Ionophore A and thrombin-induced platelet aggregation:

Transcription

1 Pro. Nati. ad. Si. US Vol. 7, No. 2, pp , February 1978 Biohemistry Ionophore and thrombin-indued platelet aggregation: Independene rom yloygenase produts (phospholipase/arahidonate/thromboanes/phosphatidate/eiosatetraynoi aid). G. LPIN, K.. CHNDRBOS, ND P. CURCSS Department o Moleular Biology, Wellome Researh Laboratories, Researh riangle Park, North Carolina 2779 Communiated by K. M. Brinkhous, Deember 9,1977 BSRC Stimulation o platelets labeled with ["C]- arahidonate by ionophore or thrombin produes rapid degradation o speii membrane phospholipids. his is also releted by the release o ['4C]arahidonate, whih is immediately transormed into produts o the yloygenase and lipoygenase enzyme systems, and by inreased labeling o phosphatidi aid. rahidonate metabolism an be eetively prevented by preinubation with indomethain and eiosatetraynoi aid, but platelet aggregation indued by ionophore or thrombin is not bloked under these onditions. Nevertheless, in the virtually total absene o metabolism o arahidonate, platelet aggregation still ours onomitantly with phospholipid breakdown and with inreased labeling o phosphatidi aid. Inreased levels o yli MP blok both phospholipase ativation and aggregation indued by ionophore and thrombin. hese data suggest that some early onsequene o phospholipase ativation, independent o a metaboli produt o arahidonate but possibly related to the prodution o phosphatidi aid, may play a entral, ausative role in mediating platelet aggregation. Metabolism o arahidoni aid by the yloygenase enzyme system leads to prodution o the ative yli endoperoides, prostaglandins G2 and H2 (PGG2 and PGH2), and o thromboane 2 (X2). lthough these produts are apable o induing platelet aggregation (1-), their relevane in mediating the normal proess o aggregation indued by physiologial stimuli has been questioned (4, 6, 7). rahidonate is released rom phospholipids when platelets are stimulated by thrombin, ionophore 23187, and ollagen. his arahidonate is utilized immediately by the yloygenase and lipoygenase enzyme systems (8-1). hrombin- and ionophore indued platelet aggregation an be bloked by dibutyryl adenosine 3':'-yli monophosphate (Bt2MP), yli MP (MP) phosphodiesterase inhibitors, and stimulators o adenylate ylase (1, 12, 14-17). Very reently, it has been shown that elevation o MP inhibits release o arahidonate rom phospholipids, and it was suggested that this ation o MP might, at least in part, eplain its inhibitory eet on platelet aggregation (1, 12, 14, 1). It seems, however, that the yloygenase produts that appear during platelet stimulation may not be involved in mediating the aggregation or release reation sine aspirin and indomethain do not blok thrombin- and ionophore indued aggregation (16-19). I this interpretation is orret, then it is apparent that i the phospholipid/arahidonate system is to be involved at all in a ausative role, some step proimal to arahidonate metabolism must be onsidered. We have shown that phospholipase ativation and aggregation indued by thrombin or ionophore are bloked he osts o publiation o this artile were derayed in part by the payment o page harges. his artile must thereore be hereby marked "advertisement" in aordane with 18 U. S. C solely to indiate this at. 818 by inreased levels o MP. Indomethain inhibits yloygenase ativity and eiosatetraynoi aid inhibits both yloygenase ativity. Sine neither o these inhibitors prevents aggregation by thrombin or ionophore 23187, the possible role o arahidonate metabolites, but not o other produts o membrane phospholipases, must be urther questioned as ausative agents. MRILS ND MHODS Most o these have already been essentially desribed (1). Platelet-rih plasma was obtained rom 1 unit o horse blood antioagulated with antioagulant itrate detrose. Samples ( ml) o the plasma were inubated with 2 ici o [1-14C]arahidonate at 37 or 2 hr. Platelets were then isolated by entriugation, resuspended in ris/saline/d buer (NaCl, 134 mm/1 mm ris-hci, ph 7.4/1' mm D/ mm D-gluose), entriuged again, and inally resuspended in 1-2 ml o the same buer. Samples o ml (3-6 mg o protein) were inubated with ionophore (2,gM) (li Lilly and Co.), or thrombin (1 unit/ml) or min. Preinubations with or without Bt2MP, aminophylline plus methylisobutylanthine, or eiosatetraynoi aid (Homan-La Rohe, In.) were or 1-1 min ollowed by a -min inubation with or without thrombin or ionophore Platelets were treated with indomethain during the 2 hr o [14C]arahidonate-labeling or they were not treated with indomethain. Dupliate inubations were stopped with hloroorm/methanol and partitioned with hloroorm and water. Lipids rom the lower phases were dried under N2 and redissolved in hloroorm (1). One o the dupliate inubation mitures was plaed on thin-layer hromatography plates or phospholipid separation (hloroorm/ methanol/aeti aid/water, :3:8:4, vol/vol) (1, 2) and the other one or separation o phosphatidi aid, XB2, 12- hydroy-,8,1,-heptadeatrienoi aid (HH), 12-hydroy-,8,1,14-eiosatetraenoi aid (H), and arahidonate (top phase o ethyl aetate/2,2,4-trimethylpentane/aeti aid/ water, 9::2:1, vol/vol) (1). In all eperiments o thrombin-indued release o [14C]arahidonate metabolites we notied a marked inrease o radioativity in a spot that runs very lose to the origin. his spot does not hromatograph with known standard prostaglandins and it is degraded by phospholipase 2 with release o [14C]arahidonate. he ompound inorporates 32P, and it is quantitatively reovered with the solvent ront in our usual phospholipid separations where phosphatidi aid is present. uthenti phosphatidi aid ohromatographs with this ative spot and not with other phospholipds, whih remain together bbreviations: MP, yli MP; Bt2MP, dibutyryl MP; X, thromboane; HH, 12-hydroy-,8,1-heptadeatrienoi aid; H, 12-hydroy-,8,1,14-eiosatetraenoi aid.

2 Biohemistry: Lapetina et al. 3! Pro. Natl. ad. Si. US 7 (1978) ' - l Q. C.) * lonophore 2318, jum FIG. 1. et o dierent onentrations o ionophore on [14C]arahidonate-labeled platelets., H;, HH; *, XB2; o, phosphatidi aid. - _ ir 2 at the origin (phosphatidylholine, phosphatidylethanolamine, phosphatidylinositol, and 9phingomyelin). Radioative spots were loalized by radioautography and quantitated by liquid sintillation ounting. Platelet aggregation o -ml samples o platelet-rih plasma or washed platelets (-2 mg o protein) was measured by ontinuous reording o light transmission at 37 (Chronolog Corp., Broomall, P) RSULS et o Ionophore on Redistribution o Radioativity rom [14CJrahidonate-Labeled Platelets. s shown previously (1, 14), inubation o platelet-rih plasma with [14C]arahidonate or 2 hr leads to its inorporation into the phospholipids o horse platelets. When the labeled platelets are washed and treated with ionophore 23187, radioativity is deteted in arahidonate and produts o lipoygenase (H) and yloygenase (HH and XB2), and there is inreased inorporation o label in phosphatidi aid (Fig. 1). ept or,) ( C 6 me, se 12 FIG. 2. me-ourse o ionophore (2 MM) eet on [14C]arahidonate-labeled platelets. *, rahidonate; other symbols as in legend o Fig. 1. Phosphotidyk- Phosphatidyl- Phosphatidyl- Phosphatidi holine ethanolamine inositol aid FIG. 3. et o MP phosphodiesterase inhibitors and Bt2MP on ionophore and thrombin ation on [14C]arahidonate-labeled phospholipids. Preinubations with or without aminophylline (2. mm) plus methylisobutylanthine (22 mm) or Bt2MP (1 mm) were or 1 min ollowed by a -min inubation with or without ionophore (2 1M) or thrombin (1 unit/ml). C, ontrol without additions; l, ation o ionophore 23187; 2, preinubation with aminophylline plus methylisobutylanthine ollowed by ionophore 23187; 3, preinubation o Bt2MP ollowed by ionophore , 2, and 3 are as in l, 2, and 3, respetively, but inubations were with thrombin. H, between 2 and,um o ionophore gives the maimal inorporation into the [14C]arahidonate produts. t very short time intervals (e.g., 1 se) there is release o [14C]arahidonate whih is immediately metabolized to H, HH, and XB2 (Fig. 2). Inrease labeling o phosphatidi aid is also observed at short time intervals. ter 3 se there is virtually no urther inrease in HH, XB2, and phosphatidi aid, but there is ontinued inorporation o radioativity into H. MP Inhibits hrombin- and Ionophore Indued Phospholipid Breakdown. Ionophore dereases the ['4C]arahidonate radioativity present as phosphatidylholine and phosphatidylinositol, but not phosphatidylethanolamine (Fig. 3). his ionophore-indued degradation is substantially inhibited by aminophylline and methylisobutylanthine and by Bt2MP (Fig, 3). hrombin degrades all these phospholipid lasses, and this is prevented by Bt2MP, aminophylline plus methylbutylanthine, and by prostaylin, a potent stimulant o adenylate ylase (res. 1 and 14; Fig. 3). hrombin also inreases inorporation o [14C]arahidonate into phosphatidi aid, perhaps even more eetively than the ionophore, and this is also bloked by MP (Figs. 3 and ). he ionophore and thrombin-indued phospholipid degradation ours in parallel to ormation o H, HH, XB2, and

3 82 Biohemistry: Lapetina et al. Pro. Natl. ad. Si. US 7 (1978) in 6 1 1O l I 3 H r l 2 I C) U o 2~ C C h 23 o L _ XB2 HH H FIG. 4. et o MP phosphodiesterase inhibitors and Bt2MP on ionophore and thrombin-indued prodution o oygenated metabolites rom [14C]arahidonate-labeled platelets. ll details as in legend o Fig. 3., arahidonate. arahidonate, the ormation o whih is also onomitantly bloked by Bt2MP and by aminophylline plus methylisobutylanthine (Fig. 4) in a onentration-dependent manner (Fig. ). ets o Indomethain and iosatetraynoi id. Pretreatment o platelet-rih plasma with indomethain is eetive in bloking the yloygenase produts, as releted by HH and XB2 when platelets are stimulated with ionophore and thrombin (Fig. 6). he derease, in HH and XB2 is aompanied by inreased labeling in H. Further addition o indomethain to washed platelets beore or during 4) a: o I I M, mm MIX, mm FIG.. tion o dierent onentrations o MP phosphodiesterase inhibitors on the ionophore eet on [14C]arahidonate-labeled platelets. Preinubations with aminophylline (M) plus methylisobutylanthine (MIX) were or 1 min ollowed by ionophore or min. Symbols: see legend o Fig. 1. C P I XB2 I HH L H I I FIG. 6. et o ionophore and thrombin on [14C]arahidonate-labeled platelets pretreated with indomethain. Platelet-rih plasma was labeled with [14C]arahidonate or 2 hr with and without indomethain (1M4M). Platelets were then isolated and inubated without urther additions (C) or with 2 IM ionophore () or 1 unit o thrombin per ml () or min. Subsript I reers to platelets that have been pretreated with indomethain. P, phosphatidi aid;, arahidonate. treatment with ionophore and thrombin has no urther eet. he inreased 14C labeling into phosphatidi aid indued by ionophore or by thrombin is not aeted by indomethain. Pretreatment o washed platelets with eiosatetraynoi aid (7-28,gM) inhibits nearly ompletely the ormation o produts derived rom lipoygenase and yloygenase ativities indued by ionophore and thrombin (Fig. 7). Radioative H, HH, and XB2 are prooundly dereased, whereas ree ["4C]arahidonate is inreased. iosatetraynoi aid also slightly inhibits phospholipase ativity, as releted by the total phospholipid radioativity (Fig. 7). et o Indomethain and iosatetraynoi id on Platelet ggregation. Ionophore (-4,uM) indues aggregation o horse blood platelet-rih plasma, and this aggregation is not bloked by preinubating the plasma with 1 1M indomethain or 2 hr (data not shown). Ionophore (-1,uM) also indues aggregation o washed platelets that have been isolated rom platelet-rih plasma treated or not treated with 1 1M indomethain or 2 hr. In both ases aggregation is inhibited by aminophylline plus methylisobutylanthine or Bt2MP (Fig. 8). lthough thrombin-indued aggregation (washed platelets) is also similarly inhibited by MP (1, 14), aggregation is not prevented by preinubation o the platelet-rih plasma with indomethain (Fig. 8). Further addition o indomethain (1- jim) to the washed platelets rom the pretreated platelet-rih plasma also does not prevent aggregation by 2-4 unit o thrombin per ml (data not shown). Furthermore, ionophore and thrombinindued aggregation is also not bloked by preinubation (1-4

4 12 Biohemistry: 1 _ Lapetina et al. n 3V a()~~~~~~~ 2. Pro. Nati. ad. Si. US 7 (1978) 821 I.OHM O. M IHM B (I FM) C (I FM) D (I IM) *, 6 *6 a ~~~~~~~~ ii L otol phospholipids P XB2 HH H FIG. 7. et o eiosatetraynoi aid on ionophore and thrombin ation on 14C-labeled platelets. Preinubation with or without eiosatetraynoi aid (28 MM) was or 1 min ollowed by a -min inubation with or without ionophore (2 MM) or thrombin (1 unit/ml). C, Control without additions;, ation o ionophore 23187;, ation o thrombin. Subsript e reers to platelets preinubated with eiosatetraynoi aid. P, phosphatidi aid;, arahidonate. min) with 1MM eiosatetraynoi aid under onditions under whih all arahidonate metabolism is virtually bloked (Fig. 8). DISCUSSION Indomethain, whih is learly shown to blok yloygenase produt ormation (re. 8 and Fig. 6), does not inhibit thrombinor ionophore indued aggregation. iosatetraynoi aid inhibits both yloygenase and lipoygenase ativities prooundly (re. 8 and Fig. 7) but does not aet aggregation. Several reports rom other laboratories desribe the inability o aspirin and indomethain to blok thrombin- and ionophore-indued aggregation (16-19). ll these observations suggest that produts derived rom known metaboli routes o arahidonate metabolism are not essential mediators o platelet aggregation. * However, these data do not ompletely elude the possibility that the prodution o only minute or undetetable amounts o endoperoides or thromboanes are suiient to initiate aggregation. Despite the lak o evidene or an obligatory role o an arahidonate metaboli produt or aggregation, muh evidene still suggests that some proess related to phospholipid degradation may be an early and speii event. he early epression o phospholipase ativity, whih also leads to the ormation o produts other than arahidonate, is greatly avored by thrombin and ionophore as well as virtually all other stimuli o primary platelet aggregation. Further, inhibition o phospholipase ativity by MP orrelates well with the blokade o platelet aggregation. Sine phospholipase ativity * It is well known that indomethain inhibits platelet "aggregation," but, as summarized reently (21), this indomethain-sensitive aggregation is o a "seondary" (i.e., "seond wave") type and thus is dierent rom the "primary" aggregation events studied here. 1 ) l~~~~ 1 'a 1 JO hrombin hrombin hrombin (13 unit/ml) (13 unit/ml) ( unit/ml) (1 MM) FIG. 8. tion o MP phosphodiesterase inhibitors, Bt2MP, indomethain, and eiosatetraynoi aid, on ionophore and thrombin-indued aggregation o washed platelets. () ggregometer traings o dierent onentrations o ionophore 23187; (B) eet o a 1-min preinubation with aminophylline (2. mm) plus methylisobutylanthine (22 mm); (C) eet o a 1-min preinubation with Bt2MP (1 mm); (D) as in B but platelets were pretreated with 1,MM indomethain as indiated in Fig. 6; () thrombin; (F) thrombin-indued aggregation o indomethain-pretreated platelets; (G) ation o a 1-min preinubation with 1 MM eiosatetraynoi aid () on thrombin-indued aggregation; (H) ation o a 4-min preinubation with 1 MM eiosatetraynoi aid on ionophore indued aggregation. is alium dependent (22), the ation o MP ould in theory be eplained by a redution o this divalent ation. It has reently been shown that platelet vesiles an onentrate alium when inubated in the presene o MP, P, and protein kinase (23). In order to eamine the possible role o phospholipase ativity independent o alium mobilization, we tried a more speii phospholipase 2 inhibitor, bromophenaylbromide (24), whih produes inhibition o aggregation in rabbit platelets (7). However, under our usual eperimental design, bromophenaylbromide (1- gm) ails to inhibit thrombin- and ionophore indued phospholipid degradation. Meparine is reputedly another potential phospholipase 2 inhibitor that also inhibits rabbit platelet aggregation, but it is questionable whether its ation on phospholipase is speii (7, 9). t present, the preise physiologial role o this phospholipase ativity annot be asertained. It is quite possible, as has been supposed beore (11, 13), that the phospholipase ativation and phospholipid breakdown during platelet stimulation is simply seondary to this stimulation or an independent onsequene o Ca2+ mobilization. However, virtually no inormation is urrently available about the alium requirements, i indeed any suh eist, or ativation o a ritial phospholipase during platelet stimulation. he ativity o a phospholipase that speiially releases arahidonate in platelet homogenates and membranes is greatly stimulated in the presene o 1 mm Ca2+ (22). hrombin and ionophore inrease ['4C]arahidonate labeling o phosphatidi aid (Figs. 1-3 and -7) almost ertainly on the basis o phospholipid degradation sine washed, labeled platelets are virtually devoid o ree [14C]arahidonate. Inreased labeling o phosphatidi aid is deteted 2 se ater addition o thrombin, and probably relets the prodution o diaylglyerol and its phosphorylation by diaylglyerol kinase.

5 822 Biohemistry: Lapetina et al. Notably, MP also inhibits thrombin- and ionophore indued labeling o phosphatidi aid in parallel with the inhibition o aggregation. It is reasonable to speulate that the appearane o phosphatidi aid may be releting the ativity o a very speii phospholipase (probably o the C- type) possibly related in an important way to the early events o platelet aggregation. It has also been reported that DP, thrombin, and ollagen inrease 32P labeling into phosphatidi aid in rabbit platelets (, 26). Very reently, the same group has also reported that [14C]arahidonate labeling o phosphatidi is inreased by thrombin (27). 1. Hamberg, M. & Samuelsson, B. (1973) Pro. Nati. ad. Si. US 7, Hamberg, M., Svensson, J. & Samuelsson, B. (197) Pro. Natl. ad. Si. US 72, Needleman, P., Monada, S., Bunting, S., Vane, J. R., Hamberg, M. & Samuelsson, B. (1976) Nature 261, Needleman, P., Minkes, M. & Raz,. (1976) Siene 193, Needleman, P., Raz,., Ferrendelli, J.. & Minkes, M. (1977) Pro. Natl. ad. Si. US 74, Bult, H. & Bonta, I. L. (1976) Nature 264, Vargatig, B. B. (1977) J. Pharm. Pharmaol. 29, Bills,. K., Smith, J. B. & Silver, M. J. (1976) Biohim. Biophys. ta 424, Blakwell, G. L., Dunombe, W. G., Flower, R. J., Parsons, M. F. & Vane, J. R. (1977) Br. J. Pharmaol. 9, Lapetina,. G., Shmitges, C. J., Chandrabose, K. & Cuatreasas, P. (1977) Biohem. Biophys. Res. Commun. 76, Pikett, W. C., Jesse, R. L. & Cohen, P. (1977) Biohim. Blophys. ta 486, Pro. Nati. ad. Si. US 7 (1978) 12. Minkes, M., Stanord, N., Shi, M. M. Y., Roth, G. J., Raz,., Needleman, P. & Majerus, P. N. (1977) J. Clin. Invest. 9, Rittenhouse-Simmons, S. & Deykin, D. (1977) J. Clin. Invest. 6, Lapetina,. G., Shmitges, C. J., Chandrabose, K. & Cuatreasas, P. (1978) in dvanes in Prostaglandin and hromboane Researh, eds. Galli, C., Galli, G. & Porellati G. (Raven Press, New York), Vol. 3, pp Gerrard, J. M., Peller, J. D., Krik,. P. & White, J. G. (1977) Prostaglandins 14, White, J. G., Rao, G. H. R. & Gerrard, J. M. (1974) m. J. Pathol. 77, Feinstein, M. B. & Fraser, C. (197) J. Gen. Physiol. 66, O'Brien, J. R. (1968) Lanet i, Smith, J. B. & Willis,. L. (1971) Nature 231, Skipski, V. P., Peterson, R. F. & Barlay, M. (1964) Biohem. J. 9, Charo, I. F., Feinman, R. D. & Detwiler,. C. (1977) J. Clin. Invest. 6, Derksen,. & Cohen, P. (197) J. Biol. Chem., Kaser-Glanzmann, R., Jakabova, M., George, J. N. & Luisher,. F. (1977) Biohim. Biophys. ta 466, Volwerk, J. J., Pieterson, W.. & De Haas, G. H. (1974) Biohemistry 13, Lloyd, J. V., Nishizawa,.., Joist, J. H. & Mustard, J. F. (1973) Br. J. Haematol. 24, Lloyd, J. V. & Mustard, J. F. (1974) Br. J. Haematol. 26, Leung, N. L., Kinlough-Rathbone, R. L. & Mustard, J. F. (1977) hromb. Haematol. 38,7 (bstr.)