(Circulation. 1997;96:3860-3866.)
© 1997 American Heart Association, Inc.
Articles |
Rapid Assessment of Platelet Function With a Modified Whole-Blood Aggregometer in Percutaneous Transluminal Coronary Angioplasty Patients Receiving Anti-GP IIb/IIIa Therapy
From the Pharmaceutical Division, Centocor Inc, Malvern, Pa (M.A.M., E.L., S.M., T.S., H.F.W., R.J.); Chrono-log Corp, Havertown, Pa (N.J.V.); and the Lancaster Heart Foundation, Lancaster, Pa (S.W.).
Correspondence to Mary Ann Mascelli, PhD, Department of Clinical Pharmacology, Centocor, Inc, Malvern, PA 19355-1307.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background The glycoprotein (GP) IIb/IIIa receptor antagonist abciximab (c7E3 Fab, ReoPro) is approved for use in high-risk percutaneous transluminal coronary angioplasty (PTCA). At present, no "point of care" exists for measuring pharmacological GP IIb/IIIa blockade. To address this need, the Chrono-log Whole Blood Aggregometer, which measures platelet aggregation by electrical impedance, was adapted to test platelet function at the bedside.
Methods and Results GP IIb/IIIa receptor blockade, impedance (5
µg/mL collagen), and turbidimetric aggregation (5 and 20
µmol/L ADP) measurements were obtained on 14 PTCA patients who
received the standard bolus plus a 12-hour infusion of abciximab.
During abciximab administration, mean GP IIb/IIIa receptor blockade was
>91%, and both impedance and turbidimetric aggregation were inhibited
by 
90%. At 12 hours after abciximab treatment, the mean inhibition
of turbidimetric platelet aggregation to 5 and 20 µmol/L ADP
was 65±20% and 49±14%, respectively, and inhibition of impedance
aggregation was 69±12%. GP IIb/IIIa receptor blockade was 67±8%. At
36 hours after abciximab treatment (n=8), the mean inhibition of
turbidimetric platelet aggregation to 5 and 20 µmol/L ADP
was 44±21% and 30±14%, respectively, whereas impedance aggregation
was inhibited by 60±14%. GP IIb/IIIa receptor blockade was
57±7%.
Conclusions During and at 12 hours after abciximab therapy, impedance and turbidimetric platelet aggregation to 5 µmol/L ADP were comparable and closely correlated with GP IIb/IIIa receptor blockade. However, at 36 hours after abciximab treatment, impedance platelet aggregation more closely paralleled GP IIb/IIIa receptor blockade and indicated a slower recovery of platelet function than turbidimetric aggregometry.
Key Words: platelet aggregation inhibitors • glycoproteins • receptors
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The antiplatelet agent abciximab (c7E3 Fab, ReoPro) reduces ischemic complications in patients undergoing high-risk PTCA1 and is approved for use in patients undergoing PTCA who are at high risk of abrupt closure. Abciximab is a human-murine chimeric Fab fragment of the monoclonal antibody 7E3 that was derived from immunization of a mouse with human platelets.2 The drug inhibits platelet function by engaging the GP IIb/IIIa receptor, thereby preventing the binding of adhesive ligands, such as fibrinogen and von Willebrand factor, to activated platelets. The use of abciximab, as well as the continuing development of other parenteral3 and oral4 GP IIb/IIIa antagonists, may necessitate rapid and accurate measurement of platelet function to optimize and monitor the effects of these therapies. In patients receiving abciximab5,6 and in animal models of coronary thrombosis,7–9 the ex vivo platelet aggregation response was virtually abolished when GP IIb/IIIa receptor blockade was maintained
80%. However, in some
individuals, differences in peripheral and total body
platelet count as well as in the number of GP IIb/IIIa receptors
per platelet may influence optimal dosing of a GP IIb/IIIa
antagonist. Consequently, it may be advantageous to monitor
antiplatelet therapy to ensure that a therapeutic effect has been
achieved initially and is sustained throughout the course of treatment.
Also, under certain circumstances after discontinuation of therapy, it
may be important to know if or when platelet function has been
restored. Currently available methods for assessing platelet function (eg, light transmittance aggregometry, bleeding time) were developed primarily to detect inherited and acquired platelet abnormalities and are not readily adaptable to a point-of-care setting. Bleeding time measurements are impractical in an intensive care or catheterization laboratory setting because of the need for prolonged monitoring periods under circumstances of profound GP IIb/IIIa blockade, and they are not highly reproducible.10 Major limitations of the current light transmittance (turbidimetric) platelet aggregation assays are the multicomponent equipment requirements, the relatively long time required to perform these analyses, and the need for technologists experienced in preparation of PRP and cell counting techniques. In contrast, electrical impedance aggregometry requires no cell separations and minimal preparation time (only 1:1 dilution of the blood with saline and a 5-minute incubation before the initiation of the assay) and is an approved clinical method for evaluating platelet function.11–13 The technique measures aggregation as an increase in the electrical impedance across two precious metal wires resulting from the accumulation of platelets in response to an agonist.14 Impedance aggregation can be completed within 15 minutes after a blood sample is obtained, and the method provides accurate results up to 3 hours after the sample has been attained.15 Comparisons of turbidimetric and impedance aggregation responses on blood samples from healthy donors show a good correlation between the techniques.15
The purpose of the present study was to evaluate electrical impedance aggregometry for measuring inhibition of platelet function produced by the pharmacological blockade of the platelet GP IIb/IIIa receptor in PTCA patients receiving standard abciximab therapy. Whole-blood aggregation was measured with a modified version of the Chrono-log Whole Blood Aggregometer that has been engineered with automated calibration and readout functions that minimize the amount of operator attention and time. The sensitivity of electrical impedance aggregation for measuring the effects of abciximab on platelet function was compared with the standard turbidimetric platelet aggregation technique. Estimates of platelet function determined by both aggregation techniques were compared to the extent of GP IIb/IIIa receptor blockade.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Reagents
ADP and collagen were purchased from Chrono-log. Stabilized isotonic saline was from Baxter. The modified whole-blood platelet aggregometer was designed by Chrono-log Corp. All supplies needed for performing whole-blood aggregation (ie, cuvettes, stir bars) were purchased from Chrono-Log Corp. Microsequencing pipette tips (Seratips) were purchased from Marsh. Turbidimetric aggregation measurements were performed on a PAP-4C aggregometer (BioData Inc). The 11-amino-acid TRAP16 was synthesized at Centocor.
Study Populations
The study was conducted in two stages. The first stage was used
to establish an appropriate agonist for use in the aggregation studies.
The second stage was conducted to validate the use of electrical
impedance aggregation in patients treated with abciximab who were
undergoing PTCA. GP IIb/IIIa receptor blockade, turbidimetric, and
electrical impedance aggregation measurements were performed on both
study groups. Electrical impedance aggregation measurements were
completed within 30 minutes of sample collection. Turbidimetric
platelet aggregation and GP IIb/IIIa receptor blockade measurements
were normally completed within 1 and 2 hours, respectively, after the
blood samples were obtained. All platelet function measurements
were performed in citrate anticoagulant because independent studies
established that the binding and inhibitory profile of
abciximab were not affected by the presence of chelating anticoagulant
(unpublished observations).
Stage 1: Electrical Impedance Agonist Selection Study
The electrical impedance platelet aggregation
responses to the agonists ADP (20 µmol/L) and TRAP
(6 µmol/L) were obtained in a group of 8 subjects who
were given abciximab. The electrical impedance aggregation response to
collagen was also obtained in 2 of these patients for 72 hours after
abciximab administration. All subjects received the 0.25-mg/kg
bolus and the 12-hour infusion of abciximab. Three individuals received
a 10-µg/min infusion, and 5 received a 0.125-µg ·
kg-1 · min-1
infusion regimen. All individuals received an oral dose of aspirin (325
mg) 
12 hours before abciximab administration. The study was approved
by the institutional review board of the National Medical Research
Corp, and all patients gave written informed consent.
Stage 2: Electrical Impedance Aggregation Assessment Study
The trial was a single-center, open-label, nonrandomized study.
Individuals participating in the trial were patients undergoing
high-risk PTCA and received standard abciximab therapy (bolus dose of
0.25 mg/kg plus a 12-hour infusion of 10 µg/min). The
study was approved by the institutional review board of Lancaster
General Hospital, and all patients gave written informed consent.
PTCA Procedure
Patients were pretreated with aspirin (325 mg) before the PTCA
procedure. Within 15 minutes before balloon inflation, bolus increments
of heparin were given to achieve an ACT of >300 seconds. After the
procedure, heparin was continued at the discretion of the clinician;
however, an infusion of 1000 IU/h was normally continued for 24 hours
after the procedure and was adjusted to maintain the activated
partial thromboplastin time within a range of 50 to 70 seconds. Within
1 to 2 hours after the procedure, the heparin infusion was temporarily
discontinued to pull the arterial sheath, which was
accomplished after the ACT was <180 seconds.
Blood Collection
Stage 1: Agonist Selection Study
Blood samples were collected at five time points: predose
and 2, 6, 12, and 24 hours after abciximab bolus (12 hours after
infusion). Blood samples were drawn through the cap of the in-dwelling
catheter through an 18-gauge needle into polypropylene syringes
containing 1/100 volume of 40% trisodium citrate. Blood (5 mL) was
drawn from the in-dwelling catheter and discarded before the test
samples were obtained. After collection, the syringes were gently
inverted two or three times to ensure complete mixing of the
anticoagulant. After each blood draw, the catheter was flushed with 3
mL of normal saline solution and reflushed every 2 to 3 hours with
saline until it was removed.
Stage 2: Electrical Impedance Aggregation Assessment Study
Blood samples were collected at five designated times:
immediately before administration of abciximab but after the bolus dose
of heparin was received, after abciximab bolus and 5 minutes before the
first balloon inflation, 2 hours after abciximab bolus, 24 hours after
abciximab bolus (12 hours after abciximab infusion), and 48 hours after
abciximab bolus (36 hours after abciximab infusion). The predose sample
was drawn 5 minutes before administration of the abciximab bolus, and
the postabciximab, preballoon inflation blood samples were drawn within
10 to 15 minutes after the bolus dose of the drug was received. During
times in which heparin dosing was being monitored (ie, the first three
time points), blood samples were collected from the same access site as
samples drawn for ACT measurements. Otherwise, blood was drawn by
direct venipuncture from a peripheral vessel.
Samples were collected into standard blue-top vacutainers (citrate
anticoagulant). After collection, the blood tubes were gently inverted
two or three times to ensure complete mixing of the anticoagulant.
Preparation of PRP and Platelet-Poor Plasma
PRP was prepared by centrifugation at
600g for 10 minutes in a Sorvall GLC4 tabletop
centrifuge. Platelet-poor plasma was prepared by spinning
the remaining blood at 1200g for 15 minutes. The
platelet count of the PRP was determined by use of a Coulter
counter ZM resistance particle counter. Platelet counts of the PRP
were adjusted to 200 000±25 000 cells per µL with autologous
platelet-poor plasma, except for samples (n=9) with platelet
counts 
175 000 cells per µL, in which no adjustments were
made.
Platelet Aggregation Measurements
Light Transmittance Technique
Platelet aggregations of PRP samples were evaluated by the
turbidimetric method.17 PRP samples (500 µL)
were prewarmed to 37°C for 10 minutes before the aggregation studies
were performed. For each PRP sample, the extent of light transmittance
was monitored for 1 minute to ensure the absence of autoaggregation.
Next, ADP was added to the cuvette (final concentration, 5 or 20
µmol/L), and aggregation response was monitored for 4 minutes.
The extent of aggregation was defined as the maximal amount of light
transmittance reached within 4 minutes after addition of the agonist.
For each postabciximab administration sampling time and for each ADP
concentration used, the percent of baseline aggregation was determined
by the following calculation: maximum change in light transmission of
test sample divided by the maximum change in light transmission of
baseline sample. Finally, the product of the above calculation was
multiplied by 100.
Impedance Method
Electrical impedance aggregation measurements were
performed on the modified whole-blood aggregometer, which was equipped
with automated calibration and readout functions. The instrument has
received a premarket 510(k) notification from the Food and Drug
Administration Center for Devices and Radiologic Health, indicating
that the modified Whole Blood Aggregometer is equivalent to the
Chrono-log Corp Whole Blood Aggregometer (model
540).18 The technicians followed manufacturer
instructions for proper cleaning and maintenance of the
electrode. An aliquot of whole blood (0.5 mL) was diluted with an
equivalent volume of isotonic saline and incubated for 5 minutes at
37°C. The impedance of each sample was monitored in sequential
1-minute intervals until a stable baseline was established (<5 mV
drift per minute). After a stable baseline was established, the agonist
was then added to the sample, aggregation was monitored for 6 minutes,
and the final increase in ohms over this period was displayed as a
numeric LED readout. In addition, a graphical printout (ie, chart
tracing) of each electrical impedance aggregometry tracing was
obtained. For each postabciximab administration sampling time, the
percent of baseline aggregation was determined by the following
calculation: maximum change in ohms of test sample divided by the
maximum change in ohms of the baseline sample. Finally, the product of
the above calculation was multiplied by 100.
GP IIb/IIIa Radioimmune Assay
A direct-binding radioimmune assay was used to measure the
extent of abciximab-mediated GP IIb/IIIa receptor
blockade.2 Briefly, PRP was incubated with
125I-labeled murine 7E3 IgG (18 µg/mL)
for 30 minutes at room temperature. Replicate3
100-µL aliquots of the PRP incubation were then centrifuged
through 200 µL of 30% sucrose to separate free from
platelet-bound antibody. The number of molecules of antibody per
platelet was calculated as the fraction of platelet-bound
125I-m7E3 IgG times the total antibody
concentration (1.8 µg/mL) times the number of molecules per 1
µg (Avogadro's number divided by the molecular weight of m7E3 IgG;
155 000). Finally, the previous product of the calculation was
divided by the number of platelets in the sample.
Statistical Analysis
Patient demographics and baseline platelet function data are
expressed as means, medians, ranges, and ±1 SD of the mean. For
continuous measurements, the primary descriptive statistics were means
and ±1 SD of the mean. An ANOVA was applied to assess for differences
between GP IIb/IIIa receptor blockade, turbidimetric, and electrical
impedance aggregation measurements.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Stage 1: Electrical Impedance Agonist Selection Study
The electrical impedance aggregation responses to the agonists ADP, TRAP and collagen, the light transmittance aggregation responses to ADP and GP IIb/IIIa receptor blockade measurements were obtained on eight subjects who received a 0.25-mg/kg bolus and a 12-hour infusion of abciximab. The mean levels of GP IIb/IIIa receptor blockade, inhibition of the electrical impedance, and turbidimetric aggregation responses for the test population are summarized in Fig 1
. During abciximab treatment, mean GP
IIb/IIIa receptor blockade was maintained above 80%, and the light
transmittance platelet aggregation response to both 5 and 20
µmol/L ADP was 80% inhibited, except at 2 hours, when
average percent inhibition with 20 µmol/L ADP was 65%.
Also, throughout abciximab treatment, average level of inhibition of
the impedance platelet aggregation responses to ADP and TRAP was
100%. At 12 hours after abciximab infusion, the average level of GP
IIb/IIIa receptor blockade dropped to 65%, and the level of inhibition
of turbidimetric aggregation to 5 and 20 µmol/L ADP was
65% and 56%, respectively. However, no recovery in the impedance
aggregation response to either TRAP or ADP was detected.
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-] and 6 µmol/L [-
-] TRAP) and
turbidimetric (5 µmol/L [-
-] and 20 µmol/L [-
-]
ADP) platelet aggregation as well as GP IIb/IIIa receptor blockade
[-
-] in eight stable subjects who received the 0.25-mg/kg bolus
and a 12-hour infusion of abciximab. Five subjects received a
0.125-µg · kg-1 · min-1
infusion, and three received a 10-µg/min infusion of abciximab.
Degree of GP IIb/IIIa receptor blockade is expressed as the percentage
of baseline GP IIb/IIIa receptors that are blocked by abciximab. Levels
of inhibition of electrical impedance and light transmission
aggregation are expressed as the percent inhibition of aggregation
present after abciximab administration. Results represent
the mean values at each time point.
In addition to ADP and TRAP, the impedance aggregation response
to collagen was obtained on 2 patients (Fig 2). In both individuals, GP IIb/IIIa
receptor blockade was maintained >80%, and both turbidimetric
aggregation responses to ADP and the impedance aggregation responses to
all agonists were profoundly inhibited during abciximab administration.
At 12 hours after abciximab treatment, when GP IIb/IIIa receptor
blockade in both patients was 68%, comparable decreases were observed
in the inhibition of the turbidimetric aggregation responses to 5 and
20 µmol/L ADP (72% and 70% inhibition, respectively)
and the electrical impedance aggregation responses to 5 µg/mL
collagen (72% inhibition). At this time, however, the impedance
aggregation response to both TRAP and ADP were still completely
inhibited. At 72 hours after treatment, inhibition of the impedance
aggregation response to collagen decreased to 20%. Because collagen
was the most sensitive agonist for detecting recovery of platelet
function by impedance aggregometry, it was chosen for monitoring the
impedance aggregation response in abciximab-treated PTCA patients in
the second stage of the study.
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]) and turbidimetric (5 µmol/L [-
Stage 2: Electrical Impedance Aggregation Assessment Study
Patient Demographics and Baseline Platelet Function
Data
Eleven men and 3 women were enrolled in the study. The average age
of the group was 66 years (range, 43 to 80 years). The average weight
for the group was 85 kg (range, 66 to 137 kg). The baseline values for
platelet aggregation, abciximab binding sites per platelet, and
platelet count for the study population (n=16) are shown in the
Table. The baseline platelet counts
of each patient were within the normal range of 150 000 to 450 000
cells per µL. The mean platelet count was 228 000 cells per µL
and ranged from 150 000 to 337 000 cells per µL. The average number
of abciximab receptors was 101 200±26 400 molecules per
platelet, matching previously reported
estimates.19 The predose platelet samples
from each patient generated a vigorous turbidimetric aggregation
response to both concentrations of ADP. The mean percent increases in
light transmittance to 5 and 20 µmol/L ADP were 63±7.8%
and 73±6.9%, respectively.
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Pharmacodynamic Responses
In contrast to the study group in stage 1, cardiac patients
undergoing PTCA are routinely administered heparin and other
medications before and during the intervention. Therapeutic levels of
heparin used during PTCA have been shown both in vitro and ex vivo to
enhance platelet reactivity.20,21 Because
heparin and other concomitant medications that are normally
administered to PTCA patients might alter platelet function, all
baseline pharmacodynamic measurements were performed on blood that was
drawn 5 minutes after a bolus administration of heparin. In addition,
baseline electrical impedance aggregation responses to a number of
collagen concentrations were measured to identify the minimal collagen
concentration that would elicit the maximal aggregation response in
PTCA patients. The results are shown in the Table
, in which the level
of aggregation (measured as impedance) is correlated with the amount of
collagen agonist. A 50% increase (18 versus 27 ohms) in the mean
baseline platelet aggregation response was observed with 5
µg/mL compared with 2.5 µg/mL collagen. Doubling the
concentration of collagen resulted in an additional, yet smaller,
increase (13%; 31 verses 27 ohms) in the extent of the aggregation
response. Because near-maximal electrical impedance aggregation was
achieved with 5 µg/mL collagen, the electrical impedance
aggregation results of all postabciximab treatment samples to 5
µg/mL of collagen are presented.
The turbidimetric (5 µmol/L ADP), and impedance (5
µg/mL collagen) aggregometry tracings of a
representative patient are shown in Fig 3. Before abciximab treatment, the
platelets generate a rapid and vigorous aggregation response in
both the turbidimetric and electrical impedance methods. In contrast,
during abciximab therapy (preballoon inflation and 2 hours after
bolus), both turbidimetric and impedance aggregation responses were
almost or completely abolished, as illustrated by virtually no increase
in either light transmittance or ohms during the aggregation monitoring
period. Twelve hours after cessation of abciximab therapy (24 hours
after abciximab bolus), partial recovery of platelet aggregation
was detected with both aggregation methods, and platelet function
continued to increase at 36 hours after cessation of therapy. However,
it should be noted that neither aggregation response recovered to
baseline at 36 hours after therapy (48 hours after bolus).
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The effect of abciximab administration on GP IIb/IIIa receptor
blockade, ex vivo electrical impedance, and turbidimetric platelet
aggregation are illustrated in Fig 4.
Immediately before device activation (15 minutes after abciximab
bolus), mean GP IIb/IIIa receptor blockade was 96±2.1% and ranged
from 91% to 99%. The light transmittance platelet aggregation
responses to 5 and 20 µmol/L ADP were inhibited 98±2.6%
and 94±2.5%, respectively. Comparable levels of inhibition of
platelet aggregation to collagen were also observed with impedance
aggregation (96±4.5%). The differences between the amount of
inhibition of impedance aggregation and the levels of inhibition of
turbidimetric aggregation to both concentrations of ADP
(P=.742 [5 µmol/L ADP] and 0.133 [20
µmol/L ADP], respectively), as well as the extent of GP
IIb/IIIa receptor blockade (P=.971), were not statistically
significant. These results illustrate that both the turbidimetric and
impedance aggregation responses in PTCA patients treated with the
standard dose of abciximab were well inhibited immediately before
coronary intervention.
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Comparable levels of inhibition of platelet function were also observed after 2 hours of abciximab administration. The mean level of GP IIb/IIIa receptor blockade was 92±3.1% and ranged from 86% to 96%. At this time, the turbidimetric platelet aggregation responses to 5 and 20 µmol/L ADP were inhibited by 98±2.3% and 90±3.7%, respectively. Likewise, the impedance platelet aggregation response to collagen was inhibited by 90±8.6%.
The recovery in platelet function was also assessed after cessation of abciximab treatment. At 12 hours after abciximab administration, the level of GP IIb/IIIa receptor blockade (67±8.3%) was comparable to the extent of inhibition of platelet aggregation observed with the impedance method (69±12.7%) and turbidimetrically with 5 µmol/L ADP (65±19.9%). The differences between the level of inhibition of impedance and turbidimetric aggregation responses to 5 µmol/L ADP (P=.918), as well as the level of GP IIb/IIIa receptor blockade (P=.983), were not statistically significant. At this time, however, the turbidimetric platelet aggregation response to 20 µmol/L ADP was less inhibited (49±14.2%) and was significantly different from the level of electrical impedance aggregation (P=.004).
By 36 hours after abciximab treatment (48 hours after abciximab bolus), the mean level of inhibition of impedance aggregation (60±14.7%) again coincided with the extent of GP IIb/IIIa receptor blockade (57±7.7%), and the differences between these measurements were not statistically significant (P=.983). However, a higher degree of recovery of platelet function was detected by the turbidimetric method with both concentrations of ADP. The turbidimetric platelet aggregation responses to 5 and 20 µmol/L ADP were inhibited by 44±21% (P=.193) and 30±15% (P=.004), respectively. Therefore, after cessation of abciximab therapy, the extent of GP IIb/IIIa receptor blockade correlated closely with the level of inhibition of platelet function as measured by electrical impedance. However, turbidimetric platelet aggregometry measurements recorded a greater degree of platelet functional recovery after abciximab treatment than either the recovery of GP IIb/IIIa receptor blockade or platelet aggregation as measured by electrical impedance.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The purpose of the present study was to assess the feasibility of using an electrical impedance aggregometer as a point-of-care instrument for monitoring platelet function in patients undergoing PTCA who received the potent antiplatelet agent abciximab. Impedance aggregation measurements were performed by use of standard instrumentation that was adapted for use at the bedside. The study was designed to compare electrical impedance platelet aggregation to both the radiometric GP IIb/IIIa receptor blockade assay and ADP-mediated turbidimetric platelet aggregation, which have been broadly used to monitor the pharmacodynamic effects of abciximab in both animal7–9 and clinical studies.5,6 On the basis of these previous studies, the therapeutic target level for coronary artery angioplasty was defined as
80% GP IIb/IIIa receptor blockade. The
results of the present study show that in the patient population
evaluated, electrical impedance and turbidimetric methods yielded
comparable levels of inhibition of platelet aggregation during
abciximab treatment. Profound inhibition of both light transmittance
and electrical impedance aggregation was observed during abciximab
treatment when GP IIb/IIIa receptor blockade was >90% inhibited.
Inhibition of both electrical impedance and turbidimetric platelet
aggregation was observed within 15 minutes of abciximab administration,
thereby illustrating the rapid onset of the pharmacological effects of
the drug. The level of inhibition of platelet function with both
aggregation techniques was sustained at 2 hours after abciximab bolus.
These observations suggest that electrical impedance aggregation can be
used by clinicians to monitor the antiplatelet effects of abciximab
to ensure that a therapeutic benefit has been attained initially and is
sustained during the 12-hour course of treatment.
In the posttreatment phase, impedance and light transmittance
aggregation both displayed a gradual recovery in platelet function.
However, the level of turbidimetric aggregation recovered to a faster
extent than the decline in GP IIb/IIIa blockade, and the rate of
recovery was proportional to the strength of the agonist. In contrast,
recovery of electrical impedance aggregation was slower during the
postabciximab infusion phase and closely paralleled the extent of
GP IIb/IIIa receptor blockade. Twelve hours after cessation of
abciximab therapy, when GP IIb/IIIa receptor blockade was 67%, the
extent inhibition of platelet function with electrical impedance
(69%) and turbidimetric aggregation to 5 µmol/L ADP
(65%) were equivalent. At 36 hours after therapy, inhibition of
electrical impedance aggregation was again comparable to the level of
GP IIb/IIIa receptor blockade, while lower levels of inhibition of
turbidimetric platelet aggregation were detected with both 5 and
20 µmol/L concentrations of ADP. Because the degree of
recovery of electrical impedance aggregation more closely matched the
amount of GP IIb/IIIa receptor blockade, the electrical impedance
aggregation response seemed more sensitive to the
inhibitory effects of abciximab than turbidimetric
platelet aggregation and may be a more precise
physiological indicator of platelet activity.
This increased sensitivity at intermediate GP IIb/IIIa receptor
blockade is not unique to electrical impedance aggregation. The effects
of abciximab administration on shear-induced formation of large
platelet aggregate formation were still apparent 1 week after
treatment, whereas light transmittance aggregation responses to high
levels of ADP (20 µmol/L) are normally fully restored
within 36 to 48 hours after treatment.22 Thus,
although all methods of assessment indicate nearly complete inhibition
of platelet function during abciximab treatment, the different
methods of platelet function testing lead to different estimates
for the extent and duration of inhibition after cessation of treatment.
The increased sensitivity of electrical impedance aggregometry to the
antiplatelet effects of abciximab was not the result of
platelets receiving less agonist stimulation than in turbidimetric
measurements. In the postabciximab treatment phase of the agonist
selection (stage 1) study, the same concentration of ADP (20
µmol/L) reported a partial recovery of platelet function
turbidimetrically, whereas no restoration of platelet aggregation
was observed with impedance aggregometry (Fig 1). The concentration of
collagen used in the study was shown to elicit the maximal impedance
aggregation response and release of dense granule constituents in
platelets from normal human donors.13 In
addition, 5 µg/mL of collagen demonstrated near-maximal
impedance aggregation responses from the test population. Collagen,
unlike ADP, also is classified as a strong platelet agonist in that
it can stimulate granule release in the absence of
platelet-platelet contact,23 and an in
vitro dose titration study demonstrated that abciximab elicited
comparable platelet aggregation inhibitory capacities
by light transmission aggregation to both 5 µg/mL collagen and
5 µmol/L ADP (data not shown). Rather, it is more likely
that technical differences between the two aggregation techniques are
responsible for the different estimations of antiplatelet effects
of abciximab during the posttreatment recovery phase. Turbidimetric
platelet aggregation requires the separation of platelets from
other blood cells that also may play an important role in modulating
platelet behavior. Leukocytes release
prostacyclin24 and platelet activation
factor,25,26 and erythrocytes have receptors for
prostacyclin27 and release
ADP.28 In addition, the extensive sample
preparation that is required for turbidimetric platelet aggregation
may result in the degradation of short half-life mediators (ie,
prostacyclin and thromboxane A2).
Finally, the heterogeneous size and density of circulating
platelets make it likely that subpopulations of platelets may
be lost during the centrifugation step for preparing
PRP.29 In contrast, impedance aggregometry
requires no cell separation and minimal preparation time, and
platelets are tested in a more physiological
medium containing all blood cells that could modulate platelet
behavior. Taken in their entirety, inherent differences of impedance
and turbidimetric aggregation techniques could explain the dissimilar
recovery rates of platelet function under conditions of
intermediate GP IIb/IIIa blockade. Because electrical impedance
aggregometry requires less sample manipulation than turbidimetric
platelet aggregation, it may be more convenient for assessing the
pharmacological effects of abciximab during treatment and may more
accurately reflect the persistence of
physiologically significant GP IIb/IIIa
receptor blockade.
In conclusion, electrical impedance aggregometry is a rapid, simple, and accurate method for measuring the pharmacological effects of abciximab. The assay, therefore, may be useful for monitoring abciximab and other antiplatelet therapies. The increased sensitivity of electrical impedance aggregation at intermediate GP IIb/IIIa receptor blockade may be a more accurate reflection of platelet function in vivo than turbidimetric platelet aggregation. Electrical impedance aggregation may be a useful tool for the physician who wants to assess the degree of platelet function in an abciximab-treated patient requiring either emergency coronary bypass surgery or platelet transfusion.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We would like to thank Joann Tuzi, RN, Lou Ann Frey, RN, and Kay Nepper, RN, of the Mid Atlantic Heart Institute for their help in coordinating the study and Lakshmi Damaraju, PhD, for performing the statistical analyses.
Received May 15, 1997; revision received August 14, 1997; accepted August 22, 1997.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The EPIC Investigators. Use of a monoclonal
antibody directed against the platelet glycoprotein
IIb/IIIa receptor in high-risk coronary angioplasty.
N Engl J Med. 1994;330:956–961.
[Abstract/Free Full Text]
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Coller BS. A new murine monoclonal antibody reports an
activation-dependent change in the conformation and/or micro
environment of the platelet glycoprotein IIb/IIIa
complex. J Clin Invest. 1985;76:101–108.
-
Tcheng JE, Harrington RA, Kottke-Marchant K, Kleiman
NS, Ellis SG, Kereiakes DJ, Mick MJ, Navetta FJ, Smith JE, Worley SJ,
Miller JA, Joseph DM, Sigmon KN, Kitt MM, du Mée CP, Califf RM,
Topol EJ, for the IMPACT investigators. Multicenter, randomized,
double-blind, placebo-controlled trial of the platelet integrin
glycoprotein IIb/IIIa blocker Integrelin in elective
coronary intervention. Circulation. 1995;91:2151–2157.
[Abstract/Free Full Text]
-
Kereiakes DJ, Kleiman NS, Runyon JP, Martin LH,
Hantsberger GS, McDonald S, Anders RJ. Sustained platelet GP
IIb/IIIa blockade with oral Xemilofiban: pharmacodynamics with clinical
outcomes in 103 patients. Circulation. 1996;94(suppl
I):I-256. Abstract.
-
Tcheng JE, Ellis SG, George BS, Kereiakes DJ, Kleiman
NS, Talley D, Wang AL, Weisman HF, Califf RM, Topol EJ.
Pharmacodynamics of chimeric glycoprotein IIb/IIIa integrin
anti-platelet antibody Fab 7E3 in high risk coronary
angioplasty. Circulation. 1994;90:1757–1764.
[Abstract/Free Full Text]
-
Kleiman NS, Raizner AE, Jordan R, Wang AL, Norton A,
Mace KF, Joshi A, Coller BS, Weisman HF. Differential inhibition of
platelet aggregation induced by adenosine diphosphate or at
thrombin receptor-activating peptide in patients treated with bolus
chimeric 7E3 Fab: implications for inhibition of the internal pool of
GPIIb/IIIa receptors. J Am Coll Card. 1995;26:1665–1671.[Abstract]
-
Coller B, Folts JD, Smith SR, Scudder LE, Jordan R.
Abolition of in vivo thrombus formation in primates with monoclonal
antibodies to the platelet GPIIb/IIIa receptor.
Circulation. 1989;80:1766–774.
[Abstract/Free Full Text]
-
Gold HK, Coller BS, Yasuda T, Saito T, Fallon
JT, Guerrero JL, Leinbach RC, Ziskind AA, Collen D. Rapid and sustained
coronary recanalization with combined bolus
injection of recombinant tissue-type plasminogen
activator and monoclonal antiplatelet GPIIb/IIIa
antibody in canine preparation. Circulation. 1988;77:670–677.
[Abstract/Free Full Text]
-
Jordan RE, Wagner CA, Mascelli MA, Treacy G, Nedelman
MA, Woody JN, Weisman HF, Coller BS. Preclinical development of c7E3
Fab, a mouse/human chimeric monoclonal antibody fragment that inhibits
platelet function by blockade of GPIIb/IIIa receptors with
observations on the immunogenicity of c7E3 Fab in humans. In: Horton MA
ed. Adhesion Receptors as Therapeutic Targets. Boca Raton,
Fla: CRC Press; 1996:281–305.
-
Channing-Rodgers RP, Levin J. A critical reappraisal of
the bleeding time. Semin Thromb Hemost. 1990;16:1–20.[Medline]
[Order article via Infotrieve]
-
Sweeny JD, Hoernig LA, Michnik BS, Fitzpatrick JE.
Whole blood aggregometry: influence of sample collection and delay in
study performance on test results. J Clin
Pathol. 1989;92:676–679.
-
Sweeney JD, Hoergig MS, Michnik A, Fitzpatrick
JE. Whole blood platelet aggregation predicts in vitro and in vivo
hemostatic function in the elderly. Arterioscler Thromb Vasc
Biol. 1995;15:748–753.
[Abstract/Free Full Text]
-
Ingerman-Wojenski CM, Silver MJ. Quick method for
screening platelet dysfunctions using the whole blood
lumi-aggregometer. Thromb Haemost. 1984;51:154–156.[Medline]
[Order article via Infotrieve]
-
Cardinal DC, Flower RJ. The electronic aggregometer: a
novel device of assessing platelet behavior in blood. J
Pharmacol Meth. 1980;3:1350–1358.
-
Riess H, Braun G, Brehm G, Hiller E. Critical
evaluation of platelet aggregation in whole blood. Am J
Clin Pathol. 1986;85:50–56.[Medline]
[Order article via Infotrieve]
-
Coller BS, Ward P, Ceruos M, Scudder LE, Springer K,
Kutok J, Prestwich GD. Thrombin receptor activating peptides:
importance of the N-terminal serine and its ionization state as judged
by pH dependence, nuclear magnetic resonance spectroscopy, and cleavage
by aminopeptidase M. Biochemistry. 1992;31:11713–11720.[Medline]
[Order article via Infotrieve]
-
Born GVR. Aggregation of blood platelets by
adenosine diphosphate and its reversal. Nature. 1962;194:927–929.[Medline]
[Order article via Infotrieve]
-
Premarket Notification. Centocor Aggrestat, Centocor
Collagen Reagent, Centocor Control Reagent. 510k-001-U. S. A.
Centocor Central Documentation Center Report number 96000669. 1995:1.
-
Wagner, CL, Mascelli MA, Neblock DS, Weisman HF, Coller
BS, Jordan RE. Analysis of GPIIb/IIIa receptor number by
quantification of 7E3 binding to human platelets. Blood. 1996;88:907–914.
[Abstract/Free Full Text]
-
Thomson C, Forbes CD, Prentice CBM. The potentiation of
platelet aggregation and adhesion by heparin in vitro and in vivo.
Clin Sci Mol Med. 1973;45:485–494.[Medline]
[Order article via Infotrieve]
-
Berglund U, Wallentin L. Influence on platelet
function by heparin in men with unstable coronary artery
disease. Thromb Haemost. 1991;66:648–651.[Medline]
[Order article via Infotrieve]
-
Konstantopoulos K, Kamat SG, Schafer AI, Bãnez
EI, Jordan R, Kleiman NS, Hellums JD. Shear-induced platelet
aggregation is inhibited by in vivo infusion of an
anti-glycoprotein IIb/IIIa antibody fragment, c7E3 Fab, in
patients undergoing coronary angioplasty.
Circulation. 1995;91:1427–1431.
[Abstract/Free Full Text]
-
Holmsen H. Platelet secretion and energy
metabolism. In: Colman RW, Hirsch J, Marder VJ, Salzman EW,
eds. Hemostasis and Thrombosis: Basic Principles and Clinical
Practice. 3rd ed. Philadelphia, Pa: JB Lipincott Co;
1994:524–556.
-
Blackwell GJ, Flower RJ, Russell-Smith N, Salmon JA,
Thorogood PB, Vane JR. Prostacyclin is produced in whole blood.
Br J Pharmacol. 1978;64:436.
-
Benveniste J, Cochrane CG, Henson PM.
Leukocyte-dependent histamine release from rabbit platelets: the
role of IgE, basophils and platelet-activating factor. J Exp
Med. 1972;136:1356–1377.[Abstract]
-
Benveniste J, Jouvin E, Pirotzky E. Platelet
activating factor (PAF-acether): molecular aspects of its release and
pharmacological actions. Int Arch Allergy Immunol.
1981;66(suppl 1):121–126.
-
Willems C, Stel HV, Van Aken Wg, Van Mourik JA. Binding
and inactivation of prostacyclin (PGI2), by human
erythrocytes. Br J Haematol. 1983;54:43–52.[Medline]
[Order article via Infotrieve]
-
Gaarder A, Jonson J, Leland S. Adenosine
diphosphate in red cells as a factor in adhesiveness of human blood
platelets. Nature. 1961;192:531–532.[Medline]
[Order article via Infotrieve]
-
Karpatkin S, Charmatz A. Heterogeneity
of human platelets, 1: metabolic and kinetic evidence
suggestive of young and old platelets. J Clin
Invest. 1969;48:1073–1087.
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