Acute reduction of TxA2 platelet binding sites after in vivo administration of a TxA2 receptor inhibitor

Acute

reduction of

TxA2

platelet binding

sites after

in vivo

administration of

a

TxA2

receptor

inhibitor

PIETRO AMEDEO MODESTI, ANDREA COLELLA, ILARIA CECIONI, GIAN FRANCO GENSINI,

ROSANNAABBATE& GIAN GASTONENERISERNERI ClinicaMedicaI,UniversityofFlorence, Florence, Italy

1

Picotamide has been shown

to interfere

competitively with the thromboxane A2

(TxA2) platelet

receptor. In

the

present

study the effect of

in vivo

administration of

picotamide

on

TxA2 human platelet

receptorswas

investigated

in

10

healthy subjects.

2

Picotamide (300

mg x

3

daily)

or

placebo

were

administered

ina

double-blind,

cross-over,

placebo

controlled

study,

eachtreatment

lasting

1 week with a2weekinterval

period. TxA2

receptorswere

investigated by

adirect

radioligand binding

assaymethod

employing [1251]-PTA-OH

as labelled

ligand.

Platelet studies were

performed

onthe

first

day

oftreatment

immediately

before and

2,

4 and 8 h after the

ingestion

of the

drug.

The effects of chronic administration wereassessed on the seventh

day.

3

Two

and 4 h after the administration of

picotamide

300 mg

orally platelet TxA2

receptorswere

significantly

reduced from 1366 ± 237to957 ± 221

(P

<

0.05)

and753

+ 119

receptors/platelet (mean

±

s.d.) (P

<

0.03).

After 8 h

platelet

receptor

population

was restored

(1362

±

324, NS).

The same pattern was observed after 7

days

oftreatment. Thus

picotamide

seemsto induce a short

lasting

down

regulation

of

platelet TxA2

receptors.

Keywords

picotamide thromboxane

A2

platelets

Introduction Methods

The exposureof cellstoagonistsoften results inashort

ormediumtermdecrease in

responsiveness

of the cells

to the agonist

(Benovic

et al., 1985; Harden,

1983),

frequentlyaccompanied byalossin the

specific

membrane receptorsfor theagonist.Thisagonist-induced desensitiz-ation has also been reportedforplatelets. In fact anti-platelet drugs with specific agonist effects on

PGI2

or PGD2 receptors have been reported to induce

down-regulation ofspecificmembrane

platelet

receptors either after in vitro incubation

(Alt

etal.,

1986;

Jaschonek et

al.,

1988)

orin vivoadministration

(Modesti

etal.,

1987)

and thisphenomenonlimits theefficacyand theusefulness

of these drugs. This

down-regulation

patternwas also observedto occurin vitroafter

platelet

incubationwith

aTxA2analogue

(Crouch

&Lapetina, 1989;Lieletal.,

1988; Murray & FitzGerald,

1989).

The reduction in TxA2 receptor numberin vivo after theadministration ofTxA2 inhibitors might represent a useful additional effect fordrugsinteractingwith

specific

TxA2receptors. This study was undertaken to investigate whether the administration ofadrugwithTxA2receptor

antagonist

activity

(picotamide)

tohealthy

subjects

induced

changes

inaffinityand/or densityof theTxA2

platelet

receptors, assessedbythebindingof the labelled

antagonist

[125I]_

PTA-OH.

Materials

ONO11120 (9,11-dimethylmethano-11,12-methane-16-phenyl-13,14-dihydro-13-aza-15-tetranor-TxA2)

(Naru-miyaetal.,1986)was akind gift of Dr Narumiya (Kyoto,

Japan).

[125I]-PTA-OH

(2000Ci

mmol-1)

(9,11-dimethyl-methano-11,12-methano-16-(3

[1231]-4-hydroxyphenyl)-13,14-dihydro-13-aza-15-tetranor-TxA2), the labelled

hydroxylated form of

ON011120,

was obtained from Amersham(Buckinghamshire, GB).Prostacycin (PGI2)

wasobtained from Upjohn(Kalamazoo, Mi, USA). Pico-tamide (N,N'bis(3-picolyl)-4-methoxy-isophthalamide)

(batch

no 870311) was provided by the SAMIL inc.-SandozGroup,Rome,Italy. All the other reagents were obtained from Merck(Darmstadt, F.R.G.) and were of analytical grade.

Experimentalprocedure

Ten healthy subjects aged 44 to 62 years were treated with placebo or picotamide (Plactidil, Samil-Sandoz,

Italy)300 mg threetimesdaily orally in a double-blind,

cross-over placebo controlled clinical study. The two treatments, eachlasting 1 week, were separated by a 1

Correspondence:DrPietro AmedeoModesti,Clinica MedicaI,UniversityofFlorence,VialeMorgagni 85,50134, Florence,Italy 439

week wash-out interval. Nosubjecthad takenany other drug for at least 15 days preceding the study. Blood samples forbinding studies were collectedon the first and on the lastdayof eachtreatment

period

before the first administration (08.00 h) ofpicotamide orplacebo

respectivelyand2,4 and8 h afterpicotamideorplacebo administration.

Bloodsamplingandplateletisolation

Blood was withdrawn by clean venepuncture and anti-coagulated with 15% (v/v) acid citrate dextrose (NIH formulaA).

Platelet-rich plasma wasprepared as

previously

des-cribed (Neri Serneri etal.,

1984).

Platelet countswere performed by an automatic counter (Platelet analyzer 810,Baker

Instruments,

Allentown, PA, USA).

Platelet-richplasma,adjustedtopH6.5 with acidcitricdextrose,

wascentrifugedat1,800gfor30 min at room temperature

(20-22° C). Platelets were then resuspended in 10 ml of phosphate buffer pH 7.2 (8 mmol 1-1 Na2HPO4,

2 mmol

1-1

NaH2PO4, 10 mmol 171 KCI,

135 mmol

1-1

NaCl) andagainrecentrifuged at1,800g for 30 min at room temperature. The supernatant was discarded and the platelets were resuspended in assay buffercontaining 138mmol

1-1

NaCl,5mmolI1 MgCl2,

1 mmol

1-1

EGTA and 25 mmol 1-1 Tris/HCl,pH 7.5. Ifnecessaryassay buffer was added to obtain aplatelet concentration of

109

platelets ml-1.

Bindingstudies

Kinetics of 125I-PTA-OH binding The binding of0.5

nmol I-'

[1251]-PTA-OH

(final concentration, f.c.) to

108platelets atroomtemperature, in afinalvolume of

0.2 ml was preliminarily evaluated at selected time intervalsNon-specific bindingwasassessedateachtime by adding0.02 mlofONO11120(2x 10-5mol

l-1,

f.c.).

Specificbindingateachtimewasthencalculatedasthe

differencebetween total andnon-specific platelet bound

radioactivity.After 10min thereversibilityofthebinding

was assessed by adding a large amount of unlabelled ligand (0.02ml ofON011120, 2 x

10-5

mol

l-1,

f.c.)

to the reaction mixture and the residual binding was

evaluated atselected timeintervals.

[1251]-PTA-OHbinding isotherms Platelet suspension (0.1 ml)wasincubated in afinalvolume of 0.2 mlwith

[1251]-PTA-OH

0.05 nmol

1-1

(f.c.) (2000 Ci

mmol-1),

plus ON011120 at increasing selected concentrations (0to4 x 10-6 mol1-1)for 10 minatroomtemperature.

The residualradioactivityaftertheaddition ofON011120

2 x 10- mol F1 (f.c.) wasconsidered as non-specific

binding. Non-specific binding of 0.05 nmol

1-1

[1251]-PTA-OH

amounted to 25-35% of total bound

radioactivity.

After 10min incubation four4mlaliquots ofice-cold bufferwereaddedtoeachtubetostopthe reaction and

thecontentswererapidly filtered under reducedpressure

throughWhatmanGF/Cglass microfiber filters. Previous

time course experiments showed that under these

conditionsbinding equilibrium had been reached. The

entire washing procedure was completed within about

15s. Filterswere dried under airflow and counted ina Beckman gamma counterwith an overallefficiency of

50%.

The

experiments

werecarriedoutin

triplicate.

Analysis of

data

Kinetic studies The kinetic constants were calculated

by

time course

experiments

according to Weiland &

Molinoff

(1981). Briefly,

the observed association

constant

(K0b)

wascalculatedastheslopeof the

pseudo-first orderplot:

ln(LReq)/((LReq)

-

(LRt))

vstime

(t).

LReq

and LRt respectively indicate the concentration ofligand-receptor complex at equilibrium and at each time

(t).

Thedissociationrateconstant

K,1

isafirst orderrate constantbecause therateisdeterminedafterelimination

of the forward reaction following prior incubation of

ligand

and receptor. The first order integrated rate equationfordissociationis:

ln(LRt)/(LR)

=

-K_1

LR and LRt are the concentrations of

ligand-receptor

complexjust priortothe addition ofcompeting

ligand

(time 0) and at time t respectively. The association

constant

(K+,)

wasthencalculatedbytheequation:

K+1 =

(Kob-K-,)/[L]

Thekineticallydetermined dissociation constant(Kd)is finally givenby:

Kd=

K=,IK+l

Bindingisotherms The totalbinding for each concentra-tion indisplacementcurve atequilibriumwasdetermined by dividing the decay per minute (d

min-')

of each

platelet pellet by the calculated specific activity in d

min-'

mol-1

(Modesti et al., 1985). The analysis of these binding isotherms was performed according to Scatchard (1949).

Hill Plot of thedisplacementcurve and

Ki

were calcu-latedaccordingtoCheng& Prusoff(1973).

Statistical analysis

If nototherwiseindicated,alldata given in the text are mean ± 1s.d. of nexperiments. Thereceptornumbers and thedissociationconstantsateach time were compared byanalysisofvariance.

Results

Time course and binding isotherms of the

[125I]-PTA-OHbinding

[1251]-PTA-OH

rapidly bound to intact humanplatelets (Figure 1). The observed rate constant(KObS) forspecific

bindingwas0.340

min-'

(theequation for the line was y = - 0.026 + 0.340x; n = 5). The dissociation curve

-o -0 4-b 3 2 4

002

4 6 8 10 30

Time(min) Time (min)

Figure 1 a) Timecourseof the associationanddissociation phase of the binding of[1251]-PTA-OH(0.5 nmoll-1)to

washed human plateletsatroomtemperature.Dissociation

wasobtained after10 min incubationuponadditionof 20,000 nmol1-1ON011120totheincubation mixture. Data points representmeansoftriplicate determinations inatleast five independentexperiments.b)Specific binding association is plotted accordingtothepseudo-first orderrateequation(Kobs

=0.340min-'). c) Dissociation is plottedas afirst order

reaction(K-1=0.324min-1;K,=(Kobs- K-1) 1-1=0.032

nmol1-1min-';Kd=K_,/K, = 10 nmoll-1).

performed after 10 min of incubation showed a rapid

displacementof about65-75% ofthe totalradioactivity

bound. Theanalysisof the first orderrateofdissociation

showeda linearpatternwithaK-1of 0.324min-1(n=5).

The association rate constant (K1) was 0.032 nmol

1-1

min-'

andtheresultingkineticallydetermined

dissoci-ationconstantKdwas10nmoll-1.

Thesaturation of[1251]-PTA-OH binding occurredat

a ligand concentration about 100 nmol 1- (Figure 2).

TheScatchard analysisofthisbindingyieldedastraight

line, indicating asingle class ofbinding sites for[12

-PTA-OH (Figure 2). The equilibrium dissociation constant(Kd)and the maximum concentration ofbinding

sites(Bmax)determinedbyScatchardanalysis,gavevalues of21.5 ± 7.2 nmol1[1and 198 ± 63 fmol/108platelets

(1194±379binding sitesperplatelet,n=12)respectively.

Hillcoefficient(n Hill)of thebindingwas0.942±0.099,

suggestingthe existenceofhomogeneousindividual class ofbindingsites withoutcooperativity (Figure 3). Effect of picotamide administration The number or

affinityofTxA2receptorsdidnotundergoanysignificant changesafterthe administration ofplacebo.Incontrast,

a significantreduction ofplatelet membrane receptors wasfound 2h(957 ± 221 receptors/platelet,P<0.05)

and 4 h (753 ± 119receptors/platelet, P < 0.03) after

picotamide administration in comparison with placebo

administration. After 8 h platelet receptor population

wascompletelyrestored(1362 ±324receptors/platelet,

NS)(Figure 4) (Table 6). Nochangesintheaffinityfor theligandwereobservedafterpicotamide.

The chronic administration of the drug for 1 week did not induce a significant change in TxA2 platelet

receptors. In fact binding studies did not show any

significantdifferencesinpatientstreatedwithpicotamide

whencomparedwithpatientstreated withplacebobefore drug administration. O O 100 200 E) ______ X_{__X___ 0 co a ~ ~ ~ ~ ~ ~ ~ 0 100 200 300 400 [1251]PTA-OH (nM)

Figure 2 Saturationcurveof the[125I]-PTA-OHbindingto

washed human plateletsatroomtemperature.(O=total

binding;*=specificbinding; =nonspecific binding) (n=

12). Inset:Scatchard analysis of the specific binding (Bmax=

198±63fmol/108 platelets, Kd=21.5±7.2 nmol1-1).

:a 0 .0 0- I-I 0 CL N it -j -J -J 03) 0 2 4 -1 0 1

ON011120 log (nmol 1-')

Figure 3 Hillplot of the displacementcurve.Ki,calculated

accordingtoCheng &Prusoff(1973),was19 ±4nmol1-1(n = 10,nHill=0.942). 4-a) U1) cn o QL az) 1400 1200 1000 800 Basal 2 4 8 Time (h)

Figure 4 Number of TxA2receptorsperplatelet after placebo

(@)orpicotamide administration (U, 300mgorally). *P<

0.05, **P<0.01.

Discussion

Present results indicate thatpicotamide administration

to humans induces a reduction of the TxA2 binding

sites available on the platelet membrane. This effect

is reversible and is not observed 8 h after the last

administration.

Thekinetics ofplatelet TxA2receptordesensitization

observedinvivo and the kinetic studiesof

[125I]-PTA-OH

0 COU v-0._ c.QE -0 0 Q C -5 0 4 .0 Annl} ou

Table1 TxA2 receptornumberand dissociationconstantafter picotamideorplaceboadministration(mean ±s.d.)

Basal 2 h 4h 8h

Bindingsites/platelet

Picotamide 1366 ± 237 957 ± 221* 753 ± 119** 1362 ±324

Placebo 1291±211 1360 ± 324 1294±258 1312±308

Dissociation constant(nmol1-1)

Picotamide 19 ± 3.8 17 ± 5.7 23 ± 7.8 22 ± 4.2 Placebo 21 ± 4.1 19±2.9 21 ±6.2 18±3.8 *=P<0.05,**P=<0.01

a)

1400 1300 1200 X 1100_ ° 1000 _ a) 900_ i 800 700 600 6001 1 10 100 Time (min)

Figure5 To evaluate theoccupancyof thethromboxane

plateletreceptorsbypicotamide after platelet washing

accordingtotheprotocolused,separatesamplesofplatelet richplasma(PRP)wereincubated withONO11120(O,1,umol

I'),picotamide(U 2,umoll-1)orbuffer(*)at370 C.Before and after5, 10 and120min ofincubation,sampleswerewashed andbindingsites for[1251]-PTA-OHweredetermined(see

Methods).

inhibition by picotamide seem to stand against the

possibilitythat the reduction ofspecific[1251]-PTA-OH binding sites could be simply due to the incomplete removal oftheagonistwithresidualreceptoroccupation. Infactduring the platelet washing procedure performed during binding studies, picotamide is easily removed

from its specific receptors due to its relatively high dissociation constant(Modesti etal., 1989). Moreover

in vitro experiments confirmed that the TxA2 binding capacity of plateletswasunchangedafterashort

incuba-tion withpicotamide.

Desensitization ofplatelet thromboxane A2receptors

by TxA2 analogueshasbeenrecently reportedtooccur

in vitro (Crouch & Lapetina, 1989; Liel et al., 1988;

Murray & Fitzgerald, 1989; Sato etal., 1989). In fact platelets incubated with stableTxA2 analogues rapidly (within few minutes) reachadesensitized state(Liel et al., 1988;Murray&Fitzgerald, 1989).Thesequenceof

events in TxA2 receptor desensitization was found to

involve initialuncouplingofthereceptorfromaguanine

nucleotide binding (G) protein, followed byareceptor downregulationwithalossofspecific membranebinding

sites(Murray&Fitzgerald, 1989).Infact afteraninitial

rapiduncouplingoftheTxA2receptorandits associated G protein, a slow down regulation of receptors was

observed. This pattern was observed to occur in vitro afterincubation either with an agonist (U46619) (Lielet

al., 1988) or an antagonist (ONO11120) (Murray & Fitzgerald, 1989). The early desensitization after TxA2 analogue stimulation could be physiologically relevant tothe modulation of the plateletresponse toendogenous TxA2. In fact TxA2 potentiates platelet aggregation

inducedby other platelet stimulants (ADP,adrenaline)

and is released by platelets in response to different stimuli or pathophysiological conditions, so that this desensitization could represent aregulatoryconstraint. On the other hand the late receptor down-regulation inducedby TxA2analogues, which according to present data seems to occur in vivotoo,could be relevant to the

antiaggregating treatment with drugs interfering with TxA2 platelet receptors. In fact this platelet response couldpotentiate the direct effect of the treatment.

Thechangesinthesensitivityof cells to aneicosanoid

has also been studied in humans in vivo during the infusion of aPGI2 stable analogue (iloprost)(Sinzinger etal., 1981). The functionaldesensitizationafteriloprost infusion was found to be related to a reduction in the number of PGI2 platelet receptors (Jaschonek et al.,

1988; Modesti et al., 1987) and to a decrease in the amount offunctional

Gs

(Edwardset al., 1987; Jaschonek etal., 1988).The reduction of

Gs

also results in decreased responsiveness to other antiaggregating agents such as PGD2 or adenosine A2 which interact with

Gs

(Edwards etal., 1987).

The mechanism underlying the receptor

down-regulation is still under investigation, even though severalhypotheseshave been proposed such as

internal-ization, proteolysis or phosphorylation (Hollemberg, 1985). A recent paper by our group (Modesti et al.,

1990),showed theinternalizationofONO11120 after its

bindingtoplateletspecificTxA2 receptors, so suggesting that the observedreduction may be due to the internaliz-ation ofthe receptormolecule and its consequent removal from theplatelet surface.

In any case, the in vivo down regulation of TxA2

binding sites even after the stimulation with a TxA2

antagonist could represent an additional effect of the treatment, potentially enhancingthe antiplatelet effect ofTxA2antagonists.

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(Received 17May

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