Document BR1Qa4J7R2Vyvapkx07m250Kw
Defective
Platelet Function Following
the
Administration
of Penicillin Compounds
By Clarence H. Brown, Ill, Major W. Bradshaw, Ethan A. Natelson, Clarence P. Alfrey, Jr., and Temple W. Williams, Jr.
Platelet function and blood coagulation
were studied in five human volunteers
receiving penicillin-G
in incremental
doses
of 1.2-48 million U/day, in six volunteers
receiving ampicillin in incremental
doses
of 60-300 mg/kg/day
(4-20 g/day), and
in six volunteers
receiving
methicillin
in
incremental
doses of 60-300
mg/kg/day.
Coagulation
tests remained normal in all
17 volunteers.
However,
ADP-induced
platelet aggregation became abnormal in
every subject except one receiving ampi-
cillin and one receiving methicillin.
De-
fective aggregation
occurred with pre-
dictability
with the following
doses:
penicillin-G, 24 million U/day; ampicillin,
300 mg/kg/day;
methicillin,
300 mg/kg/
day. All volunteers given penicillin-G and
all given ampicillin
experienced
dose-
related
prolongation
of bleeding
time
which did not occur with methicillin.
Striking prolongation
of bleeding time
occurred only with penicillin-G
in doses of
48 million U/day.
Other tests of platelet
function including
clot retraction,
platelet
factor 3 availability,
and collagen-induced
or epinephrine-induced
aggregation re-
mained normal during the administration
of these drugs. Measurement
of intracellu-
lar adenine nucleotides revealed that the
ADP and ATP content of platelets was un-
affected. It appears that at least one
mechanism
by which the penicillin
com-
pounds alter platelet behavior is by inter-
fering with activation of these cells by
ADP.
R ECENT
ADDITIONS
to the list of pharmacologic
agents capable of in-
hibiting platelet function ` include the closely related semisynthetic
pen i-
cillins, carbenicillin,2
and ticarcillin.5
Indeed, bleeding ascribed to defective
platelet function has been reported
The most predictable
abnormality
in several patients receiving carbenicillin.2'4 of platelet function in these subjects is a
reduced aggregation
response to adenosine
diphosphate
(ADP).2'4'5 Since the
degree ofderangement
is dose dependent,
it is not surprising
that carbenicillin,
which is usually prescribed
in very large doses of 300 600 mg/kg/day
(20-40
g/day), was the first penicillin compound
to be recognized
as having an effect
on platelets.2
Now it is clear that even penicillin-G
in extremely
large doses
alters platelet function.3'6
Since all pen icillins share the same basic structure,
6-am ino-penicillanic
acid
(differing
only in the radical attached
to the free amino group of this acid
moiety 7), it may be that all penicillin compounds
have the capability
of altering
platelet function but that very high doses must be administered
before the de-
fect becomes apparent.
The present study was carried out to examine this possi-
From the Hematology
and Infectious Disease Sections of the Department
of Medicine. Methodist
Hospital and Baylor College of Medicine, Houston. Tex.
Submitted June 17, 1975; accepted January 12. 1976.
Supported by Grant HL 16938 from the National Heart and Lung Institute and Grant RR 00350
from the General Clinical Research Centers Program of the Division of Research Resources, Na-
tional Institutes of Health, and by Bristol Laboratories,
Division of Bristol-Myers
Co.. Syracuse,
N. Y. During this study, Dr. Brown was an Investigator,
Howard Hughes Medical Institute.
Address for reprint requests: Clarence H. Brown, III, M.D., 85 West Miller St., Suite 403,
Orlando, Fla. 32806.
(c) 1976 by Grune & Stratton, Inc.
Blood, Vol. 47, No. 6 (June), 1976
949
950 BROWN ET AL.
bility. The effects of three commonly
used penicillin
compounds
on platelet
function and blood coagulation
were investigated
using human volunteer
sub-
jects. The penicillins employed were: penicillin-G,
since it is the penicillin most
commonly
prescribed
and is not infrequently
given in very large doses; ampi-
cillin, a widely used broad-spectrum
penicillin; and, methicillin,
a penicillinase-
resistant semisynthetic
penicillin.
Another objective of the study was to investigate
further the mechanism
whereby these drugs alter platelet function.
MATERIALS
AND METHODS
Subjects
For this study, human volunteers
obtained
through the Texas Department
of Corrections
were
admitted to the General Clinical Research Center of The Methodist
Hospital.
The purpose, meth-
ods, and hazards of the study were explained
in detail and presented
in writing to each volunteer.
The option to withdraw
from the study at any time was emphasized.
All subjects signed written,
informed consent forms before receiving any study drug. A detailed investigational
protocol
was
submitted
and approved
by both committees
on research
involving
human beings from the two
institutions
involved in the study. All volunteers
were in good health as judged by physical cx-
amination,
complete
blood counts, blood chemical
studies, urinalysis,
stool examination
for
occult blood, electrocardiography,
and radiographic
examination
of the upper gastrointestinal
tract. Each gave a negative history for bleeding tendency,
peptic ulcer disease, renal disease, or
previous allergic reaction to penicillin.
Drugs other than those under investigation
were not ad-
ministered
to any volunteer
during the study and no aspirin-containing
medications
for the month
prior to the study.
Drug Administration
Following
the establishment
of baseline values for tests of blood coagulation
and platelet func-
tion, five volunteers
received incremental
doses of penicillin-G
intravenously
beginning
with I .2
million U/day5 for 3 days. This period was followed with 6 million U/day for 4 days, 12 million
U/day for 3 days, 24 million U/day for 4 days, and finally 48 million U/day for 3 days. Two
groups with six volunteers
in each received incremental
doses of either ampicillin
or methicillin
intravenously.
These two drugs were given sequentially
as follows: 60 mg/kg/day
for 3 days, 100
mg/kg/day
for 4 days, 200 mg/kg/day
for 3 days, and finally 300 mg/kg/day
for 4 days. Daily
doses of each of the drugs were given in divided doses on a 4-6-hr basis. Otherwise,
there were
no interruptions
in drug administration
for any volunteer
in the study. The duration
of drug
administration
for different doses was chosen for convenience.
Blood Coagulation and Platelet Function Studies
Before, during, and after drug administration
numerous
tests of blood coagulation
and platelet
function were performed.
The methods employed,
previously
described,4
included
platelet count,
template bleeding time, prothrombin
time, partial thromboplastin
time, thrombin
time, plasma
fibrinogen, kaolin-induced
prothrombin platelet
consumption,
thrombin-induced
platelet-rich
factor 3 availability,
and platelet aggregation.
plasma Platelet
clot retraction,
aggregation
was
studied in a Bryston Aggregometer
(Bryston
using collagen (bovine tendon), epinephrine,
Manufacturing,
Ltd., Scarborough,
Ont.,
or ADP as aggregating
agents. All platelet
Canada) aggrega-
tion studies were carried out with a standard
platelet concentration
of 300 x 10 /liter. Aggrega-
tion to ADP and epinephrine
was assessed by determining
the presence or absence of a secondary
wave and by quantification
of primary aggregation.
ADP-induced
primary aggregation
was quanti-
fied by measuring
the light transmission
of platelet-rich
plasma (LTPRP) and platelet-poor
plasma
*ln keeping with international
convention,
doses of penicillin-G
in this report are given in
terms of units and doses of the other penicillin compounds
cilitate a comparison
of the doses given, see Fig. 2.
are given in terms of weight. To fa-
PLATELET FUNCTION
AND PENICILLIN
951
(LTp)
and after completion
of the primary
wave of aggregation
(LT,.) and then applying
the following
equation:
I' aggregation
= (LT,- - LTPRP) #{24(L7}T
- LTpRp).4'5 In all aggre-
gation studies, unless indicated
otherwise,
the final concentrations
of ADP and epinephrine
were
2 zM and 20 MM, respectively.
The final concentration
of collagen
used in aggregation
stud-
ies was the greatest dilution that gave a maximum
aggregation
response
with control
platelets.
Control platelets were obtained, on each day of testing, from a small pool of normal donors with
known aggregation
responses.
Collagen
was kept frozen at -20'C in small quantities
until
thawed for use, at which time serial twofold dilutions were made. All collagen that was thawed
and/or diluted was discarded after a single day's use.
Platelet Adenine Nucleotides
For the purpose ofassessing
the effects of penicillin compounds
on the adenine nucleotide
con-
tent ofplatelets,
the concentrations
of ADP and adenosine
triphosphate
(ATP) in platelets before
and after drug administration
were measured
in three volunteers
receiving
penicillin-G,
two
receiving ampicillin,
and two receiving
methicillin.
For these determinations,
ethanol extracts
ofplatelets
in platelet-rich
plasma were prepared according
to the method of Holmsen
and Weiss.5
ATP and ADP (after its conversion
in the presence of phosphoenolpyruvate
and pyruvate
kinase
to ATP8) in extracts were measured
by a bioluminescence
technique9
utilizing
luciferase
en-
zyme (firefly lantern extract, Sigma FLE-50, Sigma Chemical
Co., St. Louis, Mo.) and a Packard
574 liquid scintillation
spectrometer
(Packard Instrument
Co., Downer's
Grove, Ill.). Results were
expressed in terms ofzmoles/lO'1
platelets. Results were analyzed by the Student's t test.
RESULTS
Coagulation and Platelet Function Studies
Tests of plasma coagulation
including
prothrombin
time, partial thrombo-
plastin time, thrombin
time, and plasma fibrinogen
levels were unaffected
by
the administration
of penicillin-G
in doses as high as 48 million U/day or by
ampicillin
or methicillin
in doses as high as 300 mg/kg/day
(approximately
20g/day).
No significant
changes in platelet count, clot retraction,
kaolin-induced
platelet factor 3 availability,
or platelet-dependent
prothrombin
consumption
were observed
during this study. However,
significant
alterations
in bleeding
time and platelet aggregation
occurred.
Progressive
lengthening
of bleeding time occurred in all subjects receiving
penicillin-G
or ampicillin (Fig. 1). No significant
change in bleeding time oc-
curred with methicillin
administration.
Following
4 days of penicillin-G
at a
dose of 24 million U/day, the mean (SEM)
bleeding time was 6.13 0.82
mm (range = 3.5-7.5 mm), a value significantly
longer than the baseline value
of 3.20 0.46 mm (range = 2.0-5.0 mm). Following
3 days of 48 million
U/day, the bleeding time was 12.25 1.97 mm (range = 5.5-15 mm). Ampi-
cillin doses of 100 mg/kg/day
for 4 days resulted in a significant
prolongation
of bleeding time (4.97 0.64 mm versus 2.96 0.27 mm prior to drug admin-
istration). After 200 mg/kg/day,
the difference
between the treatment
mean and
control mean dropped
to a level not significantly
different from the baseline
value (p = 0.05). This finding was the result of one volunteer
experiencing
a
shortening
of bleeding time from 7.5 to 4.0 mm in spite of an increase in the
dosage of ampicillin.
When the dose was raised to 300 mg/kg/day,
the same
volunteer once again demonstrated
a bleeding time of 7.5 mm and the mean
value for the ampicillin group (6.0 0.46 mm; range 5.0-7.5 mm) was again
significantly
prolonged.
Three days after discontinuation
of penicillin-G
and
952
lb 14 I? 10
PENICILLIN
C
BROWN
ET AL.
005
wu v's-
1.2
-
: :
x 106 u `day
... . . . .
AMP IC ILI IN
. 02 MITHICILI IN
]MJ I day 18
20
DAY Of STUDY
Fig. 1 . Bleeding times ( mean SEM)
(U), ampicillin
(.), and methicillin
(o).
significantly from baseline are shown.
in volunteers The levelsof
receiving incremental
doses of penicillin-G
significance (p values) for data that differ
ampicillin
administration,
bleeding times were beginning
to shorten but were
still significantly
prolonged
(Fig. 1). Further
follow-up
studies were not
performed.
Platelet aggregation
studies revealed defects in the capacity of platelets to
aggregate when ADP was employed as the aggregating
agent but not when col-
lagen or epinephrine
was used. This effect was noted with all three drugs. A
summary of the results of ADP-induced
aggregation
studies is shown in Table
1. A small but significant
decrease in the degree of primary aggregation
ac-
companied
the administration
of pen icillin-G in doses of 24 million U/day or
more. This change was also seen with ampicillin
and methicillin
at a dose level
of 300 mg/kg/day.
All subjects given penicillin-G
eventually
demonstrated
a
loss of secondary
aggregation.
One of the five volunteers
given this drug ex-
hibited a loss of secondary
aggregation
while receiving as little as 1.2 million
U/day, whereas another volunteer in the penicillin-G
group received 48 million
U/day before exhibiting
this manifestation
of altered platelet function.
One
volunteer given 300 mg/kg/day
of ampicillin
and one given the same dose of
methicillin
showed persistent
but reduced secondary
aggregation.
PLATELET FUNCTION AND PENICILLIN
953
Table 1. Summa ry of ADP-induced Platelet A ggregation Studies
Drug and Dose
Degree of Primary Aggregation (Relative
Light Transmission) (mean SEM)
No. of Volunteers Demonstrating Loss of Secondary Aggregation/
No. at Risk'
Penicillin-G 0 1.2 6
12 24 48
(million U/day)
0.56 0.02 0.50 0.04 0.50 0.05 0.57 0.02 0.46 0.03t 0.45 0.03t
0/5 1/5 1/4 0/3 2/3 1/1
Ampicillin 0
60 100 200 300
(mg/kg/day)
0.58 0.02 0.58 0.01 0.56 0.02 0.50 0.03 0.48 0.02t
0/6 2/6 0/4 1/4 2/3
Methicillin (mg/kg/day)
0 60 100 200 300
* Denominator
represents
number
0.57 0.02 0.59 0.01 0.51 0.04 0.54 0.01 0.52 0.01 t
of volunteers
who retained
secondary
0/6 0/6
2/6
3/4
0/it
aggregation
after drug was
administered at the previous dose level, and therefore during administration of the dosage shown.
were at risk to lose secondary aggregation
tSignificantly less than baseline value (p <0.05). The single volunteer who demonstrated a persistence of secondary aggregation at this dose level
did exhibit a reduction in the magnitude
of the secondary wave.
Platelet Adenine Nucleotides
The concentrations
ofATP and ADP in platelets obtained from two subjects
receiving a daily dose of 48 million units of penicillin-G,
from one receiving
a daily dose of 24 million units of penicillin-G,
from two receiving ampicillin
at a dose level of 200 mg/kg/day,
and from two receiving methicillin
at 300
mg/kg/day
did not differ significantly
from values determined
for these volun-
teers during the baseline period (Fig. 2). Untoward
bleeding was not noted in
any ofthe 17 volunteers
included in this study.
DISCUSSION
The penicillin compounds
are among the most widely used drugs in medicine.
It is not uncommon
for patients being treated for severe infection to receive
very large doses of these agents. For example, carbenicillin
must be given in
doses of 20-40 g/day to be maximally
effective in the treatment
of gram-nega-
tive septicemia)#{176} While such therapy is usually tolerated
without significant
toxicity or untoward
effects, some patients receiving carbenicillin
have experi-
enced bleeding.2'4'M'3
Admittedly,
most of these patients have had some degree
of renal insufficiency
which by itself can cause a hemorrhagic
disorder or they
have been receiving other drugs that might have contributed
to a bleeding dis-
954 10 9 8
PENICILLIN AMPICILLIN METHICILLIN
CU #{149} o
AlP ADP(----)
BROWN ET AL.
26
05
E
2
Fig. 2. Concentrations
of
platelet ATP and ADP before and
S. .-_- `..
S - -o - -U
after drug administration
in
three volunteers given penicil-
lin-G (.), two given ampicillin
(#{149a}n),d two given methicillin
(o). Doses of penicillin-G
in mil-
lions of units per day and of
ampicillin
and methicillin
in
mg/kg per day are shown on
the abscissa. Horizontal
lines
represent
the lower limits of
normal (mean - 2 SD) for ATP
9 1/
36
48 siC. u/day{Penicillin
C (-)
and ADP (----)
as de-
termined in 1 8 normal control
0 100 200 300 400 Mg/Kg /day{AmMMenthicillin
subjects.
order. When the carbenicillin
is stopped, however, so does the bleeding)3
Fur-
ther evidence that carbenicillin
can create a bleeding tendency is that untoward
bleeding has been noted in several patients (and human volunteers)
with normal
renal function who have received only carbenicillin
in clinically
applicable
doses.4 Ticarcillin,
an investigational
semisynthetic
penicillin similar in struc-
ture and antibacterial
spectrum
to carbenicillin
but different in its greater bac-
tericidal activity against Pseudomonas,'4
has not produced
any evidence of
bleeding when given in a controlled
study to human volunteers.5
Bleeding does
not occur in spite of clear evidence that, like carbenicillin,
ticarcillin
causes a
dose-related
inhibition
of platelet function.
The absence of any evidence of
bleeding in the ticarcillin
study is explained
by the fact that the largest dose of
ticarcillin employed
was 300 mg/kg/day,
which is below the dose level of car-
benicillin that is associated
with bleeding in patients or volunteers
with normal
renal function.4
As a result of our earlier studies4'5 and those of Cazenave
and co-workers3
who showed that large in vitro concentrations
of penicillin-G
affected platelet
function and of Lacombe and associates6
who noted the effect in vivo, we felt
that the present controlled
studies would help to delineate:
(I) the effects of
other penicillin compounds
on platelets as well as other components
of the
hemostatic
system; (2) the dose level at which such effects occur; and (3) the
mechanism
of effect.
The results indicated that as with carbenicillin4
and ticarcillin,5
platelet func-
tion but not coagulation
was altered by the penicillin compounds
under study
PLATELET FUNCTION
AND PENICILLIN
955
and the character
of the platelet defect was essentially
the same as that pro-
duced by carbenicillin
and ticarcillin. That is, the effect appeared to last for the
life span of the exposed platelets (as evidenced
by persistent
prolongation
of
bleeding time for several days after drug was stopped) and ADP-induced
plate-
let aggregation
was the single most sensitive test for measuring
the effect of
these agents on platelets.
While doses of penicillin-G
as small as 1 .2 million U/day affected the plate-
lets of some volunteers,
significant
prolongation
of bleeding time required doses
of24 million U/day or more. Ampicillin
in doses of 100 mg/kg/day
or more
caused prolongation
of bleeding time but methicillin
even at 300 mg/kg/day
did not. Thus, it would appear that methicillin
exerted less of an effect on plate-
lets than did the other two agents. Aggregation
studies, however, revealed sim-
ilar degrees of effect by methicillin,
ampicillin,
and penicillin-G
in equivalent
doses. It may be that other components
of platelet function such as adhesive-
ness were less affected by methicillin.
Since we did not examine all aspects of
platelet function, this remains an unresolved
point.
Differences
between the results of this study and those in which carbenicillin
and ticarcillin
were used4'5 were that platelet-dependent
prothrombin
con-
sumption
and epinephrine-
and collagen-induced
aggregation
tests remained
normal in volunteers
given penicillin-G,
ampicillin,
or methicillin,
whereas
these tests became abnormal
in most volunteers
receiving comparable
doses of
carbenicillin
or ticarcillin.
We have no explanation
for these disparate
results,
except for the obvious one related to the structural
differences
among these
compounds.
It has been suggested
that the penicillin
compounds
alter platelet behavior
by coating the cell membrane,
thereby interfering
with sites for activation
of the
cell by compounds
such as ADP.3'4 One alternative
explanation
for the loss of
secondary
aggregation
is that the intracellular
content of adenine nucleotides
is in some manner reduced to an extent that the amount of ADP released from
the platelet during the release reaction is insufficient
for sustaining
and aug-
menting aggregation.'5
This alternative
explanation
seems untenable
in view of
the results of the present study that showed that the intracellular
content of
platelet ATP and ADP was not significantly
changed by administration
of these
drugs. While the degree of abnormality
of platelet function observed in the subjects
receiving penicillin-G,
ampicillin,
and methicillin
did not result in an apparent
bleeding tendency, there was sufficient inhibition
in platelet function in several
volunteers
receiving high doses of pen icillin-G to lengthen the bleeding time to
15 mm or greater. Therefore,
like aspirin and other antiaggregating
agents,
penicillin compounds
should be considered
potentially
hazardous
from the
standpoint
of bleeding when given in extremely
high doses to patients with
underlying
disorders
of hemostasis,
especially
those with throm bocytopenia.
Furthermore,
when the penicillins
are given to patients
with compromised
renal function (who are likely to possess platelet function derangements
related
to uremia), careful attention
should be given to the dosage regimen, since in
such patients the blood levels of these drugs can become exceedingly
high,
which could potentiate
the hazard of bleeding due to faulty platelet function.
956 BROWN ET AL.
ACKNOWLEDGMENT
The authors thank P. McPherson
and D. Smith for their technical
assistance,
the nursing
staff of the Clinical Research
Center for assistance
in conducting
the volunteer
studies, Dr. D.
Kirkpatrick,
Director
of Treatment,
Mr. C. Jefferies,
Warden
L. Beaird, and Medical
Officer
0. F. Donnell of the Texas Department
of Corrections
for their cooperation,
and the volunteers
for their participation
in the research program.
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1972, p 199
2. McClure
PD, Casserly
iG, Monsier
C,
et al: Carbenicillin-induced
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1970
3. Cazenave
J-P, Packham
MA, Guccione
MA, et al: Effect of penicillin
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