1382

ANTI-PLATELET ACTIVITY OFBETA-ADRENERGIC ANTAGONISTS:

INHIBITION OF THROMBOXANE SYNTHESISAND PLATELET AGGREGATION IN PATIENTSRECEIVING LONG-TERM PROPRANOLOL

TREATMENT

WILLIAM B. CAMPBELL,KARLEEN S. CALLAHAN,

ALICE R. JOHNSON,ROBERT M. GRAHAM

Departments of Pharmacology and Internal Medicine,University of Texas Health Science Center,

Dallas, Texas 75235, U.S.A.

Summary Treatment of hypertensive patients withdl-propranolol (640 mg/day) significantly

inhibited thromboxane synthesis by their platelets and

platelet aggregation induced by thrombin or arachidonicacid. The effects were dose-related and were also caused bythe stereoisomer, d-propranolol (640 mg/day), which has verylittle beta-blocking activity. These findings suggest that thecardioprotective effects of propranolol may be due partly tothis anti-platelet activity, to a reduction in thromboxane-induced coronary-artery vasoconstriction, or to both.

d-Propranolol treatment may be particularly useful, since thisisomer provides similar benefits without causing pronouncedbeta-adrenergic blockade.

Introduction

BETA-ADRENERGIC antagonists are widely used in themanagement of hypertension and angina pectoris. They mayalso be useful in limiting infarct size after coronary-arteryocclusion2 and may have cardioprotective effects inthreatened myocardial infarction.3 However, the basis forthese therapeutic effects remains unclear. Propranololinhibits the second phase of platelet aggregation induced byadenosine diphosphate, collagen, thrombin, adrenaline, orthe ionophore A23187.4 Therapeutic concentrations (10 - 8 to10-6 mol/1) of dl-propranolol, d-propranolol, timolol (a beta-blocker with no membrane-stabilising activity), and

metoprolol (a beta1-selective antagonist) inhibited plateletaggregation in vitro. Furthermore, these drugs also reducedthe generation of thromboxane A2 (TxA2) in platelets byinhibiting the thromboxane synthetase reaction of the TxA2biosynthetic pathway. Since TxA2 is an endogenousmediator of secondary platelet aggregation, it was proposedthat beta-adrenergic antagonists exert their anti plateletaction by inhibiting thromboxane synthesis. We decided toevaluate the clinical relevance of our in-vitro findings byexamining the effects of long-term oral treatment with

dl-propranolol or d-propranolol, alone or in combination withindomethacin, on platelet thromboxane synthesis and

-platelet aggregation in patients with essential hypertension.

Patients and Methods

Patients

Fifteen patients, aged 21-63 years (8 women, 7 men), with mildessential hypertension (World Health Organisation grades I-II;average diastolic pressures, 95-115 mmHg) gave written informedconsent to participate in the study. They underwent detailedevaluation including history, physical examination,electrocardiogram, complete blood count, biochemical tests, andstandard laboratory tests to exclude secondary causes of

hypertension. None of the patients had a history of bleedingdiathesis or dyspeptic symptoms, and renal function was normal inall cases.

The patients were randomly assigned to receive either 160 mgdl-propranolol/day, 640 mg dl-propranolol/day, or 640 mgd-propranolol/day. The drug-administration protocol consisted ofplacebo for 2 weeks, followed by the various propranolol regimensfor 4 weeks, the propranolol treatments plus 100 mg indomethacin/day for 2 weeks, and finally 100 mg indomethacin/day for 2 weeks.Some patients took part in more than one study. In these cases, adrug-free period of at least 2 weeks was allowed before the placeboperiod of the next study began. All drugs were given twice daily inequal doses. To avoid gastrointestinal side-effects, the patients wereinstructed to take all medications after meals. dl-Propranololregimens were initiated at 160 mg/day and, for those assigned toreceive 640 mg/day, increased by 80 mg daily. At the end of eachplacebo or drug period, blood samples were taken for plateletstudies, and blood pressure, serum propranolol, and other

haemodynamic and neuroendocrine measurements were carriedout. The results of the latter studies will be reported fully elsewhere.

Platelet Studies

20 ml samples of blood were taken for platelet studies and wereimmediately mixed with 2 ml 3 - 5% sodium citrate, pH 7 - 4. Theblood was centrifuged at 150 g for 10 min, and the platelet-richplasma (PRP) was removed. Platelet-poor plasma (PPP) wasobtained after centrifugation at 1500 g for 10 min and was used as ablank in the aggregation studies.

Platelet aggregation was determined in vitro by the method ofBorn 7 with a dual-channel ’Sienco Aggregometer’. 1’ 5 ml PRP wasincubated at 370C with stirring at 1000 rpm. Aggregation wasmeasured by an increase in light transmission. 0, 0 - 5,1’ 0, 1 - 5,2 - 0,and 4 - 0 min after the addition of thrombin (1 U) or arachidonic acid(0’ 2 mg), 50 µl of PRP was removed from the cuvette, diluted in450 µl of ice-cold phosphate-buffered saline, pH 7 - 4, and quicklyfrozen in an alcohol/dry-ice bath. These samples were subsequentlyassayed for thromboxane B2 (TxB2) by radioimmunoassay. Theplatelet concentrations of the PRP were determined by counting ina haemocytometer after dilution in a ’Unopette’ reservoir (Becton-Dickenson). The lowest concentration of thrombin that induced asecondary aggregation response (the thrombin threshold) wasquantified by monitoring the induction of platelet aggregation afterthe addition of increasing amounts of thrombin to 0.4 4 ml of PRP.Radioimmunoassay of TxB2 was carried out by the method of

Dray et al. 8 as previously described.5 Anti-TxB2 serum washarvested from rabbits immunised with a TxB2/thyroglobulincomplex. There was less than 0-003% cross-reaction with

prostaglandins A2, B2, E2, and F2a and with 6-keto-prostaglandinFla. For the assay 0’ 1 ml tritium-labelled TxB2 (3000 cpm) wasadded to 0.1 ml of TxB2 standard, or 0.1 ml of diluted PRP plus0.1 ml of a 1 :60 000 dilution of the anti-TxB2 serum. All dilutionswere made in 0 1 mol/1 phosphate buffer, pH 7 - 4, containing 0 - 9%sodium chloride and 0 - 1% polyvinylpyrrolidone. The mixture wasincubated overnight at 4°C. Bound and free TxB2 were separatedby means of dextran-coated charcoal, and the bound fraction wascounted in a Beckman liquid scintillation spectrometer. The

sensitivity of the assay was 5 pg and the interassay variability 8 %.The drugs and chemicals used were: dl-propranolol (Ayerst),

d-propranolol (ICI Ltd.), indomethacin (Merck), arachidonic acid(Sigma), thrombin (Parke-Davis) 3H-TxB2 (New England Nuclear),and TxB2 (Upjohn Company).

Statistical AnalysisTxB2-generation curves for the various treatment groups were

compared by means of an analysis of covariance. The effects of thedrug treatments on aggregation were compared by the Fisher’s exact.test.9 Results are expressed as mean ± 1 SD.

Results

The effects of treatment with propranolol alone,indomethacin alone, and both drugs together on thrombin-induced platelet TxB2 formation are shown in the

1383

TIME (min.)Effect of long-term administration of propranolol, indomethacin,

and both drugs together on thrombin-induced plateletthromboxane B2 production.Each point represents mean for 7 or 8 patients. T thrombin.

accompanying figure. In placebo-treated patients, thrombinstimulated the release of TxB2. dl-Propranolol (160 mg/day)did not significantly alter Tx synthesis but the higher dose ofdl-propranolol (640 mg/day) reduced TxB2 formation by53 % (p < 0 - 025). d-Propranolol (640 mg/day) also reducedTxB2 synthesis by approximately the same proportion as theracemic mixture. Indomethacin (100 mg/day) reduced thebasal levels of TxB2 in PRP and reduced the rate of TxB2synthesis by 77% (p <0’ 001). When indomethacin and eitherdl-propranolol or d-propranolol were given together, theeffects on TxB2 generation were similar to those observedwith indomethacin alone and were not additive. The lowerdose of dl-propranolol appeared to reduce the effects of

indomethacin, but this trend was not significant.

Similar results were observed with arachidonic acid, whichcaused a rapid increase in the rate of platelet TxB2 synthesisin the placebo group. This stimulation was reduced by 35 %(not significant) by the 160 mg/day dose ofdl-propranolol andby 56% (p <0 - 05) and 60% (p <0 - 05) by treatment with 640mg/day of dl-propranolol and d-propranolol, respectively.Indomethacin alone also reduced the rate of TxB2 synthesis(p <0’0001). When indomethacin and propranolol weregiven together, there were different effects in each group.The combination of indomethacin and either dose of

dl-propranolol produced effects similar to those ofindomethacin alone; however, combination of indomethacinand d-propranolol produced greater inhibition of TxB2synthesis than indomethacin alone (p <0-05).

Platelet concentrations measured during any of the drugtreatment periods did not differ from those measured duringthe placebo period (199 . 6&plusmn;10.2.10x3 /ml PRP), theyaveraged 183 .4&plusmn;16.4x103/ml PRP (range, 171.4&plusmn;17.1x 103/ml PRP to 215 - .7&plusmn;20 .8 x 103 /ml PRP). However, theanti-aggregatory effects of indomethacin, dl-propranolol (640mg/day), d-propranolol (640 mg/day), or the combined drugtreatments were shown by the increased concentrations ofthrombin that were required to induce secondary aggregationof platelets, and by the lower numbers of aggregatoryresponses observed with the various treatment regimens. Thethrombin threshold was increased from 0-35 :&plusmn; 0 - 04 U/0 - 4ml with placebo to 0.85&plusmn;0.17 U/0-4 ml (p < 0-001),0 - .67&plusmn;0 25 U/0.4 ml (p <0’05), and 1 - .19&plusmn;0. 35 U/0. 4 ml

(p <0-001) with the indomethacin, dl-propranolol (640 mg)and d-propranolol treatments, respectively. Similarly, therewere increases in the thrombin threshold during the .

combined indomethacin plus dl-propranolol (160 mg,p <0’05, 640 mg, p <0-01) or d-propranolol (p <0’001)treatment periods. In 31 tests of patients receiving placeboplatelet aggregation was induced by thrombin in 16 and byarachidonic acid in 9. Indomethacin alone significantlyinhibited aggregation induced by arachidonic acid (0 of 22aggregatory responses, p <0-025) but not that induced bythrombin (8 of 22 aggregatory responses, p <0’025).dl-Propranolol (640 mg/day) and d-propranolol (640 mg/day)reduced the frequency of aggregation induced by boththrombin and arachidonic acid, and similar effects wereobserved when these drugs were given with indomethacin.

Discussion

We found that the platelets of hypertensive patientsaggregated less readily in response to thrombin or

arachidonic acid when the patients were receiving eitherdl-propranolol or d-propranolol than when they were

receiving placebo. Platelets from patients taking propranololalso produced less of the pro-aggregatory hormone, TxA2, asassessed by the measurement of its stable metabolite, TxB2.The dose of propranolol required to inhibit aggregation andTxB2 generation was greater than 160 mg/day; this dose wasonly partially effective, whereas a dose of 640 mg/dayproduced significant inhibition. Indomethacin treatment

slightly decreased TxB2 generation induced by boththrombin and arachidonic acid but significantly inhibitedonly aggregation induced by arachidonic acid. Thecombination of propranolol and indomethacin produced noeffect on these responses additional to those for either drugalone.

These findings in patients given long-term propranololtreatment confirm our in-vitro observations that several beta-

adrenergic antagonists, including (in order of potency)timolol, dl-propranolol and d-propranolol, metoprolol, andH 35/25, inhibit platelet aggregation and TxA2 generation. 5At least one of these beta-antagonists has no intrinsic

sympathomimetic, membrane-stabilising, or beta-

adrenoceptor-blocking activity; therefore, it appears thatthese properties are not related to the anti-platelet effects. It ismore likely that the inhibitory effects of these compounds onplatelet aggregation are due to a previously unrecognisedproperty-inhibition of the synthesis of the pro-aggregatoryhormone TxA2.5 Cyclo oxygenase inhibitors non-specificallyblock the synthesis of both the pro-aggregatory TxA2 andanti-aggregatory prostacyclin (PGIz); in contrast propranololinhibits thromboxane synthesis without inhibiting PGI2production by endothelial cells.’&deg; Indeed, propranololenhances the anti-aggregatory effects of PGI2 on humanplatelets. 10The specific inhibition of TxA2 synthesis but not PGI2

synthesis by propranolol and possibly other beta-blockersmay contribute to the cardioprotective effects of these agentsobserved in animal models of myocardial ischaemia wherethe formation of intravascular platelet aggregatesll andintracoronary thrombi 12 may be increased. Similarly, in

patients with unstable angina pectoris or myocardialinfarction, the number of platelet aggregates in the coronarycirculation 13 and the peripheral blood 14 is higher than inpatients without coronary artery disease. As a corollary,

1384

Hirsh et al. 15 found that the release ofTxB2 into the coronarycirculation is higher in patients with unstable angina pectorisand recent chest pain than in patients with stable anginapectoris without recent chest pain, or patients with valvularheart disease. If TxA2 helps to.promote intravascular plateletaggregation, coronary vasoconstriction, or both in theseconditions, the potential therapeutic effects of propranolol inangina pectoris and threatened myocardial infarction 2,5 maybe due partly to the drug’s ability to block platelet TxA2synthesis without affecting endothelial PGlz production.d-Propranolol is as effective as dl-propranolol in inhibiting

platelet aggregation, inhibiting TxB2 synthesis,5 and

enhancing the anti-aggregatory activity of PGlz.1O Thed-isomer has only 2’ 5 % of the beta-blocking activity of theracemic mixture;’ it may, therefore, be superior to dl-

propranolol in treating patients with threatened myocardialinfarction by inhibiting platelet aggregation and TxA2production without substantially reducing heart rate or

myocardial contractility. Similarly, in patients with asthmaor peripheral vascular disease, d-propranolol may be thepreferred treatment.

We thank Ms Stephanie Wooten for secretarial assistance, Ms BeverleyAdams, Ms Judy Zimmer, and Ms Sue-Jean Tsai for technical assistance, MsShirley Anderson and Ms Carolyn Muckleroy for their nursing assistance, DrJames Conway and ICI Ltd. (U.K.) for their interest and for supplyingd-propranolol, and Dr J. E. Pike of the Upjohn Company for supplyingthromboxane B2. The study was supported by grants from the Texas Affiliateof the American Heart Association, the Merck Foundation, and the NationalHeart, Lung, and Blood Institute (HL-25471 and HL-18826). W. B. C. is arecipient of a National Institutes of Health research center development award(1K04 HL 00801), R. M. G. is a recipient ofa Pharmaceutical ManufacturersAssociation faculty development award in clinical pharmacology, and K. S. C.is a National Institutes of Health predoctoral trainee (5T32GM-07062-06).

Correspondence should be addressed to R. M. G., Department ofPharmacology, University of Texas Health Science Center, 5323 Harry HinesBoulevard, Dallas, Texas 75235, U.S.A.

REFERENCES

1. Graham RM. The physiology and pharmacology of alpha- and beta-blockade.Cardiovasc Med 1981; Special suppl April 7-22.

2. Reimer KA, Rasmussen MM, Jennings RB. Reduction by propranolol of myocardialnecrosis following temporary coronary artery occlusion in dogs. Circ Res 1973; 33:353-63.

3. Norris RM, Sammel NL, Clarke ED, Smith WM, Williams B. Protective effect ofpropranolol in threatened myocardial infarction. Lancet 1978; ii: 907-09.

4. Weksler BB, Gillick M, Pink J. Effect of propranolol on platelet function. Blood 1977;49: 185-96.

5. Campbell WB, Callahan KS, Johnson AR, Hirsh PD. Mechanism of the antiplateletactivity of the &bgr;-adrenergic antagonists. Int J Clin Pharmacol (in press).

6. Hamberg M, Svensson J, Samuelsson B. Thromboxanes: a new group of biologicallyactive compounds derived from prostaglandin endoperoxides. Proc Natl Acad SciU.S.A. 1975; 72: 2294-98.

7. Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal.Nature 1962; 194: 972.

8. Dray F, Charbonnel B, Maclouf J. Radioimmunoassay of prostaglandins F&agr;, E1 andE2 in human plasma. Eur J Clin Invest 1975; 5: 311-18.

9. Zar JH. Biostatistical analysis. Englewood Cliffs: Prentice-Hall, 1974: 228.10. Callahan KS, Campbell WB, Johnson AR. Antiplatelet activity of propranolol:

interaction with PGI2 and endothelial cells. Fed Proc 1980; 39: 392.11. Folts JD, Crowell EB, Rowe GG. Platelet aggregation in partially obstructed vessels

and its elimination with aspirin. Circulation 1976; 54: 365-70.12. Aiken JW, Shebuski RJ, Gorman RR. Blockage of partially obstructed coronary

arteries with platelet thrombi: Comparison between its prevention with

cyclooxygenase inhibitors versus prostacyclin. Adv Prostag Thromb Res 1980; 7:635-39.

13. El-Maraghi N, Genton E. The relevance of platelet and fibrin thromboembolism ofthecoronary microcirculation, with special reference to sudden cardiac death.Circulation 1960; 62: 936-44.

14. Schwartz MB, Hawiger J, Timmons S, Firesinger GC. Platelet aggregates in ischemicheart disease. J Int Soc Thromb Haemost 1980; 43: 185-88.

15. Hirsh PD, Hills LD, Campbell WB, Firth BG, Willerson JT. Release of

prostaglandins and thromboxane into the coronary circulation in patient withischemic heart disease. N Engl J Med 1981; 304: 685-91.

RE-ANALYSIS OF DATA IN TWO LANCETPAPERS ON THE EFFECT OF DIETARY SODIUM

AND POTASSIUM ON BLOOD PRESSURE

J. M. P. HOLLYS. J. W. EVANS

F. J. GOODWINM. J. VANDENBURG

J. M. LEDINGHAM

Medical and Steroid Units, London Hospital Medical College,London E1

As promised in our recent letter to the Editor, we present afull re-analysis of the data which were published in erroneousform in The Lancet earlier this year. 2,3 The re-analysis hasbeen based on the original clinic and laboratory records.

First Paper215 patients with mild essential hypertension (average

diastolic blood pressure 90-110 mm Hg) and 8 normotensivesubjects (diastolic pressure less than 85 Iim Hg), all withinthe age-range 26-49, were studied in a randomly allocatedcross-over, observer-blind trial in which they received theirnormal diets supplemented by 100 mmol sodium (Na) dailyfor 12 weeks and a no added sodium diet supplemented by100 mmol potassium (K) daily for the same period. Blood-pressure (BP) and hormonal data are shown in figs 1 and 2.

[] Hypertensive [] NormotensiveFig. 1-Blood pressure and heart-rate (mean&plusmn;SE, measured after

resting supine for 5 min) of hypertensive patients andnormotensive control subjects during various stages of the trial.