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Thrombocytopenia due to digitoxin
Case Reports Thrombocytopenia
Due to Digitoxin*
Demonstration of Antibody and Mechanisms of Action ROBERT C. YOUNG, M.D., RALPH L. NACHMAN, M.D. and HERBERT I. HOROWITZ, M.D.
New York, New York preparation, salt restriction and mercurial diuretics. On November 7 the patient was admitted to the New York Hospital for the first time because of progressive dyspnea. Auricular fibrillation was present with a ventricular rate of 100 per minute, and digitoxin was administered. Doses of 0.2 and 0.4 mg. on alternate days were required to control the tachycardia. When he left the hospital on November 17, his condition was improved. The patient entered the hospital on January 7, 1963, for cardiac catheterization which revealed severe mitral insufficiency. During this hospital course he experienced epistaxis and passed guaiacpositive stools. The platelet count was 49,000 per cu. mm. and the bleeding time was greater than 30 minutes. The thrombocytopenia was thought to be secondary to hydrochlorothiazide and this medication was discontinued. The patient was discharged to await readmission for insertion of a mitral valve prosthesis. He continued to take 0.4 mg. of digitoxin per day and noted frequent epistaxis, easy bruisability and tarry stools. During this period his private physician inadvertently gave him a prescription for digoxin, 0.5 mg. per day, in place of digitoxin. The hemorrhagic symptoms disappeared and a platelet count was within normal limits. The patient reentered the New York Hospital on May 2, 1963. The admitting house officer, unaware of the change in digitalis preparations, reinstituted digitoxin, 0.4 mg. per day. The tachycardia with auricular fibrillation proved difficult to control and the dosage of digitoxin was increased to 0.6 mg. per day. Purpura was not present at this time, but the platelet count was 71,000 per cu. mm. and the bleeding time was 15 minutes. A bone marrow aspiration revealed hypercellnlarity with erythroid and myeloid hyperplasia, and the number and maturation of the megakaryocytes were within normal limits. The patient was given Mercuhydrin@, meperidine and ammonium chloride in addition to digitoxin.
HROMBOCYTOPENIA has been reported to complicate the administration of a large number of drugs. In vitro tests have provided supporting evidence of the presence of drug antibody reactions in cases due to Sedormid@ [7-51, quinidine [6-701, quinine [77] and stibophen [72]. A mixture consisting of human platelets, antiserum and drug was essential for the demonstration of the drug-induced antibody in each case. The results of similar in vitro test systems employed in investigating thrombocytopenias presumed to be due to such drugs as chlorothiazides [ 73,741, arsenobenzol compounds [ 751 and sulfonamides [ 761 have been negative. Despite the negative reactions to in vitro tests, the relationship of the drug to thrombocytopenia in such cases has been confirmed in vivo by rechallenging the patient with the suspected drug, with recurrence of thrombocytopenia. In vivo experiments of this type have been used to substantiate the diagnosis in the two previously reported cases of tbrombocytopenia due to digitoxin [ 77,781, The purpose of this paper is to describe a third patient with digitoxin-induced thrombocytopenia. By using a number of in vitro tests, we have demonstrated the presence of antibody which reacted with digitoxin and secondarily resulted in thrombocytopenia.
T
CASE
REPORT
The patient was a fifty-five year old white man with an eight year history of congestive heart failure, presumably secondary to rheumatic heart disease. For the eight years prior to November 7, 1962, the patient was maintained on an unknown digitalis
* From the Department of Medicine, New York Hospital-Cornell Medical Center, New York, New York. This work was supported by U. S. Public Health Service Grant HE-08744 and U.S. Public Health Service Training Grant 2A-5337. Manuscript received November 22, 1965. VOL.
41,
OCTOBER
1966
605
Thrombocytopenia
606
Due to Digitoxin-Young
TABLE I PLATELET
COUNTS
AFTER
WITHDRAWAL
OF DIGITOXIN
Date
Platelet Count (per cu. mm.)
5/4/63 5/10/63* 5/13/63 5/14/63 5/15/63 5/17/63
71,000 84,000 176,000 178,000 226,000 241,000
* Digitoxin
discontinued.
In citro studies (to be described) indicated that digitoxin was responsible for the thrombocytopenia. On May 10, 1963, digitoxin therapy was discontinued and the patient was given 0.5 mg. digoxin per day. The other medication was continued and 100 mg. of prednisone was added. Platelet counts (Table I) returned to normal within three days. On May 23, 1963, the patient underwent open-heart surgery and the damaged mitral valve was replaced with a StarrEdwards ball valve prosthesis. On July 11 he was discharged from the hospital on a regimen of 0.25 mg. digoxin per day and has since remained free of thrombocytopenia. MATERIALS
AND METHODS
Methods for siliconization of glassware, collection and processing of blood specimens for serum and for platelet-rich plasma have been described previously [ 791. Platelet Factor 3 Test. The principles and technics used in this test have been described elsewhere [ZO]. In brief, the test consists of incubation of normal intact platelet-rich plasma, inactivated serum or plasma-containing antibody, and drug in appropriate concentrations at 37’~. for 1 and 2 hours. Samples of the incubation mixture are taken at intervals and tested for platelet factor 3 activity using the Russell’s viper venom (Stypven@) time test. Control incubation mixtures lacking platelets, drug or antibody are sampled and tested at the same time. The results are considered to be positive when the Stypven time of the test incubation mixtures is reproducibly 10 seconds or more shorter than for the control mixtures. Stypven Time Test. To 0.05 ml. of test material was added 0.05 ml. of normal platelet-poor plasma and 1.0 ml. of 1 : 100,000 dilution of Stypven* in 0.025 M calcium chloride; the time for clot formation was determined. Clot Retraction Inhibition. These studies were carried out as previously described [ZO]. Amino Acid Generation. Platelets participating in certain in vitro immune reactions undergo changes in amino acid composition which result in an increase in * Burroughs Welcome
Co., Tuckahoe,
New York.
et al.
total free amino acids present in the incubation mixture [27]. This phenomenon was employed to demonstrate antibody to digitoxin in the patient’s serum. The method previously described [27] was modified as follows: Normal human platelets were separated from ethylenediaminetetracetic acid whole blood, washed and suspended in Verona1 buffer. A suspension of 1 X log platelets was incubated with barium sulfate-adsorbed serum from the patient in the presence of 0.2 mg. per ml. of digitoxin. Controls included incubation of (1) platelets in adsorbed normal serum, (2) platelets in digitoxin alone, (3) platelets in normal serum plus digitoxin and (4) platelets in patient’s serum plus digoxin. After 1 hour of incubation the alpha amino nitrogen [ZZ] in the system was compared to the amount present before incubation. Digitalis Preparations. Commercial preparations of digitoxin for intravenous use were employed. The drug was dissolved in varying concentrations of alcohol; further dilutions were made in normal saline solution. Preparations of digoxin, ouabain and Cedilanid@ were handled in a similar way. Pure crystalline digitoxin, digoxin, and their glycosides were kindly supplied by Dr. Daniel Lucas (Department of Medicine, Cornell Medical School). They were weighed, taken up in small amounts of absolute ethanol and further dissolved in normal saline solution to give a concentration of less than 1 per cent ethanol in the final test mixtures. Tritium-labeled digitoxin, dissolved in absolute ethanol with a specific activity of lo6 c.p.m. per ml., was made available by Dr. Lucas, and was diluted 1 : 100 to provide 104 c.p.m. per ml. and less than 1 per cent ethanol in the final text mixtures. Latex Particles. A 0.52 weight per volume suspension of polystyrene latex particles,-/ 2.64 =t 0.06 p in diameter, in platelet-poor plasma produced a suspension containing approximately 600,000 particles per cu. mm., with a diameter approximating that of normal platelets. Platelet Sonicate. Washed platelets from 120 cc. of blood from a patient with polycythemia vera were resuspended in 5 cc. of Verona1 buffer. The final suspension was solubilized by sonification$ 30 seconds at full amplitude and was concentrated to 1 ml. by ultrafiltration. This was used as a source of soluble platelet protein. Starch Gel Electrophoresis. Horizontal electrophoresis was performed by the method of Smithies 1231. Materials to be electrophoresed, in 0.4 ml. volumes, were made into a paste with dry, hydrolyzed starch, and introduced into a transverse cut into the gel. Electrophoresis was carried out at 10”~. for 16 hours at a pH of 8.6 and constant voltage of 5 volts per cm. The starch gel strips were cut into 1 cm. segments. In order to dissolve the starch, each segment was incut Dow Chemical Co., Midlands, Michigan. 1 Branson Sonifier, Heat Systems, Great Neck, New York. AMERICAN
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Thrornbocytopenia
Due
bated for 12 hours at room temperature with 2 ml. of a saturated solution of Cotazym@* digestant containing amylase equivalent to at least 1.5 gm. of pancreatin N.F. per capsule. This incubation produced a thick solution which was then extractable by the following method. Extraction and Counting Procedures. Solutions were transferred to 30 ml. conical centrifuge tubes. Isotonic saline solution was used to rinse the original tubes and added to the solutions to bring the total volume to 5 ml. Methylene chloride, 25 ml., was added and mixed thoroughly with the aqueous layer by thirty to fifty inversions. The aqueous layer was discarded. To saponify the fats 2.5 ml. of 0.1 N sodium hydroxide was added; after the inversion, the aqueous layer was again discarded. To neutralize the sodium hydroxide, 2.5 ml. of 0.1 N acetic acid was then added, and after mixing, the aqueous layer was again discarded. A 5 ml. aliquot of the methylene chloride extract containing the recovered digitoxin was placed in a counting vial and evaporated to dryness. The residue was resuspended in 5 ml. of a phosphor-toluene solution (40 ml. Liquiflour@t and 1,000 ml. toluene) and counted for 30 minutes in a Packard liquid scintillation spectrometer. A phosphor-toluene blank gave background counts for each run. RESULTS In Vitro Demonstration of a Digitoxin-Dependent Factor in the Patient’s Blood Which Interacted with Normal Platelets. Clot retraction inhibition: 1 .O ml. of platelet-rich plasma, taken from the patient after recovery from thrombocytopenia, was incubated with 0.02 mg. per ml. digitoxin. Suitable controls were used and attempts to show reactivity with Cedilanid, ouabain and
* Organon
Inc., West Orange, New Jersey. t T. M. Pilot Chemicals, Watertown, Massachusetts.
A FIG. tion. (0.02 only VOL.
41,
B
C
to Digitoxin-Young
607
et al.
FIG. 1. Clot retraction inhibition. A, no inhibition was observed when the patient’s platelet-rich plasma was recalcified in the presence of saline solution. B, inhibition was observed in the presence of digitoxin (0.02 mg. per ml.) and the patient’s platelet-rich plasma. C, there was no inhibition when normal platelet-rich plasma was calcified in the presence of digitoxin (0.02 mg. per ml.).
digoxin were unsuccessful. The results appear in Figures 1 and 2. Marked clot retraction inhibition was observed with the patient’s plasma, platelets and digitoxin, but normal clot retraction was observed with all control mixtures. Complement jxation: Quantitative complement fixation studies using patient’s serum, normal platelets and digitoxin were kindly performed by Dr. Peter Miescher (Department of Medicine, New York University School of Medicine). No complement fixation was demonstrated. Platelet factor 3 activation: On incubation of a suspension of titrated platelet-rich plasma, a progressive shortening of the Stypven time, indicating an increase in platelet factor 3 activity, was observed only in the tube containing digitoxin and patient’s serum. Table II. Platelet factor 3 activation occurred with as little as 0.0004 mg. per ml. (final concentration) of
D
E
2. Incubation of the patient’s platelet-rich plasma with various agents. A, with saline soluB, with Cedilanid (0.02 mg. per ml.). C, with ouabain (0.02 mg. per ml.). D, with digitoxin mg. per ml.). E, with digoxin (0.02 mg. per ml.). Clot retraction inhibition was demonstrated in the presence of digitoxin. OCTOBER
1966
608
Thrombocytopenia
Due to Digitoxin-Young
TABLE II INCREASE IN PLATELET FACTOR 3 PRODUCED BY INCUBATION OF NORMAL PLATELET-RICH PLASMA WITH DIGITOXIN AND PATIENT’S
Incubation
Stypven Time (sec.) Before After Incubation* Incubation
Mixture
Platelet-rich plasma f normal saline solution Platelet-rich plasma + normal digit&n (0.02 mg./ml.) Platelet-rich plasma + normal mercuhydrin (0.12 mg./ml.) Platelet-rich plasma + normal meperidine (0.1 mg./ml.) Platelet-rich plasma i- patient’s saline solution Platelet-rich plasma + patient’s digit&n (0.02 mg./ml.) Platelet-rich plasma i- patient’s mercuhydrin (0.12 mg./ml.) Platelet-rich plasma + patient’s meperidine (0.1 mg./ml.) * Incubation
SERUM
serum + 22
23
serum + 23
18
27
19
serum + serum + 22
23
serum + 24
18
serum + 24
12
serum + 23
21
23
18
serum +
digitoxin. (Table III.) The system was specific for digitoxin. Tests with other cardiac glycosides (digoxin, ouabain and Cedilanid) gave negative results, (Table IV.) Pure crystalline digitoxin gave positive results but a solution of digitoxigenin, the steroid portion of the glycoside, was inactive. (Table v.) After the patient recovered from thrombocytopenia, it was possible to test for sensitivity to digitoxin by adding the drug directly to the patient’s platelet-rich plasma. TABLE Iv OF PLATELET FACTOR 3 INCREASE FOR DIGITOXIN
SPECIFICITY
Incubation Platelet-rich plasma saline solution Platelet-rich plasma digoxin Platelet-rich plasma Cedilanid Platelet-rich plasma ouabain Platelet-rich plasma digitoxin Platelet-rich plasma saline solution Platelet-rich plasma + dig&n Platelet-rich plasma Cedilanid Platelet-rich plasma ouabain _ Platelet-rich plasma
Stypven Time (sec.) Before After Incubation* Incubation
Mixture
TABLE III EFFECT OF DIGITOXIN CONCENTRATION ON PLATELET FACTOR 3 INCREASE PRODUCED BY INCUBATION OF NORMAL PLATELET-RICH PLASMA WITH DRUG AND PATIENT’S SERUM
Incubation
+ normal serum + 38
29
39
28
35
27
37
29
36
28
36
31
Stypven Time (sec.) Before After Incubation* Incubation
Mixture
Platelet-rich plasma + normal saline solution Platelet-rich plasma-t normal digit&n (0.04 mg./ml.) Platelet-rich plasma f normal digitoxin (0.004 mg./ml.) Platelet-rich plasma + normal digit&n $ (0.0004 mg./ml.) Platelet-rich plasma + patient’s saline solution Platelet-rich plasma + patient’ digitoxin (0.04 mg./ml.) Platelet-rich plasma -I- patient’s dieitoxin (0.004 me./ml.) Plat&t-rich’plasma 7 patient’s digit&n (0.0004 mg./ml.) * Incubation
was carried out for 2 hours at 37’~.
et al.
serum +
normal
serum +
28
31
25
32
28
35
29
27
23
serum + serum C serum + serum + 27
14
29
17
29
15
serum + serum +
was carried out for 2 hours at 37Oc.
The striking increase in platelet factor 3 which resulted from incubation is shown in Figure 3. Generation of amino acid nitrogen: An increase in alpha amino nitrogen after 60 minutes of incubation was observed in the incubation system composed of normal platelets, patient’s serum and digitoxin (Table VI) ; 10.3 fig. of alpha amino nitrogen were generated. In the control mixtures a decrease of alpha amino nitrogen was noted after 60 minutes of incubation. This phenomenon has been observed previously [21]. Increased alpha amino nitrogen was not observed in the incubation system consisting of normal platelets, patient’s serum and digitoxin. There was no change in alpha amino TABLE v FAILURE OF DIGITOXIGENIN TO INCREASE PLATELETFACTOR
+ normal serum + +
30 serum +
Incubation
Mixture
Stypven Time (sec.) Before After Incubation* Incubation
+ normal serum + f
normal serum -I-
+ patient’s serum -I+
patient’s
serum 37
31
35
33
+ patient’s serum f + patient’s serum + 35
29
35
19
+ patient’s serum +
NOTE: Digodn and ouabain were added in ammum of 0.005 mg. per ml., digitoxin and Cedilanid in amounts of 0.004 mg. per ml. *Incubations were carried out for 1 hour at 37Oc.
Platelet-rich plasma saline solution Platelet-rich plasma digit&n Platelet-rich plasma genin Platelet-rich plasma saline solution Platelet-rich plasma digit&n Platelet-rich plasma digitoxigenin
+ normal serum + 44 f
40
normal serum + 47
35
47
36
48
37
44
23
48
35
+ normal + digitoxii- patient’s serum + + patient’s serum+ + patient’s serum +
NOTE: Digitoxin was a pure crystalline preparation with a concea t&ion of 0.0002 mg. per ml. Digitoxigenin concentration was 0.1 mg. per ml. * Incubation was carried out for 1 hour at 37Oc. AMERICAN
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Thrombocytopenia
Due to Digitoxin-Young TABLE
ALPHA
AMINO NITROGEN
Incubation
GENERATION
VI
AFTER INCUBATION OF NORMAL
PLATELETS, PATIENT'S SERUM AND DIGITOXIN
Alpha Amino Nitrogen (pg.) Before Incubationt After Incubation Supernate : Supernate : Total Cells Cells Total
Mixture*
Normal platelets + patient’s serum •l- digitoxin Normal platelets + patient’s serum f saline solution Normal platelets + patient’s serum + digoxin Normal platelet + saline solution f digitoxin Normal platelets + normal serum + digitoxin * Reactants: t Incubation
609
et al.
20.7~7.7
28.4
37.0:1.7
38.7
24.017.7 23.017.7 1.317.7 27.5:7.1
31.7 30.7 9.0 28.6
28.0:1.8 26.5:1.8 8.0:11 25.5:l.l
29.8 28.3 9.0 26.6
.O
Difference Total
f10.3 -
1 X 109 washed platelets, 0.02 mg. per ml. digitoxin, 0.05 mg. per ml. digoxin. 60 minutes at 37"~.
nitrogen when platelets were incubated in digitoxin alone. Studies of the Mechanism of Antibody and Drug Interaction with Platelets Using Tritiated Digitoxin. 0.2 ml. of Adsorption of digitoxin by the platelets: patient’s serum or control serum were incubated with 1.0 ml. of platelet-rich plasma and 0.2 ml. of tritiated digitoxin (20,000 c.p.m.). separated by centrifugation, The platelets, were washed three times with isotonic saline solution in some experiments, but not in others. All platelet buttons were finally resuspended in 1.0 ml. of isotonic saline solution. Both supernatants and platelets were extracted and counted in the manner described. The results are given in Table VII. When exposed to patient’s serum and digitoxin the platelets adsorbed the drug,
but the drug could be washed off by three washings of isotonic saline solution. The experiment was repeated with aliquots of platelets extracted and counted after serial washings. The results shown in Table VIII indicate that digitoxin was very 1ooseIy bound to the platelets and was easily removed by one saline wash. Failure of the tritiated digitoxin to be adsorbed by red blood cells or latex particles: In order to determine the specificity of adsorption of radioactive drug by platelets, red cells from blood compatible with the patient’s serum were substituted for the platelets and a similar experiment was performed. Red blood cells were washed TABLE VII CONCENTRATION OF TRITIATED DIGITOXIN ABOUT PLATELETS IN THEPRESENCE OFPATIENT'S SERUM % Total
Recovered
Counts = Incubation
SECONDS
‘-._
20_
-.
‘.B ‘0C
\,
rD
FIG. 3. Plateletfactor 3 release with patient'splateletpoor plasma and drug. A is normal platelet-rich plasma and saline solution;B, normal platelet-rich plasma and digitoxin (0.02 mg. per ml.); C, patient'splatelet-rich plasma and saline solution; D, patient's platelet-rich plasma and digitoxin (0.02 mg. per ml.).
41,
OCTOBER
1966
from
Counts* platelets
Counts from PIa te1et.Y+ Supernate x 100
Mixture
Platelets + patient’s serum $ tritiated digitmint Unwashed Activity after 3 isotonic saline
13.1 0.3
WaSheS
\,
VOL.
1.9 2.4 0 2.0
Platelets + normal Serum Unwashed Activity after 3 isotonic saline washes Platelet5 + control serum, + tritiated digitoxin Unwashed Activity after 1 isotonic saline wash
*Recovered digitoxin as t Specific mg. (20,000 $ From a the sensitive peni&
4.4 0.4
5.0 1.1
counts = number of beta emissions from the tritiated recorded by a liquid scintillation specoomcter. activity of 36,000 platelets per mg. concentration of 0.0006 c.p.m.). patient given large doses of digit&n of the same order as patient. This control patient did not have tbromhocyto-
_
-
610
Thrombocytopenia
Due
to Digitoxin--Young
et al.
TABLE v n EFFECT
OF SERIAL
TRITlATED
WASHING
DIGITOXIN
Incubation
TABLE
ON CONCENTRATION ABOUT
OF
PLATELETS
PAILURE
RED BLOOD
70 Total Recovered counts*
Mixture
Platelets + patient’s serum + tritiated digitoxin Unwashed 1 saline wash 2 saline washes 3 saline washes Platelets + normal serum + tritiated digitoxin Unwashed 1 saline wash 2 saline washes 3 saline washes
OF TRITIATED
10.2 1.7 1.1 0.5
5.0 0.4 0.4 0.1
IX
DIGITOXIN
TO CONCENTRATE
CELLS Ii-4 THE PRESENCE
Incubation
OF PATIENT’S
ABOUT SERUM
% Total Recovered Counts*
Mixture
Red blood cells + patient’s serum tritiated digitoxin Unwashed 3 saline washes Red blood cells + normal serum tritiated digitoxin Unwashed 3 saline washes
+ 6.4 1.0 + 7.0 0.8
NOTE: Tritiated digitoxin concentration 0.0006 mg. (20,000 c.p.m.) * See Table VII.
* See Table VII.
saline solution and resuspended in five times their original volume of platelet-poor plasma so that the number of red cells would correspond to the normal number of platelets. Then 0.2 ml. of patient’s serum, 1.0 ml. of red cell-rich plasma and 0.2 ml. of tritiated digitoxin were incubated, the red cells were separated and radioactive digitoxin was extracted and counted as in the platelet experiments. The results are given in Table IX. Polystyrene latex particles of the same approximate diameter (2.64 I*) as platelets were substituted in equivalent concentrations in the experiment, The results are shown in Table x. Like red cells, these particles did not adsorb digitoxin in the presence of the patient’s serum. Failure of tritiated digitoxin to be adsorbed by platelets treated with sodium cyanide: Sodium four
times
with
isotonic
cyanide has been shown to inhibit platelet viscous metamorphosis and clot retraction [24]. An attempt was made to determine whether platelets, damaged in this manner, were capable of participating in the reaction leading to the concentration of drug about the cell surface. Platelets were incubated for 60 minutes at 37 ‘c. with one-tenth volume of sodium cyanide (O.OlM). After three saline washes, these platelets were resuspended in platelet-poor plasma and were incubated in a mixture of the patient’s serum and tritiated digitoxin. The results are shown in Table XI. Platelets exposed to sodium cyanide did not absorb the drug in the presence of the patient’s serum. Binding of tritiated digitoxin by patient’s serum demonstrated by starch gel electrophoresis: This experiment was designed to determine whether TABLE
TABLE FAILURE LATEX
OF TRITIATED PARTICLES
DIGITOXIN
IN THE
Incubation
FAILURE
x TO CONCENTRATE
PRESENCE
OF PATIENT’S
Mixture
Latex particles + patient’s serum + tritiated digitoxin Unwashed 1 saline wash Latex particles + normal serum and tritiated digitoxin Unwashed 1 saline wash * See Table VII.
ABOUT
5.5 0.5
NONVIABLE
XI
DIGITOXIN
PLATELETS OF PATIENT’S
SERUM
To Total Recovered Counts*
6.5 2.4
OF TRITIATED
Incubation
TO CONCENTRATE
ABOUT
IN THE PRESENCE SERUM
% Total Recovered Counts *
Mixture
Platelets (treated with sodium cyanide + patient’s serum + tritiated digitoxin Unwashed 1 saline wash Platelets (treated with sodium cyanide) + normal serum + tritiated digitoxin Unwashed 1 saline wash
3.8 1.0
5.1 0.7
* See Table VII. AMERICAN
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Thrombocytopenia
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TABLE TRITIATED
DIGITOXIN
FRACTION
OF NORMAL
VOL.
41,
OCTOBER
1966
AND
IN GAMMA
PATIENT’S
GLOBULIN
SERUM
TOTotal Recovered Counts* in Gamma Globulin Fraction Patient’s Normal Serum Serum
1 2 3 4
digitoxin was bound to the antibody-containing gamma globulin fraction of the patient’s serum. The patient’s serum (total protein 5.3 gm., albumin 3.3 gm. per cent, globulin 2.0 gm. per cent) was compared in the following experiments with normal serum, with the same total protein and albumin: globulin ratio. Paper electrophoresis of the serum revealed 15 per cent gamma globulin in the normal serum and 14 per cent in the patient’s serum. 0.2 ml. of patient’s serum was incubated with 0.2 ml. of tritiated digitoxin (0.0006 mg., 20,000 c.p.m.). Simultaneous control studies were performed with normal serum and serum from patients given digitoxin. At the end of the 16-hour electrophoresis, the gel was removed and one-fourth of the strip was cut longitudinally and stained with amido black to determine the distances traveled by each of the serum proteins. Serial 1 cm. segments were prepared. Processing, extraction and counting procedures have been described. The results of a typical experiment experiments appear in Figure 4. Repeated showed consistently increased binding of tritiated digitoxin in the gamma globulin fraction of the patient’s serum. (Table XII). Failure of platelet sonicate to bind tritiated digitoxin An attempt was in the presence of patient’s serum: made to determine whether soluble platelet protein would bind digitoxin in the presence of the patient’s serum. Soluble platelet material, 0.1 ml., was incubated with 0.1 ml. of tritiated digitoxin and 0.1 ml. of patient’s serum for 60 minutes at 37”~. Previous electrophoresis of a serum-platelet sonicate mixture indicated that the soluble platelet material
XII
CONCENTRATION
Electrophoresis Run
FIG. 4. Starch gel electrophoresis of tritiated digitoxin and serum. Eighty-four per cent of the recovered radioactivity is represented. The remaining 16 percent was distributed uniformly over the gel without any association to a particular protein fraction.
611
et al.
14 10 14 11
7 4 7 5
* See Table VII.
appeared in a discrete band in the region of the bk;a globulins [25 1. After electrophoresis the gel strip was removed and one-fourth of the strip was cut longitudinally and stained to locate the platelet sonicate. A sharp band in the beta globulin region which was not present in normal serum was observed. This was completely contained within a 1 cm. segment of the starch gel. The gel was processed, extracted and counted as described. The results appear in Figure 5. Platelet sonicate protein in the presence of patient’s serum and digitoxin did not bind the drug to any significant degree. Studies with the serum of a patient requiring high The serum of a patient redoses of digitoxin: 60-
Origin sonicate
fractions
FIG. 5. Starch gel ekctrophoresis of the platelet sonicate protein incubated with tritiated digitoxin and serum. Binding of the tritiated digitoxin to the patient’s gamma globulin was again demonstrated. No specific binding to the platelet sonicate was noted. Eighty-four per cent of the recovered radioactivity is represented. The remaining 16 per cent was distributed uniformIy over the gel without any association to a particular protein fraction.
612
Thrombocytopenia
Due to D&toxin-Young
ceiving doses of digitoxin in the range of those administered to our patient was electrophoresed as already described. Results of a single determination indicated that 18 per cent of the total recovered counts appeared in the gamma globulin fraction of this subject as compared to 7 per cent of the recovered counts in the gamma globulin fraction of normal serum. This patient was not thrombocytopenic. As seen in Table VII, no concentration of digitoxin was demonstrated about the platelets when this ‘serum was incubated with normal platelets and tritiated digitoxin. COMMENTS
The low degree of sensitivity of in vitro systems and low concentrations of antibody appear to be responsible for the difficulty most workers experience in demonstrating platelet antibodies. Serum from the patient described herein was active in the in vitro systems only at the peak of thrombocytopenia. Serum taken one week after’ recovery was inactive or gave inconclusive reresults in most in vitro systems. Attempts to demonstrate clot retraction inhibition by adding the patient’s serum to normal platelet-rich plasma and digitoxin were inconclusive, probably because of the dilution of antibody. Clot retraction inhibition was demonstrated only by using the patient’s own plasma after the platelet count returned to normal. The platelet factor 3 assay described previously by us in cases of quinidine purpura [ZO] proved to be a sensitive means of demonstrating the presence of digitoxin-induced thrombocytopenia. This test gave the earliest indication that the thrombocytopenia was due to digitoxin. Other drugs taken by the patient were negative in the test, as were digoxin, ouabain and Cedilanid, so it was possible to select a safe digitalis preparation for this patient early in his illness. Sensitivity in drug-induced purpuras appears to be highly specific. Quinidine and quinine, optical isomers both known to cause drug purpura, have been shown not to cross-react [6,26], and chlorothiazide has been used with impunity in cases of hydrochlorothiazide purpura 1.271. In this patient digitoxigen (the steroid portion of the glycoside) was shown to be inactive. The generation of amino acid nitrogen in the in vitro system consisting of normal platelets, patient’s serum and digitoxin is further presumptive evidence of a digitoxin antibody resulting in platelet damage. Previous studies [27]
et al.
indicate that a cytotoxic immune reaction involving platelets may induce proteolytic activity which secondarily leads to amino acid generation. The nature and specificity of this proteolytic response is not clear. It is possible that cell damage induced by antigen-antibody reactions might activate latent enzyme systems, perhaps by direct disruption of intracellular lysosomes. The use of tritium-labeled digitoxin offered an opportunity to focus on the relationship between the drug, antibody and platelets. It was shown that digitoxin, in the presence of patient’s serum, concentrated about the surface of the platelet. This association was weak and could easily be removed with a single saline wash. This relatively loose association of drug, antibody and platelets confirms the findings of others [2,7&B]. Shulman [8,9,70,26,28] has suggested that platelets are not primarily involved in the reaction but are secondarily affected by the antigen-antibody complex adsorbing to their surface. The fact that the drug complex was so easily washed off the platelets lends credence to this theory. The reaction of this particular antigen-antibody complex appears to be somewhat specific for platelets. Concentration of drug did not occur about red blood cells or latex particles of platelet size. Sodium cyanide-treated platelets and soluble platelet material in the presence of drug and immune serum did not preferentially bind the drug. This observation suggests that intact viable platelets are necessary for the drug antibody-platelet interaction to occur, and is in contrast to the findings of others who reported complement fixation with platelet fragments and “ghosts” [8]. Shulman has suggested that platelets enter the reaction by virtue of having sites on their membranes with appropriate changts and configurations to permit attachment of antibody-haptene complexes. Such adsorption might be similar to the nonspecific adsorption of various proteins on membranes of platelets and other blood cells. Haptene inhibition of antibody attachment would not be exljected to occur. If this theory is correct, one would expect to find some binding of the drug to the antibody, since this complex would be necessary for nonspecific binding with the platelet. Several investigators have been unable to demonstrate antibody-haptene binding [2,7]. Shulman, using quinidine, was unable to demonstrate any increase in the nonspecific affinity of serum for AMERICAN
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Due
quinidine in the presence of antibody [a]. However, he pointed out that normal serum proteins adsorbed quinidine so strongly that specific binding may have been masked by nonspecific adsorption. Using starch gel electrophoresis with tritiated digitoxin and patient’s serum, it was possible in this case to show increased digitoxin concentration in the gamma globulin fraction of immune serum as compared to control serum. it appears that antibody-haptene Therefore, interaction can and does occur in the absence of platelets. This finding lends additional support to the conceDt that the platelet acts as an innocent bystander in this type of immunologic purpura. A single starch gel electrophoresis of serum from a patient without thrombocytopenia who reauired digitoxin indicated that increased biiding of the drug occurred in the gamma globulin fraction of this patient’s serum as weli. Sufficient information is not available to draw anv conclusions about this isolated finding bu; further work in this area is warranted ti determine whether or not antibodies are responsible for digitoxin resistance. I
SUMMARY
A case of digitoxin-induced thrombocytopenia is reported. Clot retraction inhibition studies using patient’s platelet-rich plasma and digitoxin yielded positive results. Platelet factor 3 assay established the sensitivity of the patient to digitoxin. The sensitivity was specific for digitoxin ; negative results were obtained with diCedilanid and digitoxigenin. goxin, ouabain, Amino acid nitrogen generation was observed when platelets were incubated with the patient’s serum
and
digitoxin
digitoxin.
Studies
demonstrated
using
tritiated
concentration
of
the
d&g
on the surface of the platelet in the presence of the patient’s serum. Negative results were
obtained
with
red
blood
cells,
latex
par-
titles and platelets treated with sodium cyanide. Starch gel electrophoresis demonstrated increased
binding
lin fraction tion
of drug,
induced
of digitoxin
to the gamma
of the patient’s antibody
and
th;ombocvtoDenia
globu-
serum. The interacplatelets
in
drug-
is discussed.
REFERENCES 1. ACKROYD, J. F. The pathogenesis of thrombocytopenic purpura due to hypersensitivity to sedormid. Clin. SC., 7: 249, 1949. VOL.
41,
OCTOBER
1966
to Digitoxin-Young
et al.
613
2. ACKROYD, J. F. The role of complement in sedormid purpura. Clin. SC., 10: 185, 1951. 3. ACKROYD, J. F. The mechanism of the reduction of clot retraction by sedormid in the blood of patients who have recovered from sedormid purpura. CL. SC., 8: 235, 1949. 4. ACKROYD, J. F. The cause of thrombocytopenia in sedormid purpura. Clin. SC., 8: 269, 1949. 5. ACKROYD, J. F. Platelets, agglutinins and lysins in the pathogenesis of the thrombocytopenic purpura with a note on platelet groups. Brit. M. Bull., 11: 28, 1955. 6. BOLTON, F. G. and DAMESHEK, W. Thrombocytopenic purpura due to quinidine. 1. Clinical studies. Blood, 11: 527, 1956. 7. BOLTON, F. G. Thrombocytopenic purpura due to quinidine. II. Serologic mechanisms. Blood, 11: 547, 1956. 8. SHULMAN, N. R. Immunoreactions involving platelets. I. A steric and kinetic model for formation of a complex from a human antibody, quinidine as a haptene, and platelets, and for fixation of complement by the complex. J. Exper. Med., 107: 665, 1958. 9. SHULMAN, N. R. Immunoreactions involving platelets. III. Quantitative aspects of platelet agglutination, inhibition of clot retraction, and other reactions caused by the antibody of quinidine purpura. J. Exper. Med., 107: 697, 1958. 10. SHIJLMAN, N. R. Immunoreactions involving platelets. xv. Studies on the pathogenesis of thrombocytopenia in drug purpura using test doses of quinjdine in sensitized individuals_ their implications in idionathic thrombocvtouenic nuroura. J. E.qm. Med:, 107: 111, 1958.’ * 1 L 11. GRANDJEAN, L. C. A case of purpura haemorrhagica after administration of ouinine with soecific 1 thrombocytolysis demonstrated in vitro. Acta med. scandinau., 131 (supp. 213): 165, 1948. 12. KAHN, H. R. and BROD, K. C. Thrombocytopenia due to stibophen (1 case). Arch. Znt. Med., 108: 496, 1961. 13. BALL, P. Thrombocytopenia and purpura in patients receiving chlorothiazide and hydrochlorothiazide. J.A.M.A., 173: 663, 1960. 14. JAFFE, M. 0. and KIERLAND, R. R. Purpura due to chlorothiazide (Diuril). J.A.M.A., 1968: 2264, 1958. 15. FALCONER,E. H., EPSTEIN,N. N. and MILLS, E. S. Purpura haemorrhagia due to arsphenamine. Arch. Znt. Med., 66: 319, 1940. 16. ACKROYD, J. F. Allergic purpura, including purpura due to foods, drugs and injections. Am. J. Med., 14: 605, 1953. 17. BERGER, H. Thrombocytopenic purpura following use of digitoxin. J.A.M.A., 148: 282, 1952. 18. MIESCHER, P. and RITTER, 0. Purpura thrombopenique par allergie i la digitoxine. Internat. Arch. Allerev. __ 4: 253, 1953. 19. HOROWITZ, H. I:, WILCOX, W. P. and FUJIMOTO, M. Assay of plasma thromboplastin -antecedent (PTA) with artificially depleted normal plasma. Blood, 22: 35, 1963. 20. HOROWITZ, H. I., RAPPAPORT, H. I., YOUNG, R. and FUJIMOTO, M. Change in platelet factor 3 as a means of demonstrating immune reactions in-
614
21.
22.
23.
24.
Thrombocytopenia
Due to Digitoxin-Young
valving platelets: its use as a test for quinidine induced thrombocytopenia. Transfusion, 5: 335, 1936. NACHMAN,R. L. and ENGLE, R. L., JR. Amino acid generation following platelet antibody interaction. VOX Sanguinir, 10: 416, 1965. ROSEN, H. A modified ninhydrin calorimetric analysis for amino acids. Arch. B&hem., 67: 10, 1957. SMITHIES, 0. Zone electrophoresis in starch gels. Group variations in the serum proteins of normal human adults. Biochem. J., 61: 629, 1955. ZUCKER, M. B. and BORRELLI, J. Viscous metamorphosis, clot retraction and other morphologic
25. 26.
27.
28.
et al.
alterations of blood platelets. J. ,4ppl. Physiol., 14: 575, 1959. NACHMAN, R. L. Immunologic studies of platelet protein. Blood, 25: 703, 1965. SHULMAN,N. R. A mechanism of cell destruction in individuals sensitized to foreign antigens and its implications in auto-immunity. Ann. Znt. Med., 60 : 506, 1964. GESINK, M. H. and BRADFORD, H. A. Thrombocytopenic purpura associated with hydrochlorothiazide therapy. J.A.M.A., 172: 556, 1960. SHULMAN,N. R. and BALL, J. E. Mechanism of blood cell destruction in individuals sensitized to foreign antigens. Tr. A. Am. Physicians, 75: 72, 1962.
AMERICAN
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Demonstration of Antibody and Mechanisms of Action ROBERT C. YOUNG, M.D., RALPH L. NACHMAN, M.D. and HERBERT I. HOROWITZ, M.D.
New York, New York preparation, salt restriction and mercurial diuretics. On November 7 the patient was admitted to the New York Hospital for the first time because of progressive dyspnea. Auricular fibrillation was present with a ventricular rate of 100 per minute, and digitoxin was administered. Doses of 0.2 and 0.4 mg. on alternate days were required to control the tachycardia. When he left the hospital on November 17, his condition was improved. The patient entered the hospital on January 7, 1963, for cardiac catheterization which revealed severe mitral insufficiency. During this hospital course he experienced epistaxis and passed guaiacpositive stools. The platelet count was 49,000 per cu. mm. and the bleeding time was greater than 30 minutes. The thrombocytopenia was thought to be secondary to hydrochlorothiazide and this medication was discontinued. The patient was discharged to await readmission for insertion of a mitral valve prosthesis. He continued to take 0.4 mg. of digitoxin per day and noted frequent epistaxis, easy bruisability and tarry stools. During this period his private physician inadvertently gave him a prescription for digoxin, 0.5 mg. per day, in place of digitoxin. The hemorrhagic symptoms disappeared and a platelet count was within normal limits. The patient reentered the New York Hospital on May 2, 1963. The admitting house officer, unaware of the change in digitalis preparations, reinstituted digitoxin, 0.4 mg. per day. The tachycardia with auricular fibrillation proved difficult to control and the dosage of digitoxin was increased to 0.6 mg. per day. Purpura was not present at this time, but the platelet count was 71,000 per cu. mm. and the bleeding time was 15 minutes. A bone marrow aspiration revealed hypercellnlarity with erythroid and myeloid hyperplasia, and the number and maturation of the megakaryocytes were within normal limits. The patient was given Mercuhydrin@, meperidine and ammonium chloride in addition to digitoxin.
HROMBOCYTOPENIA has been reported to complicate the administration of a large number of drugs. In vitro tests have provided supporting evidence of the presence of drug antibody reactions in cases due to Sedormid@ [7-51, quinidine [6-701, quinine [77] and stibophen [72]. A mixture consisting of human platelets, antiserum and drug was essential for the demonstration of the drug-induced antibody in each case. The results of similar in vitro test systems employed in investigating thrombocytopenias presumed to be due to such drugs as chlorothiazides [ 73,741, arsenobenzol compounds [ 751 and sulfonamides [ 761 have been negative. Despite the negative reactions to in vitro tests, the relationship of the drug to thrombocytopenia in such cases has been confirmed in vivo by rechallenging the patient with the suspected drug, with recurrence of thrombocytopenia. In vivo experiments of this type have been used to substantiate the diagnosis in the two previously reported cases of tbrombocytopenia due to digitoxin [ 77,781, The purpose of this paper is to describe a third patient with digitoxin-induced thrombocytopenia. By using a number of in vitro tests, we have demonstrated the presence of antibody which reacted with digitoxin and secondarily resulted in thrombocytopenia.
T
CASE
REPORT
The patient was a fifty-five year old white man with an eight year history of congestive heart failure, presumably secondary to rheumatic heart disease. For the eight years prior to November 7, 1962, the patient was maintained on an unknown digitalis
* From the Department of Medicine, New York Hospital-Cornell Medical Center, New York, New York. This work was supported by U. S. Public Health Service Grant HE-08744 and U.S. Public Health Service Training Grant 2A-5337. Manuscript received November 22, 1965. VOL.
41,
OCTOBER
1966
605
Thrombocytopenia
606
Due to Digitoxin-Young
TABLE I PLATELET
COUNTS
AFTER
WITHDRAWAL
OF DIGITOXIN
Date
Platelet Count (per cu. mm.)
5/4/63 5/10/63* 5/13/63 5/14/63 5/15/63 5/17/63
71,000 84,000 176,000 178,000 226,000 241,000
* Digitoxin
discontinued.
In citro studies (to be described) indicated that digitoxin was responsible for the thrombocytopenia. On May 10, 1963, digitoxin therapy was discontinued and the patient was given 0.5 mg. digoxin per day. The other medication was continued and 100 mg. of prednisone was added. Platelet counts (Table I) returned to normal within three days. On May 23, 1963, the patient underwent open-heart surgery and the damaged mitral valve was replaced with a StarrEdwards ball valve prosthesis. On July 11 he was discharged from the hospital on a regimen of 0.25 mg. digoxin per day and has since remained free of thrombocytopenia. MATERIALS
AND METHODS
Methods for siliconization of glassware, collection and processing of blood specimens for serum and for platelet-rich plasma have been described previously [ 791. Platelet Factor 3 Test. The principles and technics used in this test have been described elsewhere [ZO]. In brief, the test consists of incubation of normal intact platelet-rich plasma, inactivated serum or plasma-containing antibody, and drug in appropriate concentrations at 37’~. for 1 and 2 hours. Samples of the incubation mixture are taken at intervals and tested for platelet factor 3 activity using the Russell’s viper venom (Stypven@) time test. Control incubation mixtures lacking platelets, drug or antibody are sampled and tested at the same time. The results are considered to be positive when the Stypven time of the test incubation mixtures is reproducibly 10 seconds or more shorter than for the control mixtures. Stypven Time Test. To 0.05 ml. of test material was added 0.05 ml. of normal platelet-poor plasma and 1.0 ml. of 1 : 100,000 dilution of Stypven* in 0.025 M calcium chloride; the time for clot formation was determined. Clot Retraction Inhibition. These studies were carried out as previously described [ZO]. Amino Acid Generation. Platelets participating in certain in vitro immune reactions undergo changes in amino acid composition which result in an increase in * Burroughs Welcome
Co., Tuckahoe,
New York.
et al.
total free amino acids present in the incubation mixture [27]. This phenomenon was employed to demonstrate antibody to digitoxin in the patient’s serum. The method previously described [27] was modified as follows: Normal human platelets were separated from ethylenediaminetetracetic acid whole blood, washed and suspended in Verona1 buffer. A suspension of 1 X log platelets was incubated with barium sulfate-adsorbed serum from the patient in the presence of 0.2 mg. per ml. of digitoxin. Controls included incubation of (1) platelets in adsorbed normal serum, (2) platelets in digitoxin alone, (3) platelets in normal serum plus digitoxin and (4) platelets in patient’s serum plus digoxin. After 1 hour of incubation the alpha amino nitrogen [ZZ] in the system was compared to the amount present before incubation. Digitalis Preparations. Commercial preparations of digitoxin for intravenous use were employed. The drug was dissolved in varying concentrations of alcohol; further dilutions were made in normal saline solution. Preparations of digoxin, ouabain and Cedilanid@ were handled in a similar way. Pure crystalline digitoxin, digoxin, and their glycosides were kindly supplied by Dr. Daniel Lucas (Department of Medicine, Cornell Medical School). They were weighed, taken up in small amounts of absolute ethanol and further dissolved in normal saline solution to give a concentration of less than 1 per cent ethanol in the final test mixtures. Tritium-labeled digitoxin, dissolved in absolute ethanol with a specific activity of lo6 c.p.m. per ml., was made available by Dr. Lucas, and was diluted 1 : 100 to provide 104 c.p.m. per ml. and less than 1 per cent ethanol in the final text mixtures. Latex Particles. A 0.52 weight per volume suspension of polystyrene latex particles,-/ 2.64 =t 0.06 p in diameter, in platelet-poor plasma produced a suspension containing approximately 600,000 particles per cu. mm., with a diameter approximating that of normal platelets. Platelet Sonicate. Washed platelets from 120 cc. of blood from a patient with polycythemia vera were resuspended in 5 cc. of Verona1 buffer. The final suspension was solubilized by sonification$ 30 seconds at full amplitude and was concentrated to 1 ml. by ultrafiltration. This was used as a source of soluble platelet protein. Starch Gel Electrophoresis. Horizontal electrophoresis was performed by the method of Smithies 1231. Materials to be electrophoresed, in 0.4 ml. volumes, were made into a paste with dry, hydrolyzed starch, and introduced into a transverse cut into the gel. Electrophoresis was carried out at 10”~. for 16 hours at a pH of 8.6 and constant voltage of 5 volts per cm. The starch gel strips were cut into 1 cm. segments. In order to dissolve the starch, each segment was incut Dow Chemical Co., Midlands, Michigan. 1 Branson Sonifier, Heat Systems, Great Neck, New York. AMERICAN
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bated for 12 hours at room temperature with 2 ml. of a saturated solution of Cotazym@* digestant containing amylase equivalent to at least 1.5 gm. of pancreatin N.F. per capsule. This incubation produced a thick solution which was then extractable by the following method. Extraction and Counting Procedures. Solutions were transferred to 30 ml. conical centrifuge tubes. Isotonic saline solution was used to rinse the original tubes and added to the solutions to bring the total volume to 5 ml. Methylene chloride, 25 ml., was added and mixed thoroughly with the aqueous layer by thirty to fifty inversions. The aqueous layer was discarded. To saponify the fats 2.5 ml. of 0.1 N sodium hydroxide was added; after the inversion, the aqueous layer was again discarded. To neutralize the sodium hydroxide, 2.5 ml. of 0.1 N acetic acid was then added, and after mixing, the aqueous layer was again discarded. A 5 ml. aliquot of the methylene chloride extract containing the recovered digitoxin was placed in a counting vial and evaporated to dryness. The residue was resuspended in 5 ml. of a phosphor-toluene solution (40 ml. Liquiflour@t and 1,000 ml. toluene) and counted for 30 minutes in a Packard liquid scintillation spectrometer. A phosphor-toluene blank gave background counts for each run. RESULTS In Vitro Demonstration of a Digitoxin-Dependent Factor in the Patient’s Blood Which Interacted with Normal Platelets. Clot retraction inhibition: 1 .O ml. of platelet-rich plasma, taken from the patient after recovery from thrombocytopenia, was incubated with 0.02 mg. per ml. digitoxin. Suitable controls were used and attempts to show reactivity with Cedilanid, ouabain and
* Organon
Inc., West Orange, New Jersey. t T. M. Pilot Chemicals, Watertown, Massachusetts.
A FIG. tion. (0.02 only VOL.
41,
B
C
to Digitoxin-Young
607
et al.
FIG. 1. Clot retraction inhibition. A, no inhibition was observed when the patient’s platelet-rich plasma was recalcified in the presence of saline solution. B, inhibition was observed in the presence of digitoxin (0.02 mg. per ml.) and the patient’s platelet-rich plasma. C, there was no inhibition when normal platelet-rich plasma was calcified in the presence of digitoxin (0.02 mg. per ml.).
digoxin were unsuccessful. The results appear in Figures 1 and 2. Marked clot retraction inhibition was observed with the patient’s plasma, platelets and digitoxin, but normal clot retraction was observed with all control mixtures. Complement jxation: Quantitative complement fixation studies using patient’s serum, normal platelets and digitoxin were kindly performed by Dr. Peter Miescher (Department of Medicine, New York University School of Medicine). No complement fixation was demonstrated. Platelet factor 3 activation: On incubation of a suspension of titrated platelet-rich plasma, a progressive shortening of the Stypven time, indicating an increase in platelet factor 3 activity, was observed only in the tube containing digitoxin and patient’s serum. Table II. Platelet factor 3 activation occurred with as little as 0.0004 mg. per ml. (final concentration) of
D
E
2. Incubation of the patient’s platelet-rich plasma with various agents. A, with saline soluB, with Cedilanid (0.02 mg. per ml.). C, with ouabain (0.02 mg. per ml.). D, with digitoxin mg. per ml.). E, with digoxin (0.02 mg. per ml.). Clot retraction inhibition was demonstrated in the presence of digitoxin. OCTOBER
1966
608
Thrombocytopenia
Due to Digitoxin-Young
TABLE II INCREASE IN PLATELET FACTOR 3 PRODUCED BY INCUBATION OF NORMAL PLATELET-RICH PLASMA WITH DIGITOXIN AND PATIENT’S
Incubation
Stypven Time (sec.) Before After Incubation* Incubation
Mixture
Platelet-rich plasma f normal saline solution Platelet-rich plasma + normal digit&n (0.02 mg./ml.) Platelet-rich plasma + normal mercuhydrin (0.12 mg./ml.) Platelet-rich plasma + normal meperidine (0.1 mg./ml.) Platelet-rich plasma i- patient’s saline solution Platelet-rich plasma + patient’s digit&n (0.02 mg./ml.) Platelet-rich plasma i- patient’s mercuhydrin (0.12 mg./ml.) Platelet-rich plasma + patient’s meperidine (0.1 mg./ml.) * Incubation
SERUM
serum + 22
23
serum + 23
18
27
19
serum + serum + 22
23
serum + 24
18
serum + 24
12
serum + 23
21
23
18
serum +
digitoxin. (Table III.) The system was specific for digitoxin. Tests with other cardiac glycosides (digoxin, ouabain and Cedilanid) gave negative results, (Table IV.) Pure crystalline digitoxin gave positive results but a solution of digitoxigenin, the steroid portion of the glycoside, was inactive. (Table v.) After the patient recovered from thrombocytopenia, it was possible to test for sensitivity to digitoxin by adding the drug directly to the patient’s platelet-rich plasma. TABLE Iv OF PLATELET FACTOR 3 INCREASE FOR DIGITOXIN
SPECIFICITY
Incubation Platelet-rich plasma saline solution Platelet-rich plasma digoxin Platelet-rich plasma Cedilanid Platelet-rich plasma ouabain Platelet-rich plasma digitoxin Platelet-rich plasma saline solution Platelet-rich plasma + dig&n Platelet-rich plasma Cedilanid Platelet-rich plasma ouabain _ Platelet-rich plasma
Stypven Time (sec.) Before After Incubation* Incubation
Mixture
TABLE III EFFECT OF DIGITOXIN CONCENTRATION ON PLATELET FACTOR 3 INCREASE PRODUCED BY INCUBATION OF NORMAL PLATELET-RICH PLASMA WITH DRUG AND PATIENT’S SERUM
Incubation
+ normal serum + 38
29
39
28
35
27
37
29
36
28
36
31
Stypven Time (sec.) Before After Incubation* Incubation
Mixture
Platelet-rich plasma + normal saline solution Platelet-rich plasma-t normal digit&n (0.04 mg./ml.) Platelet-rich plasma f normal digitoxin (0.004 mg./ml.) Platelet-rich plasma + normal digit&n $ (0.0004 mg./ml.) Platelet-rich plasma + patient’s saline solution Platelet-rich plasma + patient’ digitoxin (0.04 mg./ml.) Platelet-rich plasma -I- patient’s dieitoxin (0.004 me./ml.) Plat&t-rich’plasma 7 patient’s digit&n (0.0004 mg./ml.) * Incubation
was carried out for 2 hours at 37’~.
et al.
serum +
normal
serum +
28
31
25
32
28
35
29
27
23
serum + serum C serum + serum + 27
14
29
17
29
15
serum + serum +
was carried out for 2 hours at 37Oc.
The striking increase in platelet factor 3 which resulted from incubation is shown in Figure 3. Generation of amino acid nitrogen: An increase in alpha amino nitrogen after 60 minutes of incubation was observed in the incubation system composed of normal platelets, patient’s serum and digitoxin (Table VI) ; 10.3 fig. of alpha amino nitrogen were generated. In the control mixtures a decrease of alpha amino nitrogen was noted after 60 minutes of incubation. This phenomenon has been observed previously [21]. Increased alpha amino nitrogen was not observed in the incubation system consisting of normal platelets, patient’s serum and digitoxin. There was no change in alpha amino TABLE v FAILURE OF DIGITOXIGENIN TO INCREASE PLATELETFACTOR
+ normal serum + +
30 serum +
Incubation
Mixture
Stypven Time (sec.) Before After Incubation* Incubation
+ normal serum + f
normal serum -I-
+ patient’s serum -I+
patient’s
serum 37
31
35
33
+ patient’s serum f + patient’s serum + 35
29
35
19
+ patient’s serum +
NOTE: Digodn and ouabain were added in ammum of 0.005 mg. per ml., digitoxin and Cedilanid in amounts of 0.004 mg. per ml. *Incubations were carried out for 1 hour at 37Oc.
Platelet-rich plasma saline solution Platelet-rich plasma digit&n Platelet-rich plasma genin Platelet-rich plasma saline solution Platelet-rich plasma digit&n Platelet-rich plasma digitoxigenin
+ normal serum + 44 f
40
normal serum + 47
35
47
36
48
37
44
23
48
35
+ normal + digitoxii- patient’s serum + + patient’s serum+ + patient’s serum +
NOTE: Digitoxin was a pure crystalline preparation with a concea t&ion of 0.0002 mg. per ml. Digitoxigenin concentration was 0.1 mg. per ml. * Incubation was carried out for 1 hour at 37Oc. AMERICAN
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Thrombocytopenia
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ALPHA
AMINO NITROGEN
Incubation
GENERATION
VI
AFTER INCUBATION OF NORMAL
PLATELETS, PATIENT'S SERUM AND DIGITOXIN
Alpha Amino Nitrogen (pg.) Before Incubationt After Incubation Supernate : Supernate : Total Cells Cells Total
Mixture*
Normal platelets + patient’s serum •l- digitoxin Normal platelets + patient’s serum f saline solution Normal platelets + patient’s serum + digoxin Normal platelet + saline solution f digitoxin Normal platelets + normal serum + digitoxin * Reactants: t Incubation
609
et al.
20.7~7.7
28.4
37.0:1.7
38.7
24.017.7 23.017.7 1.317.7 27.5:7.1
31.7 30.7 9.0 28.6
28.0:1.8 26.5:1.8 8.0:11 25.5:l.l
29.8 28.3 9.0 26.6
.O
Difference Total
f10.3 -
1 X 109 washed platelets, 0.02 mg. per ml. digitoxin, 0.05 mg. per ml. digoxin. 60 minutes at 37"~.
nitrogen when platelets were incubated in digitoxin alone. Studies of the Mechanism of Antibody and Drug Interaction with Platelets Using Tritiated Digitoxin. 0.2 ml. of Adsorption of digitoxin by the platelets: patient’s serum or control serum were incubated with 1.0 ml. of platelet-rich plasma and 0.2 ml. of tritiated digitoxin (20,000 c.p.m.). separated by centrifugation, The platelets, were washed three times with isotonic saline solution in some experiments, but not in others. All platelet buttons were finally resuspended in 1.0 ml. of isotonic saline solution. Both supernatants and platelets were extracted and counted in the manner described. The results are given in Table VII. When exposed to patient’s serum and digitoxin the platelets adsorbed the drug,
but the drug could be washed off by three washings of isotonic saline solution. The experiment was repeated with aliquots of platelets extracted and counted after serial washings. The results shown in Table VIII indicate that digitoxin was very 1ooseIy bound to the platelets and was easily removed by one saline wash. Failure of the tritiated digitoxin to be adsorbed by red blood cells or latex particles: In order to determine the specificity of adsorption of radioactive drug by platelets, red cells from blood compatible with the patient’s serum were substituted for the platelets and a similar experiment was performed. Red blood cells were washed TABLE VII CONCENTRATION OF TRITIATED DIGITOXIN ABOUT PLATELETS IN THEPRESENCE OFPATIENT'S SERUM % Total
Recovered
Counts = Incubation
SECONDS
‘-._
20_
-.
‘.B ‘0C
\,
rD
FIG. 3. Plateletfactor 3 release with patient'splateletpoor plasma and drug. A is normal platelet-rich plasma and saline solution;B, normal platelet-rich plasma and digitoxin (0.02 mg. per ml.); C, patient'splatelet-rich plasma and saline solution; D, patient's platelet-rich plasma and digitoxin (0.02 mg. per ml.).
41,
OCTOBER
1966
from
Counts* platelets
Counts from PIa te1et.Y+ Supernate x 100
Mixture
Platelets + patient’s serum $ tritiated digitmint Unwashed Activity after 3 isotonic saline
13.1 0.3
WaSheS
\,
VOL.
1.9 2.4 0 2.0
Platelets + normal Serum Unwashed Activity after 3 isotonic saline washes Platelet5 + control serum, + tritiated digitoxin Unwashed Activity after 1 isotonic saline wash
*Recovered digitoxin as t Specific mg. (20,000 $ From a the sensitive peni&
4.4 0.4
5.0 1.1
counts = number of beta emissions from the tritiated recorded by a liquid scintillation specoomcter. activity of 36,000 platelets per mg. concentration of 0.0006 c.p.m.). patient given large doses of digit&n of the same order as patient. This control patient did not have tbromhocyto-
_
-
610
Thrombocytopenia
Due
to Digitoxin--Young
et al.
TABLE v n EFFECT
OF SERIAL
TRITlATED
WASHING
DIGITOXIN
Incubation
TABLE
ON CONCENTRATION ABOUT
OF
PLATELETS
PAILURE
RED BLOOD
70 Total Recovered counts*
Mixture
Platelets + patient’s serum + tritiated digitoxin Unwashed 1 saline wash 2 saline washes 3 saline washes Platelets + normal serum + tritiated digitoxin Unwashed 1 saline wash 2 saline washes 3 saline washes
OF TRITIATED
10.2 1.7 1.1 0.5
5.0 0.4 0.4 0.1
IX
DIGITOXIN
TO CONCENTRATE
CELLS Ii-4 THE PRESENCE
Incubation
OF PATIENT’S
ABOUT SERUM
% Total Recovered Counts*
Mixture
Red blood cells + patient’s serum tritiated digitoxin Unwashed 3 saline washes Red blood cells + normal serum tritiated digitoxin Unwashed 3 saline washes
+ 6.4 1.0 + 7.0 0.8
NOTE: Tritiated digitoxin concentration 0.0006 mg. (20,000 c.p.m.) * See Table VII.
* See Table VII.
saline solution and resuspended in five times their original volume of platelet-poor plasma so that the number of red cells would correspond to the normal number of platelets. Then 0.2 ml. of patient’s serum, 1.0 ml. of red cell-rich plasma and 0.2 ml. of tritiated digitoxin were incubated, the red cells were separated and radioactive digitoxin was extracted and counted as in the platelet experiments. The results are given in Table IX. Polystyrene latex particles of the same approximate diameter (2.64 I*) as platelets were substituted in equivalent concentrations in the experiment, The results are shown in Table x. Like red cells, these particles did not adsorb digitoxin in the presence of the patient’s serum. Failure of tritiated digitoxin to be adsorbed by platelets treated with sodium cyanide: Sodium four
times
with
isotonic
cyanide has been shown to inhibit platelet viscous metamorphosis and clot retraction [24]. An attempt was made to determine whether platelets, damaged in this manner, were capable of participating in the reaction leading to the concentration of drug about the cell surface. Platelets were incubated for 60 minutes at 37 ‘c. with one-tenth volume of sodium cyanide (O.OlM). After three saline washes, these platelets were resuspended in platelet-poor plasma and were incubated in a mixture of the patient’s serum and tritiated digitoxin. The results are shown in Table XI. Platelets exposed to sodium cyanide did not absorb the drug in the presence of the patient’s serum. Binding of tritiated digitoxin by patient’s serum demonstrated by starch gel electrophoresis: This experiment was designed to determine whether TABLE
TABLE FAILURE LATEX
OF TRITIATED PARTICLES
DIGITOXIN
IN THE
Incubation
FAILURE
x TO CONCENTRATE
PRESENCE
OF PATIENT’S
Mixture
Latex particles + patient’s serum + tritiated digitoxin Unwashed 1 saline wash Latex particles + normal serum and tritiated digitoxin Unwashed 1 saline wash * See Table VII.
ABOUT
5.5 0.5
NONVIABLE
XI
DIGITOXIN
PLATELETS OF PATIENT’S
SERUM
To Total Recovered Counts*
6.5 2.4
OF TRITIATED
Incubation
TO CONCENTRATE
ABOUT
IN THE PRESENCE SERUM
% Total Recovered Counts *
Mixture
Platelets (treated with sodium cyanide + patient’s serum + tritiated digitoxin Unwashed 1 saline wash Platelets (treated with sodium cyanide) + normal serum + tritiated digitoxin Unwashed 1 saline wash
3.8 1.0
5.1 0.7
* See Table VII. AMERICAN
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Thrombocytopenia
Due
to Digitoxin-Young
TABLE TRITIATED
DIGITOXIN
FRACTION
OF NORMAL
VOL.
41,
OCTOBER
1966
AND
IN GAMMA
PATIENT’S
GLOBULIN
SERUM
TOTotal Recovered Counts* in Gamma Globulin Fraction Patient’s Normal Serum Serum
1 2 3 4
digitoxin was bound to the antibody-containing gamma globulin fraction of the patient’s serum. The patient’s serum (total protein 5.3 gm., albumin 3.3 gm. per cent, globulin 2.0 gm. per cent) was compared in the following experiments with normal serum, with the same total protein and albumin: globulin ratio. Paper electrophoresis of the serum revealed 15 per cent gamma globulin in the normal serum and 14 per cent in the patient’s serum. 0.2 ml. of patient’s serum was incubated with 0.2 ml. of tritiated digitoxin (0.0006 mg., 20,000 c.p.m.). Simultaneous control studies were performed with normal serum and serum from patients given digitoxin. At the end of the 16-hour electrophoresis, the gel was removed and one-fourth of the strip was cut longitudinally and stained with amido black to determine the distances traveled by each of the serum proteins. Serial 1 cm. segments were prepared. Processing, extraction and counting procedures have been described. The results of a typical experiment experiments appear in Figure 4. Repeated showed consistently increased binding of tritiated digitoxin in the gamma globulin fraction of the patient’s serum. (Table XII). Failure of platelet sonicate to bind tritiated digitoxin An attempt was in the presence of patient’s serum: made to determine whether soluble platelet protein would bind digitoxin in the presence of the patient’s serum. Soluble platelet material, 0.1 ml., was incubated with 0.1 ml. of tritiated digitoxin and 0.1 ml. of patient’s serum for 60 minutes at 37”~. Previous electrophoresis of a serum-platelet sonicate mixture indicated that the soluble platelet material
XII
CONCENTRATION
Electrophoresis Run
FIG. 4. Starch gel electrophoresis of tritiated digitoxin and serum. Eighty-four per cent of the recovered radioactivity is represented. The remaining 16 percent was distributed uniformly over the gel without any association to a particular protein fraction.
611
et al.
14 10 14 11
7 4 7 5
* See Table VII.
appeared in a discrete band in the region of the bk;a globulins [25 1. After electrophoresis the gel strip was removed and one-fourth of the strip was cut longitudinally and stained to locate the platelet sonicate. A sharp band in the beta globulin region which was not present in normal serum was observed. This was completely contained within a 1 cm. segment of the starch gel. The gel was processed, extracted and counted as described. The results appear in Figure 5. Platelet sonicate protein in the presence of patient’s serum and digitoxin did not bind the drug to any significant degree. Studies with the serum of a patient requiring high The serum of a patient redoses of digitoxin: 60-
Origin sonicate
fractions
FIG. 5. Starch gel ekctrophoresis of the platelet sonicate protein incubated with tritiated digitoxin and serum. Binding of the tritiated digitoxin to the patient’s gamma globulin was again demonstrated. No specific binding to the platelet sonicate was noted. Eighty-four per cent of the recovered radioactivity is represented. The remaining 16 per cent was distributed uniformIy over the gel without any association to a particular protein fraction.
612
Thrombocytopenia
Due to D&toxin-Young
ceiving doses of digitoxin in the range of those administered to our patient was electrophoresed as already described. Results of a single determination indicated that 18 per cent of the total recovered counts appeared in the gamma globulin fraction of this subject as compared to 7 per cent of the recovered counts in the gamma globulin fraction of normal serum. This patient was not thrombocytopenic. As seen in Table VII, no concentration of digitoxin was demonstrated about the platelets when this ‘serum was incubated with normal platelets and tritiated digitoxin. COMMENTS
The low degree of sensitivity of in vitro systems and low concentrations of antibody appear to be responsible for the difficulty most workers experience in demonstrating platelet antibodies. Serum from the patient described herein was active in the in vitro systems only at the peak of thrombocytopenia. Serum taken one week after’ recovery was inactive or gave inconclusive reresults in most in vitro systems. Attempts to demonstrate clot retraction inhibition by adding the patient’s serum to normal platelet-rich plasma and digitoxin were inconclusive, probably because of the dilution of antibody. Clot retraction inhibition was demonstrated only by using the patient’s own plasma after the platelet count returned to normal. The platelet factor 3 assay described previously by us in cases of quinidine purpura [ZO] proved to be a sensitive means of demonstrating the presence of digitoxin-induced thrombocytopenia. This test gave the earliest indication that the thrombocytopenia was due to digitoxin. Other drugs taken by the patient were negative in the test, as were digoxin, ouabain and Cedilanid, so it was possible to select a safe digitalis preparation for this patient early in his illness. Sensitivity in drug-induced purpuras appears to be highly specific. Quinidine and quinine, optical isomers both known to cause drug purpura, have been shown not to cross-react [6,26], and chlorothiazide has been used with impunity in cases of hydrochlorothiazide purpura 1.271. In this patient digitoxigen (the steroid portion of the glycoside) was shown to be inactive. The generation of amino acid nitrogen in the in vitro system consisting of normal platelets, patient’s serum and digitoxin is further presumptive evidence of a digitoxin antibody resulting in platelet damage. Previous studies [27]
et al.
indicate that a cytotoxic immune reaction involving platelets may induce proteolytic activity which secondarily leads to amino acid generation. The nature and specificity of this proteolytic response is not clear. It is possible that cell damage induced by antigen-antibody reactions might activate latent enzyme systems, perhaps by direct disruption of intracellular lysosomes. The use of tritium-labeled digitoxin offered an opportunity to focus on the relationship between the drug, antibody and platelets. It was shown that digitoxin, in the presence of patient’s serum, concentrated about the surface of the platelet. This association was weak and could easily be removed with a single saline wash. This relatively loose association of drug, antibody and platelets confirms the findings of others [2,7&B]. Shulman [8,9,70,26,28] has suggested that platelets are not primarily involved in the reaction but are secondarily affected by the antigen-antibody complex adsorbing to their surface. The fact that the drug complex was so easily washed off the platelets lends credence to this theory. The reaction of this particular antigen-antibody complex appears to be somewhat specific for platelets. Concentration of drug did not occur about red blood cells or latex particles of platelet size. Sodium cyanide-treated platelets and soluble platelet material in the presence of drug and immune serum did not preferentially bind the drug. This observation suggests that intact viable platelets are necessary for the drug antibody-platelet interaction to occur, and is in contrast to the findings of others who reported complement fixation with platelet fragments and “ghosts” [8]. Shulman has suggested that platelets enter the reaction by virtue of having sites on their membranes with appropriate changts and configurations to permit attachment of antibody-haptene complexes. Such adsorption might be similar to the nonspecific adsorption of various proteins on membranes of platelets and other blood cells. Haptene inhibition of antibody attachment would not be exljected to occur. If this theory is correct, one would expect to find some binding of the drug to the antibody, since this complex would be necessary for nonspecific binding with the platelet. Several investigators have been unable to demonstrate antibody-haptene binding [2,7]. Shulman, using quinidine, was unable to demonstrate any increase in the nonspecific affinity of serum for AMERICAN
JOURNAL
OF
MEDICINE
Thrombocytopenia
Due
quinidine in the presence of antibody [a]. However, he pointed out that normal serum proteins adsorbed quinidine so strongly that specific binding may have been masked by nonspecific adsorption. Using starch gel electrophoresis with tritiated digitoxin and patient’s serum, it was possible in this case to show increased digitoxin concentration in the gamma globulin fraction of immune serum as compared to control serum. it appears that antibody-haptene Therefore, interaction can and does occur in the absence of platelets. This finding lends additional support to the conceDt that the platelet acts as an innocent bystander in this type of immunologic purpura. A single starch gel electrophoresis of serum from a patient without thrombocytopenia who reauired digitoxin indicated that increased biiding of the drug occurred in the gamma globulin fraction of this patient’s serum as weli. Sufficient information is not available to draw anv conclusions about this isolated finding bu; further work in this area is warranted ti determine whether or not antibodies are responsible for digitoxin resistance. I
SUMMARY
A case of digitoxin-induced thrombocytopenia is reported. Clot retraction inhibition studies using patient’s platelet-rich plasma and digitoxin yielded positive results. Platelet factor 3 assay established the sensitivity of the patient to digitoxin. The sensitivity was specific for digitoxin ; negative results were obtained with diCedilanid and digitoxigenin. goxin, ouabain, Amino acid nitrogen generation was observed when platelets were incubated with the patient’s serum
and
digitoxin
digitoxin.
Studies
demonstrated
using
tritiated
concentration
of
the
d&g
on the surface of the platelet in the presence of the patient’s serum. Negative results were
obtained
with
red
blood
cells,
latex
par-
titles and platelets treated with sodium cyanide. Starch gel electrophoresis demonstrated increased
binding
lin fraction tion
of drug,
induced
of digitoxin
to the gamma
of the patient’s antibody
and
th;ombocvtoDenia
globu-
serum. The interacplatelets
in
drug-
is discussed.
REFERENCES 1. ACKROYD, J. F. The pathogenesis of thrombocytopenic purpura due to hypersensitivity to sedormid. Clin. SC., 7: 249, 1949. VOL.
41,
OCTOBER
1966
to Digitoxin-Young
et al.
613
2. ACKROYD, J. F. The role of complement in sedormid purpura. Clin. SC., 10: 185, 1951. 3. ACKROYD, J. F. The mechanism of the reduction of clot retraction by sedormid in the blood of patients who have recovered from sedormid purpura. CL. SC., 8: 235, 1949. 4. ACKROYD, J. F. The cause of thrombocytopenia in sedormid purpura. Clin. SC., 8: 269, 1949. 5. ACKROYD, J. F. Platelets, agglutinins and lysins in the pathogenesis of the thrombocytopenic purpura with a note on platelet groups. Brit. M. Bull., 11: 28, 1955. 6. BOLTON, F. G. and DAMESHEK, W. Thrombocytopenic purpura due to quinidine. 1. Clinical studies. Blood, 11: 527, 1956. 7. BOLTON, F. G. Thrombocytopenic purpura due to quinidine. II. Serologic mechanisms. Blood, 11: 547, 1956. 8. SHULMAN, N. R. Immunoreactions involving platelets. I. A steric and kinetic model for formation of a complex from a human antibody, quinidine as a haptene, and platelets, and for fixation of complement by the complex. J. Exper. Med., 107: 665, 1958. 9. SHULMAN, N. R. Immunoreactions involving platelets. III. Quantitative aspects of platelet agglutination, inhibition of clot retraction, and other reactions caused by the antibody of quinidine purpura. J. Exper. Med., 107: 697, 1958. 10. SHIJLMAN, N. R. Immunoreactions involving platelets. xv. Studies on the pathogenesis of thrombocytopenia in drug purpura using test doses of quinjdine in sensitized individuals_ their implications in idionathic thrombocvtouenic nuroura. J. E.qm. Med:, 107: 111, 1958.’ * 1 L 11. GRANDJEAN, L. C. A case of purpura haemorrhagica after administration of ouinine with soecific 1 thrombocytolysis demonstrated in vitro. Acta med. scandinau., 131 (supp. 213): 165, 1948. 12. KAHN, H. R. and BROD, K. C. Thrombocytopenia due to stibophen (1 case). Arch. Znt. Med., 108: 496, 1961. 13. BALL, P. Thrombocytopenia and purpura in patients receiving chlorothiazide and hydrochlorothiazide. J.A.M.A., 173: 663, 1960. 14. JAFFE, M. 0. and KIERLAND, R. R. Purpura due to chlorothiazide (Diuril). J.A.M.A., 1968: 2264, 1958. 15. FALCONER,E. H., EPSTEIN,N. N. and MILLS, E. S. Purpura haemorrhagia due to arsphenamine. Arch. Znt. Med., 66: 319, 1940. 16. ACKROYD, J. F. Allergic purpura, including purpura due to foods, drugs and injections. Am. J. Med., 14: 605, 1953. 17. BERGER, H. Thrombocytopenic purpura following use of digitoxin. J.A.M.A., 148: 282, 1952. 18. MIESCHER, P. and RITTER, 0. Purpura thrombopenique par allergie i la digitoxine. Internat. Arch. Allerev. __ 4: 253, 1953. 19. HOROWITZ, H. I:, WILCOX, W. P. and FUJIMOTO, M. Assay of plasma thromboplastin -antecedent (PTA) with artificially depleted normal plasma. Blood, 22: 35, 1963. 20. HOROWITZ, H. I., RAPPAPORT, H. I., YOUNG, R. and FUJIMOTO, M. Change in platelet factor 3 as a means of demonstrating immune reactions in-
614
21.
22.
23.
24.
Thrombocytopenia
Due to Digitoxin-Young
valving platelets: its use as a test for quinidine induced thrombocytopenia. Transfusion, 5: 335, 1936. NACHMAN,R. L. and ENGLE, R. L., JR. Amino acid generation following platelet antibody interaction. VOX Sanguinir, 10: 416, 1965. ROSEN, H. A modified ninhydrin calorimetric analysis for amino acids. Arch. B&hem., 67: 10, 1957. SMITHIES, 0. Zone electrophoresis in starch gels. Group variations in the serum proteins of normal human adults. Biochem. J., 61: 629, 1955. ZUCKER, M. B. and BORRELLI, J. Viscous metamorphosis, clot retraction and other morphologic
25. 26.
27.
28.
et al.
alterations of blood platelets. J. ,4ppl. Physiol., 14: 575, 1959. NACHMAN, R. L. Immunologic studies of platelet protein. Blood, 25: 703, 1965. SHULMAN,N. R. A mechanism of cell destruction in individuals sensitized to foreign antigens and its implications in auto-immunity. Ann. Znt. Med., 60 : 506, 1964. GESINK, M. H. and BRADFORD, H. A. Thrombocytopenic purpura associated with hydrochlorothiazide therapy. J.A.M.A., 172: 556, 1960. SHULMAN,N. R. and BALL, J. E. Mechanism of blood cell destruction in individuals sensitized to foreign antigens. Tr. A. Am. Physicians, 75: 72, 1962.
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