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(Circulation. 1997;95:860-867.)
© 1997 American Heart Association, Inc.

Articles

Rapid and Simple Platelet Function Assay to Assess Glycoprotein IIb/IIIa Receptor Blockade

Barry S. Coller, MD; Daniel Lang, MD; Lesley E. Scudder, BS

the Division of Hematology, Department of Medicine, Mount Sinai School of Medicine, New York, NY.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background The platelet glycoprotein (GP) IIb/IIIa receptor antagonist c7E3 Fab (abciximab; ReoPro) has been approved as an antithrombotic agent, and other GP IIb/IIIa antagonists, including oral agents, are under development. At present, there are no rapid and simple assays to monitor GP IIb/IIIa receptor blockade.

Methods and Results An assay was devised based on the ability of platelets in whole blood to rapidly agglutinate fibrinogen-coated beads when stimulated with a peptide, (iso-S)FLLRN, that activates a platelet thrombin receptor but resists inactivation by plasma aminopeptidase M. Preincubation of normal blood with increasing concentrations of c7E3 Fab led to increasing inhibition of the assay, correlating with increased GP IIb/IIIa receptor blockade. Assay conditions were chosen so that agglutination was inhibited at 2 minutes when >82% of the receptors were blocked. Similar results were obtained with the use of GP IIb/IIIa antagonists based on the arginine-glycine-aspartic acid (RGD) cell-recognition sequence.

Conclusions A simple and rapid assay sensitive to GP IIb/IIIa receptor blockade has been developed that may be helpful in optimizing GP IIb/IIIa antagonist therapy.

Key Words: glycoproteins • receptors • platelets • fibrinogen

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
New agents that block the platelet glycoprotein (GP) IIb/IIIa receptor are being developed for use as antithrombotic agents, including a recombinant murine/human chimeric antibody Fab fragment (c7E3 Fab; abciximab, ReoPro) and both peptides and peptidomimetics based on the arginine-glycine-aspartic acid (RGD) and related cell-recognition sequences.^{1 2} On the basis of a phase III study,^{3 4} c7E3 Fab has been approved for use as adjunctive therapy in high-risk angioplasty in the United States, Great Britain, Europe, Australia, New Zealand, and Scandinavia. A number of the other agents are currently in early and advanced clinical trials, including agents that are orally active.^{1 2}

Because the efficacy of GP IIb/IIIa antagonists appears to correlate with the extent of receptor blockade in animal models,^{5 6} and because there may be significant interindividual variations in response to the agents based on differences in drug pharmacokinetics and pharmacodynamics, especially with long-term use of oral agents, it would be desirable to be able to monitor the effect of therapy. Currently available assays for evaluating GP IIb/IIIa receptor blockade, including bleeding time, platelet aggregation, thromboelastography, clot retraction, radiolabeled antibody binding, and flow cytometry, are time-consuming, require extensive standardization, or require specialized equipment.

It would be desirable, therefore, to have a rapid and simple monitoring assay that did not require specialized equipment. In the setting of coronary artery angioplasty, it would be desirable to have a bedside assay that reflected GP IIb/IIIa receptor blockade that could be conducted at the same time as the activated clotting time assay, which is performed to assess the adequacy of heparinization. During long-term infusions of GP IIb/IIIa antagonists or with long-term oral therapy, periodic monitoring in the hospital, physician's office, or even at home may also be desirable. Finally, in certain circumstances, for example, before surgery or an invasive procedure, it may be desirable to rapidly determine whether the effect of the GP IIb/IIIa antagonist has worn off sufficiently to allow the surgery or procedure to be performed without additional interventions to reverse the effect of the GP IIb/IIIa inhibitor.

To address the need to monitor GP IIb/IIIa receptor blockade, we have developed an assay to assess platelet function based on our previous observations that platelets will agglutinate fibrinogen-coated beads⁷ via interactions between GP IIb/IIIa receptors and the fibrinogen and that blockade of GP IIb/IIIa receptors with monoclonal antibodies prevents these interactions.^{8 9} The speed of bead agglutination is more rapid and reproducible if platelets are activated,⁷ and so we have incorporated a thrombin receptor–activating peptide [(iso-S)FLLRN], which produces reproducible platelet activation without the confounding effect of fibrin formation.¹⁰ We chose the peptide derivative we previously described,¹⁰ with an N-terminal isoserine rather than serine, because this derivative resists cleavage and inactivation by plasma aminopeptidase M, thus eliminating a potential source of interindividual variability.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
GP IIb/IIIa Antagonists
The chimeric mouse/human monoclonal antibody fragment c7E3 Fab (abciximab; ReoPro) was obtained from Centocor, Inc.¹¹ The peptide RGDF was synthesized by use of the techniques previously described.¹² RGD-based peptidomimetics identified only by the letters A, B, C, D, and E were obtained in solution from Dr Larry Feigen, Searle, Skokie, Ill. The peptidomimetic GR144053, which inhibits platelet GP IIb/IIIa but not endothelial cell _vß₃ receptors,¹³ was obtained from Dr Philip Lumley of Glaxo Research and Development Limited, Hertfordshire, UK, and dissolved at 5.1 mmol/L in 0.15 mol/L NaCl, 0.01 mol/L Tris/HCl, 0.05% azide, pH 7.4. This compound inhibits platelet aggregation induced by ADP (10 µmol/L), collagen (1 µg/mL), U-46619 (0.3 µmol/L), platelet-activating factor (0.3 µmol/L), and a thrombin receptor–activating peptide, with IC₅₀ values of 40 to 180 nmol/L.¹³

Blood Donors and Medications
This study was approved by the Mount Sinai School of Medicine Institutional Review Board. Blood was obtained from normal donors who, unless otherwise noted, had not taken aspirin or other drugs known to interfere with platelet function for at least 7 days. Blood was anticoagulated with 12.9 mmol/L citrate with the use of a vacuum tube (Vacutainer No. 6418, Becton Dickinson) or 13.6 mmol/L citrate with the use of a polypropylene tube (Falcon 2059, Becton Dickinson) containing 1/100th volume 40% trisodium citrate. In experiments designed to compare the effects of citrate and heparin on the assay, blood was collected into citrate-containing vacuum tube(s) as above as well as into a heparin-containing vacuum tube (15 U/mL heparin final concentration; Vacutainer No. 6387, Becton Dickinson).

To assess the contribution of platelet GP IIb/IIIa receptors to bead agglutination, blood was obtained from two patients, each from a different kindred, with Glanzmann's thrombasthenia and anticoagulated with citrate. Their platelets had reduced levels of GP IIb/IIIa and normal or increased numbers of _vß₃ receptors as assessed with the use of techniques previously described (data not shown).¹⁴

To assess the effects of in vitro addition of aspirin on the assay, blood samples were incubated with 2 mmol/L aspirin for 10 minutes at 22°C before testing.

To assess the impact of GP IIb/IIIa receptor blockade on the assay, aliquots of whole blood were incubated with different concentrations (0.8, 1.2, 1.5, 1.8, 2.4, and 3.0 µg/mL) of c7E3 Fab or various doses of the peptidomimetic agents for 10 minutes at 22°C before testing in the assay.

To assess the impact of in vivo administration of heparin on the assay, blood was also obtained from seven patients scheduled for cardiac catheterization and possible coronary artery intervention. All patients were receiving aspirin therapy at the time of the catheterization, and two of the patients also received dipyridamole and low-molecular-weight dextran (dextran-40, Rheomacrodex, Baxter Healthcare; 100 mL/h). After informed consent was obtained, an arterial sheath was inserted and 15 mL of blood was immediately taken into a 20-mL syringe; a 19-gauge needle was then placed on the syringe, and the blood was distributed into four citrate-containing vacuum tubes as above. The patients then received a bolus intravenous injection of 10 000 U porcine heparin (ESI Pharma, Inc), and 5 minutes later, another sample was obtained from the arterial sheath. Samples were tested in the assay either directly or after preincubation with different concentrations of c7E3 Fab.

Procedure for Coupling Fibrinogen to Carboxylated Polystyrene Beads
Blue carboxylated polystyrene beads (3 µm; 2.5% slurry; 15 mL; No. 19123, Polysciences, Inc) were washed twice in 35 mL of 0.1 mol/L carbonate-bicarbonate, pH 9.6, and washed three times in 25 mL 0.02 mol/L sodium phosphate, pH 4.5. The pellet was resuspended in 18.7 mL of 0.02 mol/L sodium phosphate, and 18.7 mL of fresh 2% carbodiimide [1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride] (Eastman Kodak) in 0.02 mol/L sodium phosphate was added by drops and rocked 3.25 hours at 22°C. The mixture was centrifuged (2000g for 10 minutes at 22°C) to pellet the beads, and the beads were then washed three times with the above phosphate buffer and once with 0.2 mol/L sodium borate, pH 8.5. The beads were resuspended in 18 mL sodium borate buffer and added to 18 mL fibrinogen solution containing 11 mg purified fraction I-2 fibrinogen (a generous gift from Dr Dennis Galanakis, State University of New York at Stony Brook), producing a final fibrinogen-to–original bead slurry ratio of 0.73 mg/mL. The suspension was rocked for 17 hours at 22°C, and then 1.5 mL of 0.25 mol/L ethanolamine in 0.2 mol/L sodium borate, pH 8.5, was added and the suspension was incubated 30 minutes more at 22°C with rocking. The mixture was centrifuged, and the bead pellet was resuspended in 30 mL of 10 mg/mL BSA (Sigma Chemical Co) in 0.2 mol/L sodium borate buffer, pH 8.5, and rocked for 30 minutes at 22°C. The beads were washed once more in 30 mL BSA solution. The beads were then resuspended in 0.02 mol/L sodium phosphate, 0.15 mol/L NaCl, 1% BSA, 0.1% azide, pH 7.4, at 6.0 to 7.5x10⁸ beads/mL and stored at 4°C. In other fibrinogen coupling reactions, the fibrinogen was coupled at concentrations of 0.77 and 0.92 mg/mL of original bead slurry.

Platelet Aggregation
Platelet aggregation on citrated platelet-rich plasma was performed with the use of a Chronolog aggregometer (model 540-VS) as previously described,^{8 9} with ADP (8 to 10 µmol/L) used to initiate aggregation.

Assay Procedure
Fibrinogen-coated beads (20 µL), buffer (0.15 mol/L NaCl, 10 mmol/L HEPES, 0.5 mmol/L CaCl₂, pH 7.4; 105 µL), and (iso-S)FLLRN-NH₂ (5 to 10 µL; 2 µmol/L final concentration) were placed in a glass 12x75-mm test tube. Fresh, citrated whole blood (70 µL) was added to the test tube, and the tube was capped and placed on a rocking platform (Vari-Mix model 48725; Barnstead/Thermolyne) set to rock at 16 cpm at room temperature. The agglutinated beads were readily seen in the stream of blood as the tube was tilted back and forth, and the extent of agglutination was rated from 0+ (no agglutination) to 4+ (extensive agglutination). In studies in which a 2-minute end point was used, after 2 minutes of rocking, the tubes were placed in a vertical position for 5 seconds to allow the blood to drain, and then they were viewed and rated for the extent of bead agglutination. A negative control, in which the tube contained EDTA (10 mmol/L final concentration), was included in each assay.

Platelet GP IIb/IIIa Receptor Blockade Assay
To correlate the functional assay with GP IIb/IIIa receptor blockade produced by incubating whole blood with increasing concentrations of c7E3 Fab, samples tested in the functional assay were also analyzed for GP IIb/IIIa receptor blockade as previously described.^{5 15}

Statistical Analysis
Two-way ANOVA was used to test the effect of different anticoagulants on the time to the end points of the assay with the use of 3- to 7-day-old beads. Interobserver variability was tested by paired t test with the use of data obtained from the citrated blood sample and the 3- to 7-day-old beads.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Assay Characteristics, Reproducibility, and Interindividual Variations Among Patients and Observers
Strong bead agglutination (4+) was observed by 2 minutes in all 180 assays conducted with citrated blood obtained from the 19 normal donors tested (Tables 1 and 2; Figure). In dramatic contrast, the blood from two patients with Glanzmann's thrombasthenia, whose platelets were deficient in GP IIb/IIIa receptor function but had normal or increased numbers of _vß₃ receptors, failed to agglutinate the beads even with prolonged rocking (data not shown).

View this table: [in this window] [in a new window]   Table 1. Time After Adding Blood to Bead-Peptide Mixture Until Appearance of 1+ and 4+ Bead Agglutination

To assess the reproducibility of the assay and observer variability, two observers performed replicate assays on citrated blood from six normal donors (Table 1). Each observer recorded the time of onset of 1+ agglutination (slight graininess) and 4+ agglutination. The average times to 4+ agglutination varied between 39 to 46 seconds for observer 1 and 31 to 41 seconds for observer 2. There was no significant correlation between the donor's platelet count and the time to 4+ agglutination when the end-point times recorded by observer 1 (r=.18) were used, whereas there was a trend toward a negative correlation using the end-point times recorded by observer 2 (r=-.69).

The average difference between observers in assessing the time until the onset of 1+ agglutination was 2 seconds (P=.06), and the average difference in assessing the time until 4+ agglutination was 5 seconds (43 seconds for observer 1 and 38 seconds for observer 2; P=.01), with observer 2 consistently recording slightly shorter times than observer 1. The average coefficients of variation for determining the time to 4+ agglutination for observers 1 and 2 were 8% and 9%, respectively.

Blood from donor 1 was also used to assess several other features of the assay. The beads used for the studies reported in the first section of Table 1 were prepared 3 to 7 days before the studies and kept in solution at 4°C. To assess the impact of prolonged storage of the beads in solution, beads prepared 2, 4, and 19 months before use were also tested (Table 1). The times required to achieve the 4+ end point were longer with the 2- and 4-month-old beads than with the 3- to 7-day-old beads, and the 19-month-old beads did not consistently reach a 4+ end point even after >102 seconds.

We also assessed the impact of delaying testing after blood drawing. There was no observable temporal drift during the 180 minutes required for observer 1 to perform the 16 sequential replicates of the assay included in Table 1 for beads 2 months old (data not shown). Moreover, even when the citrated blood obtained from donor 1 was tested 5.5, 12, and 24 hours after venipuncture, the 4+ end points occurred before the 2-minute assay end point (66, 60, and 105 seconds).

The effect of heparin (15 U/mL) on the assay was tested by adding blood to heparin-containing tubes and comparing the results to the same blood anticoagulated with citrate (Table 1). In six of eight experiments, the heparin-anticoagulated sample reached a 4+ end point more rapidly (2 to 9 seconds) than did the citrate-anticoagulated sample; in one experiment, the values were the same, and in one experiment, observer 1 indicated that it took 8 seconds longer for the heparin-anticoagulated sample to reach the 4+ end point. ANOVA, however, failed to reveal a significant difference between anticoagulants with either the 1+ or 4+ end points (P=.81 for 1+ and P=.25 for 4+ end points). Thus, there was a trend toward a reduction in the time needed to reach the 4+ end point with heparinized samples, but the differences were modest. The agglutinates in the heparinized samples appeared somewhat coarser than those in the citrated samples.

We also tested blood obtained from seven patients who were scheduled to undergo a coronary intervention before and after they received a 10 000-U intravenous bolus injection of heparin. When the 2-minute end point was used, the blood from all patients gave 4+ agglutination both before and after heparin administration. Blood from these patients was also incubated with increasing concentrations of c7E3 Fab for 10 minutes at 22°C before testing. The minimal dose of c7E3 Fab needed to eliminate bead agglutination at 2 minutes produced by blood obtained from seven patients before receiving the bolus injection of heparin was 1.67±1.6 µg/mL (mean±SD). Blood obtained after heparin was administered required a slightly higher dose (1.80±0.35 µg/mL) of c7E3 Fab, but the difference was not significant.

Aspirin had no consistent effect on the time required to reach 4+ agglutination, with the mean effect in eight experiments being less than a 2-second prolongation (Table 1).

Effect of GP IIb/IIIa Receptor Blockade by c7E3 Fab on Assay
The effect of GP IIb/IIIa receptor blockade by c7E3 Fab on the assay was assessed by incubating aliquots of citrate-anticoagulated blood with increasing concentrations of c7E3 Fab and then directly measuring both the extent of GP IIb/IIIa blockade with the use of ¹²⁵I-c7E3 Fab and the assay end point. Two different types of experiments were performed.

The Figure shows the relation between the amount of c7E3 Fab added to the blood obtained from seven donors (one of whom was taking aspirin), the GP IIb/IIIa blockade produced by the c7E3 Fab, and the extent of bead agglutination at the 2-minute end point. The beads used for this experiment had been stored for 1 to 1.5 months before use. In the absence of c7E3 Fab, blood from all donors gave a 4+ response at 2 minutes. Adding increasing concentrations of c7E3 Fab to the donor's blood resulted in increasing GP IIb/IIIa receptor blockade. The highest dose, 3 µg/mL (which is slightly less than the theoretical maximal blood level of 3.5 µg/mL produced by injecting the recommended dose of 0.25 mg/kg into a patient with a blood volume of 72 mL/kg), produced 89% blockade in all donors. There was no obvious correlation between the patients' platelet counts and the amount of antibody required to abolish bead agglutination because both the donor with the highest platelet count (580 000/µL) and the donor with the lowest platelet count (130 000/µL) required the highest dose of antibody (2.4 µg/mL). After excluding the donor with the lowest platelet count, however, there was a trend toward a relation between the platelet count and the amount of antibody required to abolish bead agglutination (r=.88). The data in Table 2 relating platelet count to the concentration of antibody needed to inhibit agglutination also showed little correlation.

View larger version (25K): [in this window] [in a new window]   Figure 1. Relation between glycoprotein (GP) IIb/IIIa receptor blockade, amount of c7E3 Fab added to blood, and extent of bead agglutination after 2 minutes. Blood from seven donors was anticoagulated with citrate and then tested in the assay after incubation with buffer or various doses of c7E3 Fab. After 2 minutes of rocking, the agglutination was graded 0+ to 4+. Various amounts of antibody were required to achieve 80% blockade of GP IIb/IIIa receptors. When receptor blockade was 83%, all of the samples failed to agglutinate (0+), whereas none of the samples with <77% GP IIb/IIIa receptor blockade failed to agglutinate.

View this table: [in this window] [in a new window]   Table 2. Effect of c7E3 Fab on Platelet Glycoprotein IIb/IIIa Receptor Blockade and Time Required to Achieve 4+ Agglutination

Further analysis of the data points in the Figure revealed that when 83% of the GP IIb/IIIa receptors were blocked by preincubation with c7E3 Fab, 100% of the specimens tested (21/21) gave a completely negative response (0+). When 75% of the GP IIb/IIIa receptors were blocked by c7E3 Fab, 17 (94%) of 18 samples gave 3+ or 4+ reactions (11% 3+ and 83% 4+), whereas only 1 (6%) of 18 gave a weak, 1+ reaction. Various responses occurred when there was between 77% and 82% receptor blockade, with 1 of 3 samples giving a 3+ reaction, 1 giving a 2+ reaction, and 1 giving a 0+ reaction.

To obtain additional detailed information on the relation between receptor blockade and assay end points, citrated blood samples from nine donors were incubated with increasing concentrations of c7E3 Fab, and both the GP IIb/IIIa receptor blockade percentage and time to reach different extents of agglutination were determined for each sample (Table 2). The beads used for these experiments were from a different batch than the ones used for the above experiments and had been stored for 14 days before use.

When >80% of the GP IIb/IIIa receptors were blocked, 25 of 26 specimens from nine donors failed to achieve 4+ agglutination in <120 seconds. The remaining specimen, which had 85% receptor blockade, produced 4+ agglutination after 110 seconds. When <80% of the receptors were blocked, 4+ agglutination was achieved in <120 seconds in 25 of 27 specimens from nine donors. In the other two specimens, which had 62% and 71% receptor blockade, 4+ agglutination occurred at 130 seconds.

Inhibition of Agglutination Assay by RGDF and Peptidomimetic Inhibitors of GP IIb/IIIa
The peptidomimetic agents obtained from Searle were tested on three occasions in the bead agglutination assay, each time with the use of different final concentrations of the agents. After the range of concentrations needed to partially and completely inhibit the assay was determined, serial dilutions were tested to narrow the range. The same compounds were tested for their ability to inhibit the initial slope of platelet aggregation induced by 8 µmol/L ADP. As shown in Table 3, the compounds varied considerably in their potency in inhibiting the agglutination assay, but there was an excellent correlation between the concentrations required to partially inhibit the agglutination assay and the concentrations required to partially inhibit ADP-induced platelet aggregation. Similar results were obtained with the RGDF peptide and the peptidomimetic GR144053.

View this table: [in this window] [in a new window]   Table 3. Effect of RGDF and Peptidomimetic Glycoprotein IIb/IIIa Antagonists on the Bead Agglutination Assay and Platelet Aggregation Induced by ADP (8 to 10 µmol/L*)

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Intrinsic differences among the GP IIb/IIIa antagonists currently under investigation in pharmacodynamics and pharmacokinetics may affect the dose required to achieve a therapeutic antiplatelet effect. Beyond these overall differences in the drugs, however, interindividual variations in drug excretion and metabolism, as well as peripheral blood platelet count, total body platelet content, and the number of GP IIb/IIIa receptors per platelet, may have an impact on optimal drug dosing. Prolonged therapy, either with parenteral or oral agents, is likely to magnify the importance of interindividual differences, especially with agents that rely on renal excretion or hepatic metabolism.

It would be desirable, therefore, to be able to monitor the antiplatelet effects of GP IIb/IIIa receptor antagonists to ensure that a therapeutic effect is achieved initially and that it remains sustained throughout the course of therapy. After discontinuing the drug, it would also be desirable in certain circumstances to know when the antiplatelet effect is no longer likely to have a significant impact on hemostasis.

Several assays are currently available to evaluate the effects of GP IIb/IIIa receptor antagonists. A functional assessment can be obtained with the bleeding time assay or platelet aggregometry. Use of bleeding time, however, suffers from difficulties in reproducibility,¹⁶ and when it is prolonged, it takes a considerable amount of time to perform. Platelet aggregometry requires expensive equipment that is not easily portable and requires extensive standardization to ensure accurate quantitative results.¹⁷ In addition, unless performed on site with the use of whole blood,¹⁸ results are unlikely to be available for 1 hour. Thromboelastography,¹⁹ an assay involving occlusion of an aperture in an epinephrine- and collagen-coated membrane by platelet thrombi,²⁰ as well as assays of platelet adhesion and platelet-induced thrombin generation time²¹ have been reported to be affected by GP IIb/IIIa blockade, but they also require specialized equipment.

Direct measurement of GP IIb/IIIa receptor blockade has been reported with only a few of the GP IIb/IIIa antagonists. An assay to measure GP IIb/IIIa receptor blockade by murine 7E3 and c7E3 Fab on the basis of inhibition of platelet binding of radiolabeled 7E3 has been used extensively to correlate GP IIb/IIIa receptor blockade, inhibition of platelet aggregation, prolongation of the bleeding time, and antithrombotic efficacy in animal models.^{5 6 22} On the basis of these results, the therapeutic target level for coronary artery angioplasty was defined as 80% GP IIb/IIIa receptor blockade (abciximab package insert). Similar data on c7E3 Fab binding have been obtained by use of flow cytometry.²³ Although flow cytometry does not provide as reliable a measure of mean platelet GP IIb/IIIa number as does the radiolabeled antibody assay, it does provide unique information on whether subpopulations of platelets differ in their extent of receptor blockade by c7E3 Fab. Both of these techniques depend on the relatively slow in vitro off rate of murine 7E3 and c7E3 Fab; thus, the radiolabeled or fluorescently labeled antibody added to detect free GP IIb/IIIa receptor sites does not substantially displace the unlabeled antibody during the time required for the labeled antibody to reach equilibrium. Other GP IIb/IIIa antagonists have more rapid off rates in vitro, making it difficult to apply analogous techniques. In a preliminary report,²⁴ however, a fluorescent derivative of a peptidomimetic GP IIb/IIIa inhibitor (RO48) has been used to estimate the number of receptors blocked by another peptidomimetic inhibitor (RO44-9883). An alternative approach to quantifying GP IIb/IIIa receptor blockade, reported by Murphy et al,²⁵ relies on the change induced in the receptor by the binding of the peptidomimetic R043-5054. Thus, the binding of antibody D3, which recognizes an epitope on GP IIb/IIIa that is only produced when the drug binds to the receptor (ie, a ligand-induced binding site, or LIBS),²⁶ could be used to infer the binding of R043-5054.²⁵

Although all of the above techniques provide important information, none is likely to be adaptable for routine dose monitoring. As a result, we have developed a simple and rapid assay that potentially can be used to monitor GP IIb/IIIa antagonist therapy. It is based on the ability of activated platelets to interact with immobilized fibrinogen, resulting in macroscopically visible bead agglutination. Although the assay requires a visual determination of the end point, and although there are minor variations in end-point determination by different observers, the overall agreement between the two observers in the present study was very good, with coefficients of variation of <10%. It would, however, be desirable to develop an objective detection system that would not rely on an observer's judgment. It will also be necessary to establish conditions of storage of the beads that will prolong their shelf life.

The time to reach 4+ agglutination was only minimally affected by the platelet count of the donors, indicting that the assay should be applicable to patients who have a wide range of platelet counts. Aspirin did not affect the assay, most likely because activation of GP IIb/IIIa via the thrombin receptor is primarily independent of thromboxane A₂ production. Heparin demonstrated a tendency to modestly speed up and enhance bead agglutination, but even with high doses of heparin, the assay was sensitive to GP IIb/IIIa blockade by c7E3 Fab. The enhancing effect of heparin is consistent with its known effects on platelet aggregation,²⁷ raising the possibility that heparin, despite its anticoagulant effect, may facilitate platelet-mediated thrombosis.²⁸

We chose a functional assay rather than one based on GP IIb/IIIa receptor occupancy or percentage blockade because we believe that functional impairment is most likely to reflect the biologically important antithrombotic effect of the GP IIb/IIIa receptor antagonists. The assay will therefore be sensitive to platelet function abnormalities independent of GP IIb/IIIa blockade (eg, hyporesponsiveness to stimulation by the thrombin-activating peptide) as well as severe thrombocytopenia, and this needs to be considered when the results of the assay are assessed. Despite this, there is a strong correlation between GP IIb/IIIa receptor blockade produced by c7E3 Fab and the results of the assay, expressed either as the strength of agglutination after 2 minutes (Figure) or the time required to achieve 4+ agglutination (Table 2). Thus, with the use of data derived from studies of c7E3 Fab, it may be possible to construct an idealized standard curve of GP IIb/IIIa receptor blockade versus functional impairment measured in the assay. This standard curve could then be used to estimate the equivalent GP IIb/IIIa receptor blockade on the basis of the functional impairment of the assay produced by any of the GP IIb/IIIa antagonists. This would be particularly helpful in assessing GP IIb/IIIa receptor blockade by peptidomimetic agents for which no direct measure of GP IIb/IIIa receptor blockade is available. Our studies with several such agents obtained from Searle and Glaxo, as well as the peptide RGDF, indicate that the assay is also sensitive to GP IIb/IIIa inhibition by these agents and that there is a good correlation between the inhibitory effects of these agents on the assay and on platelet aggregation.

At present, the clinical dosing of c7E3 Fab is designed to achieve a threshold of 80% receptor blockade. Because there is no upper limit of acceptable receptor blockade, we designed the assay to show significant inhibition of the agglutination response when 80% GP IIb/IIIa receptor blockade of normal platelets was achieved. This involved adjusting the proportions of the reagents and the concentration of the agonist peptide used. In studies not reported, we have been able to make the assay more sensitive to GP IIb/IIIa receptor blockade by reducing the dose of the agonist peptide such that marked inhibition of agglutination occurs with 50% GP IIb/IIIa receptor blockade. It may be possible, therefore, to use this modification of the assay to assess the point at which sufficient numbers of unblocked GP IIb/IIIa receptors have become available after stopping GP IIb/IIIa antagonist therapy to support hemostasis if an invasive procedure is required. Because carriers of Glanzmann's thrombasthenia have 50% to 60% of the normal numbers of GP IIb/IIIa receptors and do not have a bleeding diathesis,²⁹ this may be a reasonable level for judging when it is not necessary to try to reverse the effect of the agent (as, for example, by platelet transfusion for c7E3 Fab³⁰ ) before surgery or an invasive procedure. Finally, unlike short-term therapy with c7E3 Fab, in which the goal is to exceed a threshold effect of 80% GP IIb/IIIa receptor blockade, long-term oral GP IIb/IIIa antagonist therapy is likely to be prescribed to achieve a window of GP IIb/IIIa receptor blockade, with upper and lower limits designed to maximize both safety and efficacy. It should be possible to design two versions of the assay of different sensitivities that can be used to assess whether the dose is achieving GP IIb/IIIa receptor blockade within the prescribed window.

In conclusion, we have devised a rapid and simple assay to assess impairment of platelet GP IIb/IIIa function that correlates with GP IIb/IIIa receptor blockade. The assay may have potential for monitoring GP IIb/IIIa antagonist therapy. Moreover, because the assay measures the competence of both platelet activation and the GP IIb/IIIa receptor, modifications involving different agonists may be more generally applicable to assessing platelet function.

	Acknowledgments

 
This study was supported in part by grant 19278 from the National Heart, Lung, and Blood Institute. We would like to thank Dr Harvey Weiss of St Luke's–Roosevelt Hospital, New York, NY, for allowing us to study his patients with Glanzmann's thrombasthenia; Dr David Cheresh of Scripps Clinic for supplying antibody LM609; Drs Larry Feigen and Philip Lumley for supplying platelet GP IIb/IIIa receptor antagonists; Dr Dennis Galanakis for supplying purified fibrinogen; Dr Carol Bodian for statistical analysis; and Melanie Alvarez and Suzanne Rivera for outstanding secretarial assistance.

	Footnotes

 
Reprint requests to Barry S. Coller, MD, Department of Medicine Box 1118, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. E-mail BCOLLER@SMTPLINK.MSSM.EDU.

Received April 9, 1996; revision received September 25, 1996; accepted October 7, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

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12. Beer JH, Springer KT, Coller BS. Immobilized Arg-Gly-Asp (RGD) peptides of varying lengths as structural probes of the platelet GPIIb/IIIa receptor. Blood. 1992;79:117-128.[Abstract/Free Full Text]
    
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