding site.
STONE ET AL.
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TABLE 2. Effect of 6-MPRBP and 8-SH A B P on the aggregation sf human platelets induced by ADP in platelet-rich plasma
Talues calculated as: 100 - ([(rate (or extent) of aggregation in the presence of ana1sgiae)l (rate (or extent) of aggregation in the absence of analogue)] x 188q7,). bMeasurements f rate and extent of aggregation were as described earlier (16). CMeanvalue sf two or three experiments, as indicated in parentheses. Control experiments omitting the analogue, were carried out immediately before and after each determination and' the relevant rate and extent in the absence of inhibitor obtained by interpolation. *@oncentrationsthat caused shape change or slight aggregation effects.
12. Steiner, M., Ando, Y. & Eswenstein, S. R. (1973) In Etythrocytes, Thrombocytes, Leukocytes (Gerlach, The excellent technical assistance of Mr. C. Gibbs E., Moser, K., Beutseh, E. & Wilmanns, W., eds), and Ms. B. Guspie is gratefully acknowledged. The Georg Thieme Verlag K.G., Sbuttgarb work was supported by grants from the Medical 13, Brox, L. W. & ~ a & ~ t o A. n , (1968) Biochemistry 7, 398-485 Research Council of Canada and the Alberta Heart Foundation. 3. V. S. and H. H. held f e l ] o w s h i ~ ~ 14. Murphy, A. J. Morales, M.F. (I9743)~iochemistty 9,1528-1532 from the Canadian Heart Foundation. The authoks Protein Chem. 22,8-54 15. Singer, S- J. (1967) are indebted to D ~J . ~R. . ill, S. A. ~ l iand , Judith 16. Horik, H. & Barton, P. G . (1974) Biochian Biophys. Hannon from the Department of Haematology, Acta 373,47 1480 University of Alberta Hospital9 for their help in the 17. Fiske, C. H. & Subbarow, Y. (1925)J . Bioi. Chem. 66, collection of human blood samples. 375400 18. Way, J. L. & Parks, R. E. (1958) J. Bdol. Chern. 231, 467480 1. Born,G.V.R.(1965)Matus.e(London)206, 8121-1122 19. Gough, G., Helen, F. 8e Michal, F. (1969) J. Med. 2. Boullim, B.I., Green, A. R. & Price, K. S. (1972) J . Chern. H 12,494498 Physiol. 221,415-526 20. Ikehara, M. & Yarnada, S. (1978) Chern. Pharrn. Bull. 3. Nachman, R. k. & Ferris, B. (1974) J. Bid. Chem. 19, 164-109 249,708-710 21. Kikugawa, K.,Suehiro, H. & [chino, M. (1974) J . 4. Weiss, H. S. (1972) Ann. N.Y. Acad. Sci. 201, 3-450 Med. Chern. 16, 1381-1388 5. Mustard, 9. F. & Packham, M.A. (1978) Pharmacol. 22. Hoard, D. E. & Qtt, D. G. (1965) J. Am. Chem. Soc. Rev. 22,97-187 87,1785-1788 6. Bssyse, F. M. & Wafelson, M. E., Jr. (1972) Ann. 23. Murphy, A. J., Duke, J. A. & Stowring, k. (1970) N . Y.Arad. Sci. 281,37-60 Arch. Biochem. Bioph-ys. 137,297-298 7. h s z k i n , S., h s z k i n , E., Katz, A. M. & AOedort, L. 24. Hampton, A. (1963) J . Bid. @hem. 238,30624-3074 M. (1974)Biochim. Biophys. Acta 347, 802-1 12 25. Born, G . V . R. ( 1970) J . Physiol. 209,437-5 11 8. Elgsaeter, A. & Branton, D. (1974) J. Cell Bid. 63, 26. Youmt, R. G., Fsye, J. S. &B'Keefe, W.R.(1972)CoM 1818 Spring Harbor Symp. Quant. Biol. 37,113-1 19 9. Verrna, S. B. & Wallaeh, D. F. H. (1975) Biochim. 27. Gaarder, A. & Laland, S. (19618) Mature (London) 202, Biophys. Acta 382,73-82 989-9 18 10. Barton, P. G. (1976) In Mernbacane-Bound E;Pmzymes 28. Maguire, M. H. & Miehal, F. 6 19681Mafare (London) (Martonosi, A., ed.), Plenum Publishing Corporation, 217,571-573 New Ysrk, N.Y. in press 29. Gsugh, G o ,Maguire, M. H.& Penglis, F. (8972) Mol. 11. Naehman, R. L. & Ferris, B. (1972) J . BioI. Chem. Pharrnucsl. 8, 170-177 247,44684475
Acknowldgments -
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Department ofBioclzemistiy, University ofAlberta, Edmonton, Alhertcr T6G 2H7 Received October 6, 1975
Stone, J. V . , Singh, R . KO, Horhk, H. & Barton, P. G. (1976)Sulfhydryl analogues of adenosine Biochrm. . 54, diphosphate: chemical synthesis and activity as platelet-aggregating agents. Can. .I 529-533 2-Thioadenosine 5'-diphosphrate (2-SH WDP), 2,2'-dithiobisadenosine 5'-diphosphate (2,2'(S-ADP),), 8-thioadenosine 5 'diphosphate (8-SH ADP),and6-mercaptopurine riboside 5 '-disphosphate (6-MPRDP) were synthesized as potential affinity labels for ADP receptors on the bloodplatelet membrane. The mean relative activities of these compounds in aggregating human platelets suspended in homologous plasma were 155% (2,2'-(S-ADP),), 74% (2-SH ADP), 0.65% (8-SH ADP), and 0.08% (dMPRDP). The mean relative activities against washed platelets were 249% (2,2'-(S-ADP),) and 115% (2-SH ADP), whereas no aggregation occurred with 8-SH ADP or 6-MBRDP. The last two compounds were found to be weak inhibitors of ADP-induced aggregation. Therefore, thio-substitution at position 2 followed by oxidation to a disulfide appears to be the most promising approach to further studies of affinity labelling of membrane ADP-receptors. Stone, J. V., Singh, R . K . , Horak, H. $ Barton, P. G . (1976) Sulfhydryl analoguesof adenosine diphosphate: chemical synthesis and activity as pHateIet aggregating agents. Can. J . Bitac-hem.54, 529-533 Nous avons synthetise le 2-thioadenosine 5'-diphosphate (2-SH ADPI, Be 2,2'-dithiobisadenosine 5'-diphosphate (2,2'-(S-ADP),), Be 8-thioadknosine 5'-diphosphate (8-SH ADP) et le Cmercaptopurine riboside 5'-diphssphate (6-MPRDP) et nous les avons utilises comme marqueurs possibles de B'affinite pour les ADP rkceptetars sur la membrane des plaquettes sanguines. L'activite relative msyenne de ces substances dans 19agr6gationdes plaquettes humaines suspendues dam le plasma homologue est de 155% (2.2'-(S-ADP),), 74% (2-SW ADP), 0.65% ((8-SH ADP) et 0.88% (6-MPRDPb. L'activite moyewne relative contre les plaquettes 1avCes est de 249% (2,2'-(S-ADP),) et 115% (2-%PI ADP). k e &SH ADP et le 6-MPRDP ne prduisent aucune agregation et ils inhibent faiblement l'agregation induite par I'ADP. La this-substitution en position 2 suivie Be l'oxydation en disulfure seraable donc l'approche la plus prometteuse pour les prochaines etudes du rnarquage de I'affinitC des recepteurs ADP membranaires. [Traduit gar le journal]
Introduction A D P causes aggregation of blood platelets. Binding o f A D P t o receptor sites on the platelet membrane is likely t o be a n important initial s t e p in this process ( 1-31. T h e nature of these receptors and the mechanism by which A D P causes the platelets t o aggregate are currently under investigation in many laboratories (4, 5). O n e possibility is that platelet A ~ s a ~ v r a ~ a 2-SH s ~ s : ADP, 2-thioadenosine 5'-diphosphate; 2,2'-(S-ADP),, 2,2'-dithiobisadenosine 5'-diphosphate; 8-SH ADP, tkthioadenosine 5'-diphosphate; 4-MPRDP, 6-mercaptopurine riboside 5'-diphosphate; ADB, adenosine 5'-diphosphate; 6-MPRMP, 6-thiopurine riboside 5'-monophosphate; 8-Br AMP, 8-bromoadenosine 5'-monophosphate; 2-SH AMP, Zthioadenosine 5'monsphosphate; 2-CI AMP, 2-chloroadenosine 5'-monophosphate; DMF, N,M-dimethylformamide; tlc, thinlayer chromatography. 'Present address: Department of Zoology and Applied Entomology, Inaperia! College of Science and Technology, London SW7, U.K.
actomyosin may be implicated in the early stages of ADP-induced aggregation ( 6 )and could furnish binding sites for ADB ('7). Since membrane-associated components of actomyc~slncould control t h e distribution of intrinsic membrane proteins in some cells (8, 91, w e have suggested that in platelets A D P may dissociate a network of filamentous protein, thereby permitting more rapid lateral and rotational diffusion o f intrinsic membrane proteins. This would allow specific protein-protein interactions t o occur in the membrane and could account for the development of prothrcsmbin-converting activity a t the cell surface during secondary aggregation (18). T o verify t h e nature of t h e A D P receptors o n the platelet membrane, one possible approach is t o label them covalently using reactive analogues of ADP. Sulfkydryl groups may be implicated in t h e ADP binding sites o n the platelet membrane since N-ethylmaleimide, p-chloromercuribenzoate. and pkaenyl mercuric acetate react with specific membrane components t o inhibit platelet aggregation ( B I ,
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CAN. 9. BHOCHEM. V8L. 54, 8976
12). Since sulfhydryl groups can undergo nucleophilic displacement or mixed disulfide formation with cysteine residues in proteins (13, 141, sulfiydry1 analogues sf ADB could be very useful for affinity Babelling (15) of A D P receptors. BPI this paper we report the synthesis of 2-SH ADP, 2,%'-(S-ADP),, 8-SH ADP, and 6-MPWDP, together with their effects on pIatelet aggregation in virro.
in V ~ C U Oto a yeilow oily residue, dissolved in isopropansl0.25 M N ~ H C O (2: , I), and chromatographed on a Whatman cellulose CF 1 1 column (2.5 x 4.0cm) in isopropanol0.25 M NI-$HCO, (2: 1). Appropriate fractions were collected, evaporated in vacuo, and the evaporation repeated with H,O to remove NH,HC03 and give a yellow oily residue sF2-C1AMP NH4+salt (155 mg). A single spot was obtained after tlc on silica gel (solvent system A) (A,,, (0.1 N HCI) 265 nm).
2-SH A MP (20,21) For tlc, silica gel plates (13181) and cellulose plates (6064) were obtained from Eastman K d a k Company, Rochester, N.Y. Whatman cellulose CF 11 and Whatman cellulose BE 32 were purchased from Reeve Angel, Clifton, N.J., BioRad AG50W-X8 resin (100-208 mesh, H9 form) was purchased from BioRad Laboratories (Canada) Ltd., Mississauga, Ont. GMPRMP and 8-Br AMP were purchased from PL Biochemicals, Milwaukee, Wisc,, 2-chloroadenosine was purchased from Cyclochemicals, Los Angeles, Calif. and 1,l'-carbonyldiimidazole was from Aldrich Chemical Co. Inc., Milwaukee, Wisc. Silica gel and cellulose tlc plates were developed in CH,CN : B M NH40H (7.3) (solvent system A) and in isobutyric acid : 1 M N&OH (5:3) (solvent system B), respectively. Spots were visualized under uv light. Fractions from the coiurnns were monitored by uv spectrophotometer at appropriate wavelength. Evaporations were carried out under reduced pressure at temperatures less than 35 C . Reaction products were dried under vacuum over P205.POCI,, DMF, and pyridine were all dried and freshly distilled. Samples were stored under N, at -20 "C. The preparation of platelet-rich plasma and washedplatelet suspensions and the assessment of the rate and extent of aggregation induced by nucleotides were as described before (16). Phosphate analyses were carried out by the method of Fiske and Subbarow (17) using the modification of Way and Parks (18). Synthesis of2-SH ADP sodium salt 2-C/ A MP (19) To an ice-cold solution of triethylphosphate (28 ml) and 2-chloroademosine (505 mg, 1.68 mmol), cold POCB, (1.65 ml, 17.2 mmol) and H 2 0 (30 ,al) were added with magnetic stirring. The reaction mixture was held at 4 T for 48 h. The solution was then poured over ice (66 g) with stirring and the pH was adjusted to 9 with concentrated aqueous LiOH. The solution was kept for 3 h and maintained at pH 9 by further addition of LiBH. The precipitate was removed and washed with H,O (five times) by centrifugation. The combined supernatants were extracted with chloroform (2 x BOO ml). The supernatant was diluted to 200 mP with H28, barium acetate (0.87 g, 3.4 mmol) was added with continuous stirring, and the solution was left overnight. The precipitate was removed by centrifugation and the supernatant was evaporated under vacuum to about 50 mmB. Ethanol (100 ml) was added and the precipitated Ba salt was centrifuged and washed with cold ethanol-H20 (2: l), ethanol, acetone, and ether. A slurry of the Ba salt was made with BioRad AG50W-X8 (PI9 form, 2 ml) and poured onto an ion-exchange column ( l x 40cm) of the same resin. The effluent was neutralized with N&OH and evaporated
The ammonium salt of 2-Cl AMP (150 n~g,0.32 mmol) was dissolved in 7 ml of dry DMF and then saturated with H,S. NaSM (179 mg, 3.2 rnmol) was added and the reaction IC for 24 h with continuous mixture was heated at %O stirring under anhydrous conditions. The soHution was cooled in ice and H 2 0 (10 ml) was added, neutralized with acetic acid, and evaporated under vacuum. The residue was dissolved in H 2 0and chromatographed on a Whatman cellulose DE 32 column (2.5 x 30 crn) with a linear gradient of 4 l of triethylammonium bicarbonate (0.024.4 M, pH 9.5). Appropriate fractions were collected, evaporated under vacuum, and the triethylamrnonium bicarbonate was removed by repeated addition and evaporation of ethanol. The residue was dissolved in methanol (6 mi, 0.05 M )and five volumes of an acetone solution of sodiuq perchlorate (15 equivalents) were added. The precipitated sodium salt was centrifuged and washed with acetone (2 x 3 ml) and then dried over P205under vacuum (I I6 mg). A single spot was obtained after tlc on cellulose (solvent system B) (h,,, (0. I N HCl) 293 and 238 nm, and La,(8.1 N NaOH) 283 and 240 nm (21)).
a - s ADB ~ 422) Pyridinium Salt o f 2 - S H AMP-An aqueous solution of the sodium salt of 2-SH AMP (71 mg, 175 pmol) was passed through a column of BioRad AGSOW-X8 (pyridinium) resin (50 ml). The effluent was evaporated to a small volume. Tribcdtylamrnoniusn Salt-Tributylamine (0.842 ml, 175 pmol) was added to the above solution and concentrated under vacuum. The residue was dried by repeated addition and evaporation of dry pyridine (3 x 7 ml) followed by dry DMF (3 x 5 ml). 2-SM ADP-The anhydrous tributylammonilarn salt of 2-SH AMP was dissolved in dry DM%;(2 ml) and 1,1 '-carbonyldiirnidazole (141 mg, 8.87 mmol) in dry DM%: (2 mu was added dropwise with continuous stirring. The mixture was stirred in a tightly stoppered container for 30 min, then kept overnight in vacuo over P205.Methanol (55 pl) was added and, after 30 min at room temperature, tributy8ammonium orthophosphate (8.87 mmol) in dry DMF ('7 ml) was added dropwise with vigorous stirring. The tributylammoniurn orthophosphate had been prepared by adding tributylarnine (209 pl, 0.87 mrnoi) to a solution of 85% o-phosphoric acid (56 pl, 6.87 mmol) in dry pyridine (3 mil), stirring for 15 min, and drying under vacuum (22). The o 1 day. The prestoppered mixture was kept in ~ ~ n c ufor cipitate was removed and washed with dry BMF (2 x 3 ml) by centrifugation. The supernatant was treated with an equal volume of methanol, and the solution was evaporated to dryness. The residue was chromatographed on a Whatman cellulose DE 32 column (2.5 x 30 cm) with a linear gradient of 4 1 of triethylammonium bicarbonate (0.04-0.4 M ,pH 7.5). Appropriate fractions were evaps-
53 1
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STONE ET AE.
rated under vacuum and triethylammonium bicarbonate was removed by addition and evaporation of ethanol. The salt was dissolved in methanol (2.5 ml, 0.05 M) and an acetone solution (12.5 ml) of sodium perchlorate (180 rng, 15 equivalents) was added. The precipitate of the sodium salt of 2-SH ADP was collected by centrifugation and washed with acetone (2 x 3 ml). It was dried over PaOg under vacuum. Yield was 53 mg as determined from E 18400 at 293 nm for 2-SH adenosine (21). A single spot was obtained after tlc on cellulose with solvent system B, Rf 0.46 (A,, (0.1 N HC1) 293 and 238 nm, and A,, (0.1 N NaOH) 284 and 242 wm). The ratio of labile phosphate total phosphate was 1.02:2.17.
2,2 '-(S- ADP)2 The sodium salt of 2-SH ADP (0.1 pmol in 50 pLi1 HzO) and iodine (0.05 pmol in 18 pl H,O) were mixed with 0. I M phosphate bufler (1 ml, pH 7.5) and the solution was stirred for 5 min at room temperature. The resulting solution (0.1 N HCl) of 270 nm, close to the reported showed ha, value of A,, of 273 (0.1 N HCI) for 2,2'-dithiobisadenosine (21). There was no absorption at 293 nm expected for 2-SH ADP. Synthesis of 8 S H A D P 8-SH A M P (20,21) Dry DMF (4 ml) was added to 8-Br AMP (51 mg, 0.1 mmol) and saturated with H,S. NaSH (56 mg, 1 mmol) was added and the reaction mixture was heated at 90 'T for 24 h under anhydrous conditions. It was cooled in ice and H,O (10 ml) was added, neutralized with acetic acid, and evaporated under vacuum. The residue was chromatographed on a Whatman cellulose DE 32 cs8rnmn (2.5 x 38 cm) with linear gradient of 3 1 of triethylammonium bicarbonate (0.844.4 M), pH 7.5. Appropriate fractions were evaporated under vacuum and triethylammonium bicarbonate was removed by addition and evaporation of ethanol to give the triethylammoniurn sa!t of 8-SH AMP, yield 45 mg. A single spot was obtained on tlc on cellulose with solvent system B (A,, (0.1 N HCI) 308,241, and 222 nm, Amax (0.1 N NaOH) 297 and 228 nm). 8-SHADP (22) Pyridiniunt Salt of &SHAMP-An aqueous solution of 8-SH AMP triethylammonium salt was passed through a The column of BioRad SOW-X8 (pyridinium) resin (40d). effluent was evaporated under vacuum to a small volume. Tributylamrnonium Salt-Tributylamine (19 p1, 80 pmol) was added to the pyridiniurn salt and the solution was concentrated under vacuum. The residue was dried by repeated addition and evaporation of dry pyridine (3 x 6 ml) followed by dry DMF (3 x 5 mf). 8-SH ADP-Anhydrous tributylammonium salt of 8-SH AMP was dissolved in dry DMF (1 ml) and l,lf-carbonyldiimidazole (65 mg, 0.4 mmol) in dry DMF (1 ml) was added dropwise with continuous stirring. The mixture was stoppered and stirred for SO wain and kept under vacuum overnight and then treated with methanol (34 pl). After 30 min at room temperature, tributylammonium orthophos; phate (0.5 mmol, prepared as described above) in dry DMF (3 ml) was added dropwise with vigorous stirring. The stoppered reaction mixture was kept under vacuum for 1 day. The precipitate was removed and washed with DMF (3 x 1ml) and collected by centrifugation. The supernatant
was treated with an equal volume of methanol and the solution was evaporated under vacuum to dryness. The residue was chromatographed on a Whatman cellulose DE 32 column (2 x 30 cm) with 4 1 of a linear gradient of triethylammonium bicarbonate (0.088.4 M, pH 7.5). Appropriate fractions were evaporated under vacuum and triethylammonium bicarbonate was removed by addition and evaporation of ethanol. The residue was dried under vacuum over P20g.The yield of triethylmmonium salt of 8-SH ADP was 31 mg (Bm). A single spot was obtained after tlc on cellulose (solvent system B), W f0.57 (Amax(0. I N HCI) 308,244, and 222 nm, A,, (0. I N NaBH) 297 and 227 nm). The ratio of labile phosphate - total phosphate was l.OQ:2.1. Synthesis of 6- MPWBP (23) Pyridiniurn Salt of 6-MPRMP-An aqueous solution of 6MPRMP sodium salt (100 mg, 224 pmol) was passed through a column of BioRad 5QW-X8(pyridinium) resin (50 ml). The effluent was evaporated under vacuum t o a small volume. Triburylarnmoraium Salt-Tributylamine (54 pA, 224 pmol) was added t o the pyridinium salt and the solution was concentrated under vacuum. The residue was dried by repeated addition and evaporation of dry pyridine (3 x 12 ml) followed by dry DMF (3 x 6 ml). 4-MPRDP-The residue of the anhydrous tributylammonium salt of 6-MPRMP was dissolved In dry DMF (2 ml), and I,I1-carbonyldiimidazole(143 mg, 0.88 mmol) in dry DMF (2 mi) was added dropwisc with continuous stirring. The mixture was stoppered and stirred for 30 min, kept under vacuum for 4 h, and then treated with methanol (75 pl). After 30 min at room temperature, tributylarnmonium orthophosphate (1.03 mmoB, prepared as above) in dry DMF (6 ml) was added dropwise with vigorous stirring. The stoppered reaction mixture was kept under vacuum for 1 day. The precipitate was removed and washed with dry DMF (3 x 2 ml) by centrifugation. The supernatant was treated with an equal volume of methanol and the solution was evaporated under vacuum to dryness. The residue was chromatographed on a Whatman cellulose DE 32 column (2.5 x 30 cm) with 4 1 of a Binear gradient of triethylammonium bicarbonate (0.005-0.4 M, p H 7.5). Appropriate fractions were evaporated under vacuum and triethylammonium bicarbonate was removed by addition and evaporation of ethanol. The salt was dissolved in methanol (4 ml, 0.85 M) and 20 ml of an acetone solution of sodium perchlorate (474 mg,15 equivalents) was added. The precipitate of sodium salt was collected by centrifugation and washed with acetone (3 x 2 ml). Finally, it was dried over P20, under vacuum. The yield was 94.2 mg (85.6%) as determined from E 2%100 a t 322 nm for 6mercaptopurine riboside 5'-triphosphate (23). A single spot was obtained after tlc om cellulose (with solvent system B), Rf 0.46 (A, (0.81 M acetate buffer, pH 4.4) 322 and 210 nm). The ratio of labile phosphate - total phosphate was 1.0~2.2.
Results and Dbcussion Synthesis 2-@1 AMP was synthesized from 2-cklsroadenosine by the method of Gough et aE. (19) and was
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CAN. J. BBOCHEM. VOL. 54, 1976
converted to 2-SH AMP by a modification of the method of Ikehara and Yamada (20) and Kikugawa L'P 618. (2%). In this modification, substitution of DMF for methanol as soivent increased the yield from 58% t 90%). In the second phosphorylation step (22), orthophosphate and the carbonyldiimidazolium derivative of ZSH AMP were used to yield 2-SH ADP. These sulfhydryl compounds dimerize very easily by aerial oxidation. The dimer can be reduced by passing it through - H,S - (24). - ~ - B ~ A M was P converted to 8-SH AMP by NaSH in DMF (20, 21). The second phosphate group was introduced as described for 2-SH ADP (22), which gave 8-SH ADP in 60% overall yield. The sodium salt of 8-SH ADP could not be prepared from acetone solution of sodium perchlorate, since the product was soluble in acetone. 8-SH ADP was used as triethylammonium salt, since triethylammonium ions do not affect the aggregation of platelets (Stone, J . V . . Singh, Ha. K., Horhk, PI., and Barton, P. G . : unpublished resuBt). 6-MPRMP was phosphoryBated as above (22) to give 6-MPWDP in $5% yield. A c-fii9iq%,To~i'ords P~LZPC~POS
The activities of these compounds were studied in human platelet-rich plasma and in suspensions of washed human platelets. The results are presented in Table I . The possibility of ernplc~yingsulfhydryl analogues of ADP as affinity labels was suggested by the work of Brox and Hampton (13) and Murphy and Morales (14), who used drnercaptopurine riboside 5 '-monophosphate and 6mercaptopurine riboside 5'-triphosphate to Babel guanosine 5'-phosphate reductase and myosin, respectively. The sulfhydryl groups in these analogues undergo slow nucleophilic displacement or disulfide exchange with cysteine side chains in proximity to the active sites of these enzymes. A more rapid reaction of 6-tkioinosinyHimidodiphosphate with myosin fragments was reported (26). In all cases, &substituted compounds were found to be suitable for the enzymes concerned. In contrast, Tables I and 2 show that 6-MP RDP was ineffective in causing platelet aggregation and was only a weak inhibitor of aggregation induced by ADP. This is consistent with an earliesobservation that removal of the $-amino group of ADP causes a loss of activity (27). Therefore, alternative analogues, retaining the 6-amino group were sought. The next compound synthesized, 2-SH ADP, was found to possess activity quantitatively similar to that of ADP. This is also consistent with the reported high activity of 2-Cl ADP (19, 28) and other 2-substituted analogues (29). High activity of 2-Cl
TABLE BA. Wdative activitiesa of ring-substituted analogues of ABP in causing aggregation sf human platelets suspended in hornslogous plasma (PRP)
Analogue 2,2'-(S-ADP)Z 2-SH ADP 8-SH ADP 6-MPRDP
ratee#" 1542 14 (6) 66+ 16 (18) 0 . 7 % + Q . W(5) 0.06 (2)
% extenteed
+
155 14 (5) 442 16 (18) 0.65+0.08 (5)
0.08 (2)
TABLE1B. Relative activitiesa sf ring-substituted analogues of ADP in causing aggregationb
of washed human platelets suspended in Tyrode's albumin bufFer (1 6) Analogue
% ratecod
2,2'-(S-ADP)2 2-C1 ADP 2-SH ADP
229 52 (4) 131 (2) 98221 (16)
+
% extentc** 249k 33 (4) 142 (2) 115+22 (16)
aCalculated assording to Ref. 25 as [([ADPI required to produse a given rate (extent) of aggregation)/([analogud required to produce the same rate (extent))] x BOO%',. bNucleotide added after 3-min preincubation of the platelet suapension with 0.3 mg fibrinogen per millilitre. cMeasurement of the rate and extent s f aggregation were as described earlier (16). dMean values + SD. The number of measurements is given in parentheses.
ADP was confirmed in this study ('Fable 1). Furthermore, the disulfide derivative, 2,2'-(S-ADB),, was even more active (Table 1). Yount et a/. (26) have reported that bis compounds of this type are extremely sensitive to disulfide cleavage, especially in the presence of Ca2+ and MgZ+and in the case of 6,6'-dithiobisinosinylimidodiphosphate this enhances the biologicai activity considerably. Therefore, 2.2'-(S-ADP), appears to be potentially the most useful reagent of those tested for specific labelHing of any reactive sulfiydryl group that may be present at the ADP binding site. In the presence of 4 rnM EDTA, 2-SH ADP (2 x IO-W)caused only the characteristic shape change without subsequent aggregation, as is the case for ADP (25). In washed platelets, both Ca2+ and fibrinogen are required for aggregation induced by 2-SH ABP (Table 1B). Somewhat surprisingly, 8-SH ADP was much less active thaw 2-SH ADP. It is possibie that the conformation of the ADP receptor is such that substitution at position 8 wc~uldproduce a steric hinderance to binding. In support of this speculation, 8-SH ABP was also found to be a poor inhibitor of ADPinduced aggregation (Table 2), implying that it failed to bind tightly at the ADB binding site.
STONE ET AL.
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TABLE 2. Effect of 6-MPRBP and 8-SH A B P on the aggregation sf human platelets induced by ADP in platelet-rich plasma
Talues calculated as: 100 - ([(rate (or extent) of aggregation in the presence of ana1sgiae)l (rate (or extent) of aggregation in the absence of analogue)] x 188q7,). bMeasurements f rate and extent of aggregation were as described earlier (16). CMeanvalue sf two or three experiments, as indicated in parentheses. Control experiments omitting the analogue, were carried out immediately before and after each determination and' the relevant rate and extent in the absence of inhibitor obtained by interpolation. *@oncentrationsthat caused shape change or slight aggregation effects.
12. Steiner, M., Ando, Y. & Eswenstein, S. R. (1973) In Etythrocytes, Thrombocytes, Leukocytes (Gerlach, The excellent technical assistance of Mr. C. Gibbs E., Moser, K., Beutseh, E. & Wilmanns, W., eds), and Ms. B. Guspie is gratefully acknowledged. The Georg Thieme Verlag K.G., Sbuttgarb work was supported by grants from the Medical 13, Brox, L. W. & ~ a & ~ t o A. n , (1968) Biochemistry 7, 398-485 Research Council of Canada and the Alberta Heart Foundation. 3. V. S. and H. H. held f e l ] o w s h i ~ ~ 14. Murphy, A. J. Morales, M.F. (I9743)~iochemistty 9,1528-1532 from the Canadian Heart Foundation. The authoks Protein Chem. 22,8-54 15. Singer, S- J. (1967) are indebted to D ~J . ~R. . ill, S. A. ~ l iand , Judith 16. Horik, H. & Barton, P. G . (1974) Biochian Biophys. Hannon from the Department of Haematology, Acta 373,47 1480 University of Alberta Hospital9 for their help in the 17. Fiske, C. H. & Subbarow, Y. (1925)J . Bioi. Chem. 66, collection of human blood samples. 375400 18. Way, J. L. & Parks, R. E. (1958) J. Bdol. Chern. 231, 467480 1. Born,G.V.R.(1965)Matus.e(London)206, 8121-1122 19. Gough, G., Helen, F. 8e Michal, F. (1969) J. Med. 2. Boullim, B.I., Green, A. R. & Price, K. S. (1972) J . Chern. H 12,494498 Physiol. 221,415-526 20. Ikehara, M. & Yarnada, S. (1978) Chern. Pharrn. Bull. 3. Nachman, R. k. & Ferris, B. (1974) J. Bid. Chem. 19, 164-109 249,708-710 21. Kikugawa, K.,Suehiro, H. & [chino, M. (1974) J . 4. Weiss, H. S. (1972) Ann. N.Y. Acad. Sci. 201, 3-450 Med. Chern. 16, 1381-1388 5. Mustard, 9. F. & Packham, M.A. (1978) Pharmacol. 22. Hoard, D. E. & Qtt, D. G. (1965) J. Am. Chem. Soc. Rev. 22,97-187 87,1785-1788 6. Bssyse, F. M. & Wafelson, M. E., Jr. (1972) Ann. 23. Murphy, A. J., Duke, J. A. & Stowring, k. (1970) N . Y.Arad. Sci. 281,37-60 Arch. Biochem. Bioph-ys. 137,297-298 7. h s z k i n , S., h s z k i n , E., Katz, A. M. & AOedort, L. 24. Hampton, A. (1963) J . Bid. @hem. 238,30624-3074 M. (1974)Biochim. Biophys. Acta 347, 802-1 12 25. Born, G . V . R. ( 1970) J . Physiol. 209,437-5 11 8. Elgsaeter, A. & Branton, D. (1974) J. Cell Bid. 63, 26. Youmt, R. G., Fsye, J. S. &B'Keefe, W.R.(1972)CoM 1818 Spring Harbor Symp. Quant. Biol. 37,113-1 19 9. Verrna, S. B. & Wallaeh, D. F. H. (1975) Biochim. 27. Gaarder, A. & Laland, S. (19618) Mature (London) 202, Biophys. Acta 382,73-82 989-9 18 10. Barton, P. G. (1976) In Mernbacane-Bound E;Pmzymes 28. Maguire, M. H. & Miehal, F. 6 19681Mafare (London) (Martonosi, A., ed.), Plenum Publishing Corporation, 217,571-573 New Ysrk, N.Y. in press 29. Gsugh, G o ,Maguire, M. H.& Penglis, F. (8972) Mol. 11. Naehman, R. L. & Ferris, B. (1972) J . BioI. Chem. Pharrnucsl. 8, 170-177 247,44684475
Acknowldgments -
