reversal of abnormal platelet aggregability and change in...

CHRISTODOULOU, CHARLES SMITHEN and THOMAS KILLIPWILLIAM H. FRISHMAN, BABETTE WEKSLER, JAMES P.in Patients with Angina Pectoris Following Oral Propranolol

Reversal of Abnormal Platelet Aggregability and Change in Exercise Tolerance

ISSN: 1524-4539 Copyright © 1974 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online

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Reversal of Abnormal Platelet Aggregability andChange in Exercise Tolerance in Patients withAngina Pectoris Following Oral Propranolol

By WILLIAM H. FRISHMAN, M. D., BABETTE WEKSLER, M.D., JAMES P. CHRISTODOULOU, M.D.,

CHARLES SMITHEN, M.D., AND THOMAS KILLIP, M.D.

SUMMARYNineteen patients with severe but stable angina pectoris who had positive ECG response to exercise on a

bicycle ergometer and 11 normal subjects were studied. Patients received placebo for six weeks and were

then randomized into placebo (n = 9) and propranolol (n = 10) treatment groups. Threshold for plateletaggregation in response to adenosine diphosphate (ADP) was measured in fresh platelet rich plasma.Mean concentration of ADP necessary for a biphasic threshold aggregation response was 1.56 AM

(geometric mean) in patients and 3.85 ,uM in normals (P < 0.01). Serial studies with placebo showed no

significant change in ADP threshold. With propranolol, 80 mg/day, platelet aggregation in response to ADPwas entirely normalized; 3.79 ,uM ADP produced maximal aggregation compared to 1.32 gM before therapy(P < 0.01). No additional changes were noted with propranolol, 160 mg/day. In the propranolol-treatedpatients (80 mg/day) who demonstrated reduction in ADP-induced platelet aggregation, total work duringexercise increased by 128%, from 765 ± 125 (standard error of mean) kpm during the control period to 1,-792 ± 285 kpm (P < 0.01).Thus patients with angina and abnormal exercise tolerance demonstrate increased platelet aggregability

in vitro which is restored toward normal with propranolol in dosage sufficient to improve exercise tolerance.

Additional Indexing Words:Beta-adrenergic blockadeCoronary artery disease Arteriosclerosis

Exercise tolerance

SEVERAL OBSERVERS have suggested thatplatelet dysfunction may be a factor in the

pathogenesis of coronary artery disease." 2 Plateletsinitiate thrombosis by aggregating at the site ofprevious vascular injury,3'6 and it is speculated thataltered or accelerated platelet aggregation may play a

significant, albeit not yet fully defined role in thedevelopment and progression of arteriosclerotic lesions.There are conflicting reports concerning the status

of abnormal platelet function in patients with cor-

onary artery disease. 12 Reliable and reproduciblestudies of human platelet aggregation are difficult

From the Divisions of Cardiology and Hematology, Departmentof Medicine, The New York Hospital-Cornell Medical Center, NewYork, New York.

Supported by U.S.P.H.S. Contract PH 43-67-1439; by a Grantfrom The New York Heart Association, and a Grant from the AyerstLaboratories, New York, New York.

Presented in part at the 46th Scientific Sessions of the AmericanHeart Association, Atlantic City, New Jersey, November 9, 1973.

Address for reprints. Dr. William H. Frishman, The New YorkHospital-Cornell Medical Center, 525 East 68th Street, New York,New York 10021.

Received February 27, 1974; revision accepted for publicationJune 21, 1974.

C(irctulation, Volume 50, N,ovember 1974

because of the variety of factors which alter plateletresponsiveness such as age,"3 sex, diet,'4 blood lipidand glucose levels,'5 16 and smoking history.`7 Manycommonly used drugs significantly influence plateletaggregability in vivo.'8-21 Venipuncture techniques,rapidity of sample processing, and ambient changes inpH can all contribute to altering platelet respon-

siveness with any of the standard laboratory methodsmeasuring aggregation threshold.22

Platelet aggregates have been observed in the cor-

onary microvasculature in an animal model followingcatecholamine infusion and after acute, severe

stress.23' 24 Infusions of adenosine diphosphate (ADP)induce coronary arterial platelet thrombi associatedwith myocardial necrosis in swine similar to that notedfollowing catecholamine administration.2' It has beenpostulated that therapy directed at altering theplatelet response to endogenous release ofcatecholamine or local release of ADP might alter thelikelihood of acute thrombotic events in patients withcoronary artery disease.The present study was designed to test the

hypothesis that patients with angina pectoris havealtered platelet aggregability when compared to a

normal group similar with regard to age and sex. In

887

Thrombosis



FRISHMAN ET AL.

addition, the effects of treatment with therapeuticdoses of oral propranolol, a beta-adrenergic blockingagent with known anti-anginal properties but inade-quately defined effects on platelet function, were alsoassessed.

Methods

Nineteen patients with presumed ischemic heart diseasewere studied. Criteria for inclusion were: 1) At least threeattacks of angina pectoris per week, responsive tonitroglycerin, with no evidence of an accelerated course; 2)absence of valvular heart disease, hypertension, congestiveheart failure, chronic obstructive pulmonary disease,diabetes, lipoprotein abnormalities, anemia, smokinghistory, or myocardial infarction within six months; 3)definite electrocardiographic (ECG) evidence of myocardialischemia associated with chest pain during submaximal ex-

ercise stress testing.Pertinent clinical data of these 19 patients are presented

in tables 1 and 2. There were eleven male and eight femalepatients with an average age of 54. Four patients had ahistory of previous myocardial infarction. Three patientswithout previous myocardial infarction had undergone cor-

onary angiography. In each case, narrowing of at least 70%of one or more of the major coronary arteries was seen.

Eleven normal subjects with a negative stress test were

studied along with the patient group. These subjects had no

evidence of ischemic vascular disease, lipid abnormalities or

diabetes, and each had a negative history for cigarette smok-ing and drug ingestion of any sort. Pertinent clinical data ofthe patients with angina are compared with those of the nor-

mal subjects in table 2.

Protocol

All normal subjects had platelet aggregation studies andexercise tests as described below.The patients with angina pectoris were followed

bimonthly by the same physician (W. F). Informed consentwas obtained, and all cardiovascular medications were dis-continued except for nitroglycerin. An oral placebo* was ad-ministered for six weeks. Every patient received a restingelectrocardiogram, chest roentgenogram, urinalysis, andblood analysis for cholesterol, triglycerides, glucose, bloodurea nitrogen, creatine phosphokinase (CPK), lacticdehydrogenase (LDH), serum glutamic oxaloacetic trans-aminase (SGOT), hematocrit, hemoglobin, white blood cellcount, and platelet count. Platelet aggregation studies were

performed and exercise testing on a bicycle ergometercarried out as described below.

After the six week placebo period, patients were ran-

domized into placebo and propranolol treatment groups.

The placebo group (n = 9) remained on the same placeboregimen while the drug treatment group (n = 10) was

started on 80 mg of oral propranolol daily in four divideddoses. Neither patient nor physician knew whether therapywas placebo or drug. All patients continued takingnitroglycerin as needed. After two weeks, platelet aggrega-

tion studies were repeated. The propranolol dosage was thenraised to 160 mg/day, again "double blind" as the pill wasidentical in appearance to the placebo. The drug was given

*Mannitol, 225 mg, (Ayerst Laboratories).

in four divided doses and two weeks thereafter studies wererepeated in both groups of patients.

Platelet Aggregation Studies

Blood was obtained after a 12-hour fast. After an initialresting period of 30 min, a 19 gauge needle was inserted,without use of a tourniquet, into an antecubital vein and aslow infusion of physiological saline begun. After 15 min ofrest, with the patient relaxed, blood was sampled by freeflow into plastic tubes containing 1/10 volume acid citratedextrose (ACD). The blood specimens were then im-mediately processed for analysis. The specimens were codedso that those performing the aggregation studies had noknowledge of the patient's identity, the diagnosis, or type ofdrug therapy.

Platelet aggregation studies were performed using theturbidometric method of Born,26 as modified by Mustard etal.27 The blood sample collected a few minutes earlier wasplaced in sterile polypropylene tubes and platelet richplasma separated (PRP) by immediate centrifugation for 15min at room temperature at 225 g. After removal of the up-per 2/3 of the PRP, the remaining PRP was recentrifuged at4,300 g for 10 min at 40C to yield platelet poor plasma(PPP). Platelet counts were performed on a Coulter Model Fcounter, the count of the PRP was adjusted to the range300-400,000/mm3 when necessary with the autologous PPP.

Platelet aggregation studies were performed in duplicatewithin 90 min of venipuncture. The sample was kept tightlycovered at room temperature until analyzed. Samples of0.45 ml PRP were prewarmed for 1 min with stirring insiliconized cuvettes placed in a Payton dual channelaggregation module (Payton Associates, Buffalo, N.Y.)before aggregating agents were added. The light transmit-tance was recorded on a Riken Denshi linear recorder. Theblank for each study was a similarly treated sample of PPP.The aggregating agent tested was adenosine diphosphate(ADP) (Sigma, Inc., St. Louis, Mo.), maintained as a 10-3Mfrozen stock and diluted essentially twofold just before useover a range of 10-7M to 10-4M (0.1 to 100 ,uM). The ADPconcentrations producing aggregation were 0.5, 1, 2, 5, 10,and 20 ,M. The lowest concentration producing a fullbiphasic response was recorded as the threshold dose. Theoptimal ADP concentration was assessed without extrapola-tion between different concentrations. The change in lighttransmittance to increasing concentrations of ADP and thecharacteristic biphasic response at the threshold dose are il-lustrated in figure 1. In repeated determinations usingcoded samples, reproducibility was within 10% of mean.

Exercise Testing

Bicycle ergometry testing was performed withoutknowledge of the patients' drug status during the study. Amodification of the ear-ensiform system of Abarquez et al.28was used with three apical leads recorded in the supine posi-tion and on the bicycle ergometer prior to exercise. Theplacement of electrodes was as follows: right arm in the leftV6 position, left arm on the forehead, left leg over the en-siform (xiphoid) process and right leg over the right V6 posi-tion. Simultaneous recordings were taken of aVR, aVL, andaVF, yielding modified orthogonal X, Y, and Z leads. Theprecordial leads were placed in the V, and V4 position andthe third placed either in the V3 or V5 positions so as torecord the transitional QRS over the precordium. Themodified X, Y, and Z leads were monitored continuously on

Circulation, Volume 50, November 1974

888



PROPRANOLOL AND PLATELET ACTIVITY

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FRISHMAN ET AL.

a three-channel oscilloscope throughout the study. Tracingswere recorded of both lead groups every minute during theprocedure.

Ar 10 + ooMultistage graded exercise was performed utilizing abicycle ergometer (Schwinn ergometric exerciser) as thepatient pedaled against a predetermined load. After bloodfor platelet studies had been obtained, patients were started

.q C: Gg n cat a work load of 150 kpm/min. The work load was in-creased by 150 (kilo-pond meters) kpm/min every threeminutes until either chest pain, fatigue, or a heart rate of150 beats/mm occurred. An abnormal electrocardiographicresponse was defined as a flat or downsloping ST-segmentdepression, 1 mm, measured 0.08 seconds after the termina-tion of the QRS complex with the P-Ta segment as thebaseline of reference.

Blood pressure in the brachial artery was recorded bysphygmomanometer prior to and immediately after exercise.

d ~ The heart rate-blood pressure product, an approximation of:Hsti i myocardial oxygen consumption, was calculated from

8-4.1 gmeasurements obtained immediately after cessation of exer-cise.

Propranolol Blood Levels

The concentration of propranolol in blood was measuredfrom coded samples without knowledge of previous therapy.The fluorometric method of Black and associates,30 asmodified by Shand et al.'3 was used. Four milliliters of

o oo oo plasma, obtained approximately two hours after the most re-cent dose of propranolol or placebo, were alkanized with 1

o g o ml of 1 N NaOH and extracted into 12 ml of heptane con-c v c taining 1.5% isoamyl alcohol. After centrifugation, 10 ml of

the organic phase was extracted into 1.5 ml of 0.1 N HCIand the fluorescence of the acid phase measured in an

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890




Aminco-Bowman spectrophotometer (Maximum excitation,295 ,u; maximum emission, 360 g, uncorrected). For eachpatient duplicate plasma blanks and a standard of 100 ng

per ml of known propranolol added to the plasma blankwere determined.

Statistical Analysis

For all of the variables except ADP, arithmetic group

means with the standard error of the mean are presented.For ADP data, geometric means are presented. Geometricmeans are preferred since the concentrations of ADP usedwere essentially geometric dilutions. Tests of significance forresults obtained in normals and untreated patients, as wellas between the two groups of untreated patients, were per-

formed utilizing the Wilcoxon Rank Sum Test. Comparisonof data from the same group with various experimentalprotocols were performed using the Wilcoxon Signed RankTest or by the Sign Test.

Results

Platelet Aggregation Studies

Patients with angina pectoris demonstrated in-creased platelet sensitivity to aggregating concen-

trations of ADP when compared to the control sub-jects exposed to the same concentration of ADP. Meanconcentration of ADP necessary for a biphasicaggregation response was 1.56* ,uM in the group withangina pectoris, a value significantly less than the3.85* ,uM observed in the normal controls (P < 0.01)(table 3, figure 2).

Serial studies performed on the patients withangina pectoris receiving placebo showed no changein the increased platelet sensitivity to ADP (table 3).

Administration of propranolol in a dose of 80 mg/day had a dramatic effect in reducing platelet sen-

sitivity to ADP in patients with angina pectoris. Afterpropranolol a mean of 3.79* ,uM ADP was required toproduce a biphasic aggregation response compared to1.32* jM before therapy (P < 0.01). No additionalchanges in ADP concentration to effect aggregationthreshold were noted after the propranolol dosage was

increased to 160 mg/day, (fig. 3, table 3). The mean

concentration of ADP required to produce thethreshold aggregation response after 80 mg/day ofpropranolol was not significantly different from thevalue observed in the normal controls, but was

significantly different from placebo-treated patients(P < 0.01) (table 3). An example of the effect ofpropranolol in increasing the dose of ADP required forplatelet aggregation in a patient with angina pectorisis shown in figure 4.Mean serum concentration of propranolol was

21 ± 4.0 ng/ml (range: 11-49 ng/ml) at a dose of 80mg/day and 49 ± 9 ng/ml (range: 25-110 ng/ml) at a

dose of 160 mg/day.

*Geometric mean.


There were no significant changes from control inthe mean level of blood glucose, urea nitrogen,SGOT, CPK, LDH, cholesterol, triglycerides,hematocrit, or platelet count (table 4) during the serialsampling periods in either the control, placebo or drugtreatment group.

Exercise Tests

During the control period, all 19 patients withangina had a positive electrocardiographic response to

Table 3

Individual and Group Mean Concentrations of ADPInducing Platelet Aggregation in Patients with AnginaPectoris (Treated and Untreated) and Normal Subjects

Normalsubjects Age ADP gM*

1. H.G. 47 22. N.B. 46 5)3. F.P. 00 0i4. K.B. 45 25. F.Z. 56 56. L.A. 34 57. M.G. .52 108. H.Y. 48 a9. J.S. 60 1

10. S.S. 0).o 'D11. W.P. 41. 2

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1.L.H. 54 2 2 22.M.G. 60 2 2 23.C.P. 48 2 2 54.J.H. 38 .5 2 25. B.S. 54 2 0 26. N.P. 55 2 1 17.B.M. 40 2 2 28.A.K. 59 1 1 19. M.M. 57 1 1 1

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treated ADP pM ADP ,M ADP MM

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1.32t 3.79t 3.46t

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tGeometric mean.

891



FRISHMAN ET AL.

ANGINA

Figure 2

Comparison of ADP-induced platelet aggregability in normalsubjects and patients with angina pectoris. Platelets of normal sub-jects require significantly higher concentrations of ADP (geometricmean) to attain aggregation threshold than do platelets of patientswith angina pectoris.

exercise. Administration of propranolol was followedby a significant increase in exercise tolerance in thepatients with angina pectoris. In the ten patients whoreceived propranolol (80 mg/day), total work duringexercise increased by 128%, from 765 ± 125 kpm dur-ing the control period to 1,790 + 285 afterpropranolol (P < 0.01) (fig. 5). This beneficial effecton work performance was associated with a significantdrop in the heart rate-blood pressure product from16,800 ± 1,500 before to 12,000 ± 890 afterpropranolol (P < 0.01) (fig. 6). In each case typicalanginal pain was the end-point. In contrast, there wasno significant change in exertional tolerance inpatients with angina pectoris who received a placebo.When the dose of propranolol was increased to 160mg/day, work performed during maximal toleratedexercise decreased slightly with either pain or fatiguethe end-point, but the mean for the group was notsignificantly different from that observed with 80mg/day.

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Effect of propranolol on platelet aggregability in patients withangina pectoris. After propranolol, 80 mg/day, platelets of treatedpatients are less sensitive to ADP (geometric mean) compared tobefore treatment and not significantly different from normal.Platelet responsiveness was not further changed when the dose ofpropranolol was doubled to 160 mg/day.

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Aggregation with and without propranolol in a patient with angina pectoris. Left panel, during placebo therapy, 2 AMADP required for aggregation threshold. Right panel, after 80 mg/day propranolol orally, 2 AM ADP is insufficient to

achieve aggregation threshold. Five gM ADP is now required and response is within normal limits.

Discussion

A number of abnormalities in platelet function havebeen reported in patients with ischemic heart dis-ease.7-11, 32-34 Platelet hyperresponsiveness has beendescribed in myocardial infarction7-9, 32 and

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cerebrovascular disease.35 However, other in-vestigators have failed to detect differences in plateletfunction in patients with arteriosclerotic heart diseasewhen compared to normal subjects.9' 10, 12, 36 Widedifferences in methodology and patient selection mayaccount in part for these divergent findings.22 36

Some factors that influence the reliability andreproducibility of platelet aggregation analyses havebeen identified. Repeated measurements of human

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Changes in heart rate-systolic arterial pressure product (HR X BP)after propranolol in patients with angina pectoris. Circles depictvalues before and the squares after propranolol treatment. A signifi-cant decrease in HR X BP at the onset of angina, even though exer-

cise level was higher after treatment, is demonstrated at the end ofexercise with 80 mg/day of oral propranolol.

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FRISHMAN ET AL.

platelet aggregation induced by ADP vary from day today depending on the subject's emotional state, smok-ing history,'7 or with acute changes in blood lipids,14-'8glucose,15 free fatty acids,37 38 catecholamines,39hematocrit, and platelet count. Differences in age'3and sex of the subjects studied may also affect thecomparability of previously reported studies. Thetechnique of blood sampling and processing and therapidity with which studies are performed is also im-portant.22 Since many drugs influence platelet func-tion, it is imperative that this factor be rigidly con-trolled. In our study an attempt was made to minimizethe effects of the variable factors influencing plateletfunction by carefully selecting and closely following apopulation of reliable patients with documentedischemic heart disease and comparing them to a groupof normal controls, similar in age and sex.The present study demonstrates a highly significant

increase in ADP-induced platelet aggregability inpatients with angina pectoris when compared to nor-mal subjects. There are several possible explanationsfor this observation. Platelets are known to react withcomponents of injured vascular endothelium such asexposed basement membrane and collagen by adhe-sion and aggregation, an effect mediated by ADP.'3, 6Platelets passing through narrow and sclerotic vesselsmay thus be rendered hyperaggregable. Plateletaggregation induced by these or other mechanismsmight cause intravascular thrombi further narrowingthe lumen. Thrombi produced by platelet aggregationon ruptured arteriosclerotic plaques may induce acutemyocardial infarction or ischemia by obstruction ordistal embolization of the aggregates.' Our resultsmay be reflecting the secondary effect of vascular dis-ease on platelet aggregation. We demonstrated cor-onary artery disease in three of our patients by cor-onary arteriography. The other patients had typicalangina pectoris with abnormal exercise electrocar-diograms and it seems probable that most of them hadsignificant coronary vascular disease as well.

Since catecholamines are known to alter plateletfunction, an elevation of plasma catecholamine levelsin the patient population could have influenced ourfindings. Experimentally, catecholamine infusion oracute stress induce platelet aggregation in the cor-onary microcirculation with subsequent myocardialnecrosis. Antiaggregation substances protect againstthis effect."2 40 Catecholamine levels were notmeasured in either the normal or patient group, and itis possible that anticipation of exercise, particularly inthose patients prone to pain, might have acutelyelevated plasma catecholamines and affected plateletaggregability. However, in the patient group noevidence of excessive catecholamine release could bedetected by gross measurements such as heart rate,

and all studies were done with the patients relaxedand reassured. Furthermore, platelet aggregabilitystudies were performed in other clinical settings,when establishing reproducibility of assay, and theresulsts were consistent with pre-exercise values.

Free fatty acids can also alter platelet aggregabilitybut increased levels are generally secondary to stress-induced rise in catecholamines. An alpha-globulinthat inhibits platelet aggregation has recently beendescribed in normal plasma.4' The role of this factor inplatelet aggregability of patients with angina pectorisis conjectural.

Alternatively, our findings may be a reflection of aprimary platelet abnormality in patients with anginapectoris. This was suggested by Hampton and Gorlin42who showed abnormalities in platelet function notonly in patients with symptomatic coronary vasculardisease, but also in their healthy relatives, raising thepossibility of a familial defect.Although the exact mechanism for the increased

platelet aggregability in patients with angina pectorisremains unknown, the clinical implications of thisobservation are of interest. Experimental myocardialinfarction has been observed secondary to plateletaggregation although the aggregates persisted foronly a few minutes. Haerem reported that plateletaggregates in the epicardial coronary arteries ofpatients who died suddenly of cardiac causes weremore numerous and larger than those found inpatients without cardiac disease.43 Extension or rein-farction is common in patients dying with cardiogenicshock44 45 and may be due to formation ofmicroaggregates mediated by a local release of ADPfrom ischemic myocardial cells, circulating platelets,and red blood cells. Agents which could alter plateletaggregability may have a role in the treatment of cor-onary artery disease, possibly in the prevention ofacute ischemic complications. Further clinical datawill be required, however, to evaluate the significanceof platelet dysfunction in the infarction syndrome.

In this study, ten patients who received propranololin dosage of 80 mg/day showed a dramatic and signifi-cant increase in performance during standard exer-cise. Propranolol improved the exercise response andrestored the increased platelet aggregability inducedby ADP toward normal in most patients with anginapectoris. The platelet effect was noted with serumlevels of propranolol as low as 11 ng/ml, was notfurther influenced by increasing the dose, and per-sisted over a long period of time with chronic ad-ministration of the drug. It is also of interest that theimprovement in exercise tolerance was also maximalat the lower dose of propranolol and was not enhancedby increasing dosage.The improved exercise tolerance in patients with

Circutlation V'olume 50, Novrember 1974

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angina pectoris receiving propranolol was associated,in all cases, with a decrease in the heart rate-bloodpressure product, an effect which has beendemonstrated by several investigators.46' 47 While theimproved exercise performance in patients withangina pectoris receiving propranolol can be cor-

related with reduction of myocardial oxygen con-

sumption,48 49 the possibility that the decrease inplatelet aggregability influenced cardiac performance,perhaps by improving capillary blood flow, meritsconsideration.The effect of propranolol itself on platelet function

is controversial.18' 50-54 It has been suggested thatcatecholamine-induced aggregation is an alpha-adrenergic response since phentolamine preventsepinephrine-induced platelet aggregation.`9 Othershave speculated that with beta-adrenergic blockade,the alpha receptor on the platelet could be unmasked,and aggregation responsiveness heightened.5'Thomas, however, showed that propranolol, in vitro,using a concentration of 10 ,M, reduced plateletaggregability.52 Haft and his associates have recentlydemonstrated that propranolol prevents isoproterenol-induced intravascular platelet in the rat.54

Propranolol has multiple physiologic actions andmay be of potential value in patients with ischemicheart disease through several mechanisms. In additionto reducing cardiac work during exercise and thusreducing myocardial oxygen demand,5" the drug shiftsthe oxyhemoglobin dissociation curve to the right56and also has an antirenin effect.'7 In addition, thepresent study documents that propranolol abolishesplatelet hyperresponsiveness to ADP in patients withangina pectoris.

If hyperaggregable platelets play a role in thepathogenesis of myocardial ischemia, an agent whichreverses this abnormality may be an important ad-junct in chronic treatment. The mechanism of actionof propranolol in reducing platelet hyperaggregabilityand the long-term effectiveness of oral propranolol inpreventing thrombotic events in patients with cor-

onary artery disease remains to be determined.Acknowledgments

The authors wish to extend their thanks to Miss Barbara Befler,M.A., Miss Elaine Coupal, BA., and Mrs. Muriel Gilick, B.A., fortheir unfailing technical assistance; and to Mrs. Carmen Retz, B.S.,and Mrs. Joan Steinbach for their valuable help in preparing themanuscript.A special note of appreciation is extended to John Fertig, Ph.D.,

Professor of Statistics, Columbia University, who assisted in thestatistical evaluation, and to Dr. Stephen Scheidt for his review ofthe manuscript.

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