From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
The Platelet Function Defect of Cardiopulmonary Bypass
By Anita S . Kestin, C. Robert Valeri, Shukri F. Khuri, Joseph Loscalzo, Patricia A . Ellis, Hollace MacGregor,
Vladimir Birjiniuk, Helene Ouimet, Boris Pasche, Martha J . Nelson, Stephen E. Benoit,
Louis J. Rodino, Marc R. Barnard, and Alan D. Michelson
The use of cardiopulmonary bypass (CPB) during cardiac
surgery is associated with a hemostatic defect, the hallmark of which is a markedly prolonged bleeding time. However, the nature of the putative platelet function defect is
controversial. In this study, blood was analyzed at 10 time
points before, during, and after CPB. We used a wholeblood flow cytometric assay to study platelet surface glycoproteins in (1) peripheral blood, (2)peripheral blood activated in vitro by either phorbol myristate acetate, the
thromboxane (TX)A, analog U46619,or a combination of
adenosine diphosphate and epinephrine, and (3)the blood
emerging from a bleeding-time wound (shed blood). Activation-dependent changes were detected by monoclonal
antibodies directed against the glycoprotein (GP)lb-IX and
GPllb-llla complexes and P-selectin. In addition, w e measured plasma glycocalicin (a proteolytic fragment of GPlb)
and shed-blood TXB, (a stable breakdown product of
TXA,). In shed blood emerging from a bleeding-time
wound, the usual time-dependent increase in platelet surface P-selectin was absent during CPB, but returned t o
normal within 2 hours. This abnormality paralleled both the
CPB-induced prolongation of the bleeding time and a CPBinduced marked reduction in shed-blood TXB, generation.
In contrast, there was no loss of platelet reactivity t o in
vitro agonists during or after CPB. In peripheral blood,
platelet surface P-selectin was negligible at every time
point, demonstrating that CPB resulted in a minimal number of circulating degranulated platelets. CPB did not
change the platelet surface expression of GPlb in peripheral blood, as determined by the platelet binding of a panel
of monoclonal antibodies, ristocetin-induced binding of von
Willebrand factor, and a lack of increase in plasma glycocalicin. CPB did not change the platelet surface expression
of the GPllb-llla complex in peripheral blood, as determined by the platelet binding of fibrinogen and a panel of
monoclonal antibodies. In summary, CPB resulted in (1)
markedly deficient platelet reactivity in response t o an in
vivo wound, (2) normal platelet reactivity in vitro, (3)no
loss of the platelet surface GPlb-IX and GPllb-llla coma minimal number of circulating degranuplexes, and (4)
lated platelets. These data suggest that the "platelet function defect" of CPB is not a defect intrinsic to the platelet,
but is an extrinsic defect such as an in vivo lack of availability of platelet agonists. The near universal use of heparin
during CPB is likely to contribute substantially t o this defect via its inhibition of thrombin, the preeminent platelet
activator.
0 1993 by The American Society of Hematology.
T
HE USE OF cardiopulmonary bypass (CPB) during car(For logistical reasons, only 14 of these patients were able to be
studied for the experiments shown in Fig 2.) As a result of our
diac surgery is associated with a generalized hemorfindings in these 16 patients, we studied an additional four patients.
rhagic defect.' Although thrombocytopenia of mild degree
The
data for these four patients are presented in Figs 5 and 6. All
and alterations in the fibrinolytic and coagulation systems
occur during CPB,' a platelet function defect is generally
considered to be the primary CPB-induced hemostatic defiFrom the Department of Medicine, The Medical Center of CenCPB consistently results in a reversible marked protral Massachusetts, Worcester: the Naval Blood Research Laboralongation of the bleeding time'-4 and many:-"
but not
tory, Boston University School of Medicine, Boston; the Departstudies have reported defects of in vitro platelet agments of Surgery and Medicine, Brockton/West Roxbury Veterans
gregation. However, the nature of the putative platelet funcAdministration
Medical Center, Brigham and Women S Hospital,
tion defect associated with CPB remains controversial.
and Harvard Medical School, Boston: and the Departments ofPediThree types of intrinsic platelet defects have been reported
atrics and Surgery, University of Massachusetts Medical School,
in association with CPB. First, SO",^'^''^ but not other,15-'*
Worcester, MA.
studies have reported that CPB results in partial platelet
Submitted November 12, 1992: accepted February 12, 1993.
degranulation. Second, ~ o m e , ~ , 'but
~ , ' not
~
studies
A.S.K. was supported by agrantfvom the Foundation ofthe Medihave reported CPB-induced defects in the platelet surface
cal Center of Central Massachusetts. J.L. was supported by Grant
glycoprotein (GP)Ib-IX complex. Third, s o r r ~ e , ~ , ' ~but
, ' ~ , ' ~ Nos. HL40411, HL43344, and RCDA HLO2273from the National
Institutes ofHealth and a Merit Review Awardfrom the Veterans
not ~ t h e r , ' ~studies
, ' ~ have reported defects in the platelet
Administration. A.D.M. was supported by Grant No. HL38138
surface GPIIb-IIIa complex. In the present study, wholefrom the National Institutes of Health. Supported by the US Navy
blood flow cytometry was used to circumvent many of the
(Ofice of Naval Research Contract No. NOOO14-88-C-0118,with
potential methodologic problems of other assays. Evidence
funds provided by the Naval Medical Research and Development
is presented that the CPB-induced platelet dysfunction is
Command). The opinions or assertions contained herein are those
the result of a defect extrinsic to the platelet.
ofthe authors and are not to be construed as ojicial or reflecting the
MATERIALS AND METHODS
Study population. The study was approved by the Human Subjects Committee of the Brockton/West Roxbury Veterans Administration Medical Center. Twenty patients with angiographically documented coronary artery disease referred to the Brockton/West
Roxbury Veterans Administration Medical Center for coronary artery revascularization were entered into the study after written informed consent was obtained. The original study was of I6 patients.
The data for these 16 patients are presented in Figs 2, 3, 4, and 7.
Blood, VOI 82, NO 1 (July 1). 1993: pp 107-1 17
views of the Navy Department or Naval Service at large.
Address reprint requests to Alan D. Michelson, MD, Department
of Pediatrics, University ofMassachusetts Medical School, 55 Lake
Ave N, Worcester, M A 01655.
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 I734 solely to
indicate this fact.
0 1993 by The American Society qfHematology.
0006-49 71/93/8201 -0031$3.00/0
107
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
KESTIN ET AL
108
patients were undergoing their first open heart procedure. All patients required coronary artery bypass grafting, while one required
concomitant valvular replacement. No patient had a history suggestive ofan underlying hemostatic disorder. The age ofthe subjects in
the study was 62 f 2.3 years (mean f SEM; n = 20). Nineteen ofthe
20 subjects were male. All patients underwent a standard CPB procedure using a membrane oxygenator. The mean aortic crossclamp time was 50.4 4.4 minutes and the mean duration of CPB
was 109.9 f 7.2 minutes. Patients received intravenous heparin at
an initial dose of 4 mg/kg, followed by additional doses as necessary
to maintain the activated clotting time (ACT) greater than or equal
to 999 seconds. At the completion of CPB, heparin was reversed
with protamine sulfate. Heparin reversal was verified by confirming
that the ACT had returned to the preoperative value. Maximal hypothermia during CPB was a core temperature of 26.8 f 1.O”C, as
determined by a bladder thermometer (Bard, Boston, MA). Seven
patients required no exogenous blood components intraoperatively, but most patients received autologous blood harvested intraoperatively using a cell saver (Haemonetics, Braintree, MA). None
of the patients had excessive perioperative bleeding, as defined by
transfusion of greater than 10 U of blood in the perioperative period.’ The number of units of packed red blood cells transfused in
the perioperative period was 1.8 f 0.4 (mean k SEM). The maximum number of units of packed red blood cells transfused into any
one patient was 6 and this patient was found to have surgical bleeding. Data from this patient did not differ statistically from that of
the other subjects and were therefore included in the analysis. No
patient received a platelet transfusion before or during CPB.
Blood sampling time points. Peripheral venous blood and the
shed blood emerging from a standardized bleeding time wound
(Simplate 11, Organon Teknika, Durham, NC) were collected before, during, and after CPB at the time points listed in Table l. Not
every patient had all studies performed at all time points.
Murine monoclonal antibodies. SI 2 (provided by Dr Rodger P.
McEver, University of Oklahoma) is a monoclonal antibody directed against P-selectin.z‘~2z
P-selectin, also referred to as GMP140,” platelet activation-dependent granule-external membrane
(PADGEM) pr~tein,~’
and CD62,24is a component of the a-granule membrane ofresting platelets that is only expressed on the platelet plasma membrane after platelet secretion.”
A panel of platelet GPIb-IX-specific monoclonal antibodies was
used. 6DI (provided by Dr Barry S. Coller, SUNY, Stony Brook) is
directed against the von Willebrand factor binding site on the glycocalicin portion of the a-chain of GPIb.25.26TM60 (provided by Dr
Naomasa Yamamoto, Tokyo Metropolitan Institute of Medical
*
Table 1. Blood Sampling Time Points Before,
During, and After CPB
PRE OP
Preoperative
PRE HEP
CPB END
After t h e start of anesthesia and surgery, but before
heparin and CPB
5 minutes after heparin administration,but before t h e
start of CPB
After the start of CPB (normothermicconditions)
Beginning of maximal hypothermia on CPB
45 minutes after the start of CPB (hypothermic
conditions)
Completion of CPB, immediately following
POST 2
POST 24
POST 48
administration of protamine
2 hours after completion of CPB
24 h o u r s after completion of CPB
48 hours after comtietion of CPB
HEP
CPB NT
CPB HT
CPB 45
Science) is directed against the thrombin-binding site on the amino
terminal domain of the a-chain of GPIb.Z7AKI (provided by Dr
Michael C. Berndt, Baker Institute, Melbourne, Australia) is directed against the GPlb-IX complex.28AK 1 only binds to the intact
GPIb-IX complex, not to uncomplexed GPIb or GPIX.28FMC25
(provided by Dr Berndt) is directed against GPIX.29
A panel of platelet GPIIb-IIla-specific monoclonal antibodies
was used. Y2/5 I (DAKO, Carpinteria, CA) is directed against platelet membrane GPIIIa.’’ 7E3 (provided by Dr Coller), 10E5 (provided by Dr Coller), and M 148 (Cymbus Bioscience, Southampton,
UK) are directed against different epitopes near the fibrinogenbinding site on the GPIIb-IIla c ~ m p l e x . ~
PAC1
~ ” ~(provided by Dr
Sanford J. Shattil, University of Pennsylvania, Philadelphia) is directed against the fibrinogen-binding site exposed on the GPIIb-Illa
complex of activated platelets.’’ Unlike Y2/5I, M148, 7E3, and
10E5.3’”3 PAC1 does not bind to resting platelets.35
OKM5 (provided by Dr Patricia Rao, Ortho Diagnostic Systems,
Raritan, NJ) is directed against platelet membrane GPIV.36
Flow cytometric analysis gf platelet siivface glycoproteins in peripheral blood. A whole-blood flow cytometric method was used.
The method has previously been described in detail37and includes
no centrifugation, gel filtration, vortexing, or stirring steps that
could artifactually activate platelets. Briefly, the method was as follows: the first 2 mL of blood drawn was discarded and then blood
was drawn into a sodium citrate Vacutainer (Becton Dickinson,
Rutherford, NJ) and, within 15 minutes, diluted in modified Tyrode’s buffer (137 mmol/L NaCI, 2.8 mmol/L KCI, 1 mmol/L
MgCI,, 12 mmol/L NaHCO,, 0.4 mmol/L Na,HP04, 0.35% bovine
serum albumin, I O mmol/L HEPES, 5.5 mmol/Lglucose, pH 7.4).
This method of dilution of blood into buffer before the addition of
agonist was based on the method of Shattil et ai.” The blood is
diluted into buffer to decrease the platelet concentration, thereby
preventing platelet-to-platelet aggregation after the addition of agonist. This dilution is critical because quantitation of the amount of
surface antigen per platelet by flow cytometry requires that platelets
by analyzed individually. After dilution, the sample was incubated
at 22°C with buffer only or an agonist: either phorbol myristate
acetate (PMA; Sigma, St Louis, MO), the stable thromboxane
(TX)A, analog U466 I9 (Cayman Chemical, Ann Arbor, MI), purified human a-thrombin (provided by Dr John Fenton 11, New York
Department of Health, Albany) together with 2.5 mmol/L glycyl-Lprolyl-L-arginyl-L-proline(Calbiochem, San Diego, CA) (an inhibitor of fibrin p~lymerization),’~
or a combination of adenosine diphosphate (ADP; Bio/Data, Hatboro, PA) and epinephrine
(Sigma). In kinetic studies, the samples were then fixed with formaldehyde ( I % final concentration) at various time points after the
addition of the agonist. In all other studies, fixation with formaldehyde ( I % final concentration) was performed 15 minutes after the
addition of the agonist, as previously described.’? After fixation, all
samples were diluted and incubated at 22°C for 15 minutes with ( I )
a saturating concentration of a biotinylated monoclonal antibody
(directed against either P-selectin, the GPIIb-lIIa complex, or the
GPIb-IX complex) and (2) a saturating concentration of either fluorescein isothiocyanate (F1TC)-conjugated GPIV-specific monoclonal antibody OKM5 or FITC-conjugated GPIIIa-specific monoclonal antibody Y2/51. The samples were then incubated at 22°C for
I5 minutes with phycoerythrin-streptavidin (Jackson ImmunoResearch, West Grove, PA). (When monoclonal antibody MI48 was
used, the incubation step with phycoerythrin-streptavidin was unnecessary because the antibody was purchased directly conjugated
with R-phycoerythrin.) Within 24 hours of antibody tagging, the
samples were analyzed in an EPICS Profile flow cytometer (Coulter
Cytometry, Hialeah, FL). After identification of platelets by gating
on both FITC positivity and their characteristic light scatter. bind-
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
109
PLATELET FUNCTION IN CARDIOPULMONARY BYPASS
ing of the biotinylated monoclonal antibody was determined by
analyzing 5,000 individual platelets for phycoerythrin fluorescence.
In addition to platelets, OKM5 binds to monocyte^,'^ but these
were completely gated out by size (forward-light scatter).
It has previously been shown that this method (fixation before
antibody incubation) results in no significant differences in fluorescence intensity between samples analyzed immediately and samples analyzed within 24 hours of antibody tagging.39We confirmed
this finding, and also demonstrated that collection of blood into a
Vacutainer did not result in platelet activation (Fig 1).
In addition to our standard method of determination of binding
by fluorescence intensity relative to a maximally degranulated platelet,37some samples were analyzed, as indicated in the Results, by
the percentage of P-selectin-positive platelets. The percentage of
P-selectin-positive platelets at different time points during CPB
was defined as the percentage of platelets that had an SI2 fluorescence greater than 98%ofthe platelets in samples from the preoperative time point (without an added exogenous platelet agonist).
In a subgroup of patients, the response of washed platelets to
thrombin was studied. Peripheral blood was collected as described
above and the platelets separated and washed in modified Tyrode’s
buffer as previously described.40The platelets (75,OOO/pL) were incubated with 0.05, 0.1, or I U/mL of purified human a-thrombin
or buffer only, and then analyzed by flow cytometry for the binding
of a biotinylated monoclonal antibody (either S12, PACI, 7E3, or
6D1) as previously described?’ PACl does not bind well to fixed
platelets. For this reason, as distinct from all other antibodies used
in this study, PACl was incubated with platelets before fixation, as
previously de~cribed.’~
Flow cytometric analysis of platelet suvface P-selectin in shed
blood. The platelet surface expression of P-selectin in blood
emerging from a standardized bleeding time wound was analyzed
by the whole-blood flow cytometric method. The method has been
previously described in
Duplicate standardized bleeding
times were performed on the forearm of patients with the Simplate
I1 device. The blood emerging from the bleeding-time wound (shed
blood) was collected with a micropipet at 2-minute intervals until
the bleeding stopped. After each pipetting, any residual blood at the
bleeding-time wound site was removed with filter paper. Immediately after collection at each time point, the pipetted blood was
added to a microfuge tube containing sodium citrate, fixed for 30
minutes at 22°C with formaldehyde (1% final concentration), and
diluted 1 :I O by volume in modified Tyrode’s buffer. As described
above, the fixed diluted whole-blood samples were then labeled
with the FITC-conjugated GPIV-specific monoclonal antibody
OKM5 and the biotinylated P-selectin-specific monoclonal antibody S12, and the individual platelets analyzed in an EPICS Profile
flow cytometer to assess S 12 binding.
Radioimmunoassay ofshed-blood TXB,. TXB, is a stable metabolite of TXA, and an important marker of platelet activation?,
A
The shed-blood assay method has been previously de~cribed.4~
standardized bleeding-time wound was performed, as described
above. The shed blood emerging from the wound was aspirated
through a blunt needle into a tuberculin syringe coated with heparin (1,000 U/mL) and containing 20 pL of ibuprofen for each 1 mL
of blood (1.9 mg/mL). Samples were collected every 30 seconds
until a 600-pL aliquot of blood was obtained. The TXB, concentration of the plasma was determined with an RIA kit (New England
Nuclear, Boston, MA).
Plasma glycocalicin assay. Plasma glycocalicin was determined
by a modified version of a previously described competitive inhibition assay.44 A subsaturating concentration of FTTC-conjugated
monoclonal antibody 6DI (1.2 pg/mL) was incubated at 22°C for
20 minutes with either ( I ) test plasma that had been filtered through
120
100
80
60
40
20
0
100
I
T
80
60
40
RESTING
PMA
Fig 1. Stability of antibody binding. Peripheral blood samples
were collected into either a Vacutainer or a polypropylene syringe
that contained the same citrate anticoagulant. As described in detail in the Methods, whole blood samples were incubated with or
without 10 pmol/L PMA, fixed in 1 % formaldehyde, stained with
biotinylated monoclonal antibody, and incubated with phycoerythrin-streptavidin. The samples were analyzed by whole-blood
flow cytometry immediately (day 0) and then again after 24 hours
at 4°C (day 1). (0)Day 0 Vacutainer; (E!) day 0 syringe; (
Vacutainer; (m) day 1 syringe. (A) Platelet binding of monoclonal
antibody S12 (P-selectin-specific). The fluorescence intensity of
platelets collected into a syringe, incubated with PMA, and analyzed immediatelywere assigned a value of 100 U. (B) Platelet binding of monoclonal antibody 6D1 (GPlb-specific).The fluorescence
intensity of platelets collected into a syringe, incubated without
PMA, and analyzed immediately were assigned a value of 100 U.
Data are the mean ’ 2 SEM; n ’= 4.
a 0.22-wm Acrodisc (Gelman, Ann Arbor, MI) and the pH buffered
to 7.4; or (2) various concentrations of purified glycocalicin (prepared as previously describedz6). Samples were then incubated at
22°C for 20 minutes with fixed, washed platelets (final concentration, lOO,OOO/pL) and diluted 20-fold in modified Tyrode’s buffer,
pH 7.4, before the platelet binding of 6D1 was analyzed by flow
cytometry. Linear regression analysis was used to generate a stan-
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
KESTIN ET AL
110
dard curve from 0 to 70 nmol/L from the purified glycocalicin
samples. The plasma glycocalicin concentration of unknown
plasma samples was derived from this standard curve.
Ristocetin-induced binding of von Willebrand factor to platelets. The ristocetin-induced binding of normal von Willebrand
factor to washed platelets from the patient was determined by the
following method: the patient's platelets were washed as previously
described,"' fixed in 1% formaldehyde, and resuspended at a concentration of 75,00O/pL in modified Tyrode's buffer, pH 7.4.
Twenty microliters of the platelet suspension was incubated at
22°C for 15 minutes with 20 pL of pooled platelet-poor plasma
from normal donors (as a source of von Willebrand factor) and 5 pL
of ristocetin (BioData, Horsham, PA; final concentration, 1.4 mg/
mL). The mixture was then incubated at 22°C for 15 minutes with
0.028 mg/mL of either polyclonal FITC-conjugated anti-von Willebrand factor goat IgG antibody (Atlantic Antibodies, Stillwater,
MN) or FITC-conjugated nonspecific goat IgG (Atlantic Antibodies). The sample was then diluted 16-fold in modified Tyrode's
buffer, pH 7.4, and analyzed by flow cytometry. The fluorescence
of the sample incubated with the nonspecific goat IgG was subtracted from the sample incubated with the anti-von Willebrand
factor antibody.
Fibrinogen binding to platelets. Fibrinogen binding to ADPstimulated washed platelets was determined by a direct binding
assay using radioiodinated fibrinogen, as previously de~cribed.~'
Hematocrit and platelet count. Hematocrit and platelet counts
were measured using a J.T. Electronic Particle Counter (Coulter).
Time
Points
IO0
0
0
:l
0
0
20
10
30
40
1 / 7 1
0
L-1
40
0
40
20
10
20
10
30
30
PRE HEP
HEP
CPB 45
POST 2
0
RESULTS
E f e c t of CPB on platelet reuctivity in vivo. To determine
the effect of CPB on platelet reactivity in vivo, the time-dependent up-regulation of platelet surface P-selectin was analyzed by whole-blood flow cytometry in the blood emerging
from a standardized bleeding-time wound (Fig 2). The previously
time-dependent up-regulation of platelet
surface P-selectin was observed in samples obtained from
bleeding time incisions performed preoperatively (PRE OP
time point in Fig 2) and after the start of anesthesia and
surgery but before heparin and CPB (PRE HEP time point
in Fig 2). However, this in vivo up-regulation of P-selectin
was almost abolished 5 minutes after heparin administration but before the start of CPB (HEP time point in Fig 2)
and 45 minutes after the start of CPB (CPB 45 time point in
Fig 2). The in vivo up-regulation of P-selectin returned to
near normal within 2 hours after the completion of CPB
(POST 2 time point in Fig 2) and was completely normal
within 24 hours after the completion ofCPB (POST 24 time
point in Fig 2). The maximal up-regulation of P-selectin in
shed blood collected during bypass and postbypass was
19.3% f 3.2% (mean f SEM; n = 14) and I 1 1.9% f 16.8'70,
respectively, of the prebypass value. Thus, the maximum
up-regulation of P-selectin in shed blood collected during
bypass was significantly decreased as compared with the
mean values in shed blood collected before and after bypass
(F statistic = 33.97, df = 2,223, P < ,001, analysis of variance
for repeated measures).
To provide further evidence that platelet reactivity in
vivo is deficient during CPB, TXB, (a stable metabolite of
TXA,) was assayed in the shed blood emerging from the
standardized bleeding-time wound. TXB2 generation in
shed blood was reduced 5 minutes after heparin administra-
PRE OP
50
E
10
20
30
40
I
0 0
POST 2 4
10
20
30
40
TIME
(minutes)
Fig 2. The effect of CPB on activation-induced up-regulation of
the platelet surface expression of P-selectin in whole blood in vivo.
Abbreviations listed to the right refer to the blood sampling time
points before, during, and after CPB, as defined in Table 1. A standardized bleeding time was performed and, without touching the
wound, the shed blood was collected with a micropipet directly
from the wound site at 2-minute intervals (as shown on the horizontal axis) until bleeding cessation. The sample was immediatelyanticoagulated and fixed, then analyzed by whole-blood flow cytometry with monoclonal antibody S12. The maximal binding of SI 2 to
platelets at the preoperative (PRE OP) time point was assigned a
value of 100 U of fluorescence. This experiment is representative
of 14 separate experiments.
tion but before the start of CPB (HEP time point in Fig 3)
and decreased further 45 minutes after the start of CPB
(CPB 45 time point in Fig 3). Generation of TXB, in shed
blood returned toward normal after the completion of CPB
(CPB END, POST 2, and POST 24 time points in Fig 3).
The CPB-induced inhibition of shed-blood P-selectin upregulation and TXB, generation paralleled the effect of CPB
on the bleeding time. The bleeding time (minutes iSEM; n
= 16) at the indicated time points was 6.2 f 0.3 (PRE OP),
6.7 f 0.3 (PRE HEP), 10.4 f 0.5 (HEP), 32.8 f 3.2 (CPB
4 3 , 14.3 k 1.0 (POST 2), and 11.5 f 1.8 (POST 24). This
CPB-induced marked prolongation of the bleeding time was
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
PLATELET FUNCTION IN CARDIOPULMONARY BYPASS
111
To assess the reactivity of platelets to thrombin, the patients' platelets were washed free of heparin. CPB did not
inhibit the thrombin-induced up-regulation of platelet surface P-selectin (Fig 5A), up-regulation of the GPIIb-IIIa
complex (Fig 5B), or down-regulation of the GPIb-IX complex (data not shown). However, as expected, when the
thrombin reactivity of platelets in whole blood containing
the therapeutically administered heparin was measured,
platelets were completely unreactive to thrombin at the following time points: 5 minutes after heparin administration,
but before the start of CPB; after the start of CPB (normo-
N
m
w
1600
5x
1400
m
1200
0
I
0 3
E
E
1000
I -
n
W
150
400
I
v)
200
I
I
I
I
I
I
I
T
Fig 3. The effect of CPB on TXB2 generation in response to a
standardizedvascular injury in vivo. The abbreviations listed on the
horizontal axis refer to the perioperative time points, as defined in
Table 1. At each of these perioperative time points, a standardized
bleeding-time wound was performed and the shed blood was collected every 30 seconds into a heparinized syringe containing ibuprofen. The TXB, concentration of the plasma was determined by
SEM; n = 16.
radioimmunoassay. Data are the mean
*
not accounted for by thrombocytopenia, because the platelet count declined to 166.1 12.7 X 103/pLafter the start of
CPB and never decreased below I40 X 103/pL.
Eflect of CPB on platelet reactivity in vitro. In contrast
to the inhibitory effect of CPB on platelet reactivity in vivo,
CPB did not inhibit platelet reactivity in vitro, as determined by samples obtained from the same set of patients at
the same time points using the same assays run in parallel
with the same reagents. Thus, CPB did not inhibit up-regulation of platelet surface P-selectin, or down-regulation of the
platelet surface GPIb-IX complex, induced in vitro by either 0.25 pmol/L or 10 pmol/L PMA (Fig 4). Similarly,
CPB did not inhibit up-regulation of platelet surface P-selectin, up-regulation of the platelet surface GPIIb-IIIa complex, or down-regulation of the platelet surface GPIb-IX
complex induced in whole blood in vitro by any of the following: 0.5 pmol/L of the stable TXA, analog U46619, 5
pmol/L U46619, a combination of 0.5 pmol/L ADP and 5
pmol/L epinephrine, and a combination of 10 pmol/L ADP
and 5 pmol/L epinephrine (n = 4, data not shown). CPB did
not alter the kinetics of up-regulation of P-selectin, downregulation of GPIb-IX complex, or up-regulation of the
GPIIb-IIIa complex induced in whole blood in vitro in response to U466 19 (n = 4, data not shown).
Interestingly, administration of heparin appeared to augment both the PMA-induced up-regulation of P-selectin
(Fig 4A), and down-regulation ofthe GPIb-IX complex (Fig
4B). Protamine sulfate administration appeared to reverse
this augmentation (Fig 4A and B).
*
110
-B
100 go
-
80 -
70 60 -
50
-
40
-
30
TPoints
iye
n
0
w
~
a
n
w
I
-
I
z
w n
m
~
~
w
P
1 0
o
I
-
t
CJ
~
4.10
Z
N
m tVI- t -v)l - v)
n
n
n
n
o o n n n
m
N
o
Fig 4. Effect of CPB on platelet reactivity to PMA, as determined by whole-blood flow cytometry. Abbreviations listed on the
horizontal axis refer to the perioperative blood sampling time
points, as defined in Table 1. Peripheral blood samples were incubated with PMA 0 (e),0.25 (VI,or 10 pmol/L (B).(A) Platelet surface expression of P-selectin. as determined by monoclonal antibody S12. The fluorescence intensity of platelets incubated with
PMA 10 pmol/L at the preoperative (PRE OP) time point was assigned a value of 100 U. (B) Platelet surface GPlb, as determined by
monoclonal antibody 6D1. The fluorescence intensity of platelets
incubated without PMA at the preoperative time point was asSEM; n = 16.
signed a value of 100 U. Data are the mean
*
~
W
o
o
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
112
KESTIN ET AL
tor binding site on GPIb (Fig 4B, closed circles, and Fig 6A),
the thrombin binding site on GPIb (Fig 6B), GPIX (Fig 6C),
or the GPIb-IX complex (Fig 6D).
In addition, we examined the ristocetin-induced binding
of von Willebrand factor to platelets. This binding, which
reflects the availability ofGPIb as a platelet receptor for von
Willebrand factor,'" was unchanged during and after CPB
60
(Fig 6E).
Furthermore, we assayed the patients' plasma for glyco40
calicin, a proteolytic product 0fGP1b.~'Plasma glycocalicin
did not rise above the preoperative concentration at any
time point during or after CPB (Fig 7). The observed reduc20
tion in plasma glycocalicin during CPB was probably dilutional, because the reduction paralleled the reductions in
0
hematocrit (Fig 7) and serum albumin (data not shown).
Effect of CPB on the platelet surface GPIIb-lIIa comT
plex. The platelet surface GPIIb-IIIa complex was anaB
lyzed by whole-blood flow cytometry. CPB did not result in
100 any significant change in the platelet surface expression of
the GPIIb-IIIa complex, as determined by monoclonal anti80 bodies (7E3, 10E5, and M148) directed against different
epitopes near the fibrinogen-binding site on the GPIlb-IIIa
complex
and a monoclonal antibody (Y2/51) directed
60 against GPIIIa (Fig 6F, G, H, and I).
Direct measurement of the binding of exogenous fibrino40 gen to platelets activated in vitro with ADP (reflecting exposure of the fibrinogen-binding site on the GPIIb-1IIa comp l e ~demonstrated
~~)
an increase in the KDfrom 0.43 to 0.54
20 pmol/L and no loss of binding sites during CPB (Fig 65).
Effect of CPB on thepresence of circulatingactivatedplatelets. The presence of activated platelets circulating in pe0
ripheral blood was investigated by the platelet binding of
n
Y)
z
N
Time
o
t
w
S 12, a monoclonal antibody directed against P-selectin.
IIn
m
Pointa
;
Again, to avoid artifactual platelet activation during the sep0
n
n
n
n
0
0
aration of platelets, a whole-blood flow cytometric assay
The effect of CPB on thrombin reactivity of washed platewas used. CPB did not result in any significant overall inlets-Abbreviations listed on the horizontalaxis refer to the periopercrease in P-selectin expression on the surface of circulating
ative peripheral blood sampling time points, as defined in Table 1.
platelets (Fig 4A, closed circles). Analysis of the same data
The platelets were washed, incubated with thrombin (U/mL) 0 (0).
in terms of the percentage of circulating platelets that were
0.05 (e),0.1 (V), 1 .O (VI,and analyzed by flow cytometry. (A) PlateP-selectin-positive resulted in a minimal increase during
let surface expression of P-selectin, as determined by monoclonal
antibody S12. (B) Platelet surface expression of the GPllb-llla comCPB. Thus, the percentage of circulating platelets that were
plex, as determined by monoclonal antibody PACl . The fluoresP-selectin-positive was 2.0% k 0.0%(mean & SEM; n = 14)
cence intensity of platelets incubated with thrombin 1 U/mL at the
preoperatively,
3.1% f 0.3% at 45 minutes after the start of
preoperative (PRE OP) time point was assigned a value of 100 U.
CPB, and 2.9% f 0.3% at the completion of CPB, immediData are mean f SEM; n = 4.
ately following the administration of protamine.
Monoclonal antibody PACl does not bind to resting
platelets, only to activated platelets that have exposed fithermic conditions); beginning of maximal hypothermia on
brinogen-binding sites on the GPIIb-IIIa complex.35ThereCPB; and 45 minutes after the start of CPB (hypothermic
fore, the studies with PACl (Fig 5B, open circles) demonconditions) (data not shown).
Eflect of CPB on the platelet surface GPIb-IX comstrate that CPB did not result in significant exposure of
fibrinogen-binding sites on the GPIIb-IIIa complex of circuplex. The platelet surface expression of the GPIb-IX complex can be markedly reduced by platelet a c t i v a t i ~ nand
~ ~ . ~ lating platelets.
washing.46 To avoid these potential artifacts, we used a
DISCUSSION
whole-blood flow cytometric method that did not require
The most important factor contributing to the hemostatic
any centrifugation, gel filtration, vortexing, or stimng
defect associated with CPB is generally considered to be a
steps.37As determined by this method, CPB did not result in
platelet function defect.'-4 However, the precise nature of
significant change in the platelet surface expression of the
the putative CPB-induced platelet function defect is controGPIb-IX complex, irrespective of whether the monoclonal
~ersia1.l.~
In this study, we demonstrate that CPB results in
antibody used was directed against the von Willebrand fac-
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
113
PLATELET FUNCTION IN CARDIOPULMONARY BYPASS
G P I I b / l II a
GPlb/lX
110
6D1 100
A
110
io0
i
90
Fig 6. Effect of CPB on the platelet surface expression of the GPlb-IX and GPllb-llla complexes.
Abbreviations listed on the horizontal axis refer to
the perioperative peripheral blood sampling time
points, as defined in Table 1. (A-D) Platelet binding
of a panel of GPlb-IX-specific monoclonal antibodies, as determined by whole-blood flow cytometry.
The antibodies are directed against the von Willebrand factor-binding site on GPlb (6D1). the
thrombin-binding site on GPlb (TM60). GPlX
(FMC25). and the GPlb-IX complex (AK1). (E) Ristocetin-induced binding of exogenous von Willebrand factor (vWf) to washed platelets, as determined by flow cytometry with a polyclonal
anti-vWf antibody. (F-I) Platelet binding of a panel
of GPllb-llla-specific monoclonal antibodies, as
determined by whole-blood flow cytometry. Antibodies 7E3, 10E5, and M148 are directed against
different epitopes near the fibrinogen-binding site
on the GPllb-llla complex. Y2/51 is directed
against the GPllla subunit. (A-I) Antibody binding
at the preoperative (PRE OP) time point was assigned a value of 100 U of fluorescence. (J)Binding
of exogenous radioiodinated fibrinogen to ADPstimulated washed platelets. Fibrinogen binding is
expressed as molecules X lo” per platelet. Data
are the mean f SEM; n = 4.
t
,
I
7E3
t
1 OE5
I
110
AK1
100
90
vWf 100
110
90
I-ii-l ::I!/: JI
,
,
FIBRINOGEN
40
0
Time
Points
( 1) markedly deficient platelet reactivity in response to an in
vivo wound, (2) normal platelet reactivity in vitro, (3) no
loss of the platelet surface GPIb-IX and GPIIb-IIIa complexes, and (4) a minimal number of circulating degranulated platelets. These data suggest that the “platelet function
defect” of CPB is not a defect intrinsic to the platelet, but is
an extrinsic defect such as an in vivo lack of availability of
platelet agonists. The near universal use of heparin during
CPB is likely to contribute substantially to this defect via its
inhibition of thrombin, the preeminent platelet activator.
Effect of CPB on platelet reactivity in vivo. The shed
blood emerging from a standardized bleeding-time wound
has been used in a number of previous studies as a reflection
of in vivo activation of platelets and other components of
the hemostatic ~ y s t e m . ’ ~ , ~In
~ ,this
~ @study,
~ ’ we have demonstrated that CPB inhibits platelet reactivity in vivo, as
determined by two independent markers of platelet activation in shed blood: up-regulation of platelet surface P-selectin and TXB’ generation. These defects paralleled the CPBinduced prolongation of the bleeding time, which is
generally accepted to be the hallmark of the hemostatic defect of CPB.’-3
Effect of CPB on platelet reactivity in vitro. The results
of studies of the effect of CPB on platelet aggregation, as
determined by standard nephelometric methods, are incons i ~ t e n t . ~ - These
’ ~ , ’ ~inconsistencies
~~~
probably result in part
from the fact that platelet aggregation, especially in a complex clinical setting, is semiquantitative and subject to standardization problem^.^^,^^ Furthermore, most of the re-
w
E
m
t
:
m
=
g
ported platelet aggregometry studies during CPB were
performed in platelet-rich plasma without normalizing the
platelet count. The CPB-induced “platelet aggregation defect” may therefore simply reflect the CPB-induced thrombocytopenia.
In this study, platelet reactivity in vitro was analyzed not
by aggregometry, but by whole-blood flow cytometric assay
of peripheral blood samples obtained before, during, and
after CPB. CPB did not result in a defect in platelet reactivity in vitro, as determined by agonist-induced up-regulation
of platelet surface P-selectin and the GPIIb-IIIa complex,
and down-regulation of the GPIb-IX complex. The results
were the same irrespective of whether the in vitro platelet
agonist was PMA, the stable TXA, agonist U466 19, a combination ofADP and epinephrine, or thrombin (in a washed
platelet system), thereby excluding the possibility of a CPBinduced signal transduction defect.
Effect of CPB on the platelet surface GPIb-IX complex. S o ~ n e , ~ but
~ ’ ~not
. ’ ~all,” previous studies have concluded that a CPB-induced modest decrease in platelet surface GPIb, a receptor for von Willebrand factor,47may play
a role in the pathogenesis of the CPB-induced platelet dysfunction. The study by George et all7has been widely interpreted as evidence for a CPB-induced reduction in platelet
surface GPIb. However, in George’s studyI7 all values for
platelet surface GPIb during CPB were within or close to the
normal range. van Oeveren et all9 reported a 25% reduction
in GPIb during CPB, but, unlike the study by George et aii7
and the present study, these investigators centrifuged and
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
KESTIN ET AL
114
45
45
T
40
40
n
0
35
35
30
30
a
ZE
W
I
25
25
20
Time
Points
I
1
1
1
1
1
1
1
I
I
20
Fig 7. Effect of CPB on the
plasma concentration of glycocalicin, a proteolytic fragment
of GPlb. Abbreviations listed on
the horizontal axis refer to the
perioperative peripheral blood
sampling time points, as defined in Table l . Plasma glycowas measured by
calicin (-1
a competitive inhibition assay.
To assess the effect of hemodilution during the bypass procedure, hematocrit (-- -) was
also measured. Data are the
mean f SEM: n = 16.
Wenger et all3 reported a decrease of 25% in the whole plategel-filtered the platelets before assay, thereby introducing
let GPIIIa content during CPB and a decrease of 4 I % in the
the possibility of an artefactual in vitro decrease in platelet
binding of exogenous fibrinogen. Dechavanne et
surface GPIb as a result of proteolysis46or a c t i ~ a t i o n . ~ ~ , ~platelet
'
all6 also used a washed platelet method and reported a 32%
Rinder et al' reported a maximal reduction of platelet surdecrease in the platelet surface GPIIb-IIIa complex during
face GPIb of 28%, but the only significant reduction in
CPB. Using a whole-blood method, George et all7reported
GPIb was at the completion of and after CPB. Thus, Rinder
only a slight decrease in platelet surface GPIIb during CPB.
et a1' did not observe a significant reduction in platelet surRinder et al,7 using a whole-blood method, reported a 2 1%
face GPIb during CPB, when the bleeding time is most pro(but statistically insignificant) decrease in the platelet surlonged.
face GPIIb-IIIa complex after CPB. However, van Oeveren
In this study, we used a flow cytometric method that alet all9 detected a modest increase in both platelet surface
lowed us to study the platelet GPIb-IX complex in whole
GPIIb-IIIa and ADP-induced fibrinogen-binding during
blood, thereby avoiding potential artifactual reductions in
CPB. Finally, using a whole-blood flow cytometric method,
platelet surface GPIb.37 As demonstrated by this method,
Abrams et all8 reported that fibrinogen binding to the plateCPB did not result in a decrease in the platelet surface exlet GPIIb-IIIa complex was slightly increased during CPB,
pression of the GPIb-IX complex. The possibility of a CPBinduced conformational change in the GPIb-IX complex,
as determined by the binding of a monoclonal antibody
(PACI) directed against the fibrinogen binding site on the
or CPB-induced proteolysis of a fragment of the GPIb-IX
GPIIb-IIIa complex and by the binding of a monoclonal
complex, was excluded by the lack of change in the platelet
antibody (9F9) directed against platelet-bound fibrinogen.
binding of a panel of monoclonal antibodies (6D1, TM60,
AKI, and FMC25) known to be directed against different
In the present study, as demonstrated by a whole-blood
flow cytometric assay, CPB did not result in a decrease in
epitopes on the GPIb-IX complex. Furthermore, CPB neither reduced the ristocetin-induced binding of von Willethe platelet surface expression of the GPIIb-IIIa complex.
The possibility of a CPB-induced conformational change in
brand factor to GPIb nor increased the plasma concentrathe GPIIb-IIIa complex, or CPB-induced proteolysis of a
tion of glycocalicin, a proteolytic fragment of GPIb. Thus,
by a combination of methods, the present study demonfragment of the GPIIb-IIIa complex, was excluded by the
strates that CPB is not associated with a loss of platelet surlack of change in the platelet binding of a panel of monoclonal antibodies (7E3, lOE5, M 148, and Y2/51) known to be
face GPIb.
Effect of CPB on the platelet surface GPIIb-IIIu comdirected against different epitopes on the GPIIb-IIIa complex. S ~ m e , ' ~ ' ~ . ' ~but
, ' ' not
previous studies
plex. In addition, we directly studied the binding of exogehave concluded that a CPB-induced modest decrease in the
nous fibrinogen to the GPIIb-IIIa complex and found no
platelet surface GPIIb-IIIa complex, the fibrinogen recepCPB-induced reduction. Finally, we demonstrated that irretor,49plays a role in the pathogenesis of the CPB-induced
spective of whether peripheral blood samples were drawn
platelet dysfunction. Using washed platelet methods,
before, during, or after CPB, maximal in vitro stimulation
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
115
PLATELET FUNCTION IN CARDIOPULMONARY BYPASS
of washed platelets by thrombin resulted in the same exposure of the fibrinogen-binding site on the GPIIb-IIIa complex, as determined by monoclonal antibody PAC 1. Thus,
by a combination of methods, the present study demonstrates that CPB is not associated with a loss of the platelet
surface GPIIb-IIIa complex.
Effect ofCPB on platelet degranulation. Electron microscopic methods, fraught with the possibility of artifactual in
vitro platelet degranulation, have resulted in reports of circulating partially degranulated platelets during CPB in
some: but not other,l5,I6studies. More recently, a number
of investigators have studied the effect of CPB on the
binding of monoclonal antibodies directed against granule
antigens that are only present on the platelet surface after
degranulati~n.~~'~,'~-'~
In washed platelet systems, Nieuwenhuis et all4found a modest increase during CPB ofthe platelet binding of a monoclonal antibody directed against a 53Kd lysosomal antigen, whereas Dechavanne et all6 found
that CPB did not result in an increase in the platelet surface
expression of P-selectin. Using whole-blood methods that
are much less likely than washed platelet methods to result
in artifactual in vitro platelet d e g r a n ~ l a t i o nboth
, ~ ~ George
et all7and Abrams et all8demonstrated that CPB resulted in
no significant increase in the platelet surface expression of
P-selectin. By comparable methods, C ~ r a s hreported
~~
a
modest, transient increase in P-selectin-positive platelets
during CPB. Rinder et ai7used a whole-blood flow cytometric assay and found a 29% increase in P-selectin-positive
platelets at the end of CPB, but the amount of increase in
P-selectin on the P-selectin-positive platelets was modest.
Consistent with these previous studies, the present wholeblood flow cytometric study of peripheral blood samples
during CPB showed that although there was a 55% increase
(2.0% to 3.1%) in the number of circulating degranulated
platelets during CPB, the overall P-selectin expression on
the surface of the platelets was minimal (Fig 4A, closed circles). These data suggest that the reported CPB-induced increases in plasma P-thromboglobulin and platelet factor
44,6,15,58-60 originate from either (1) degranulated platelets
that are rapidly cleared from the circulation6' (possibly by
circulating monocytes and neutrophil^^^^^^), (2) noncirculating degranulated platelets adherent to synthetic surfaces or
vessel walls, (3) platelet lysis in vivoL5or in vitro, and/or (4)
artifactual in vitro degranulation and secretion as a result of
separation of plasma from platelets before the performance
of the assays.64
The role of heparin in the platelet function defect of
CPB. Thrombin, the most important platelet agonist in
vivo,65-67
is functionally deficient during CPB because of the
presence of high circulating concentrations of heparin. Although thrombin bound to fibrin clots is relatively protected from inhibition by heparin-antithrombin 111,68369 the
high concentrations of heparin used during CPB (4.5 U/mL
with an ACT 2999 seconds in this study) are sufficient to
completely overcome this protection.68 The importance of
heparin inhibition of thrombin in the platelet function defect of CPB is suggested by the finding that 5 minutes after
heparin administration, but before the start of CPB, (1) the
in vivo up-regulation of platelet surface P-selectin was al-
most abolished (HEP time point in Fig 2), (2) the in vivo
generation of TXA, was almost maximally inhibited (HEP
time point in Fig 3), (3) the bleeding time was prolonged,
and (4) platelets in whole blood were completely unreactive
to thrombin in vitro.
The present study provides evidence for two distinct effects of heparin on platelet function during CPB. First, heparin augments platelet activation in whole blood exposed to
an exogenous platelet agonist in vitro (Fig 4). Second, as
discussed above, heparin suppresses platelet activation in
vivo via inhibition of endogenous thrombin. Thus, although heparin augments the activatability of platelets, the
platelets are not in fact activatable in vivo because thrombin, the preeminent a g o r ~ i s t ,is~ ~
unavailable.
-~~
In addition to heparin, other extrinsic factors such as hyp ~ t h e r m i a ~ 'and
- ~ ~fibrinolytic a ~ t i v i t y ~ may
' , ~ ~ contribute
to the platelet function defect associated with CPB. These
factors are currently under investigation in our laboratory.
ACKNOWLEDGMENT
The authors thank Drs Michael C. Berndt, Barry S. Coller, John
W. Fenton 11, Rodger P. McEver, Patricia Rao, Sanford J. Shattil,
and Naomasa Yamamoto for generously providing reagents, Drs
Peter H. Levine and James J. Hoogasian for their support, and
Nancy Healy, Mheir Doursounian, and Stephanie Francis for their
essential contributions. Dr Kestin dedicates this report to Dr Joseph
Kestin.
REFERENCES
1. Woodman RC, Harker LA: Bleeding complications associated with cardiopulmonary bypass. Blood 76: 1680, 1990
2. Harker LA: Bleeding after cardiopulmonary bypass. N Engl J
Med 314:1446, 1986
3. Michelson AD: Pathomechanism of defective haemostasis
during and after extracorporeal circulation: The role of platelets, in
Hetzer R (ed): Blood Use in Cardiac Surgery, Darmstadt, Germany,
Steinkopff, 1990
4. Harker LA, Malpass TW, Branson HE, Hessel EA, Slichter SJ:
Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: Acquired transient platelet dysfunction associated
with selective a-granule release. Blood 565324, 1980
5. Bick RL: Hemostasis defects associated with cardiac surgery,
prosthetic devices, and other extracorporeal circuits. Semin
Thromb Hemost I1:249, 1985
6. Mammen EF, Koets MH, Washington BC, Wolk LW, Brown
JM, Burdick M, Selik NR, Wilson R F Hemostasis changes during
cardiopulmonary bypass surgery. Semin Thromb Hemost 11:28 I ,
1985
7. Rinder CS, Mathew JP, Rinder HM, Bonan J, Ault KA,
Smith B R Modulation of platelet surface adhesion receptors during cardiopulmonary bypass. Anesthesiology 75563, 1991
8. Rinder CS, Bohnert J, Rinder HM, Mitchell J, Ault K, Hillman R: Platelet activation and aggregation during cardiopulmonary bypass. Anesthesiology 75:388, 1991
9. Edmunds LH, Ellison N, Colman RW, Niewiarowski S, Rao
AK, Addonizio VP, Stephenson LW, Edie RN: Platelet function
during open heart surgery: Comparison to the membrane and bubble oxygenators. J Thorac Cardiovasc Surg 83:805, 1982
10. Mammen EF, Koets MH, Washington BC, Wolk LW,
Brown JM, Burdick M, Selik NR, Wilson RF: Hemostasis changes
during cardiopulmonary bypass surgery. Semin Thromb Hemost
281:292, 1985
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
116
11. Wachtfogel YT, Musial J, Jenkin B, Niewiarowski S, Edmunds LH, Colman RW: Loss of platelet cu,-adrenergic receptors
during simulated extracorporeal circulation: Prevention with prostaglandin E,. J Lab Clin Med 105:601, 1985
12. Holloway DS, Summaria L, Sandesara J, Vagher JP, Alexander JC, Caprini JA: Decreased platelet number and function and
increased fibrinolysis contribute to postoperative bleeding in cardiopulmonary bypass patients. Thromb Haemost 59:62, 1988
13. Wenger RK, Lukasiewicz H, Mikuta BS, Niewiarowski S,
Edmunds LH: Loss of platelet fibrinogen receptors during clinical
cardiopulmonary bypass. J Thorac Cardiovasc Surg 97:235, 1989
14. Nieuwenhuis HK, van Oosterhout JJG, Rozemuller E, van
Iwaarden F, Sixma JJ: Studies with a monoclonal antibody against
activated platelets: Evidence that a secreted 53,000-molecular
weight lysosome-like granule protein is exposed on the surface of
activated platelets in the circulation. Blood 70:838, 1987
15. Zilla P, Fasol R, Groscurth P, Klepetko W, Reichenspurner
H, Wolner E: Blood platelets in cardiopulmonary bypass. Recovery
occurs after initial stimulation, rather than continual activation. J
Thorac Cardiovasc Surg 97:379, 1989
16. Dechavanne M, Ffrench M, Pages J, Ffrench P, Boukerche
H, Bryon PA, McCregor JL: Significant reduction in the binding of
a monoclonal antibody (LYP 18) directed against the IIb-llla glycoprotein complex to platelets of patients having undergone extracorporeal circulation. Thromb Haemost 57: 106, 1987
17. George JN, Pickett EB, Saucerman S, McEver RP, Kunicki
TJ, Kieffer N, Newman PJ: Platelet surface glycoproteins. Studies
on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult
respiratory distress syndrome and cardiac surgery. J Clin Invest
78:340, 1986
18. Abrams CS, Ellison N, Budzynski AZ, Shattil SJ: Direct detection of activated platelets and platelet-derived microparticles in
humans. Blood 75:128, 1990
19. van Oeveren W, Harder MP, Roozendaal KJ, Eijsman L,
Wildevuur CRH: Aprotinin protects platelets against the initial effect of cardiopulmonary bypass. J Thorac Cardiovasc Surg 99:788,
1990
20. Vandenveld C, Fondu P, Dubois-Primo J: Low-dose aprotinin for reduction of blood loss after cardiopulmonary bypass.
Lancet 337:1157, 1991
21. Stenberg PE, McEver RP. Shuman MA, Jacques YV. Bainton DF: A platelet alpha-granule membrane protein (GMP-140) is
expressed on the plasma membrane after activation. J Cell Biol
101:880, 1985
22. Bevilacqua M, Butcher E, Furie B, Gallatin M, Gimbrone
M, Harlan J, Kishimoto K, Lasky L, McEver R, Paulson J, Rosen
S , Seed B, Siegelman M, Springer T, Stoolman L, Tedder T, Varki
A, Wagner D, Weissman I. Zimmerman G: Selectins: A family of
adhesion receptors. Cell 67:233, 199 I (letter)
23. Hsu-Lin S-C, Berman CL, Furie BC, August D, Furie B: A
platelet membrane protein expressed during platelet activation and
secretion. Studies using a monoclonal antibody specific for thrombin-activated platelets. J Biol Chem 259:912 I , 1984
24. Knapp W, Dorken B, Rieber P, Schmidt RE, Stein H, von
dem Borne AEGKr: CD antigens 1989. Blood 74:1448, 1989
25. Coller BS, Peerschke EI, Scudder LE, Sullivan CA: Studies
with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: Additional
evidence in support of GPlb as a platelet receptor for von Willebrand factor. Blood 6 1 :99, I983
26. Michelson AD, Loscalzo J, Melnick B, Coller BS, Handin
RI: Partial characterization of a binding site for von Willebrand
factor on glycocalicin. Blood 67: 19, 1986
KESTIN ET AL
27. Katagiri Y, Hayashi Y, Yamamoto K, Tanoue K, Kosaki G,
Yamazaki H: Localization of von Willebrand factor and thrombininteractive domains on human platelet glycoprotein Ib. Thromb
Haemost 63: 122, 1990
28. Du X, Beutler L, Ruan C, Castaldi PA, Berndt MC: Glycoprotein Ib and glycoprotein IX are fully complexed in the intact
platelet membrane. Blood 69:1524. 1987
29. Berndt MC, Chong BH, Bull HA, Zola H, Castaldi PA: Molecular characterization of quinine/quinidine drug-dependent antibody platelet interaction using monoclonal antibodies. Blood
66:1292, 1985
30. Gatter KC, Cordell JL, Turley H, Heryet A, Kieffer N, Anstee DJ, Mason DY: The immunohistological detection ofplatelets,
megakaryocytes and thrombi in routinely processed specimens. Histopathology l3:257, 1988
3 1. Coller BS: A new murine monoclonal antibody reports an
activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/llla complex. J Clin Invest 76:101, 1985
32. Coller BS, Peerschke El, Scudder LE, Sullivan CA: A murine
monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal
platelets and binds to glycoproteins IIb and/or IIIa. J Clin Invest
72:325, 1983
33. Jones D, Fritschy J, Carson J. Nokes TJC, Kemshead JT,
Hardisty RM: A monoclonal antibody binding to human medulloblastoma cells and to the platelet glycoprotein Ilb-IIIa complex. Br
J Haematol 57:62 I , 1984
34. Coller BS: Activation-specific platelet antigens, in Kunicki
TJ, George JN (eds): Platelet Immunobiology: Molecular and Clinical Aspects. Philadelphia. PA, Lippincott, 1989, p I66
35. Shattil SJ, Hoxie JA, Cunningham M, Brass L F Changes in
the platelet membrane glycoprotein IIb-llla complex during platelet activation. J Biol Chem 260:11107, 1985
36. Asch AS, Barnwell J, Silverstein RL, Nachman RL: Isolation
of the thrombospondin membrane receptor. J Clin Invest 79: 1054,
1987
37. Michelson AD, Ellis PA, Barnard MR, Matic GB, Viles AF,
Kestin AS: Downregulation of the platelet surface glycoprotein IbIX complex in whole blood stimulated by thrombin, ADP or an in
vivo wound. Blood 77:770. 1991
38. Shattil SJ, Cunningham M, Hoxie JA: Detection ofactivated
platelets in whole blood using activation-dependent monoclonal
antibodies and flow cytometry. Blood 70:307, 1987
39. Ault KA, Rinder HM, Mitchell JG, Rinder CS, Lambrew
CT, Hillman RS: Correlated measurement of platelet release and
aggregation in whole blood. Cytometry 10:448, 1989
40. Michelson AD, Barnard MR: Thrombin-induced changes in
platelet membrane glycoproteins Ib. IX, and IIb-IIIa complex.
Blood 70:1673, 1987
41. Yamamoto N, Greco NJ, Barnard MR, Tanoue K, Yamazaki H, Jamieson GA, Michelson AD: Glycoprotein Ib (GP1b)-dependent and GPIb-independent pathways of thrombin-induced
platelet activation. Blood 77: 1740, 199 I
42. Marcus AJ: Platelet eicosanoid metabolism, in Colman RW,
Hirsh J, Marder VJ, Salzman EW (eds): Hemostasis and Thrombosis. Basic Principles and Clinical Practice. Philadelphia, PA, Lippincott, 1987, p 676
43. Valeri CR, Cassidy G, Khuri S, Feingold H, Ragno G, Altschule MD: Hypothermia-induced reversible platelet dysfunction.
Ann Surg 205: 175, 1987
44. Michelson AD, Adelman B. Barnard MR, Carroll E, Handin
RI: Platelet storage results in a redistribution of glycoprotein Ib
molecules. Evidence for a large intraplatelet pool ofglycoprotein Ib.
J Clin Invest 8 1 :1734, I988
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
PLATELET FUNCTION IN CARDIOPULMONARY BYPASS
45. Mendelsohn ME, ONeill S, George D, Loscalzo J: Inhibition of fibrinogen binding to human platelets by S-nitroso-N-acetylcysteine. J Biol Chem 265:19028, 1990
46. George JN, Thoi LL, Morgan RK: Quantitative analysis of
platelet membrane glycoproteins: Effect of platelet washing procedures and isolation of platelet density subpopulations. Thromb Res
23:69, 1981
47. Ruggeri ZM: The platelet glycoprotein Ib-IX complex. Prog
Hemost Thromb 10:35, 1991
48. Coller BS, Kalomiris E, Steinberg M, Scudder LE: Evidence
that glycocalicin circulates in normal plasma. J Clin Invest 73:794,
1984
49. Phillips DR, Charo IF, Parise LV, Fitzgerald LA: The platelet membrane glycoprotein IIb-IIIa complex. Blood 7193 1, 1988
50. Novotny WF, Girard TJ, Miletich JP, Broze GJ, Jr.: Platelets
secrete a coagulation inhibitor functionally and antigenically similar to the lipoprotein associated coagulation inhibitor. Blood
72:2020, 1988
5 I. Weiss HJ, Lages B: Evidence for tissue factor-dependent activation of the classic extrinsic coagulation mechanism in blood
obtained from bleeding time wounds. Blood 71:629, 1988
52. Kyrle PA, Stockenhuber F, Brenner B, Gossinger H, Korninger C, Pabinger I, Sunder-Plassman G, Balcke P, Lechner K:
Evidence for an increased generation of prostacyclin in the microvasculature and an impairment of the platelet a-granule release in
chronic renal failure. Thromb Haemost 60:205, 1988
53. Mohr R, Golan M, Martinowitz U, Rosner E, Goor DA,
Ramot B: Effect of cardiac operation on platelets. J Thorac Cardiovasc Surg 92:434, 1986
54. Kinlough-Rathbone RL, Packham MA, Mustard JF: Platelet aggregation, in Harker LA, Zimmerman TS (eds): Measurements of Platelet Function. New York, NY, Churchill Livingstone,
1983, p 64
55. George JN, Shattil SJ: The clinical importance of acquired
abnormalities of platelet function. N Engl J Med 324:27, 1991
56. Abrams CS, Shattil SJ: Immunological detection ofactivated
platelets in clinical disorders. Thromb Haemost 65:467, 199 1
57. Corash L Measurement of platelet activation by fluorescence-activated flow cytometry. Blood Cells I60:97, 1990
58. Aren C, Feddersen K, Radegran K: Effects of prostacyclin
infusion on platelet activation and postoperative blood loss in coronary bypass. Ann Thorac Surg 36:49, 1983
59. Mezzano D, Aranda E, Urzua J, Lema G, Habash J, Irarrazabal MJ, Pereira J: Changes in platelet /3-thromboglobulin, fibrinogen, albumin, 5-hydroxytryptamine, ATP, and ADP during and
after surgery with extracorporeal circulation in man. Am J Hematol
22:133, 1986
60. Colman RW: Platelet and neutrophil activation in cardiopulmonary bypass. Ann Thorac Surg 49:32, 1990
117
61. Rinder HM, Murphy M, Mitchell JG, Stocks J, Ault KA,
Hillman RS: Progressive platelet activation with storage: evidence
for shortened survival of activated platelets after transfusion.
Transfusion 3 1:409, 199 1
62. Larsen E, Celi A, Gilbert GE, Furie BC, Erban JK, Bonfanti
R, Wagner DD, Furie B: PADGEM protein: A receptor that mediates the interaction of activated platelets with neutrophils and
monocytes. Cell 59:305, 1989
63. Hamburger SA, McEver RP: GMP-140 mediates adhesion
of stimulated platelets to neutrophils. Blood 75550, 1990
64. Levine S P Secreted platelet proteins as markers for pathological disorders, in Phillips DR, Shuman MA (eds): Biochemistry of
Platelets. Orlando, FL, Academic, 1986, p 378
65. Hansen SR, Harker LA: Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin PPACK. Proc
Natl Acad Sci USA 85:3184, 1988
66. Eidt JF, Allison P, Nobel S, Ashton J, Golino P, McNatt J,
Buja LM, Willerson J: Thrombin is an important mediator ofplatelet aggregation in stenosed coronary arteries. J Clin Invest 84: 18,
1989
67. Kelly AB, Marzec UM, Krupski W, Bass A, Cadroy Y, Hanson SR, Harker LA: Hirudin interruption of heparin-resistant arterial thrombus formation in baboons. Blood 77: 1006, 199 I
68. Weitz JI, Huduba M, Massel D, Maragnore J, Hirsh J: Clotbound thrombin is protected from inhibition by heparin-antithrombin 111 but is susceptible to inactivation by antithrombin
111-independentinhibitors. J Clin Invest 86:385, 1990
69. Badimon L, Badimon JJ, Lassila R, Heras M, Chesebro JH,
Fuster V: Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model: Effects of hirudins, heparin, and calcium
chelation. Blood 78:423, 1991
70. Khuri SF, Wolfe JA, Josa M, Axford TC, Szymanski I, Assousa s,Ragno G, Pate1 M, Silverman A, Park M, Valeri CR: Hematologic changes during and after cardiopulmonary bypass and their
relationship to the bleeding time and nonsurgical blood loss. J
Thorac Cardiovasc Surg 104:94, 1992
7 I . Valeri CR, Kabbaz K, Khuri S, Marquardt C, Ragno G,
Feingold H, Gray A, Axford T: Effect of skin temperature on platelet function in extracorporeal bypass patients. J Thorac Cardiovasc
Surg 104:108, 1992
72. Michelson AD, MacGregor H, Kestin AS, Barnard MR,
Rohrer MJ, Valeri CR: Hypothermia-induced reversible inhibition
of human platelet activation in vitro and in vivo. Blood 78:389a,
1991 (suppl I , abstr)
73. Kowalski E, Kopec M, Wegrzynowicz Z: Influence of fibrinogen degradation products (FDP) on platelet aggregation, adhesiveness, and viscous metamorphosis. Thromb Diath Haemorrh
10:406, 1963
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
1993 82: 107-117
The platelet function defect of cardiopulmonary bypass [see
comments]
AS Kestin, CR Valeri, SF Khuri, J Loscalzo, PA Ellis, H MacGregor, V Birjiniuk, H Ouimet, B
Pasche and MJ Nelson
Updated information and services can be found at:
http://www.bloodjournal.org/content/82/1/107.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.