Proc. Nail. Acad. Sci. USAVol. 82, pp. 7434-7438, November 1985Medical Sciences

Solubilization of a thromboxane A2/prostaglandin H2 antagonistbinding site from human platelets

(icosanoid receptors/receptor solubilization/membranes)

RONALD M. BURCH*, DALE E. MAIS*t, DAVID L. SAUSSY, JR.*, AND PERRY V. HALUSHKA*1§Departments of *Pharmacology and tMedicine, Medical University of South Carolina, and the tDrug Science Foundation, Charleston, SC 29425

Communicated by Josef Fried, July 1, 1985

ABSTRACT A binding site for 9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15la4-w-tetranorthromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 antagonist, wassolubilized into the 200,000 x g supernatant from humanplatelet membranes by using the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.Binding to the solubilized site was saturable, displaceable, andreversible. Displaceable binding was not affected by sodium,potassium, or phosphate concentrations up to 50 mM or bymagnesium to 5 mM but was increased 14% (P < 0.05) by 5mM calcium. A pH optimum for displaceable binding occurredbetween pH 7.0 and 7.5. Scatchard analysis of [1251]-PTA-OHbinding to the solubilized binding site revealed a single class ofsites, having a dissociation constant (Kd) of 66 ± 16 nM (n =3) and a B.nx of 750 ± 80 fmol/mg of protein. The Kd for themembranes prior to solubilization was 47 ± 11 nM (n = 3) andthe B.. was 700 ± 90 fmol sites per mg of protein. Theassociation rate constant, kj, was 1.57 x 107 M-1 min'1 and thedissociation rate constant, kL1, was 0.61 ± 0.04 min-' (n = 4),yielding a Kd (kL/k1) of 39 nM. Several thromboxaneA2/prostaglandin H2 agonists and antagonists displaced bound[125I]-PTA-OH at concentrations similar to those at which theyaffect platelet aggregation. Collectively, these observationssuggest that the solubilized protein is the thromboxaneA2/prostaglandin H2 binding site that mediates platelet aggre-gation.

Human platelets metabolize arachidonic acid to the labileproaggregatory and vasoconstrictor compounds prostaglan-din H2 (PGH2) and thromboxane A2 (TXA2). Several analogsof PGH2 and TXA2 have been synthesized that either mimicthe parent compounds or antagonize their effects (1-5). Thisobservation has led to the suggestion that these agents actthrough specific receptor molecules. Recently, two 125i-labeled TXA2/PGH2 antagonists have been synthesized foruse in binding studies, one a derivative of 13-azaprostanoicacid (6) and the other an analog of pinane-TXA2 (5). Theseagents specifically bind to intact platelets and membranesderived from them and are displaced by TXA2 and PGH2analogs (6, 7).The first step in the purification and physical character-

ization of these putative receptor sites is their solubilizationin active form from the membranes with which they areassociated. This report describes the solubilization of ahuman platelet TXA2/PGH2 binding site by using thezwitterionic detergent 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonic acid (CHAPS) (8). This bindingsite retains binding properties similar to intact human plate-lets and membranes derived from them.

MATERIALS AND METHODSMaterials. Na125I was purchased from Amersham; PEI and

CHAPS were from Sigma; glass fiber filters (GF/C) werefrom Whatman; polyethylene glycol (Carbowax 8000) wasfrom Fisher; (15S)-hydroxy-lla,9a-(epoxymethano)prosta-(5Z,13E)-dienoic acid (U46619), prostaglandin D2 (PGD2),and prostaglandin F2a (PGF2a) were from Upjohn; 9,11-epithio-11,12-methano-TXA2 (ON011113) and 9,11-dimeth-ylmethano-11,12-methano-16-phenyl-13,14-dihydro-13-aza-15aP-&a-tetranor-TXA2 (ON011120) were gifts of Ono Phar-maceutical, Osaka, Japan; Iloprost was a gift of Schering,FRG; 9,11-dimethylmethano-11,12-methano-16-(4-hydroxy-phenyl)-13,14-dihydro-13-aza-15aB-w-tetranor-TXA2 (PTA-OH), 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-dSaI-co-tetranor-TXA2 (I-PTA-OH), 9,11-dimethylmethano-11,12-methano-16-(4-methoxyphenyl)-13 ,14-dihydro-13-aza-15a,B-w-tetranor-TXA2 (PTA-OM), and 9,11-dimethylmethano-11,12-methano-15-phenyl-13,14-dihydro-13-aza-15af3-co-pentanor-TXA2 [PTA-(w - 1)] were synthesized in this laboratory asdescribed (5). [125I]-PTA-OH was prepared and stored asdescribed (6). Other reagents were of the purest gradesavailable.

Platelet Membrane Preparation. Platelet-rich plasma con-taining 5 mM EDTA and 10 AM indomethacin was preparedas described from normal subjects (6). A platelet membranepellet was prepared as described (6) and was used eitherimmediately or stored at -70°C until use. All volunteers gaveinformed consent. This study was approved by the MedicalUniversity of South Carolina Institutional Review Board forHuman Research.

Solubilization. Platelet membranes were resuspended in 50mM Tris HCl (pH 7.4) at 4°C containing 20%o glycerol(wt/vol) and 10 ,uM indomethacin, at a final protein concen-tration of about 5 mg/ml. CHAPS was added to the finalconcentration indicated in each experiment, usually 10 mM,and the solution was mixed in a Vortex at room temperaturefor 1 min. The solutions were placed into polycarbonate tubesand centrifuged at 200,000 x g for 60 min in a Beckman 75Ti

Abbreviations: TXA2, thromboxane A2; PGH2, PGD2, PGF2,,a, andPGI2, prostaglandins H2, D2, F2,, and 12; I-PTA-OH, 9,11-dimethylmethano-11,12-methano- 16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15a,13-c-tetranor-TXA2; PTA-OH, 9,11-dimethylmethano-11,12-methano-16-(4-hydroxyphenyl)-13,14-dihydro-13-aza-15af3-w-tetranor-TXA2; PTA-OM, 9,11-dimeth-ylmethano-11,12-methano-16-(4-methoxyphenyl)-13,14-dihydro-13-aza-15ap-ai-tetranor-TXA2; PTA-(w - 1), 9,11-dimethylmethano-11,12 - methano - 15 - phenyl - 13,14 - dihydro - 13 - aza - 15at--penta-nor-TXA2; ON011120, 9,11-dimethylmethano-11,12-methano-16-phenyl-13,14-dihydro-13-aza-15ap-co-tetranor-TXA2; ON011113,9,11-epithio-11,12-methano-TXA2; U46619, (15S)-hydroxy-lla,9a-(epoxymethano)prosta-(5Z,13E)-dienoic acid; trans-13-APO, trans-7-[(2-p-hydroxyphenylethanolamino)cyclopentyl]heptanoic acid;CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-1-propanesul-fonate.§To whom reprint requests should be addressed.

7434

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 82 (1985) 7435

rotor at 40C. The transparent yellow supernatant was re-moved and diluted in 50 mM Tris HCI (pH 7.4) at 30'C to afinal concentration of 1-2 mM CHAPS and 1-2 mg of proteinper ml and immediately used in the assays. If left undilutedthe supernatant could be stored at 40C for several days withno apparent loss of activity. Protein concentration wasmeasured by the method of Lowry et al. (9).

[125T]-PTA-OH Binding Assays. Aliquots of the membranepreparation or the solubilized preparation (90-275 pug ofprotein) were added to siliconized glass tubes containing afinal volume of 200 ,pl of 50 mM Tris HC1 (pH 7.4) at 30'C,z100,000 cpm of [125T]-PTA-OH (ca. 0.2 nM), and anycompeting ligand. Nondisplaceable binding was determinedin the presence of 5 ,4M unlabeled I-PTA-OH. Incubations intriplicate were for 30 min at 30'C except in the time courseexperiments. The assays were terminated by dilution with 4ml of 50 mM Tris HCl (pH 7.4) at 40C followed by filtrationunder reduced pressure through Whatman GF/C glass fiberfilters that had been pretreated with 0.3% PEI (10) for 1 hr.The filters were washed with three additional 4-ml aliquots ofthe ice-cold buffer.

Filtration of the solubilized binding site by using untreatedGF/C filters resulted in total binding of 736 ± 84 cpm andnondisplaceable binding of 658 ± 72 cpm (mean ± SEM; n =2). For the nonsolubilized membranes total binding was 3742± 260 cpm and nondisplaceable binding was 2416 ± 180 cpm.Using PEI-treated GF/C filters to trap the solubilized bindingsites, total binding was 2284 ± 121 cpm and nondisplaceablebinding was 978 ± 36 cpm.

Using polyethylene glycol precipitation (11) the IC50 for127T-PTA-OH was 85 ± 12 nM (n = 3), similar to the resultsobtained with the PEI-treated glass fiber filters, but displace-able binding was only 35% of total binding.

Gel Filtration. An aliquot (0.5 ml) of the solubilizedpreparation was added to a column 30 x 1 cm containingSepharose 4B that had been equilibrated for several days with50 mM Tris HCl (pH 7.4) at 4°C and 1 mM CHAPS and elutedat 0.44 ml/min. Fractions were collected and aliquots wereassayed in duplicate for displaceable binding. The columnwas calibrated by using several proteins of known molecularweights.

Statistics. The equilibrium binding data were corrected fornonspecific binding and fitted to a two-parameter model byusing an iterative "Simplex" computer program (12); alter-natively, the nonspecific binding was not subtracted and thedata were fit to a four-parameter model identifying twoindependent binding sites.

100 A 1000 -,-

c

0

C) 0

li60- CE 600- I

20 1 W8/0l 0. cu.20~ 0o5-

RESULTS

Solubilization of Active Binding Sites by Using CHAPS.Increasing the concentration ofCHAPS from 0 to 20 mM wasassociated with an increased percentage of protein in the200,000 x g supernatant (Fig. 1A) to a maximum of 80% ofthe protein originally contained in the pellet. In experimentswithout CHAPS, no displaceable [125T]-PTA-OH binding wasobserved in the supernatant (Fig. 1B). By increasing CHAPSup to 6 mM, bound cpm increased in the supernatant whiledecreasing in the pellet. At higher CHAPS concentrationsbound cpm in the supernatant decreased. When binding in thesupernatant and pellets was corrected for protein concentra-tion (Fig. 1C) it was found that binding per 100 ,ug of proteinwas roughly equivalent in the supernatant and pellet.CHAPS concentration in the binding assays was varied

between 0.4 and 20 mM. Displaceable binding was maximalat CHAPS concentrations of 1-5 mM. Thus, in subsequentbinding assays CHAPS was maintained at 1-2 mM.

Role of Protein in [12511-PTA-OH Binding. Over the con-centration range of 50-400 jig of protein per tube, [125I]-PITA-OH binding to the solubilized preparation was linear.Displaceable binding was 74% ± 6% (n = 10) of total binding.In the presence of 1 mM CHAPS, but in the absence ofprotein in the assay tube, 642 ± 36 cpm (n = 6) were retainedby the filters in the presence of about 0.2 nM ['25T]-PTA-OH.In the presence of 5 ,uM [125T]-PTA-OH, 658 ± 46 cpm (n =6) were retained.

Displaceable binding of the solubilized preparation wasdecreased by boiling for 5 min, pretreatment with 100 ,ug oftrypsin for 20 min, or pretreatment with 1 mM dithioerythritolfor 10 min by 91%, 93%, and 74%, respectively.

Effect of Ionic Composition of the Buffer on [125T]-PTA-OHBinding. Displaceable binding to the solubilized preparationwas not affected by sodium, potassium, magnesium, orphosphate up to concentrations of 50 mM but was decreased14% ± 3% (P < 0.05, n = 3) by 1 mM EGTA compared to 5mM calcium (data not shown). A pH optimum for displace-able binding occurred between pH 7.0 and 7.5 and bindingdecreased to 20% of control at pH 6.5, 50% at pH 8.0, and30% at pH 8.5.

Estimation of the Kd for ['25T1-PTA-OH Binding. Binding of[125I]-PTA-OH to the solubilized preparation was saturable(Fig. 2). Scatchard analysis revealed a single class of bindingsites with a Kd of 66 ± 16 nM and a Bmax of 750 ± 80 fmol/mgof protein (Fig. 3, n = 3). In the same subjects [125T]-PTA-OHbinding to the nonsolubilized membrane preparation alsoyielded a single class of sites with a Kd of 47 ± 11 nM and a

10 1

CHAPS, mM

c0.

.O.0.

T00

EQ.00gCo00Cu0QCO0

FIG. 1. Effect of CHAPS on the appearance of ['251]-PTA-OH binding sites in the 200,000 x g supernatant of human platelet membranes.(A) Percent of protein in each tube in the supernatant as a function of CHAPS concentration. (B) Displaceable binding in the supernatant (S)and in the pellet (P) after dilution to a volume equivalent to the supernatant. (C) Displaceable binding from B normalized for the proteinconcentration in each fraction. Presented are means ± SEM for three preparations. CHAPS concentration was diluted to 1-2 mM prior to assayof [tLI]-PTA-OH binding.

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7436 Medical Sciences: Burch et al.

*,, 0.75-< ~

0.500

0)E

E

0.25CX

C.)CnCal

0

0 250 500 750 1000I-PTA-OH, nM

FIG. 2. [1251]-PTA-OH binding to the solubilized sites. Data areshown as mean ± SEM for three preparations.

Bmax of 700 ± 90 fmol/mg of protein (n = 3). If binding at 5,uM I-PTA-OH was not subtracted and the same data wereanalyzed by using the non-linear curve-fitting program, asecond component, considered nonspecific binding, wasobserved with a Kd of 225 ± 36 ttM and a Bmax of 2.16 ± 0.75nmol/mg ofprotein (n = 3). The other component was of highaffinity, with a Kd of 65 ± 18 nM and a Bmax of 790 ± 110fmol/mg of protein.

Association of [1251]-PTA-OH with the solubilized site wasrapid and the rate increased with increasing I-PTA-OHconcentration (Fig. 4 Upper). The association data werereplotted as pseudo-first-order processes (13). The true rateconstant of association (k1) was 1.57 x 107 M-1 min1 (Fig.4 Upper Inset). Dissociation was rapid and complete by 5 min(Fig. 4 Lower). A dissociation rate constant (k_1) of 0.61 +

0.04 min- (n = 4) was calculated (Fig. 4 Lower Inset). Fromthese data, Kd was determined: k-1/k, = (0.61 min-')/(1.57x 107 M-1 min1) = 39 nM.Displacement of ['25I]-PTA-OH by TXA2/PGH2 Analogs.

Competition for [1251]-PTA-OH binding to the solubilizedpreparation was also tested by using a number of analogs ofI-PTA-OH that are pharmacological antagonists ofTXA2/PGH2 agonists in human platelets as well as several

A B

0.015

-_ 0.010 0C .

0*

0.005

0 0.2 0.4 0.6 0 0.2 0.4 0.6Bound, pmol/mg of protein

FIG. 3. Scatchard analysis of I'251]-PTA-OH binding to intact andsolubilized platelet membranes from a single subject. (A) Membrane-bound site: Kd = 39 nM; Bmax = 730 fmol/mg of protein. (B)Solubilized site: Kd = 37 nM; Bmax = 605 fmol/mg of protein. Totalbinding was measured in the presence of 0.2-1000 nM I-PTA-OH.Nonspecific binding in the presence of 5 AuM I-PTA-OH wassubtracted from each point. Each point is the mean of triplicatedeterminations.

Time, min

E a0.~~~~~~~~_o ~~~~~~~~:-2.gz 750° _' I \750co0)

co

CL U

V.00 1 2 3 4 5

T , Time, mi

250

k= 0.62 min'

00 1 2 3 4 5

Time, min

FIG. 4. Time courses of association and dissociation of [L125I]-PTA-OH to the solubilized binding site at 300C. (Upper) Presentedare single experiments at each concentration. Each point is the meanof triplicate determinations. Symbols correspond to the I-PTA-OHconcentrations indicated in the Inset. (Inset) Evaluation of k, forI-PTA-OH binding to the solubilized site. Data were analyzedassuming a pseudo-first-order process and their slopes (kobserCvd) areplotted as a function of I-PTA-OH concentration. The slope of theplot is the rate constant of association (kj). (Lower) Presented is arepresentative experiment. Association at 30'C was carried out in thepresence of 0.2 nM [1251]-PTA-OH for 45 min in a single flask.Aliquots were then placed into individual tubes and 5 1AM unlabeledI-PTA-OH was added at time zero to displace [1251]-PTA-OH. Eachpoint represents triplicate determinations. (Inset) Logarithmicreplot, the negative slope of which is kL1.

other structurally dissimilar TXA2/PGH2 agonists and an-tagonists (Table 1). Each of the compounds completelydisplaced [1251]-PTA-OH to the level of nonspecific binding(Fig. 5). The potencies of the six antagonists as inhibitors ofplatelet aggregation (14) were significantly correlated withtheir potencies as inhibitors of [125I]-PTA-OH binding (r =0.94). The two PGH2/TXA2 mimetics, U46619 andON011113, also displaced [1251]-PTA-OH from its bindingsite (Fig. 5). Since PGD2 and prostaglandin I2 (PGI2) arethought to have specific receptors in human platelets andsince PGF2a has been suggested to act through TXA2 recep-tors in vascular smooth muscle, the ability of PGD2, PGF2a,and Iloprost, a stable analog of PGI2 (15), to displace[125I]-PTA-OH were also determined. IC50 values for dis-placement of [125I] -PTA-OH were 17 ± 1 ,M for PGD2 (n =3), 19 ± 1 ,M for Iloprost (n = 3), and 73 ± 14 AM for PGF2,(n = 3). At 100 ALM for each compound, PGD2 displaced 82%of specifically bound [1251]-PTA-OH, Iloprost displaced 80%,and PGF2,, displaced 56%.

Proc. Natl. Acad Sci. USA 82 (1985)

Proc. NatL Acad Sci. USA 82 (1985) 7437

Table 1. IC50 values for competition of displaceable[1251]-PTA-OH binding from the solubilized site

Agent IC50, nMTXA2/PGH2 antagonist

I-PTA-OH 56± 4PTA-OH 231 ± 15PTA-OM 356 ± 24ON011120 669 ± 26PTA-(w - 1) 1,290 ± 206trans-13-APO 11,500 ± 3,600

TXA2/PGH2 agonistON011113 159 ± 16U46619 1,662 ± 215

Other prostaglandinPGD2 17,000 ± 1,000Iloprost 19,000 ± 1,000PGF2, 73,000 ± 14,000

n- 8000

E0.c)m 600Czc

'D 400-oco00Cu.co 200

333544

34

333

Values are expressed as mean ± SEKI; n = number of prepara-tions. All compounds displaced ['25II PTA-OH (0.2 nM) to the levelof nondisplaceable binding except for PGD2 (82%o displacement at100 AM), Iloprost (80%o displacement at 100 ,4M), and PGF2,. (56%displacement at 100 AM). trans-i3-APO, trans-7-[(2-p-hydroxyphen-ylethanolamino)cyclopentyl]heptanoic acid.

Gel Filtration of the Solubilized Preparation. When thesolubilized preparation was subjected to gel filtration usingSepharose 4B the I-PTA-OH binding site eluted as a peak ofactivity with an elution volume between apoferritin (440 kDa)and gamma globulin (167 kDa) (Fig. 6). Often, but not always,a smaller peak ofI-PTA-OH binding activity was eluted in thevoid volume (Fig. 6).

DISCUSSION

The present results demonstrate that a TXA2/PGH2 antago-nist binding protein has been solubilized in active form fromhuman platelet membranes by using the detergent CHAPS.The criteria used tojudge the preparation to be solubilized are

100

~~~~~~~~~A75[-

050

0

25-

~~~~~~~~~~~~B075-

~50

6 25

-10° 101 102 103 1o4 105

Ligand, nM

FIG. 5. Displacement of [125I]-PTA-OH by several TXA2/PGH2agonists and antagonists. Displaceable binding on the ordinate is cpmin the presence of 0.2 nM [1251]-PTA-OH minus the cpm in thepresence of an additional 5 uM unlabeled I-PTA-OH. (A) Agonists.o, ON011113, IC50 = 159 + 16 nM (n = 3); e, U46619, IC50 = 1662+ 215 nM (n = 4). (B) Antagonists. 6, I-PTA-OH, IC50 = 56 4 nM(n = 3); A, PTA-OH, IC50 = 231 ± 15 nM (n = 3); *, PTA-OM, IC"0= 356 ± 24 nM (n = 3); o, ON011120, IC50 = 669 + 26 nM (n = 5);A, PTA-(w - 1), IC50 = 1290 + 206 nM (n 4); o, trans-13-APO, IC50= 11,500 ± 3600 nM (n = 4). Each point is the mean obtained in threeto five preparations.

1-100OD

Fraction

FIG. 6. Gel filtration of the solubilized preparation using Seph-arose 4B. Each fraction was assayed in duplicate for displaceableI-PTA-OH binding. Total protein in each fraction was monitored asabsorbance at 280 nm. V0, void volume; APO, apoferritin; GG,gamma globulin; BSA, bovine serum albumin; CY c, horse heartcytochrome c.

(i) the failure to sediment after centrifugation at 200,000 x gfor 1 hr, (ii) the binding activity could not be retained by~glassfiber filters unless they had been pretreated with the polycat-ion PEI, which traps solubilized membrane proteins (10), and(iii) the binding activity eluted at a time much greater than thevoid volume on gel filtration.The use ofCHAPS allowed solubilization of60-70% of the

total I-PTA-OH binding sites from platelet membranes. Thisyield was somewhat higher than the roughly 10-20% yield ofactive opiate receptors (8) and dopamine receptors (16) and40% yield of active a2-adrenergic receptors (17) recoveredafter CHAPS solubilization. The inclusion of 20% glycerol inthe present solubilization protocol may be responsible for thehigh yield since glycerol hais been reported to stabilize manysolubilized proteins (18). CHAPS (1-2 mM) was necessary inthe binding assay in order to detect specific binding of[125I]-PTA-OH to the solubilized preparation. Similar resultswith other binding sites after solubilization have been inter-preted as aggregation of the sites in the absence of detergents(18).

Protein is a necessary constituent of the binding site sinceboiling or trypsin pretreatment markedly decreased displace-able binding. Loss of binding after treatment withdithioerythritol suggests that intact disulfide bonds may alsobe required. Neither the ionic strength nor composition of theassay buffer affected [1251]-PTA-OH binding to the solubil-ized binding site with the exception of hydrogen ion concen-tration. The pH dependency for binding to the solubilizedpreparation is essentially identical to that observed for[1251]-PTA-OH binding to intact canine platelets and intacthuman platelets (7, 19).

Association and dissociation of [1251]-PTA-OH to thesolubilized site were rapid. The rates were more rapid thanthose observed in intact platelets (19), a phenomenon that hasbeen noted previously for the ,3-adrenergic receptor followingdetergent solubilization (20). The Kd for [1251]-PTA-OHdetermined kinetically was 39 nM and from equilibriumbinding was 66 nM. These values are also in the same rangeas the Kd values of 8 nM determined pharmacologically and21 nM for [1251]-PTA-OH binding to washed human platelets(19). In addition, the Bmkx is similar to that in intact humanplatelets (890 fmol/mg of protein) (19).

Several other putative TXA2/PGH2 antagonists and ago-nists of diverse structure displaced [1251]-PTA-OH from itsbinding site. The displacement curves for all of the agents

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7438 Medical Sciences: Burch et al.

were parallel and they all completely displaced specificallybound I-PTA-OH. These observations support the notionthat all of the agents displaced I-PTA-OH from the same site.The correlation coefficient for the comparison between thepharmacological activity and IC50 for displacement of [125II-PTA-OH was 0.94 for the antagonists. However, among theantagonists, ON011120 and PTA-(w - 1) are relatively lesspotent in displacement of 125I-PTA-OH than in inhibition ofplatelet aggregation. The order of potency for displacementof I-PTA-OH by the agonists was not identical to their orderfor initiation of platelet aggregation. Although ON011113displaced [1251]-PTA-OH with a potency similar to its EC50 forplatelet aggregation, U46619 was only 1/10 as potent indisplacing [1251]-PTA-OH from the solubilized site comparedto its EC50 for aggregation. The profile for displacement of[125I]-PTA-OH from membranes prepared from human plate-lets is similar to that found in this study (unpublishedobservations). In contrast, all of the agents displaced [1251]_PTA-OH bound to intact human platelets with an order ofpotency identical to that observed pharmacologically (14,19).The reason that disruption of intact platelets into mem-

branes altered the potency for displacement of [1251]-PTA-OH by only certain agonists and antagonists is unknown atpresent. However, the platelet contains many pharmacolog-ically active agents that can change the density of fibrinogenreceptors in the platelet plasma membrane (21). Thus, dis-ruption of the platelets to form membranes might be associ-ated with release of some substance(s) that can alter theTXA2/PGH2 binding site. In preliminary experiments, wehave found that if the supernatant from disrupted platelets isadded to intact platelets, the IC50 for displacement of I125I]-PTA-OH by I-PTA-OH does not change, but that for U46619is increased (22). This situation is identical to that observedin the intact and solubilized membranes.

The, human platelet also contains binding sites for PGD2and PGI2 (23). In the present experiments PGD2 displaced['25I]-PTA-OH with an IC50 - Kd of 17 ,uM. The Kd reportedfor PGD2 binding to its platelet receptor is about 400 nM (24)or 40 times more potent than that observed in the presentstudy. Iloprost, a stable analog of PGI2, had an IC50 Kd of19 uM for displacement of[1251]-PTA-OH compared to its Kdfor binding to PGI2 receptor sites of 1-15 nM (15, 25). Thus,[1251]-PTA-OH does not appear to be interacting with PGD2or PGI2 receptor sites.PGF2. has been postulated to act through the same

receptors as TXA2 and PGH2 in vascular smooth muscle (26)and in the platelet (27). PGF2a had an IC50 of 73 ,gM anddisplacement was not complete at 100,uM. Thus, it wouldappear that PGF2. is not interacting with the same bindingsite as (1251]-PTA-OH.Gel filtration revealed that most of the I-PTA-OH binding

activity elutes between apoferritin and gamma globulin,corresponding to a molecular mass of about 200 kDa. How-ever, assignment of molecular mass for a membrane proteinbased on gel filtration alone must be viewed with cautionsince many membrane proteins are asymmetric, leading tooverestimation of molecular mass (28).

In conclusion, a TXA2/PGH2 antagonist binding proteinhas been solubilized from human platelet membranes. As-suming one I-PTA-OH binding site per molecule, its concen-tration of 750 fmol/mg of solubilized protein would require5000- to 10,000-fold purification of the binding site to achieve

homogeneity. Similar purifications have been achieved forother membrane receptors and should prove feasible for theplatelet TXA2/PGH2 receptor.

We acknowledge the secretarial assistance of Marie Meadowcroftand Virginia Minchoff. D.E.M. was supported in part by the DrugScience Foundation. P.V.H. is a Burroughs-Wellcome Scholar inClinical Pharmacology. This research was supported in part byNational Institutes of Health Grants HL29566, HL07260, andRR1070.

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2. Bundy, G. L. (1975) Tetrahedron Lett. 24, 1957-1960.3. Nicolaou, K. C., Magolda, R. L., Smith, J. B., Abarony, D.,

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7. Mais, D. E., Kochel, P. J., Saussy, D. L., Jr., & Halushka,P. V. (1985) Mol. Pharmacol. 28, 163-169.

8. Hjelmeland, L. M. (1980) Proc. Natl. Acad. Sci. USA 77,6368-6370.

9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall,R. S. (1951) J. Biol. Chem. 193, 265-275.

10. Bruns, R. F., Lawson-Wendling, K. & Pugsley, T. A. (1983)Anal. Biochem. 132, 74-81.

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