CONCENTRATION OF SEROTONIN IN SERUM IN
THROMBOCYTOPATHIA, PSEUDOHEMOPHILIA,
AND THROMBOCYTOSIS
MARJORIE B. ZUCKER, P H . D . , AND JENNIE BORRELLI, M.S.
Department of Physiology, New York University College of Dentistry, New York, New York
Serotonin (5-hydroxytryptamine or enteramine), a vasoconstrictor substance
found in high concentration in blood platelets,23 is released from the platelets by
coagulation as well as by mechanical disruption or freezing. 20,3I In experimental
studies on hemostasis in animals, serotonin released from the platelet plug is
responsible for some of the vasoconstriction which aids in arresting bleeding."
The determination of the amount of serotonin in the blood of patients with
hemorrhagic disorders is of great interest, both as an indicator of platelet function and as a means of assessing the importance of serotonin in human hemostasis. A few such studies have been made which demonstrated serotonin
deficiency in the serum of patients with thrombocytopenia 3 ' 7> 13 and thrombocytosis.8 In this paper we are reporting studies on the serotonin concentration
in thrombocytopathia, pseudohemophilia, and thrombocytosis.
METHODS
Samples of serum were sent by cooperating physicians. If transportation took
more than a few hours, they were shipped in the frozen state, and in any case
were kept frozen until tested for serotonin activity. The assay was performed on
the isolated perfused rabbit ear as described previously.20 The degree of vasoconstriction elicited by the patient's serum was compared with that produced by
pooled serum derived from 11 normal persons, 10 of whom were 20 to 40 years of
age and 4 of whom were women. This pooled normal serum was designated as
having 100 per cent serotonin activity and its serotonin concentration determined by comparing its vasoconstrictor activity with that of synthetic serotonin
creatinine sulfate* on a fresh rabbit ear. Evidence that serum vasoconstrictor
activity measures serotonin concentration has been presented previously.20
Pooled normal serum was injected alternately with serum from a patient and
comparisons were made by diluting the stronger sample until it caused a vasoconstriction equal to that elicited by the weaker sample. In practice, both samples were usually diluted and their relative activity determined by the ratio of
their dilutions. In order to show that the ear was adequately responsive to
changes in dilution of the samples, the patient's serum was also often injected
in a dilution greater or less than that which equaled the normal sample.
Received, July 2S, 1955; accepted for publication October 7.
This work was supported by a grant from the American Heart Association.
Dr. Zucker was Associate Professor and Miss Borrelli was Research Assistant. The
present address of both authors is Sloan-Kettering Institute for Cancer Research, 444 East
CSth Street, New York 21, New York.
* Kindly supplied by Dr. M. E. Specter of the Upjohn Company.
13
50
«25
8 (16)§
R. R.
R. R.
R. R.
R. R. .
J. S., 45, F
C. S., 74, M
W. C.,70, Mi
N. A., 22, F
Moderate
Normal
Normal
SlightH
50% large
Normal
Normal
Few granules
Moderately
low
Normal
220
180
Large, bizarre, Normal
coarse granules
0*f
Normal
67
230
Clot
Retraction
Platelet Morphology
* Reported to be normal 6 months before at another hospital.
f Corrected by normal platelets.
| Nephritis, uremia.
§ Value in parentheses for frozen platelet-rich plasma.
UNot corrected by normal plasma.
|| Normal 3 days later.
«25
100
R. H.
J. M., 5, M
100
J. L.
N. P., 16, F
ConcentraPlatelet Count
Contribut- tion of
Patient,
Serotonin
X 10"= per
ing
Age in Years, Sex Physician in Serum,
cu. mm.
Per Cent of
Normal
5
Normal
to
>10
Long
15
>60
Bleeding
Time, in
Minutes
Negative
Usually
negative
Negative
14 s e c t
11 sec.If
Negative
11 sec.fU Negative
Negative
14 sec.
Poor
Negative
Yes
Family History
No bleeding
but
abnormal
morphology
Negative
95%
Prothrombin
Consumption
Positive
Tourniquet
Test
CONCENTRATION OP SEBUM SEROTONIN AND CLINICAL DATA IN 6 PATIENTS WITH THEOMBOCYTOPATHIA
TABLE 1
Mild; tarry
stools
Gastrointestinal; after
tooth extraction
Mild;
occ.
purpura
Mild;
occ.
purpura
Severe; skin,
gums, vagina
Severe; epistaxis, ecchymoses
Severity of
Bleeding;
Location
is
a
a
o
&
W
d
o
tJ^
Jan. 1956
15
SEROTONIN IN SERUM
All tests other than the serotonin assays were performed in the laboratories
of the cooperating physicians, employing generally accepted methods.
RESULTS
The pooled normal sample had a vasoconstrictor activity equivalent to
0.1 ng. per ml. of synthetic serotonin base. This is quite close to the value of
0.12 fig. per ml. reported by Erspamer for 14 persons aged 20 to 25 years. 5
Aliquots of each of the 11 serums which were mixed to form the pooled sample
were assayed separately. Vasoconstrictor activity ranged from 33 to 133 per cent
of the pooled sample, or 0.03 to 0.13 pg. per ml. of serotonin. No correlation with
sex or age was apparent. Because 2 of these normal persons had serotonin concentrations between 33 and 40 per cent of the pooled sample, only samples with
25 per cent or less activity were considered to fall outside of the normal range of
serotonin level.
The data on patients with thrombocytopathia are presented in Table 1. Although
this disorder has been subdivided into several types by Jiirgens9 and his classification adopted by others, 2,12 most of our cases do not fit into his categories.
Thus, patient N. P. has poor clot retraction and a long bleeding time as in
Naegeli's type of thrombocytopathia,' but this patient has morphologically normal platelets which are not found in this syndrome. The findings in patient
C. S. are similar to von Willebrand's disease,9 but patients J. M. and J. S. do
not fall into this group because of the abnormal appearance of their platelets.
It is concluded that thrombocytopathia cannot be rigidly subdivided into types.
It seems preferable, therefore, simply to determine which platelet functions are
defective, as others have done. 1 ' 15
Serum serotonin concentration was normal in a patient (N. P.) who had poor
TABLE 2
CONCENTRATION OF S E R O T O N I N I N S E R U M AND C L I N I C A L D A T A I N 11 P A T I E N T S
WITH PSEUDOHEMOPHILIA
Patient, Age in Years,
Sex
E. K., 18, F
F. H., 52, F
J. H., 35, F
J. C , 56, M
J. W., 17, F
D. Q., 52, F
A. C , 52, F
S. C , 20, F
R. L., 34, M
S. L., 6, F
D. M., 50, M
Contributing
Physician
H. F.
J. L.
M. R.
J. L.
J. L.
J. L.
J. L.
J. L.
J. L.
J. L.
J. F.
Concentration of
Serotonin in
Serum, Per cent
of Normal
18 (100)*
17
150
50
100
150
75
65
133
133
100
Tourniquet Test
Family History
Severity of
Bleeding
Negative
Negative
++
++
++++
++
+
++
+
+
++
++
++
++++
Occ. +
Negative
Negative
Occ. +
+
++
++
++
Negative
+
Negative
+)
+
+
f
:+} '
* Value in parentheses was obtained one month after first determination,
f Patients in each bracket are related to each other.
16
Vol. 26
ZUCKER AND BORRELLI
clot retraction and normal prothrombin consumption, and in 2 patients (J. M.
and J. S.) with normal or moderate clot retraction, impaired prothrombin consumption, and large platelets. Serum serotonin concentration was very low in
2 patients (C. S. and W. C.) whose only abnormality was poor prothrombin consumption, and in one patient (N. A.) who had both poor prothrombin consumption and clot retraction, whose platelets showed an unusual sparsity of granules,
and in whom the bleeding time was normal and the bleeding tendency mild. I t
is evident that in thrombocytopathia the serum serotonin concentration was not
TABLE 3
CONCENTRATION O F S E R O T O N I N I N S E R U M AND C L I N I C A L D A T A IN 9 P A T I E N T S
W I T H THROMBOCYTOSIS
Patient, Age in
Years, Sex
B., 50, F
It. G.,63,M
Primary
Diagnosis
Polycythemia
vera
Gastric
cancer
Possible
myeloid
metaplasia
M.H.,72,M Arteriosclerotic
heart
disease
Chronic
E. H.,t F
myeloid
leukemia
F. P., 55, F Thrombocytosis
J. H., 64, M Gout, polycythemia,
treated
R. C , 60, F Myeloid
metaplasia
Myeloid
M. M.,t F
metaplasia,
splenectomy
E. T.,f F
Contributing
Physician
Concentration of
Serotonin
in Serum,
Per cent
of Normal
Bleeding
Time, in
Minutes
It. It.
>200
5
J. L.
50
(370)*
Norm ill
Normal
Normal
Clot
Retraction
Prothrombin
Consumption
Severity of
Bleeding
.Normal Mild
After minor
surgery,
melena
Moderate,
postoperatively
D. J.
75
Normal
Normal
Normal
R. IL
100
Normal
Normal
Normal
Severe, after
sternal
biopsy
R. H.
33
Normal
Normal
Normal
None
R. R.
50
Normal
Normal
Normal
Normal
None; easy
bruising
Moderate
Normal
Normal
J. L.
5
Normal
(100) %
11. H.
12
D.J.
<6
(dil.)
Long
Severe, from
operative
sites
All of the patients listed in this table had platelet counts over one million.
* Value in parentheses was found !% months after first determination,
t Age unknown.
t Value in parentheses was found 2 months after first determination.
Jan.
1956
SEROTONIN IN SERUM
17
correlated with any conventional test of platelet form or function, nor with the
bleeding tendency.
Eleven patients with pseudohemophilia were studied. The term pseudohemophilia is usually reserved for patients like those presented here (Table 2) who
show normal prothrombin consumption as well as normal platelet count, morphology, clotting time, and retraction but a prolonged bleeding time. Pseudohemophilia has sometimes been regarded as synonymous with von Willebrand's
disease.'1' 6 However, Jurgens and his co-workers10' " have reported thatprothrombin consumption is defective in von Willebrand's disease. This would distinguish it
from pseudohemophilia. Although Biggs and Macfarlane4 doubted the validity
of Jurgens' results, it seems ambiguous to use the term von Willebrand's disease
until the question is settled.
In 2 patients with pseudohemophilia, the serum serotonin level was less than
normal. In one of these (E. K.) it was within the normal range when tested again
a month later. The other patient with a low serum serotonin level (F. H.) was
the only one whose capillary fragility was markedly increased. The patient with
the most severe bleeding tendency (J. H.) had a normal serotonin level.
Serotonin was determined in 9 patients with thrombocytosis whose platelet
counts were over one million; the results and data are presented in Table 3.
Because of the marked increase in platelet count one might expect serum serotonin levels above the upper limit of normal. This was found in only 2 instances
(B. and the second sample from R. G.). Serum serotonin concentration was within
the normal range in 4 patients tested once (E. T., M. H., E. H., and F. P.) and
in 1 of the 2 tests on 2 other patients (R. G. and J. H.). In view of the elevated
platelet count, the serotonin concentration per platelet is presumably low. This
subnormal concentration of platelet serotonin was more dramatically indicated
by the results on patients R. C., M. M., and the first test on J. PL, in all of whom
the serum serotonin level was far below the lower limit of the normal range. The
serum from M. M. caused very slight vasoconstriction (indicative of a very low
serotonin concentration) followed by vasodilation. In order to determine whether
a dilator substance masked serotonin activity in this patient, normal serum was
diluted 1:15 with the patient's serum and assayed. The constriction produced
by the serotonin in the normal serum was not blocked by dilator substance in
the patient's serum.
DISCUSSION
Using the isolated rabbit ear as a means of assaying serotonin activity, we
found that 3 of 6 patients with thrombocytopathia, 2 of 11 patients with pseudohemophilia and 3 of 9 patients with thrombocytosis had serum serotonin concentrations below the normal range. In thrombocytosis, the serotonin concentration
per platelet, calculated from the serum serotonin concentration and the platelet
count, was below normal in all but one patient. In general, low serotonin concentration in serum can be attributed to thrombocytopenia, subnormal liberation
of serotonin from platelets, or subnormal platelet serotonin concentration.
Thrombocytopenia was not present in the patients presented here, and serotonin
18
ZUCKBR AND BOKKELLI
Vol. 26
liberation was presumably normal since thrombin, which releases serotonin from
platelets, 3 ' 21 was formed at a rate sufficient to produce normal clotting times.
Hence, it is likely that the low concentration of serotonin in serum in these patients is the result of a low serotonin concentration in the platelets. This conclusion is supported by the fact that in patient W.C. with thrombocytopathia, subnormal amounts of serotonin were liberated when the platelets were disrupted by
freezing and thawing as well as by clotting. In thrombocytosis, similar evidence
that the platelets are deficient in serotonin was presented by Bigelow,3 using
platelets disrupted by sonic vibrations, and by Weiner and Udenfriend,18 who
employed a chemical method for the determination of serotonin in isolated platelets.17 These experiments on artificially disrupted platelets have the advantage of
determining the serotonin in the platelets directly, but they give no clue to the
normality of serotonin release. When serotonin is determined in serum as in our
experiments, a normal value indicates not only a normal platelet serotonin
concentration, but also normal release of serotonin during clotting. This added
information may bear a significant relationship to abnormal bleeding, since
presumably only serotonin liberated from the platelet plug can cause the vasoconstriction which accompanies hemostasis.19
In normal persons, values for serotonin level in serum show a wide variation—
from 0.03 to 0.13 /ug- per ml., according to our data, and from 0.07 to 0.20 ng. per
ml., according to Erspamer. 5 Serotonin content of platelets also varies—from
0.5 to 1.3 Mg- per 10' platelets according to our data, 20 and from 0.5 to 1.5 Mgper 109 platelets according to Weiner and Udenfriend.18 This variation, and the
variation in the same individual at different times (e.g., E. K., Table 2, and
J. H., Table 3) may be attributed to the capacity of human platelets22 as well
as dog platelets 8 to pick up serotonin from solutions and possibly to acquire
their serotonin as they pass through the blood vessels of the serotonin-rich
gastrointestinal tract. 6 ' 1 6 The finding of elevated blood levels of serotonin in
patients with serotonin-producing carcinoid tumors supports this view.14
I t is likely that the low level of serum serotonin in the patients presented here
indicates an impairment of the ability of platelets to take up serotonin. In thrombocytopathia, this ability had no apparent correlation with conventional tests of
platelet function. In pseudohemophilia, two patients had subnormal concentrations of serotonin in serum. On the basis of this finding, they might more properly
be classified as having thrombocytopathia, although low serotonin was the only
evidence suggestive of disturbed platelet function. In thrombocytosis, the platelet serotonin concentration was low, but clot retraction and prothrombin consumption were normal. Thus, platelet serotonin can vary independently of other
platelet constituents or functions, just as these can be dissociated from each
other.15
Since the thrombocytopathic and pseudohemophilic patients with the most
severe hemorrhagic manifestations had normal levels of serum serotonin, serotonin deficiency cannot be responsible for the bleeding in these patients. When
serotonin deficiency is present, it may contribute to the bleeding tendency. In
thrombocytopathia, the major cause of bleeding can be attributed to malfunc-
Jan.
1956
SEROTONIN' IN SERUM
19
tion of the platelets; the etiology of bleeding in pseudohemophilia remains
obscure. Our observation that most patients with pseudohemophilia have normal
levels of serum serotonin is in accord with Bigelow's report of normal values in
2 patients with pseudohemophilia associated with antihemophilic globulin deficiency.3 In thrombocytosis, Bigelow first reported low values of serum and
platelet serotonin.3 He believed that serotonin deficiency may be at least partially
responsible for the bleeding tendency in this disorder and we concur in this belief.* Evenin patients like E. T. who have normal levels of serum serotonin because
the high platelet count compensates for the low concentration of serotonin per
platelet, a subnormal amount of serotonin must be released from the platelets
which mass at the site of vascular injury. Consequently there may be insufficient
vasoconstriction for adequate hemostasis.
SUMMARY
Subnormal concentration of serotonin was found in the serum of 3 of G patients
with thrombocytopathia, 2 of 11 patients with pseudohemophilia, and 3 of 9
patients with thrombocytosis. The implications of these findings are discussed.
SUMMARIO IN lNTERLINGUA
Esseva constatate hypoconcentrationes de serotonina in le seros de 3 inter 6
patientes con thrombocytopathia, de 2 inter 11 patientes con pseudohemophilia,
e de 3 inter 9 patientes con thrombocytosis. Le iniplicationes de iste datos es
discutite.
Acknowledgments. We wish to express our appreciation to D r . Jessica Lewis, University
of Pittsburgh (formerly at University of N o r t h Carolina); D r . Robert C. H a r t m a n n , V'andcrbilt University (formerly at Johns Hopkins University); D r . Dudley Jackson, Johns
Hopkins University; D r . Martin Rosenthal and D r . Hugh Fudenberg, M t . Sinai Hospital,
New York City; D r . Robert Rosenthal, Beth Israel Hospital, N e w York City; and D r .
Joseph Flynn, University of Missouri (formerly at Columbia College of Physicians and
Surgeons) for their cooperation in the present investigation.
REFERENCES
1. ALEXANDER, B . : Coagulation, hemorrhage and thrombosis. N e w England J . Med.,
252: 432-442, 1955.
2. BASERGA, A., AND DE NICOLA, P . : Le Malattie Emorragiche. Milan: Societa Editrice
Libraria, 1950, pp. 814-823.
3. BIGELOW, F . S.: Serotonin activit}' in blood. Measurements in normal subjects and in
patients with thrombocythemia hemorrhagica and other hemorrhagic s t a t e s . J .
L a b & Clin. Med., 4 3 : 750-773, 1954.
4. B I G G S , R., AND MACFARLANE, R. G.: Human Blood Coagulation and I t s Disorders.
Oxford, E n g l a n d : Blackwell Scientific Publications, 1953, p p . 267-270.
5. ERSPAMER, V . : II sistema cellulare enterocromaffine e l'enteramina (5-idrossitript a m i n a ) . Rendiconti Scient. Farmitalia, 1: 1-193, 1954.
6. E S T I I E N , S., M E D A L , L. S., AND D A M E S H E K , W . : Pseudohemophilia.
Blood, 1: 504-533,
1946.
7. H I R O S E , K . : Relation between the platelet count of human blood and its vasoconstrictor action after clotting. Arch. I n t . Med., 2 1 : 604-612, 1918.
* F u r t h e r evidence for t h e relationship between bleeding and serotonin concentration
is the fact t h a t , according to D r . Lewis, bleeding manifestations had disappeared in patients
R.G. and J . H . when the second, normal or elevated serotonin concentration was measured
(sec Table 3).
20
ZUCKER AND BORRELLI
Vol. 26
8. HUMPHREY, J . H . , AND T O H , C. C : Absorption of serotonin (5-hydroxvtryptamine)
and histamine by dog platelets. J . Physiol., 124: 300-304, 1954.
9. JURGBNS, R . : Die erblichen Thrombopathien. Ergebn. inn. Med. u. Kinderh., 5 3 :
795-826, 1937.
10. J U R G E N S , R . AND F E R L I N ,
11.
12.
13.
14.
15.
16.
A . : Ueber den sog. P r o t h r o m b i n k o n s u m p t i o n s t e s t bei
Hiimophilie (Hiimophilie, Konduktorinnen) und bei konstitutioneller Thrombopathic (v. Willebrand-Jiirgens). Schweiz. med. Wchnschr., 80: 1098-1101, 1950.
JiJRGENS, R., AND FORSIUS, H . : Untersuchungen ilber die konstitutionelle Thrombopathie (v. Willebrand-Jiirgens) auf den Alandsinseln. Schweiz. med. Wchnschr.,
81: 1248-1253, 1951.
QUATTRIN, H . : Les thrombopathies. R6v. hemat., 6 : 101-108, 1951.
R E I D , G.: A preliminary note on t h e relationship of the blood platelets to t h e mechanism of haemostasis. M . J . Australia, 2 : 244-246, 1943.
SJOERDSMA, A., AND U D E N P R I E N D , S.: Studies on indole metabolism in patients with
malignant carcinoid (argenfcaffinoma). J . Clin. Invest., 34: 914-915, 1955.
STEPANINI, M . : Basic mechanisms of hemostasis. Bull. New York Acad. Med., 30:
239-277, 1954.
T o n , C. C . : Release of 5-hvdroxvtryptamine (serotonin) from t h e dog's gastrointestinal tract. J . Physiol., 126: 248-254, 1954.
17. U D E N P R I E N D , S., W E I S S B A C H , H . , AND C L A R K , C. T . : T h e e s t i m a t i o n of
IS.
19.
20.
21.
5-hydroxy-
t r y p t a m i n e (serotonin) in biological tissues. J. Biol. Chem., 215: 337-344, 1955.
W E I N E R , M . (New York, N . Y.), AND UDENFRIEND, S. (Bethesda, M d . ) : Personal
communication.
ZUCKER, M . B . : Platelet agglutination and vasoconstriction as factors in spontaneous
hemostasis in normal, thrombocytopenic, heparinized and hypoprothrombincmic
rats. Am. J . Physiol., 148: 275-2SS, 1947.
ZUCKER, M. B., AND BORRELLI, J . : Quantity, assav and release of serotonin in human
platelets. J . Appl. Physiol., 7 : 425-431, 1955.
ZUCKER, M . B . , AND BORRELLI, J . : Relationships of some blood clotting factors t o
serotonin release from washed platelets. J . Appl. Physiol., 7: 432-442, 1955.
22. ZUCKER, M . B . , AND B O R R E L L I , J . : Unpublished results.
23. ZUCKER, M . B . , F R I E D M A N , B . K . , AND R A P P O R T , M . M . : Identification and q u a n t i t a -
tive determination of serotonin (5-hydroxvtrvptamine) in blood platelets.
Soc. Exper. Biol. & Med., 85: 282-285, 1954.
Proc.