Testing Platelet Activation With a Shear-Dependent Platelet Function Test Versus Aggregation-Based Tests
Relevance for Monitoring Long-Term Glycoprotein IIb/IIIa Inhibition
Background—Tests developed to monitor glycoprotein (GP) IIb/IIIa blockade do not properly reflect platelet function in vivo and need a baseline (pretreatment) value. Because GP IIb/IIIa is essential in platelet aggregation and thrombosis under shear conditions, a flow-dependent approach to monitor its inhibition can be used.
Methods and Results—We compared a test based on flow-dependent platelet deposition, the Cone and Platelet Analyzer (CPA), with in vitro platelet aggregometry and the Rapid Platelet Function Assay (RPFA) on platelet function after GP IIb/IIIa inhibition. In vitro, increasing concentrations of abciximab (0% to 100% receptor occupancy) were tested. Ex vivo, platelet function was monitored with the CPA and with aggregometry for up to 1 week after abciximab administration. The CPA was better correlated with the percentage of free GP IIb/IIIa receptors than was aggregometry or the RPFA. Only the RPFA, when expressed as a ratio over baseline (pretreatment), was comparable to the CPA. Ex vivo, the CPA, but not aggregometry, showed prolonged platelet inhibition with gradual recovery from GP IIb/IIIa receptor blockade in the first week after abciximab administration.
Conclusions—Platelet function assessment by shear-induced deposition is a reliable test to monitor a wide range of GP IIb/IIIa inhibition. Its accuracy does not require a baseline reference. The effects of GP IIb/IIIa blockade on platelet function should be examined under high shear conditions.
Despite the therapeutic benefits of short-term intravenous glycoprotein (GP) IIb/IIIa receptor inhibitors, trials involving orally active compounds have shown no benefit or increased adverse events in treated groups.1 The lack of reliable bedside techniques to monitor platelet function and the uncertainty of the amount of receptor blockade required to prevent ischemic events have been postulated as limitations of these studies.1 In vitro platelet aggregometry is considered the “gold standard” and was used in the dose selection and monitoring of different GP IIb/IIIa inhibitors.2 The antithrombotic effects of GP IIb/IIIa receptor occupancy ≤50% were recently proposed as a potential target for effective long-term inhibition.3 This range of GP IIb/IIIa inhibition is achieved for 2 weeks after therapeutic abciximab administration and has been related to extended clinical benefit.4 Ideally, monitoring GP IIb/IIIa receptor inhibitors should be quick, easy, and reliable; discriminate among a wide range of receptor occupancies; and be independent of baseline values.
The purpose of the study was to compare the response to GP IIb/IIIa inhibition of a platelet test based on shear-induced platelet deposition5 with the classic turbidometric platelet aggregometry and the Ultegra Rapid Platelet Function Assay (RPFA). First, platelet function was tested after a wide range of GP IIb/IIIa receptor blockade in vitro with the 3 tests to compare the dose/response curves. Subsequently, platelet function was monitored in patients receiving abciximab during percutaneous coronary intervention (PCI) with the Cone and Platelet Analyzer (CPA) and with platelet aggregometry. The slow recovery of the GP IIb/IIIa receptor described after abciximab was used as a model to assess test performance at various levels of receptor blockade.
In Vitro Studies
Blood samples from 10 healthy volunteers were collected in vials containing 3.8% sodium citrate and were incubated for 30 minutes with abciximab (final concentrations ranging from 0 to 10 ng/mL).
Platelet Function Tests
Turbidometric platelet aggregation was performed in platelet-rich plasma (250×103 cells/mm3) in response to thrombin-receptor activating peptide (TRAP; 10 μmol/L). Aggregation was assessed as maximal aggregation at 6 minutes after adding the agonist. Results were expressed as percentage of baseline.
Rapid Platelet Function Assay
The RPFA is a semiquantitative, whole-blood automatic platelet function test that measures turbidometric platelet aggregation as an increase in light transmittance6 due to platelet agglutination. A modified TRAP peptide (iso-TRAP) is used as an agonist. Three-milliliter aliquots of blood incubated in vitro with abciximab were used. The test was performed according to the manufacturer’s instructions. The RPFA reports results in “platelet aggregation units,” a function of the rate at which platelets aggregate. Both absolute and percentage of baseline values were used.
Cone and Platelet Analyzer
The CPA tests platelet activation in whole blood under flow conditions. The test and methodology were previously described.5 In brief, 200 μL of citrated blood was placed in polystyrene wells and circulated at a high shear rate (1875 s–1) for 2 minutes with a rotating Teflon cone. Wells were washed with PBS, stained (May-Grünwald), and analyzed with an inverted-light microscope connected to an image analysis system. Results are expressed as the percentage of the well surface covered by platelets.
Flow Cytometric Assessment of GP IIb/IIIa Receptor Binding
GP IIb/IIIa receptor binding was quantified as previously described7 with slight modifications. The method relies on differential binding of 2 murine monoclonal antibodies, LYP18 and 4F8 (Biocytex) to GP IIIa (CD 61): LYP18 binding is inhibited by abciximab, but 4F8 binding is not. Whole blood was incubated with LYP18, 4F8, or a control mouse monoclonal antibody. A polyclonal anti-mouse IgG-FITC was added to each sample, incubated, and diluted in 2 mL of PBS-BSA. Samples were analyzed in a FACScalibur (Becton Dickinson), and platelets were identified according to the log-forward versus log-side scatter. Total number and the number of free GP IIb/IIIa receptors were determined by converting fluorescence intensity into corresponding number of sites per platelet on the basis of a calibrated bead standard curve. Blood samples treated without abciximab were used to determine 100% free receptors.
Monitoring of Platelet Function in Abciximab-Treated Patients
Patients (n=16) scheduled for PCI who were receiving abciximab were selected. Blood samples were placed in vials containing 3.8% sodium citrate at the following time points: before drug administration, 5 minutes after abciximab bolus, 4 hours after initiation of abciximab infusion, and 1, 3, 7, and 30 days after abciximab administration. All patients received aspirin (325 mg/d) and clopidogrel (75 mg/d) for 1 month after PCI. The institutional review committee approved the study, and subjects gave informed consent. During catheterization, heparin was administered (30 U/kg bolus followed by 10 U/kg boluses) to maintain an activated clotting time >200 s. Abciximab was administered as a 0.25 mg/kg bolus followed by a 0.125 μg · kg–1 · min–1 12-hour infusion.
Correlation coefficients were used to assess the strength of the relationship between each test and the percentage of free GP IIb/IIIa receptors. Comparisons between correlations were assessed using Fisher’s z transformation. Comparisons within the patient population were performed using paired Student’s t tests. Two-tailed significant threshold was set at <0.05.
Response of Platelet Function Tests to In Vitro GP IIb/IIIa Inhibition
Figure 1⇓ shows the correlations between the platelet tests and the percentage of free GP IIb/IIIa receptors. The correlation was r2=0.62 for optical aggregometry (Figure 1A⇓), r2=0.74 and r2=0.50 for RPFA (expressed as percentage of baseline and absolute value, respectively; Figure 1B⇓), and r2=0.90 for CPA (Figure 1C⇓). All correlations were statistically significant. No platelet count was abnormally low in the study population.
Within the range of free GP IIb/IIIa receptors ≥50%, correlations decreased to r2=0.36 for optical aggregometry, r2=0.66 and r2=0.47 for RPFA (expressed as percentage of baseline and absolute value, respectively), and r2=0.83 for CPA.
Comparison Between Tests
The responses of the CPA to GP IIb/IIIa inhibition were compared with those of both optical aggregometry and the RPFA. The correlation of the CPA was significantly better than the one for optical aggregometry (P=0.0007), including the correlation within ≥50% free GP IIb/IIIa receptors (P=0.0037). Compared with the RPFA, CPA proved to be a better predictor of free GP IIb/IIIa receptors when the RPFA data were expressed in absolute values (P=0.0015 and P=0.03 for free GP IIb/IIIa receptors ≥50%). When RPFA data were expressed as a ratio, the comparison with the CPA did not reach statistical significance (P=0.08 and P=0.24 for free GP IIb/IIIa receptors ≥50%).
Platelet Function Monitoring After Abciximab Administration
Platelet function recovery was assessed with optical aggregometry and the CPA. Optical aggregometry data (expressed as a percentage of baseline) remained significantly lower only 1 day after abciximab administration and were unchanged versus baseline at 3, 7, and 30 days (Figure 2A⇓). CPA showed a progressive platelet function recovery after abciximab administration. Function was significantly lower than baseline during the first week (P<0.0001 versus baseline, Figure 2B⇓). Platelet function at 1 month returned to baseline values (P=0.97) while patients were still receiving clopidogrel and aspirin. Furthermore, clopidogrel and aspirin administration in 3 volunteers did not significantly change CPA values (data not shown).
The main finding of this study is that testing platelet function under high shear conditions reflects GP IIb/IIIa inhibition in a more precise and convenient way than aggregation-based platelet tests. Among the tests compared, the CPA showed a better correlation with the percentage of free GP IIb/IIIa receptors and, in addition, results are accurately expressed as absolute values, making the patient’s baseline platelet function value unnecessary .
In vitro platelet aggregometry assumes that fibrinogen binding to the GP IIb/IIIa receptor represents the only relevant interaction for aggregation, a true statement in a stirred platelet suspension that can be applied to blood flowing at the velocity encountered in veins.8 Therefore, the results of optical aggregometry cannot be applied to platelet activation in arterioles or arteries, where the GP IIb/IIIa receptor is key in stabilizing platelet adhesion. Consequently, testing platelets under physiologically relevant flow reflects GP IIb/IIIa receptor function in arterial flow: initial adhesion followed by aggregation onto the adherent cells.
The RPFA results, when expressed as percentage inhibition of baseline, were comparable to the CPA, because RPFA variability is reduced when all baseline values are equally plotted. Pathophysiological changes in platelet reactivity and early therapeutic intervention, which are particularly relevant in the setting of acute coronary syndromes, may affect the baseline platelet function tests that will subsequently be used for continuous reference if data are expressed as a ratio.9 Therefore, monitoring chronic GP IIb/IIIa inhibition should provide accurate information independent of baseline values.
The ability to detect low levels of receptor blockade may be of great clinical value in monitoring long-term GP IIb/IIIa inhibition. This setting is totally different from PCI, where high but short-term antiplatelet therapy is attained to reduce the risk for acute thrombotic complications. This high antithrombotic regimen may be unsafe and even unnecessary in long-term therapy, so a different strategy might be required for long-term GP IIb/IIIa inhibition.10 Our results show, in agreement with Konstatopoulos et al,11 that the platelet inhibition obtained with GP IIb/IIIa inhibitors follows a dose-response curve rather than a threshold effect. Platelet function inhibition with levels of receptor occupancy <80% may be of clinical significance and might increase the safety of the chronic administration of GP IIb/IIIa inhibitors. Therefore, to study the amount of inhibition required to achieve clinical benefit, monitoring platelet function over a wide range of GP IIb/IIIa blockade would be important.
Smith et al6 reported results different from ours using the RPFA. Because the RPFA was marketed to assess platelet function after short-term administration of GP IIb/IIIa inhibitors, they mainly used high concentrations of abciximab. We focused on a wider range of inhibition, relevant to chronic therapy, so our results do not necessarily coincide with theirs. A fully automatic version of the CPA is currently under development.
We conclude that testing platelet deposition under high shear conditions accurately predicts platelet function after GP IIb/IIIa inhibition, without requiring a baseline reference and in a wide range of inhibition. The CPA may prove to be a valuable tool in monitoring GP IIb/IIIa dose/response and long-term inhibition.
Supported in part by a grant from the “Sociedad Española de Cardiología” to Julio I. Osende and a grant from the National Institutes of Health (SCOR HL-99-022) to Juan Jose Badimon. We thank Paul Lee, MD, FRCPC, for his assistance in the preparation of this manuscript.
November 13, 2000; revision received January 16, 2001; accepted January 22, 2001.
Dr Varon is the inventor of the CPA device and is in partnership with a company that will commercialize this device.
- Copyright © 2001 by American Heart Association
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