A LTHOUGH epinephrine is well known to cause platelet aggregation and secretion, the mechanisms of its 5everal

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1 The Response of Pltelets to Epinephrine in Storge Pool Deficiency- Evidence Pertining to the Role of Adenosine Diphosphte in Mediting Primry nd Secondry Aggregtion By Hrvey J. Weiss nd Bruce Lges Aggregtion responses nd thromboxne (Tx) formtion in ten ptients with storge pool deficiency (SPD) specific to the dense grnules (5-SPD) were studied to ssess further the role of dense grnule denosine diphosphte (ADP) in mediting pltelet ggregtion by epinephrine. The bility of epinephrine to elicit secondry ggregtion (SA) responses ws highly vrible in &-SPD when tested t 5 smol/l epinephrine. but ws consistently bnorml when tested over rnge of concentrtions. The occurrence of SA in both -SPD ptients nd norml subjects ws correlted with the mgnitude of the rte of primry ggregtion (PA). This PA rte ws norml, on verge. for the entire ptient group but ws greter in ptients with more consistent SA responses. The PA findings were relted to the K,, vlue obtined in binding studies with 3H-yohimbine. A LTHOUGH epinephrine is well known to cuse pltelet ggregtion nd secretion, the mechnisms of its 5everl ctions remin uncler. When dded in concentrtions bouve I mol/l to citrted pltelet-rich plsm (PRP), it usully elicits n immedite ggregtion response (primry ggregtion, PA) tht is followed by lrger, secondry ggregtion (SA) response2 3 ssocited with secretion nd production of thromboxne A2 (TxA2). 4 5 The role of denosine diphosphte (ADP) in both the PA nd SA responses to epinephrine is still unsettled. Mcfrlne nd Mills demonstrted tht excess ATP, which blocks the effect of ADP, hs no effect on PA.6 More recently, however, Figures et l reported tht 5 -p-fluorosulfonylbenzoyl denosine (FSBA), n irreversible inhibitor of ADPmedited pltelet ctivtion, completely bolished ggregtion by epinephnine.7 In ddition, FSBA lso blocked the exposure of fibninogen receptors,7 prerequisite for ggregtion.8 3 In contrst, Peerschke reported tht epinephrine cn expose fibninogen receptors nd induce ggregtion independently of relesed ADP.8 Thus, these nd other studies9 4 leve unnswered the question of whether ADP plys role in the PA response of pltelets to epinephnine. Eqully uncler, nd controversil, is the role of ADP in SA. Since SA is ssocited with the secretion of dense grnule ADP, 5 n ssumption tht SA is medited by secreted ADP ppered resonble. 6 7 However, Chro et nd Hung nd Detwiler 9 suggested tht ADP secretion occurs in prllel with SA, but is not its cuse. The pltelets of ptients with storge pool deficiency (SPD) re deficient in dense grnule ADP.2#{176}24Therefore, studies on the ggregtion responses of SPD pltelets to epinephrine my help to define further the role of ADP in mediting PA nd SA. Although SA ws not observed in erly studies on SPD, it hs been reported more recently in some SPD ptients with severe deficiencies of ADP In the present study, we report our findings on PA nd SA, nd the role of ADP nd TxA2 in mediting these responses, in ten ptients with SPD who re severely deficient in dense grnules but who hve norml mounts of -grnule substnces (b-spd). Prllel studies on five but not with the number of 2-receptor sites. Studies on Tx production (ssessed by rdioimmunossy of TxB2) showed tht the bility to synthesize Tx from rchidonte ws not impired in -SPD. nd tht there ws n bsolute positive correltion between epinephrine-induced SA nd Tx production. Aggregtion in -SPD pltelets in response to the Tx receptor gonist U4469 ws consistently decresed, but could be corrected by ddition of ADP. The results of the study suggest tht dense grnule-derived ADP is not required for PA by epinephrine. but medites SA s synergistic gonist with TxA2. This role of ADP in SA my be elucidted more precisely by further studies on pltelet ctivtion processes in S-SPD. S 1988 by Grune & Strtton. Inc. ptients with combined nd dense grnule deficiency (#{244}-SPD) illustrte the importnce of defining precisely the type of grnule defect in interpreting results obtined on ptients with SPD. METHODS Blood Collection nd Preprtion of PRP Blood ws obtined by venipuncture nd mixed (9:1) with 3.2% trisodium citrte dihydrte (.1 1 mol/l). Citrted PRP ws prepred by centrifugtion t 1,5 g for three minutes t 2#{176}C (undjusted PRP). For some studies, the hemtocrit ws determined nd the citrte concentrtion djusted to 19.7 mmol/l in PRP by ddition of sodium citrte. After obtining PRP, the pltelet count ws then djusted to 2,/zL with utologous pltelet-poor plsm (PPP), obtined by centrifuging PRP t 2,8 g for 3 minutes t 2#{176}C. This djusted PRP ws distributed in l-ml liquots into siliconized ggregometer cuvettes nd seled with prfilm under C2/ir (5/95) until used for ggregtion studies. The ph of the djusted PRP ws mesured before nd fter ggregtion studies nd vried fom 7.67 to 7.7. Pltelet Aggregtion Pltelet ggregtion studies on PRP were performed in Pyton Dul Chnnel Aggregtion Modules (Pyton Assocites, Bufflo) with the stirring speed set t 5 rpm. Studies on undjusted PRP From the Deprtment of Medicine (Division of Hemtology- Oncology), St. Luke s-roosevelt Hospitl Center nd Columbi University College ofphysicins nd Surgeons. New York. Submitted Februry 4, 1988; ccepted July 12, Supported in prt by Grnt No. HL27346 from the US Public Helth Service nd grnt from The Chrles Slughter Foundtion. Address reprint requests to Hrvey J. Weiss, MD. St. Luke s- Roosevelt Hospitl Center, 428 WS9th St. New York, NY 119. The publiction costs ofthis rticle were defryed in prt by pge chrge pyment. This rticle must therefore be hereby mrked dvertisement in ccordnce with /8 U.S.C. section solely to indicte this fct by Grune & Strtton, Inc /88/725-27$3./ Blood, Vol 72, No 5 (November), 1988: pp

2 1718 WEISS AND LAGES were initilly (1973 to 1978) performed with 2 ml, nd subsequently (1979 to 1987) with 1 ml of PRP. The instrument ws djusted so tht the difference in light trnsmission between PRP nd PPP ws recorded s I mm, nd the chrt speed ws 2 mm/mm. The PA response to epinephrine ws quntitted s the initil upwrd slope of the ggregtion trcing. SA ws defined s the mximum deflection (mm) from bseline, fter subtrcting the mximum deflection (mm) observed for PA. In most cses, the occurrence of SA ws pprent from the trcing s shrp increse in light trnsmission fter the smller PA response, but occsionlly this SA response ws smll. For the purpose of this study, nd to void subjective interprettion of the trcing, SA ws sid to be present when this response exceeded 2 mm. Grded concentrtions of epinephrine were used to determine the threshold concentrtion required for SA. The threshold PA ssocited with SA ws estimted from plot of SA v PA slope. In djusted PRP, the mximum PA slope ws determined from multiple studies using 5 nd 5 zmol/l concentrtions ofepinephnine, nd the EC vlue for PA ws determined from dose-response curve using concentrtions of epinephnine between.1 nd 5 mol/l. Aggregtion studies were begun pproximtely one hour fter venipuncture nd were generlly completed in one hour. Thromboxne A2 Formtion TxA2 formtion in PRP in response to gonists ws ssessed by centrifugtion of the PRP nd by rdioimmunossy of the stble metbolite TxB2 in the superntnt PPP, s previously described.#{176} Binding of3h-yohimbine to Gel-filtered Pltelets Pltelets were gel-filtered on Sephrose 2B into C, Mg-free Tyrode s buffer, ph 7.4, contining 15 mmol/l HEPES,.1% glucose, nd.2% bovine serum lbumin s described previously.3 Binding studies were conducted by incubting triplicte.5 ml liquots of gel-filtered pltelets (GFP) with.5 to 2 nmol/l [3H]-yohimbine (75 Ci/mmol, NEN Dupont) for 3 minutes t 37#{176}C in the bsence nd presence of 2,zmol/L unlbeled yohimbine. At the end ofthe incubtion period, the GFP ws diluted with 5 ml of C, Mg-free Tyrode s-hepes, buffer, rpidly filtered over vcuum on Whtmn GF/F filters, nd the filters wshed with 3 x 5 ml of buffer. Pltelet-bound 3H-yohimbine retined on the filters ws counted by liquid scintilltion, nd specific binding of 3Hyohimbine determined s the difference in rdioctivity between the smples incubted in the bsence nd presence of unlbeled yohimbine. The mount of 3H-yohimbine retined by the filters in the bsence of pltelets ws negligible. The binding prmeters of mximum binding sites (Bmx) nd dissocition constnt (K,,) were determined by Sctchrd nlysis of the binding dt.32 In ll cses, the Sctchrd plot indicted single clss of binding sites. Regents L-epinephnine (Prke-Dvis, Detroit) ws stored s 5 mmol/l solution nd diluted with.15 mol/l NCI before use. ADP ws obtined from Sigm Chemicl Co, St Louis. The TxA2 receptor gonist U4469 ws generous gift from Dr i.e. Pike, The Upjohn Co, Klmzoo, MI. Archidonic cid ws from Nuchek Prep, Elysin, MN, nd ws used s the K slt, 1 mg/ml. For in vitro studies, spirin (ASA) ws dissolved in freshly prepred. 12 mol/l NHCO3. PRP ws preincubted with 2 zmol/l ASA for two minutes t 37#{176}C before ddition of gonist. Ptients Ten ptients with b-spd were studied over period of I 5 yers. In these ptients (Tble 1), some of whom hve been described in previous studies, pltelet dense grnules nd substnces stored in dense grnules re mrkedly decresed nd the pltelet denosine triphosphte (ATP)/ADP rtio is incresed, but the number nd content of -grnule substnces re norml.27 Seven of the ten ptients hd oculocutneous lbinism (Hermnsky-Pudlk syndrome, Tble I ). Five ptients with -SPD27 were lso studied. Of these, four re from Fmily C27 with the originilly reported fmilil SPD defect,#{176} 2 nd one (ptient ic.) hs the most severe defect of -grnules. Norml Subjects Control subjects were norml mle nd femle hospitl personnel (ge 25 to 55 yers) who were studied in prllel with the ptients. Both ptients nd control subjects denied recent ingestion of drugs, such s spirin, known to ffect pltelet function for t lest I week before study. All studies were performed under protocol pproved by the Institutionl Review Bord of the St. Luke s-roosevelt Institute for the Helth Sciences. Undjusted PRP RESULTS Incidence of SA in norml subjects. Aggregtion induced by 5 tmol/l epinephrine ws mesured in 332 smples of undjusted PRP from 1 3 norml subjects (6 mle nd 7 femle) over 1 5-yer period. The number of seprte studies performed on individul subjects vried; studies were performed once in 77 subjects, nd in other subjects two to five times (n = 36), six to ten times (n = 13), nd 1 1 to 17 times (n = 4). PA ws observed in ll subjects nd, in most cses, ws followed by SA. However, SA did not occur in 29 of the 332 smples. SA occurred t lest once in 1 17 of the 1 3 subjects (9%). It ws bsent t lest once in rndom tests performed on 26 subjects (2%), but ws present on one or more other occsions in 1 3 of I 5 subjects in whom multiple studies were performed. Becuse of the considerble vrition in the number of tests performed on individul subjects, the frequency of SA in our popultion of norml subjects cn only be estimted, but figure of round 85% to 9% ppers resonble. SA responses in b-spd nd reltionship of #{163}4to PA. The frequency of SA induced by 5 mol/l epinephnine in ptients with b-spd studied over the sme time period s the controls is shown in Tble 1. In ptients no. 1, 2, nd 3, designted s group I, SA ws observed on ech of seven to 1 1 occsions. In ptients no. 4 to 7 (group II), SA on multiple testing ws observed 4% to 5% of the time, wheres in ptients no. 8, 9, nd 1 (group III), it ws rrely, if ever, seen (%to 18%). The presence or bsence of SA ws relted to the mgnitude of the PA response (slope) in both norml nd t5-spd subjects. In norml subjects, the PA slope ws.72 ±.3 when SA occurred, nd.35 ±.5 when it ws bsent. As seen in Tble 1, the sme reltionship ws observed in b-spd ptients. The PA slope ws consistently greter in ech ptient, nd in the group verges, when SA occurred (PA + SA) thn when it did not (PA only). Evidence for n bnormlity of SA in ll ptients with &-SPD. In contrst to the vrible occurrence of SA responses in b-spd observed with 5 mol/l epinephnine, SA

3 EPINEPHRINE RESPONSES IN SPD PLATELETS 1719 Tble 1. Epinephrine-induced Aggregtion in Undjusted PRP in -SPD Pltelet ADP Content (nmol/ 1 ) Responses With 5 mol/l Epinephrine Frequency of SA No. of SA PA SI #{176} Studies Occurrence (%) PA Only PA + SA eshd Vlues for SA. Epinephnne Concentrtion (jmol/l) PA Slope I (1)JD (2)WA (3)JV ±SEM 1.14±.21.9±.1 II (4) EP (5) NR (6) RZ (7)LM ± SEM.44 ±.8.78 ±.5.7 ±.1 Ill (8)LG (9) LV (1O)JA ± SEM.41 ±.12.7 ±.3 All ptients ± SE 4 ± 1.42 ±.6.91 ±.9 13 ± 5.8 ±.1 Norml subjects ± SEM 23 ± 1 332t 85-got.35 ±.5.72 ±.3.28 ±.6.15 ±.3 N Albino ptients (Hermnsky-Pudlk syndrome). tsee Results. ws consistently bnorml in ll these ptients (including those designted s type I) when ssessed over rnge of epinephnine concentrtions. As shown in Tble I, the threshold concentrtion of epinephnine required to elicit SA in norml subjects ws.28 ±.6 tmol/l, nd this ws ssocited with PA slope of.15 ±.3. In ll #{244}-SPD ptients, concentrtion of epinephnine tht elicited n SA response could, on most occsions, be found, but this response occurred t much higher concentrtion of epinephnine ( 1 to 5 smol/l) nd greter initil PA slope (.5 to I.2). These observtions explin, to some extent, the vrible frequency of SA observed in these ptients using the stndrd 5 tmol/l concentrtion of epinephnine. In group I ptients, the PA slope elicited by 5 tmol/l epinephrine ws lwys t, or bove, the threshold vlue ssocited with SA, nd SA invribly occurred (Fig 1). In other ptients, the presence or bsence of SA on ny occsion correlted well (lthough not bsolutely) with whether the PA slope ws bove or below the threshold vlue for SA in tht ptient (Fig 1). Pltelet counts nd citrte concentrtion in PRP. The men pltelet counts (±SEM) in PRP of group I (351, ± 56,), group II (445, ± 46,), nd group III (47, ± 78,) ptients were not significntly different from the control vlues of433, ± 14, (rnge, 233, to 633,). The blood hemtocnit vlues (±SEM) in the group I, II, nd III ptients (41.6 ±.8, 41. ± 1.4, nd 39.3 ± 2., respectively) were comprble, nd the clculted citrte concentrtions in the three groups were Ui. -J U) z Ui ci: ( ( >. : :. 2 IC L_L-IJ LLJ2J GROUP I GROUP II GROUP l Fig 1. Reltionship between PA nd SA with 5 smol/l epinephrine in -SPD. The PA slope in ptients no. 1 through 1 (Tbles 1 nd 2) obtined in undjusted PRP on multiple occsions. Closed circles indicte tht SA occurred; open circles. tht it did not. For ech ptient. the horizontl br indictes the threshold PA slope ssocited with SA obtined in seprte dose-response studies. Ptient subgroups (I. II. III) re defined by the incidence of SA (see Tble 1 nd Results). The men threshold PA slope ( ± SD) for SA in norml subjects is lso shown.

4 172 WEISS AND LAGES 17.5, 17.4, nd 17. mmol/l, respectively. Thus, differences in the plsm citrte concentrtion [which re known to influence SA responses33 34] re n unlikely explntion for the observed differences in SA mong the groups. The pltelet counts in the blood of the b-spd ptients (men, 255,; rnge, 216, to 36,) were lso norml. Adjusted PRP SA responses in c5-spd nd reltionship to PA Prllel, but more limited, studies were crried out to ssess SA with 5 imol/l epinephrine in PRP djusted for citrte concentrtion (19.7 mmol/l), pltelet count (2,/jL), nd ph (7.67 to 7.7) (Tble 2). As in undjusted PRP, SA ws observed most consistently (five of five occsions) in the group I ptients nd ws ssocited with greter PA slope thn observed for group II nd III ptients, in whom SA occurred on only one of 2 occsions. PA responses in 5-SPD. For ll ten ptients, the mximum PA slope (.77 ±. 1 5) (see Methods) ws entirely similr to tht in norml subjects (.75 ±.5) (Tble 2). However, s seen in Tble 2, the vlues mong the three groups vried considerbly. The PA slope ws 1.39 ±.16 in group I ptients,.61 ±.4 in group II, nd.36 ±.4 in group III (P <.1 for ech group difference). The PA slope ws not relted to the mount of ADP in the ptients pltelets (Tble 1). The EC5 vlue for PA in control subjects (N = 2) ws.83 ±.8 smol/l, nd similr verge vlue (.98 ±.27) ws observed for the ten b-spd ptients (Tble 2). The verge EC5 ws smllest in the group I ptients, but the individul vlues overlpped with those in the other two groups. In seprte studies (dt not shown) performed by mixing PPP with gel-filtered pltelets in vrying combintions, we found tht differences mong subgroups were due to pltelet, nd not plsm, differences. 3H-yohimbine Binding To ssess whether the different PA responses to epinephnine mong b-spd ptients might be relted to differences in the pltelet 2 receptor, we performed binding studies using the specific 2-ntgonist 3H-yohimbine (Fig 2A). As seen in Tble 3, the men mximum binding (Bmx) in b-spd ptients (192 ± 1 1 sites per pltelet) ws not different from tht in controls (1 59 ± 1 1). In ddition, there ws no correltion between the Bmx vlue nd the mximum PA slope determined in seprte studies (Tble 2) on djusted PRP. The Kd (2.9 ±.35 mol/l) in the t5-spd ptients ws lso similr to the control vlue (2.84 ±.18 tmol/l). However, mong the ptients, there ws strong correltion (r =.87, P <.1) between the K,, nd PA slope. As shown in Fig 2B, the greter the PA slope with epinephnine, the lrger the kd for yohimbine. Thromboxne Studies Reltionship between epinephrine-induced SA nd thromboxne formtion. Pltelet ggregtion nd TxB2 production were mesured in undjusted citrted PRP using epinephrine in concentrtions of 1, 5, nd 5 jmol/l. Among 1 7 norml subjects, SA ws bsent only once (with 1 imol/l epinephrine in one subject) nd the TxB2 vlues for the three concentrtions of epinephnine were 43.6 ± 6.6, Tble 2. Epinephrine-induced Aggregtion in Adjusted PRP in &.SPD Frequency of SA Response Wit h 5 imoi/l Epinephrine Subject No. of Studies SA PA Slope Occurrence (%) PA Only PA + SA Mximum PA Slope EC,,, PA Slope (moi/l) I (1)JD (2)WA (3)JV ± SEM 1.16 ± ±.16.4 ±.6 II (4)EP (5) NR (6) RZ (7) LM ± SEM.58 ±.5.61 ± ±.31 Ill (8)LG (9) LV (1O)JA ± SEM.28 ±.5.36 ± ±.81 All ptients ± SEM.45 ±.7.77 ± ±.27 Norml subjects ± SEM ±.8.78 ±.5.75 ±.5.83 ±.8 N PRP djusted for citrte concentrtion, pltelet count, nd ph (see Methods).

5 EPINEPHRINE RESPONSES IN SPD PLATELETS E. zd Ui z m >- I 3H-YOHIMB)NE (nm) 3.5 B 3. (I (I),,,7(2) 2.5 /(9) (71 2 #{176}#{176} MAX)MUM PA SLOPE Fig 2. Binding of 3H-yohimbine to pltelets. (A) Typicl binding of 3H-yohimbine to pltelets obtined in norml subject. Points shown were obtined fter subtrcting nonspecific binding. Insert shows Sctchrd plot of dt. (B) Reltionship (lest squre regression line) between Kd obtined for 3H-yohimbine binding (Tble 3) nd mximum primry ggregtion slope with epinephrine (Tble 2) in #{212}-SPDptients (numbered s in Tbles 1 nd 2). A. I; #{149}. group II; nd #{149}. group Ill ± 6.3, nd 53.4 ± 6.6 nmol/l pltelets, respectively. The results obtined in b-spd ptients re shown in Fig 3. SA ws gin observed most consistently in group I ptients, but in ll ptients there ws n bsolute correltion between SA nd TxB2 production (13 to 43 nmol/1 pltelets). In the bsence of SA, TxB2 ws usully undetectble, nd in no cse ws it greten thn 5 nmol/i pltelets. Thromboxne production nd response to TxA2 gonist U4469. Citrted PRP ws incubted with.8 mmol/l rchidonic cid for five minutes nd TxB2 ws mesured in the superntnt plsm. The mounts of TxB2 (522 to 1,436 nmol/i pltelets) produced in five ptients (no. 2, 3, 5, 7, 9) with b-spd were within the rnge of vlues [1,62 ± 872 (2SD)J obtined in 23 norml subjects. Although, from the bove studies, the cpcity of b-spd pltelets to synthesize Tx ws unimpired, the response to the TxA2 receptor gonist U4469 ws decresed in ptients from ech of the subgroups (two group I nd one ech group II nd III). As seen in Fig 4A, the initil ggregtion slopes were norml, but the ptients pltelets either rpidly disggregted or filed to rech norml mximum response (Fig 5 U) : 4-3 Ui E.5 2 : I- S I 5 5 EPINEPHRINE CONC (SM) Fig 3. Correltion of epinephrine-induced SA with TxB2 in undjusted PRP. The men vlues of TxB2 formtion (horizontl lines) minus 2 SD (verticl open brs) obtined in 1 7 norml subjects re shown for ech concentrtion of epinephrine. #{212}-SPD ptients re designted s group I (tringles). II (squres). nd Ill (circles). Closed figures indicte tht SA occurred; open figures. tht it did not. Note tht on dy tested. SA ws bsent in two ptients even with 5 imol/ I epinephrine. 5). Thus, the mximum ggregtion response to U4469 ws significntly decresed in b-spd (Fig 4B). Effect ofadp on c5-spd Pltelets Effect of dded ADP on epinephrine-induced SA. The cpcity of ADP to restore SA responses to epinephnine ws studied in five group II nd III ptients, nd the results of typicl experiment re shown in Fig 6. With 3 zmol/l epinephnine s gonist, sub-ggregting concentrtion of ADP could usully be found (generlly.1 to.25 zmol/l), which incresed the PA slope nd trnsformed the overll response from one in which only PA ws observed to one in which two distinct responses were seen (Fig 6A). With higher concentrtions of ADP (2. cmol/l), the PA slope incresed still further nd distinct SA response ws not observed. The chrcteristic trcing ws tht of lrge U Tble 3. 2-Adrenoceptor Vlues in -SPD B,.. Kd Subject (sites/pit) (nmol/l) IO- #{212}-SPD (1)JD (2)WA (3) JV Norml (6)RZ (7)LM (8)LG (9)LV (1)JA ±SEM 192± ±.35 ±SE 159± ±.18 N ;!UO(b U4469 CONCENTRATION (SM) 5 I Fig 4. Aggregtion responses of pltelets to TxA2 receptor gonist U4469. Vlues (±SEM) for the initil ggregtion slope (A. left) nd mximum ggregtion (B. right) re shown for 18 norml subjects (#{149}) nd four -SPD ptients (no ) (). All ptients except no. 3 were studied twice nd results for ech were verged. Vlues significntly different thn controls re indicted (#{149}P<.5. P<.1)(Student s ttest).

6 1722 WEISS AND LAGES z Cl) U) U) z 4 ci: I- I- I C-,, (II 255 ( 5 ( 5 CI 3Q NORMAL PT. 7 PT. 9 PT. 3 PT. 2 EPIPIEPIIRINE CNCENTRATlN(MI ASP CNC(MI Fig 5. Effect of ADP on pltelet ggregtion by U4469. The TxA2 receptor gonist U4469 ws dded (finl concentrtions 5 zmol/l) to citrted PRP (middle trcings) or to n liquot of the sme PRP contining.2 smol/l ADP (upper trcings). Trcings obtined with.2 smol/l ADP lone re shown in the lower trcings. Ptients no nd 9 with 6-SPD nd representtive trcings from norml subject re shown. primry wve of irreversible ggregtion. With ll concentrtions, the enhnced effects observed with ADP were ssocited with incresed production of TxB2 (Fig 6A). When Tx synthesis ws blocked with spirin, the ddition of ADP gin resulted in incresed PA responses nd single, irreversible response with 2 tmol/l ADP ws observed (Fig 6B), s previously reported in one ptient with b-spd.35 In no cse, however, were two distinct primry nd secondry ggregtion responses even seen. Effect ofdded ADP on impired U4469-induced ggregtion. The responses of &-SPD pltelets to U4469 were strikingly enhnced by the prior ddition of ADP. As seen in Fig 5, ADP in concentrtion (.2 zmol/l) tht by itself did not ggregte pltelets both prevented disggnegtion nd/ or incresed the mximum response to U4469 in ll four #{244}-SPD ptients studied. This concentrtion of ADP is pproximtely one-tenth of tht relesed from pltelet dense grnules fter mximum secretion.36 PA response of b-spd pltelets to ADP. Studies were performed to determine whether the striking differences in the PA response to epinephnine mong b-spd subjects might lso be true for the PA response to ADP. Prllel doseresponse studies were obtined with both epinephnine nd ADP in djusted PRP from three group I nd five other (group II nd III) ptients. Figure 7 gin demonstrtes the mrked differences in the PA slope with epinephnine in these A B 8) ( In C / --2IO 31.-, (.51 Fig 6. Effect of ADP on epinephrine-induced ggregtion in #{212}-SPD.(A) ADP. in concentrtions rnging from.1 to 2. zmol/l. ws dded to PRP of ptient no. 9 before ddition of 3 imol/l epinephrine. (B) Sme s in A except ASA (2 gsmol/l) ws lso dded. (C) Effect of ADP (.1 to 5. smol/l) lone. Numbers in prentheses t end of ech trcing re the TxB vlues mesured in the superntnt plsm fter centrifugtion. Fig 7. Dose-response curves for primry ggregtion with epinephrine nd ADP. Primry ggregtion slopes (i ± SEM) obtined with epinephrine (A) or ADP (B) in djusted PRP re shown for norml (N) subjects (dshed line). three group I #{212}-SPD (#{149}). nd five -SPD ptients clssified s group II or Ill (). ptients (pooled group I > group II + III). In contrst, there ws no significnt difference in the ADP-dose response curves between these two pooled groups. The greter verge sensitivity of group I ptients t the lower (.3 to.5 zmol/l) concentrtions of ADP ws due entirely to the results obtined in one ptient (no. 2), but these differences were not sttisticlly significnt. Studies on Ptients With b-spd Studies done on ptients with combined deficiencies of nd dense grnules (b-spd) re shown in Tble 4. SA ws never observed with 5 zmol/l (or 5 j.omol/l, dt not shown) epinephnine in ny of these ptients. The pltelet ADP levels were, if nything, somewht higher thn in the ptients with b-spd. The PA slope with epinephrine ws mrkedly nd consistently impired in ll ptients with b-spd. In Fmily C (but not ptient J.C.), this ws ssocited with Bmx for epinephnine binding of 82 ± 1. Finlly, the PA slope with ADP ws lso bnorml in these ptients. In dose-response curves for ADP, similr to those shown in Fig 7, the mximum initil slope (2.5 ±.5) in Fmily C nd ptient ic. (2.7) were both less thn tht in norml subjects (3.8 ±.2). DISCUSSION The PA responses to epinephnine (mximum slope nd EC,O) were, on verge, the sme in ptients with b-spd s in norml subjects (Tble 2). In ddition, lthough there ws considerble vrition in these responses mong ptients, there ws no correltion between the PA response nd the pltelet content of ADP (Tble 1). These findings in ptients whose pltelets re severely deficient in ADP re consistent with previous reports tht ADP is not involved in the primry ggregtion response to epinephnine.4 They re lso consistent with the conclusions of other studies tht ADP is not required for epinephnine-induced binding of fibninogen to GpIIb-III,8 9 prerequisite for ggregtion.8 2 However, direct mesurements of epinephnine-induced fibninogen binding in b-spd pltelets will be necessry to confirm this. Our conclusion pplies only to dense grnule ADP nd ssumes tht, if essentil, this is reduced sufficiently in b-spd to result in impired PA. Other ssumptions bout the specificity of gents such s ATP,6 CP/CPK8 #{176} nd FSBA7

7 EPINEPHRINE RESPONSES IN SPD PLATELETS 1723 Tble 4. Epinephrine Aggregtion nd 2-Adrenoceptor Vlues in #{244}-SPDPtients Responses With 5 cmol/l Epinephrine Frequency of SA inundjusted PRP 3H-yohimbine Bin5ng PA Slope Pltelet ADP No. of SA Mximum PA Slope B,,. Kd Subject Content (nmol/1o ) Studies Occurrence 1%) PA OnIy in Adjusted PRP (sites/ph) (nmol/l) Fmily Severer Norml C DC RC SC TC i±sem 6± ±.6.15±.9 82± ±.46 JC ± SEM 23 ± 1 332t 85-9t.35 ±.5.75 ± ± ±.18 N SA never occurred in these ptients. tsee Results. Ptient with most severe decrese in -grnules. for ADP my ccount for the conflicting conclusions of other studies. Another purpose of the study ws to ssess the role of ADP in mediting SA. With concentrtion of epinephnine (5 zmol/l) tht is used frequently for clinicl studies, we observed SA consistently in three ptients (group I, Tble 1), on bout 5% of occsions in four ptients (group II), nd rrely in three others (group III). The frequency of SA responses with 5 mol/l epinephnine ws not relted to the pltelet ADP content. However, for ll ptients, s for controls, there ws strong positive correltion between the occurrence of SA nd the mgnitude of the PA slope (Tble I ). When tested over rnge of epinephnine concentrtions, consistent bnormlity in SA ws observed in ll b-spd ptients, including those in group I. Thus, concentrtion of epinephnine could usully be found tht elicited SA in ll ptients, but this ws chieved t higher concentrtion of epinephnine nd t lrger PA slope thn required for norml pltelets (Tble 1). These findings of n bnorml SA response in ll b-spd ptients, when ssessed oven rnge of epinephnine concentrtions, re consistent with role for dense grnule ADP in mediting, or potentiting, SA, but leves unresolved the question of whether this ADP is secreted from the pltelet 8 or is relesed nonspecificlly during preprtion of the PRP. 9 The distinguishing feture of the group I ptients is their greten PA response in both stndrd (Tble 1) nd djusted (Tble 2) PRP. SA with 5 zmol/l epinephnine ws observed consistently in group I ptients becuse the PA slope ws invribly greten thn the threshold vlue ssocited with SA (Fig 1). An SA response occurred less frequently in group II, nd rrely in group III ptients, but when observed, it ws lmost lwys ssocited with PA slope t or bove the threshold vlue for tht ptient (Fig I). The reson for the greter PA response in the group I ptients remins to be determined. It is generlly ccepted tht epinephrine binds to pltelets through n 2-dnenengic receptor. Among ptient groups, no significnt differences in the number of pltelet 2 receptors ws observed (Tble 3). Interestingly, however, there ws strong positive correltion between the K1, for 3H-yohimbine binding nd the PA slope, suggesting possible differences in the properties of 2 receptors mong ptients with #{244}-SPD.At present, it is not cler why lrger K, (lower ffinity) for n 2-ntgonist should be ssocited with lrger PA response to epinephrine. Although reciprocl modultion of gonist nd ntgonist binding by gunine nucleotides to some receptor systems hve been described,3 current evidence suggests tht GTP does not influence the binding of yohimbine to 2 receptors in membrnes of humn pltelets. 8 More definitive studies specificlly ssessing the binding of epinephnine to b-spd pltelets my help to clrify this. The question of how ADP medites SA requires further clrifiction, but recent studies on the mechnisms involved in epinephrine ctivtion of pltelets suggest severl possibilities. These studies hve provided evidence tht the initil pltelet responses to epinephnine (fibninogen binding nd PA) ctivtes n N-H exchnge mechnism 9#{176} tht, through phospholipse A2, results in the formtion of smll mount of TxA2. 9#{176}The ltter (perhps cting synergisticlly with the epinephnine-occupied 2 receptor)4 then mitites the phospholipse C-medited formtion of lrger mounts of TxA2.42 Previous studies, in which pltelet cyclooxygense ws blocked with spirin,43 hve demonstrted tht TxA2 production is n bsolute requirement for SA, 8 nd the correltion between SA nd Tx production tht we found in b-spd ptients is consistent with this observtion. Since ADP does not pper to be required for PA responses, one possibility is tht it modultes the N-H exchnge mechnism tht is involved in the phospholipse A2- medited production of TxA2, nd studies to exmine this possibility using b-spd pltelets re nticipted. A more likely explntion is tht ADP behves s synergistic gonist with TxA2 to medite SA. Thus, lthough the formtion of TxA2 from rchidonic cid ws norml in b-spd, the response of #{244}-SPDpltelets to the thromboxne

8 1724 WEISS AND LAGES receptor gonist U4469 ws decresed in ptients from ech subgroup, nd this defect ws corrected by the ddition of ADP in concentrtions well below pproximtely one-tenth the mximum secretble mount (Fig 5). Our recent findings tht dded ADP lso corrects the impirment of thrombin-induced cid hydrolse relese in b-spd pltelets hve suggested tht dense grnule ADP my potentite this response s well.4 Thus, plusible explntion for the present observtions is tht TxA2, produced s consequence of PA, induces SA, nd ADP synergisticlly potentites this response. This might ccount for the greter frequency of SA responses in group I ptients (with lrger PA responses) thn in other SPD ptients despite comprble pltelet ADP levels. Finlly, the prllel studies tht we performed in ptients with b-spd demonstrte the striking differences in the epinephnine responsiveness of these nd 5-SPD pltelets, nd the necessity for clerly distinguishing between these groups of ptients in ssessing the functionl defects in SPD. In ptients with #{244}-SPD, the PA slope with epinephnine ws mrkedly decresed, in contrst to the norml verge response in b-spd, nd SA ws never observed. Whether these responses re relted, in some cses, to true decrese in 2 receptors (s in Fmily C, Tble 4), on to role for -grnule substnces in mediting primry ggregtion responses remins to be determined. ACKNOWLEDGMENT The uthors wish to thnk John Rogers, Crol Kruger, nd Yolnd Hernndez for expert technicl ssistnce. REFERENCES I. Shttil Si: Activtion of humn pltelets by epinephrine, in Insel PA (ed): Adrenergic Receptors in Mn. New York, Mrcel Dekker, 1987, p McMilln DC: Secondry clumping effect in humn citrted pltelet rich plsm produced by denosine diphosphte nd drenline. Nture 21 1: 14, O Brien ir: Some effects of drenline nd nti-drenline compounds on pltelets in vitro nd in vivo. Nture 2:763, Mills DCB, Robb IA, Roberts GCK: The relese of nucleotides, 5-hydroxytryptmine nd enzymes from humn blood pltelets during ggregtion. i Physiol 195:715, Zucker MB: Pltelet function, in Willims Wi, Beutler E, Enslev Ai, Lichtmn MA (eds): Hemtology (ed 3). New York, McGrw-Hill, 1983, p Mcfrlne DE, Mills DCB: The effects of ATP on pltelets: Evidence ginst the centrl role of relesed ADP in primry ggregtion. Blood 46:39, Figures WR, Scerce LM, Wchtfogel Y, Chen J, Colmn RF, Colmn RW: Pltelet ADP receptor nd,-drenoreceptor interction: Evidence for n ADP requirement for epinephrineinduced pltelet ctivtion nd influence of epinephnine on ADP binding. i Biol Chem 261:5981, Peerschke E: Induction of humn pltelet fibrinogen receptors by epinephrine in the bsence of relesed ADP. Blood 6:71, Bennett is, Vilire G, Bunch iw: A role for prostglndins nd thromboxnes in the exposure of pltelet fibrinogen receptors. J Clin Invest 68:981, Bennett is, Vilire G: Exposure of pltelet fibrinogen receptons by ADP nd epinephnine. i Clin Invest 64:1393, Mrguerie GA, Plow EF, Edgington TS: Humn pltelets possess n inducible nd sturble receptor specific for fibrinogen. J Biol Chem 254:5357, Peerschke El, Zucker MB, Grnt RA, Egn ii, Johnson MM: Correltion between fibrinogen binding to humn pltelets nd pltelet ggregbility. Blood 55:841, 198 I 3. Peerschke El: The pltelet fibrinogen receptor. Semin Hemtol 22:241, Plow EF, Mrguenie, GA: Induction of the fibrinogen receptor on humn pltelets by epinephrine nd the combintion of epinephrine nd ADP. i Biol Chem 255:1971, Holmsen H, Weiss Hi: Secretble storge pools in pltelets. Ann Rev Med 3:1 19, Hslm Ri: Mechnisms of blood pltelet ggregtion, in Johnson SA, Seegens WH (eds): Physiology of Hemostsis nd Thrombosis. Springfield, IL, Thoms, 1967, p Mustrd if, Pckhm MA: Fctors influencing pltelet function: Adhesion, relese nd ggregtion. Phrmcol Rev 22:97, Chro IF, Feinmn RD. Detwiler TC: Interreltions of pltelet ggregtion nd secretion. J Clin Invest 6:866, Hung EM, Detwiler TC: Ressessment of the evidence for the role of secreted ADP in biphsic pltelet ggregtion: Mechnism of inhibition by cretine phosphte plus cretine phosphokinse. i Lb Clin Med 95:59, Weiss Hi, Chervenick PA, Zlusky R, Fctor A: A fmilil defect in pltelet function ssocited with impired relese of denosine diphosphte. N EngI J Med 28 1: I 264, Holmsen H, Weiss Hi: Hereditry defect in the pltelet relese rection by deficiency in the storge pool of pltelet denine nucleotides. Br J Hemtol 19:643, Holmsen H, Weiss Hi: Further evidence for deficient storge pool of denine nucleotides in pltelets from some ptients with thrombocytopthi- storge pool disese. Blood 39:197, I Hrdisty RM, Mills DCB, Kets-rd K: The pltelet defect ssocited with lbinism. Br i Hemtol 23:679, Preti Fl, Dy Hi, Mills DCB: Nucleotide nd serotonin metbolism in pltelets with defective secondry ggregtion. Blood 44:789, White ig, Witkop Ci: Effects of norml nd spirin pltelets on defective secondry ggregtion in the Hermnsky-Pudlk syndrome: A test for storge pool deficient pltelets. Am J Pthol 68:57, I Weiss Hi, Rogers J: Thrombocytopthi due to bnormlities in pltelet relese rection-studies on six unrelted ptients. Blood 39:187, Weiss Hi, Witte LD, Kpln KL, Lges BA, Chernoff A, Nossel HL, Goodmn DeWS, Bumgrtner HR: Heterogeneity in storge pool deficiency: Studies on grnule-bound substnces in 18 ptients including vrints deficient in -grnules, pltelet fctor 4, f3-thromboglobulin nd pltelet-derived growth fctor. Blood 54:1296, Lges B, Weiss Hi: Biphsic ggregtion responses to ADP nd epinephrine in some storge pool deficient pltelets: Reltionship to the role of endogenous ADP in pltelet ggregtion nd secretion. Thromb Hemost 43:147, Nieuwenhuis HK, Akkermn i-wn, Sixm i: Ptients with prolonged bleeding time nd norml ggregtion tests my hve storge pool deficiency: Studies on one hundred six ptients. Blood 7:62, Lges B, Weiss Hi: Heterogeneous defects of pltelet

9 EPINEPHRINE RESPONSES IN SPD PLATELETS 1725 secretion nd responses to wek gonists in ptients with bleeding disorders. Br i Hemtol 68:53, Lges B, Scrutton MC, Holmsen H: Studies on gel-filtered humn pltelets: Isoltion nd chrcteriztion in medium contming no dded C2, Mg, or K. i Lb Clin Med 85:81 1, Sctchrd G: The ttrction of proteins for smll molecules nd ions. Ann NY Acd Sci 51:66, Mustrd if, Perry DW, Kinlough-Rthbone RL, Pckhm MA: Fctors responsible for ADP-induced relese rection of humn pltelets. Am J Physiol 228:1757, Lges BA, Weiss Hi: Dependence of humn pltelet functionl responses on divlent ctions: Aggregtion nd secretion in heprin- nd hirudin-nticogulted pltelet rich plsm nd the effect of chelting gents. Thromb Hemost 45:173, Ro GHR, Gerrrd JM, Witkop Ci, White JG: Pltelet ggregtion independent of ADP relese or prostglndin synthesis in ptients with Hermnsky-Pudlk Syndrome. Prostglndins Med 6:459, Lges B, Holmsen H, Weiss HJ, Dngelmier C: Thrombin nd ionophore A23 I 87-induced dense grnule secretion in storge pool deficient pltelets: Evidence for impired nucleotide storge s the primry dense grnule defect. Blood 6 1: 154, Burgisser E, DeLen A, Lefkowitz Ri: Reciprocl modultion of gonist nd ntgonist binding to muscrinic cholinergic receptor by gunine nucleotide. Proc NtI Acd Sci USA 79:1732, Brodde OPE, Hrdung A, Ebel H, Book KD: GTP regultes binding of gonists to 2-drenergic receptors in humn pltelets. Arch Int Prmcodyn Ther 258:193, Swett id, Johnson SL, Crgoe Ei, Limbird LE: Inhibitors of N/H exchnge block stimulus-provoked rchidonic cid relese in humn pltelets. i Biol Chem 26:1291, Swett JD, Connolly TM, Crgoe Ei, Limbird LM: Evidence tht N/H exchnge regultes receptor-medited phospholipse A2 ctivtion in humn pltelets. J Biol Chem 261 :8667, Bng HS, Simons ER, Brss LF, Rittenhouse SE: Activtion of phospholipses A nd C in humn pltelets exposed to epinephrine: Role ofglycoproteins lib/lil nd dul role of epinephrine. Proc NtI Aced Sci USA 83:9197, Swett JD, Blir IA, Crgoe Ei, Limbird LE: Inhibitors of N/H exchnge block epinephnine- nd ADP-induced stimultion of humn pltelet phospholipse C by blockge of rchidonic cid relese t prior step. i Biol Chem 261 :866, Weiss HJ, Aledort LM, Kochw 5: The effect of slicyltes on the hemosttic properites of pltelets in mn. i Clin Invest 47:2169, Zucker MB, Peterson i: Effect of cetylslicyclic cid, other nonsteroidl nti-inflmmtory gents nd dipyridmole on humn blood pltelets. i Lb Clin Med 76:66, O Brien ir: Effects of slicyltes on humn pltelets. Lncet 1:779, Roth Gi, Mjerus PW: The mechnism of the effect of spirin on humn pltelets, I. Acetyltion of prticulte frction protein. J Clin Invest 56:624, Lges B, Weiss Hi: Specific correction of impired nd hydrolse secretion in storge pool deficient pltelets by denosine diphosphte. i Clin Invest 81:1865, 1988

10 : The response of pltelets to epinephrine in storge pool deficiency-- evidence pertining to the role of denosine diphosphte in mediting primry nd secondry ggregtion HJ Weiss nd B Lges Updted informtion nd services cn be found t: Articles on similr topics cn be found in the following Blood collections Informtion bout reproducing this rticle in prts or in its entirety my be found online t: Informtion bout ordering reprints my be found online t: Informtion bout subscriptions nd ASH membership my be found online t: Blood (print ISSN , online ISSN ), is published weekly by the Americn Society of Hemtology, 221 L St, NW, Suite 9, Wshington DC 236. Copyright 211 by The Americn Society of Hemtology; ll rights reserved.