1. Healthcare

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Effect of Albumin on the Inhibition of Platelet Aggregation
by &Lactam Antibiotics
By E.M. Sloand, H.G. Klein, K.B. Pastakia, P. Pierce, and K.N. Prodouz
Platelet aggregation and bleeding time abnormalities are
reported in patients receiving p-lactam antibiotics (pLAs),
although clinical bleeding most frequently occurs in chronically ill, malnourished patients. Although most pLAs bind to
serum albumin, the relative influence of bound versus unbound pLAs on platelet function is unknown. We examined
the effect of pLAs on the aggregation of gel-filtered platelets
from normal subjects and on platelet-rich plasma (PRP) from
hypoalbuminemic patients. Therapeutic concentrations of
five BIASwere added to normal platelets at different albumin
concentrations (1.5 to 4.5 g/dL). Inhibitionof aggregation by
the pLAs was inversely proportional to the albumin concentration, and most antibiotic-treated samples showed more
than 50% inhibition at albumin levels below 2.0 g/dL. When
P
-LACTAM ANTIBIOTICS (PLAs)may contribute to
bleeding episodes in patients by depleting vitamin
K-dependent coagulation factors,’,*interfering with fibrin
polymeri~ation,~
or causing defects in platelet f~nction.~.’
Despite well-documented platelet abnormalities in both
normal and patient groups receiving PLAs,*.~clinical bleeding primarily occurs in chronically ill and/or malnourished
patients. Although platelet defects have been described at
therapeutic concentrations for many
some
antibiotics produce defective platelet function in vitro only
at concentrations greatly in excess of those used therapeutically.3,7&12
PLAs reportedly inhibit platelet function by binding to
the platelet surface, thereby preventing interaction of
platelet agonists with their receptor^.".'^ Bleeding complications in hypoalbuminemic patients receiving PJAs are
often associated with abnormal aggregation activity in vitro,
suggesting that PLAs may exert an effect on platelet
glycoprotein receptors.’’ Although most PLAs bind to
albumin as well as to other plasma proteins,’6 the effect of
albumin concentration on the PLA inhibition of platelet
function has received little attention. We investigated the
PLA effect on platelet aggregation in the presence of
varying albumin concentrations in an effort to explain the
reports of bleeding in hypoalbuminemic patients receiving
PLAs. We selected cephalothin, moxalactam, ceftriaxone,
cefamandole, and penicillin to investigate PLA-mediated
inhibition of platelet aggregation in platelets collected from
From the National Heart, Lung, and Blood Institute, and the
Department of Transfusion Medicine, National Institutes of Health;
the Warren G. Magnuson Clinical Center; the Center for Biologics
Evaluation and Research, Food and Drug Administration, Bethesda,
MD; and the Georgetown UniversityMedical Center, Washington, DC.
Submitted May 22,1991; accepted December IO, 1991.
Address reprint requests to E.M. Sloand, MD, NIH Building 31,
Room 5A21,9000 Roclcville Pike, Bethesda, MD 20892.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
This is a USgovemment work There are no restrictions on its use.
0006-49711921 7908-0019$0.00/0
2022
PRPs from hypoalbuminemic patients were incubated with
cephalothin, aggregation was completely inhibited, in contrast to samples from patients with normal albumin levels,
and this decreased platelet aggregation was partially restored (25% to 75%) by increasing the albumin concentration
above 4.0 g/ dL. Specific binding of [%]-benzylpenicillin to
normal platelets decreased proportionately as the albumin
concentration increased in the range of 1.0 to 5.0 g/dL. The
inhibitory effects of pLAs on platelets in vitro appear to be
influencedby albumin concentration.Plasma albumin concentration may influence bleeding in patients receiving BUS.
This is a US government work. There are no restrictions on
its use.
patients with normal albumin levels and from hypoalbuminemic patients. In addition, we used [35S]-benzylpenicillinto
study PLA binding to platelets and the effect of albumin on
this binding.
METHODS AND MATERIALS
Platelet sources. Platelets were obtained from three groups of
individuals. Group 1 consisted of platelet concentrates from
normal blood donors obtained by automated plateletpheresis
(Fenwal CS-3OOO; Baxter Health Care, Deerfield, IL). Group 2
consisted of platelet-rich plasma (PRP) samples from selected
oncology patients, six with normal serum albumin (range, 3.0 to 4.6
g/dL) and four with low serum albumin levels (range, 2.5 to 2.8
g/dL; mean, 2.6 g/dL), studied to determine the effect of overnight
incubation of platelets with cephalothin (200 kg/mL; Eli Lilly,
Indianapolis, IN). In group 3, PRPs from patients with low levels of
albumin (range, 2.6 to 3.0 g/dL; mean, 2.8 g/dL) receiving PLA
were studied to determine the effects of adding human serum
albumin in vitro, Two patients with uremia and one with cirrhosis
who were receiving cephalothin (1 g six times daily) and one uremic
patient receiving 250 mg of cephalothin orally four times daily were
compared with one cirrhotic and six uremic patients not receiving
PLAs. All patients and normal donors denied ingestion of aspirin,
other nonsteroidal anti-inflammatory agents, or other plateletactive drugs for at least 2 weeks before this study. The hematocrits
and platelet counts were similar in patients used for comparison
(mean hematocrit of 22.5 in hypoalbuminemic patients and a mean
of 20.0 in patients with normal albumin). Venous blood was drawn
with minimal negative pressure from the antecubital vein of the
arm not containing an arteriovenous fistula. Blood was drawn using
the double plastic syringe technique and was immediately transferred into siliconized glass tubes at room temperature, containing
sodium citrate at 3.8% (vollvol) final concentration. PRP was
prepared by centrifuging the citrated blood at 16Og for 15 minutes
at room temperature.
Measurement of the aggregation response of normalplatelets treated
with $ U s . Platelet concentrates obtained from normal individuals were gel-filtered on Sepharose 2B (Pharmacia Fine Chemicals,
Piscataway, NJ), as previously described,” and eluted with 3.8
mmol/L HEPES buffer pH 7.35 (Sigma Chemical Co, St Louis,
MO), containing NaCl 0.14 mol/L, KCI 2.7 mmol/L, dextrose 1.0
g/L, NaH,PO, 3.8 mmol/L. Samples were adjusted to a platelet
count of 2 x 105/pL(Coulter Counter, Model S; Coulter Electronics, Hialeah, FL) and fibrinogen was added (final concentration,
1.5 mg/mL). The albumin level in the gel-filtered platelets,
measured as previously described,18was less than 0.4 g/dL. SamBlood, Vol79, No 8 (April 15), 1992: pp 2022-2027
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2023
ANTIBIOTIC-MEDIATED PLATELET INHIBITION
ples were divided into paired aliquots and albumin levels were
adjusted to different levels (1.5 to 4.5 g/dL) by the addition of fatty
acid-free human albumin (25 g/dL; Sigma) or albumin for human
use (Armour Pharmaceutical, Bluebell, PA). PLAS were added to
one of the paired aliquots (at each albumin level) at concentrations
equivalent to plasma levels achieved therapeutically: cephalothin,
100 pg/mL; moxalactam, 400 pg/mL; cefamandole, 13 pg/mL (Eli
Lilly); ceftriaxone, 250 pg/mL (Roche Pharmaceutical, Nutley,
NJ); and penicillin G 10 pg/mL (Squibb and Sons, Inc, Princeton,
NJ). The interval between gel filtration and addition of PLA never
exceeded 15 minutes.
Platelet function was measured by platelet aggregation 15
minutes after BLA addition. Each sample (0.45 mL) was incubated
at 37°C for 10 minutes and aggregation was induced by adding 50
ILL of 80 pmol/L adenosine diphosphate (ADP; Sigma) and
stirring. The maximum slope of aggregation was determined for
duplicate samples and the results were averaged.” Inhibition for
each albumin level was calculated as the percent of maximum slope
of aggregation of the control sample at the same albumin level, but
without PLAs.
Aggregation response of platelets from hypoalbumrnemic patients.
Thirty milliliters of PRP from nine oncology patients (six with
normal albumin and three hypoalbuminemic) was prepared from
venous blood collected into a solution of acid-citrate dextrose (15%
[vol/vol]) and centrifuged at 160g for 15 minutes at room temperature. Platelet counts ranged from 1.4 to 2.0 x 1@/mL.Cephalothin
(in phosphate-buffered saline [PBS], pH 7.4) was added to a
sample of PRP at a concentration of 200 kg/mL and an equal
volume of PBS was added to a control sample. The PLA-treated
and untreated samples were stored overnight in modified PL 732
platelet storage bags (Fenwal; Baxter Health Care) at 22°C with
constant agitation. Platelet aggregation was measured as described
above. The maximum slope of aggregation in response to 8 Fmol/L
ADP was calculated for samples tested in duplicate.
In separate experiments, PRP from 10 hypoalbuminemic patients (four of whom were receiving cephalothin) was divided into
two aliquots. Fatty acid-free human albumin (25 g/dL in 0.9%
NaCI; Sigma) was added to one aliquot to adjust the albumin
concentration to approximately 4.0 g/dL. An equal volume of 0.9%
NaCl was added to the control aliquots. Platelet aggregation in
response to 0.5 p,mol/L ADP was measured as described above.
Penicillin brnding experiments. A 15-pL suspension of Apiezon
oil A (Biddle Co, Bluebell, PA) and n-butyl phthalate (1:9 vol/vol)
together with 4 p L of [35S]-benzylpenicillin(0.28 to 4.0 nmol/L, 27
to 162 pmol/lO’pplatelets; Du Pont Co,Wilmington, DE) in ethanol
(final concentration, < O S % ) was added to 200 pL samples of
gel-filtered albumin-free platelets obtained from normal individuals?’ Samples were inverted several times, incubated at 37°C for 15
minutes, and then centrifuged at 1,200g for 15 minutes. The
supernatant was removed and the tip of the tube containing the
pellet was excised. The pellet was solubilized in 500 pL of 1%
sodium dodecyl sulfate. Radioactivity was measured after addition
of Aquasol (Du Pont Co). Binding specificity was assessed by
measuring the displacement of [’5S]-benzylpenicillinby 1,000-fold
excess nonradioactive penicillin. Binding experiments were also
performed on three gel-filtered plateletpheresis concentrates at
different albumin levels. All samples were tested in quadruplicate.
RESULTS
Aggregation response of normal platelets treated with PLA.
Aggregation was decreased significantly in gel-filtered platelets treated with any of the five PLAs tested at albumin
concentrations less than 2.5 g/dL in the medium. An
example of the relationship of platelet inhibition to albumin
l O O r
“---f+q
90
80 0
.-C
\
‘t
70.
al
6 60
2
5 50
Albumin Levels (g/dL)
4.0
Fig 1. The albumin concentrations of gel-filtered platelets obtained from a normaldonor were adjusted to levels in the range of 1.5
to 4.5 g/dL by the addition of 25 g/dL human albumin; PES was
added, where appropriate, to maintain a constant volume. The
aggregation responses to 8 pmol/L ADP were determined on duplicate samples for each different albumin level before and 15 minutes
after the addition of five different BLAs: (0-0) cephalothin, 100
pg/mL; (A-A)moxalactam, 400 pg/mL; (0-0) ceftriaxone, 250
pg/mL; (e-+) cefamandole, 13 pg/mL; (0-0) penicillin 0, 10
pg/mL for each albumin level. These responses were compared with
those of platelets at similar albumin levels but without @LA.Inhibition
was calculated as the percent of the maximum slope of aggregation of
the control sample at the same albumin level but without PIA. Similar
results were obtained when the maximum aggregation at 3
minutes was used as an index of aggregation instead of slope of
aggregation.
level is seen in Fig 1 when 8 wmol ADP was used as an
agonist. The mean inhibition of aggregation for cephalothin
in the five different platelet samples was 64% ? 35% for an
albumin of 2 g/dL and 0% for an albumin of 4.0 g/dL. For
most PLAs tested, platelet aggregation was either slightly
affected or similar to the control sample (at the same
albumin concentration but without PLA) when albumin
levels exceeded 3.0 to 3.5 g/dL. Below this level, the
inhibitory effect of PLAs was inversely related to albumin
level in all samples. Similar effects were observed in washed
platelets when 8 Kmol/L ADP or thrombin (0.1 to 1.0
U/mL) were used as agonists and in gel filtered platelets
when collagen and epinephrine were used (Table 1).When
Table 1. Effect of Antibiotics on Platelet Aggregation Response
Agonistldose
Albumin
(%)
Antibiotic
Aggregation Rate
I%control)
Extent
(% control)
~~
Thrombin
0.08 U/mL
0.08 U/mL
0.20U/mL
0.20 U/mL
Co IIagen
5 pmol/L
5 kmol/L
Epinephrine
50 *mol/L
50 p n o l / L
1.8
2.7
1.8
2.7
2.0
3.0
2.0
4.0
Penicillin
4 mg/mL
4 mg/mL
4 mg/mL
4 mg/mL
Cephalothin
100 pg/mL
100 kg/mL
Cephalothin
100 kg/mL
100 bg/mL
0
14
94
0
50
70
82
103
88
100
16
100
35
42
100
100
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2024
SLOAND ET AL
doses of cephalothin, 10-fold in excess of those used
therapeutically, were added to gel filtered platelets, all
samples showed inhibition of function regardless of albumin level (when ADP [8 pmol/L], collagen [200 pg/mL], or
epinephrine were used as agonists).
Aggregation response of platelets from hypoalbuminemic
patients. Complete inhibition of platelet aggregation in
response to 8 pmol/L ADP was achieved when platelets
from four hypoalbuminemic individuals were incubated
overnight with 200 pg/mL cephalothin (Fig 2B). Little or
no inhibition of ADP aggregation (8 pmol/L) occurred in
platelets from six patients with albumin levels greater than
3.0 g/dL (Fig 2A). The inhibition exhibited by the platelets
in hypoalbuminemic plasma was partially reversible (25%
to 75%) when the albumin levels were increased to 4.0 to
4.8 g/dL by the addition of 25 g/dL human albumin.
Representative aggregation curves from a single subject are
shown in Fig 3.
When the albumin concentration of PRP from hypoalbuminemic patients (albumin range, 2.6 to 3.0 g/dL; mean, 2.8
g/dL) receiving cephalothin (1 g every 4 hours) was
increased to 4.0 to 4.5 g/dL by the addition of human
albumin, the platelet aggregation response to 0.5 pmol/L
ADP increased (Fig 4A). The maximum slope of platelet
aggregation increased from a mean of 0.72 to 1.6 in patients
receiving cephalothin when the albumin level was raised
from an average of 2.8 g/dL to 4.2 g/dL. In contrast, the
slope of aggregation decreased in PRP samples from
patients with hypoalbuminemia who were not receiving
PLA (Fig 4B). The maximum slope of aggregation de-
B
A
Hypoalbuminemic
Patients
Patients with
Normal Albumin
25
A
c
a
i
a
10
0
1 0 0 "
Cephalothin
Concentration (pglmll
0
1 0 0 2 0 0 3 0 0
Cephalothin
Concentration (pg/ml)
Fig 2. Samples of PRP from six patients with normal albumin
levels (A) and from four with low levels (B) were split Into two
samples and stored overnight after the addition of either cephalothin
(200 pg/mL) or an equal volume of PBS in PL 732 platelet storage
bags with constant agitation at 22°C. Samples were removed and
platelet aggregation in response to 8 pmol/L ADP was determined in
duplicate samples.
0
1
2
Minutes
3
Fig 3. A sample of PRP from a hypoalbuminemicpatient (albumin,
2.6) was storedovernight with cephalothin (200 pg/mL). The aggregation response to 8 pnol/L ADP was determined before pLA addition
(A), after storage of platelet sample with @LA (B), and after the
albumin was raised to 4.2 g/dL in this stored sample by the addition
of 25 g/dL fatty acid-free albumin (C).
creased from a mean of 1.7 to 1.2 in samples from those
individuals not receiving cephalothin. This latter observation is consistent with previous reports of the effect of
albumin concentration on platelet a g g r e g a t i ~ n . Similar
~'~~~
effects were obtained by using albumin prepared for human
use.
Penicillin binding. The binding of [35S]-labeled benzylpenicillin to albumin-free ( < 0.04 g/dL) gel-filtered platelets is shown on the Scatchard plot in Fig 5. Binding was
specific and reversible, as indicated by displacement of
radiolabeled penicillin by unlabeled penicillin. Saturation
was achieved in albumin-free platelets at 130 pmol/108
platelets. Scatchard plot analysis of these data indicates,
under these conditions, approximately 4,800 binding sites
per platelet with an apparent dissociation constant of 200
nmol/L and an affinity constant of 5 x lo6 L/mol. At a
concentration of 54 pmol of benzylpenicillin/108 platelets,
binding of penicillin to platelets was increased in samples
with lower albumin concentrations, and platelets showed a
decrease in binding with increasing albumin concentrations
(Fig 6). Similar results were obtained when stabilized
albumin prepared for transfusion was substituted for fatty
acid-free albumin. Platelets exposed overnight to radiolabeled penicillin continued to show decreased binding of
penicillin at higher albumin levels. However, platelets
incubated overnight bound less penicillin than those incubated for 15 minutes. This is most likely due to a loss of
platelet integrity that occurs after gel filtration and storage
in a non-gas-permeable container.
DISCUSSION
Bleeding episodes observed in patients who receive PLAs
have been attributed to platelet dysfunction, hypoprothrombinemia, and the inhibition of fibrin polymerization. Factors that have been implicated in the increased risk of
bleeding include thrombocytopenia, uremia, chemother-
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2025
ANTIBIOTIC-MEDIATED PLATELET INHIBITION
A
Cephalothin Treated
Hypoalbuminemic
Patients
3.0
B
Untreated
Hypoalbuminemic
Patients
i
.014
.012
t
\
.OlO[
3.0
.008
-JX+
.
\
U
2.5
2.5
$
2.0
m
2.0
C
7
m
?!
\
,002
m
8 1.5
1.5
c
0
0
100
200
0
300
400
500
600
700
800
900
Bound (fmolesll08 platelets)
0"
1.o
5 1.0
0.5
0.5
u
1 2 3 4 5
Albumin Level (g/dL)
u
1 2 3 4 5
Albumin Level (g/dL)
Fig 4. Platelet aggregation responses to 0.5 pmol/L ADP were
tested using PRP obtained from four hypoalbuminemic patients
(meanalbumin, 2.8 g/dL) receiving cephalothin (1 g every 4 hours) (A)
and six hypoalbuminemic patients receiving no antibiotics (B). Samples were split and the albumin levels of one of the pair were adjusted
to approximately 4 g/dL; a similar volume of PBS was added to the
control sample. Samples of PRP from patients not receiving antibiotics showed decreased aggregation after addition of albumin, a
well-described phenomenon.'9 while the aggregation responseof PRP
from patients receiving PIASincreased.
apy, chronic illness, and malnutrition." The latter two
conditions are often accompanied by hypoalbuminemia.
Although serum albumin concentration influences the action of many drugs in vivo," no relationship between
albumin concentration and @LA-inducedplatelet dysfunction has been reported.
In this study, the degree of inhibition of aggregation of
gel-filtered platelets by @LAswas shown to be inversely
related to albumin levels at concentrations below approximately 3.5 g/dL. This inhibition of function parallels the
increased binding of radiolabeled penicillin to platelets at
declining albumin levels. Platelet aggregation in patients
with low albumin levels was inhibited after an overnight
incubation with cephalothin (200 pg/mL), while platelets
from patients with normal albumin levels exhibited no such
inhibition. This inhibition of @LA-treated platelets was
partially reversible after albumin levels were returned to
normal by the addition of exogenous albumin.
In this study, platelets bound to the antibiotic in a specific
manner and the amount of binding was inversely related to
the albumin concentration. The protective effect of albumin is most likely a function of the avid binding of
antibiotics to
and the consequent depletion of
free PLA available to the platelets. The binding of albumin
Fig 5. The binding of [a6S]-benzylpenicillinto samples of gelfiltered, albumin-free platelets from two donors was determined as
previously described for concentrations of benzylpenicillin of 27 pmol
through 162 pmol/ lo" platelets. Saturation was previously determined to have been achieved a t 130 pmol/lo" platelets. A Scatchard
analysis is presented. Nonspecific binding was determined by the
addition of 1,000-fold excess unlabeled benzylpenicillin. A total of
4,800 sites per platelet were seen with an apparent dissociation
constant of 200 nmol/L and an affinity constant of 5 x lO'L/mol.
to the antibiotic is reversible and drug molecules are in
constant equilibrium between the bound and unbound
~ t a t e . ~For
' penicillin, one molecule of drug binds to one
molecule of albumin.%When concentrations of drug exceed
the amounts that can be bound to albumin, or when
concentrations of albumin decline, the levels of free unbound drug increase. Although it is clear that antibiotic
bound to albumin has no antimicrobial a~tivity?~*~'
the
relationship of protein binding to other drug-related actions has not been explored. All PLAs used in this study
v)
8E
0.4
0.2
0
0
1
2
3
4
5
6
Albumin (g/dL)
Fig 6. The effect of albumin concentration on the binding of
["SI-benzylpenicillin was measured for three different gel-filtered
plateletpheresis concentrates. Binding of benzylpenicillin at each
albumin level was measured in quadruplicate for each sample using a
concentration of 54 pmol of benzylpenicillin/lV platelets. Results
were averaged and plotted using linear regression analysis. The error
bars representthe standard error of the mean.
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2026
SLOAND ET AL
exhibit significant binding to p r ~ t e i n ,and
~ ~ the
. ~ ~inhibition
of platelet function by each PLA was blocked by albumin in
a similar manner.
It is likely that PLAs exert their inhibitory effect by
interfering with agonist interaction with platelet surface
receptor^.'^^'^ This mechanism is supported by evidence that
binding of an a-adrenergic antagonist, (3H)dihydroergocryptine, to platelet adrenergic receptors is reduced twofold,
and (I4C)serotonin release is completely blocked after
incubation of platelets with PLAs.I3 Evidence supporting
reversible PLA binding to the platelet surface can be found
in reports that platelet function returns to normal after
exposed platelets are resuspended in PLA-free
In our storage studies, in which platelets were stored under
standard blood banking conditions, but in the presence of a
therapeutic concentration of cephalothin, antibiotic-mediated platelet dysfunction was at least partially reversed by
the addition of albumin. However, irreversible binding of
radiolabeled penicillin to platelets and irreversible inhibition of platelet function after prolonged exposure to very
high doses of penicillin (10 to 20 mmol/L) have been
reported.” Using similar conditions, we found that 24 hours
of exposure to a high dose of penicillin G (5.4 mmol/L) in
the presence of lowered albumin levels (1.27 to 1.45 g/dL;
average, 1.36 g/L) impaired the aggregation response of
platelets to thrombin even after removal of the antibiotic by
washing, but no structural changes were observed in glycoprotein Ib or IIb by polyacrylamide gel electrophoresis and
immunoblotting (data not shown). This finding is consistent
with the report that in rat platelets treated with a variety of
P U S , the loss of platelet function does not correlate with
structural changes in surface glycoprotein^.^' This irreversible inhibition of platelet function seen at higher doses of
penicillin probably is caused by a different mechanism of
platelet membrane damage from the one responsible for
the reversible inhibition seen at lower doses.
Our observation that inhibition of platelet aggregation by
PLAs is inversely related to the albumin level suggests that
albumin-bound antibiotic is unable to bind to platelets, or
does so with less affinity than does unbound antibiotic.
While our experiments do not directly address PLAinduced bleeding, we suspect that the increased frequency
of bleeding reported in individuals with chronic illness who
are treated with PLAs may be related to low albumin
concentration. Our observations may further explain why
PLA administration to normal subjects is rarely associated
with platelet dysfunction or bleeding.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1992 79: 2022-2027
Effect of albumin on the inhibition of platelet aggregation by betalactam antibiotics
EM Sloand, HG Klein, KB Pastakia, P Pierce and KN Prodouz
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