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A Novel Method to Assess Platelet Inhibition by Eptifibatide with Thrombelastograph®

Katori, Nobuyuki MD; Szlam, Fania MMSc; Levy, Jerrold H. MD; Tanaka, Kenichi A. MD

doi: 10.1213/01.ANE.0000136476.82598.88
Technology, Computing, and Simulation: Research Report
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We examined a novel method to detect platelet inhibition with thrombelastography (TEG®). We hypothesized that this method would be suitable for monitoring the antiplatelet effects of eptifibatide (Integrilin®). Whole blood from healthy volunteers was anticoagulated with 3.2% citrate or unfractionated heparin (7 IU/mL). For the platelet aggregation test, both citrate and heparinized samples were spiked with increasing concentrations of eptifibatide (0, 0.2, 0.4, 0.8, 1.6, and 4 μg/mL). Conventional kaolin TEG® was performed with citrated samples, and batroxobin-modified TEG® was performed with heparinized samples, which were spiked with eptifibatide at concentrations of 0, 0.4, 0.8, 1.6, 4, 8, and 24 μg/mL. Adenosine 5′-diphosphate-induced platelet aggregation was reduced to 6.4% ± 2.9% (citrate) and 10.3% ± 4.8% (heparin) with eptifibatide at the concentration of 4 μg/mL. The kaolin TEG® showed a decrease in maximum amplitude (MA) only at the eptifibatide concentration of 24 μg/mL and no change in α angle, whereas with the batroxobin-based TEG®, the difference in MA and α angle was observed at concentrations ≥0.8 μg/mL. Additionally, the time to achieve maximum MA was much shorter for batroxobin TEG® than for kaolin TEG®. We conclude that the batroxobin-modified TEG® is a sensitive method that detects platelet inhibition induced by eptifibatide.

IMPLICATIONS: We examined a novel batroxobin-based activator for detecting platelet inhibition induced by eptifibatide on thrombelastography (TEG®). We concluded that batroxobin-modified TEG® was more sensitive than kaolin TEG® for detecting eptifibatide-induced platelet inhibition at clinical concentrations.

Department of Anesthesiology, Emory University School of Medicine, Atlanta, Georgia

Supported by the Department of Anesthesiology, Emory University School of Medicine, and the Bayer Research Fellowship Grant (KAT).

Accepted for publication June 3, 2004.

Address correspondence and reprint requests to Kenichi A. Tanaka, MD, Department of Anesthesiology, Emory University Hospital, 1364 Clifton Rd., N.E., Atlanta, GA 30322. Address e-mail to kenichi_tanaka@emoryhealthcare.org.

Platelet/fibrinogen interaction via glycoprotein (GP)IIb/IIIa receptors is a critical step in the pathogenesis of coronary artery thrombosis. Currently, two classes of GPIIb/IIIa antagonists are available. Class I antagonists (e.g., abciximab) are nonspecific but have high affinity (the Kd for abciximab is 5 nM) for resting and activated platelets and are slowly reversible. Because they equilibrate preferably with platelets (10:1 distribution, platelets/plasma), their antiplatelet effects are partially independent of their corresponding plasma concentration. Class II drugs are specific but rapidly reversible and have low affinity (the Kd for eptifibatide is 15 nM) to GPIIb/IIIa receptors and a rapid platelet dissociation time. Further, the efficacy of drugs in this class not only is dependent on their plasma levels (1:1 distribution, platelets/plasma), but also is affected by ionized calcium levels (1). Although treatment with GPIIb/IIIa antagonists has improved clinical outcomes for patients with acute coronary syndrome or those undergoing percutaneous coronary intervention, the risk of bleeding complications may be increased, especially when patients undergo coronary artery bypass grafting after receiving GPIIb/IIIa antagonists (2,3). GPIIb/IIIa antagonist treatment before surgery should be discontinued according to its elimination half-life (4), but recovery from platelet inhibition varies considerably in patients who receive GPIIb/IIIa drugs (5). Thus, the use of bedside platelet monitoring may be useful in predicting bleeding risks during perioperative anti-GPIIb/IIIa therapy.

In vitro monitoring of platelet function is often performed in the absence of thrombin generation. This allows for testing of specific platelet functions by using various agonists, without causing blood clotting. In regard to the whole-blood clotting monitor, the Thrombelastograph® (TEG®) measures the net product of the interaction of platelets with coagulation cascade factors. Monitoring the effect of abciximab, the Fab fragment of the chimeric monoclonal antibody (7E3), with the TEG® has been well established (6), but TEG® may be less sensitive than Sonoclot® (Sienco, Wheat Ridge, CO) in detecting the antiplatelet effects of eptifibatide, a cyclic heptapeptide, at clinically small concentrations (7). It is suggested that TEG® and Sonoclot may have different sensitivities in changes in the viscoelastic properties of clotting blood treated with antiplatelet drugs such as eptifibatide.

We hypothesized that a more sensitive platelet function assay would be feasible with TEG® only if thrombin generation were suppressed. To form clotting that is detectable by TEG®, a batroxobin-based activator was used to cleave fibrinogen in the presence of heparin and a platelet agonist (i.e., adenosine 5′-diphosphate (ADP)), and this enabled us to accomplish a separate activation of fibrinogen and platelets. Therefore, we investigated the usefulness of batroxobin-activated TEG® in comparison with conventional kaolin TEG® in monitoring the antiplatelet effects of eptifibatide, which would be helpful in minimizing the risk of bleeding and, if needed, in guiding platelet transfusion therapy in eptifibatide-treated patients.

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Methods

After institutional approval and informed, written consent, whole blood was obtained from 12 healthy volunteers with no history of aspirin use or other medication use for the previous 2 wk. The whole-blood samples were anticoagulated with 3.2% citrate (1:9 in volume) or unfractionated heparin (UH; final concentration, 7 IU/mL).

To investigate the platelet-inhibitory effect of eptifibatide, two types of platelet function tests were performed: platelet aggregation assay (Plateletworks®; Helena, Beaumont, TX) and TEG® (Haemoscope Corp., Niles, IL). Platelet aggregation was investigated with citrate and UH samples after 5 min of incubation with increasing (0, 0.2, 0.4, 0.8, 1.6, and 4 μg/mL) concentrations of eptifibatide (Integrilin; Millennium Pharmaceuticals Inc., San Francisco, CA). The extent of platelet aggregation was measured by counting platelets with Plateletworks® before and after adding ADP (final concentration, 20 μM) (8). As aggregates of platelets formed, each aggregate was still counted as one particle; hence, the ADP-induced reduction of platelet count was used as a simple estimate of platelet aggregation (% aggregation) based on the following formula:

The mean platelet count of duplicate counts was used for the calculation of % aggregation. The degree of aggregation was compared between two anticoagulants (citrate and UH).

The viscoelastic property of whole blood clotting in the presence of eptifibatide was examined by using TEG®. Citrate- or UH-anticoagulated blood was incubated with increasing concentrations of eptifibatide (0.4, 0.8, 1.6, 4, 8, and 24 μg/mL) for 5 min. For conventional TEG®, citrated blood (350 μL) was recalcified with 20 μL of 0.2M CaCl2 and activated with kaolin. For batroxobin-modified TEG® (9), 10 μL of batroxobin-based activator (a gift from Haemoscope Corp.) and 10 μL of 0.072 mM ADP (final concentration, 2 μM) were added to UH-anticoagulated blood (350 μL), which was pretreated with eptifibatide, in disposable TEG® cuvettes. The percentage change in maximum amplitude (MA) was calculated according to the following formula: MA2/MA1 ×100, where MA1 is MA at baseline (no drug added) and MA2 is MA after the addition of eptifibatide. The % aggregation after the addition of ADP, and the TEG® variables—including reaction time (R time), α angle, MA, and time to MA—were collected. R time is defined as the time from the start of a sample run until the early clot formation (amplitude of 2 mm), and α angle measures the kinetics of clot development. MA is defined as the maximum distance between two lines on the TEG® tracing. Time to MA was defined as the time needed to obtain MA. Data—including hematological values, % aggregation, and TEG® variables—are expressed as mean ± sd. The % aggregation and TEG® variable data were compared between citrate samples (kaolin TEG®) and UH samples (batroxobin TEG®) by using a nonparametric test (Friedman’s test followed by Wilcoxon’s t-test with Bonferroni’s correction) because they were not normally distributed. Paired t-tests were performed to compare platelet counts and other hematological values between citrate and UH samples. A P value of <0.01 was considered significant.

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Results

Baseline platelet counts were 241 ± 40 × 103/μL and 263 ± 36 × 103/μL for citrate and UH samples, respectively. There was no statistical difference in platelet count or other hematological data between citrate and UH samples. There was a significant decrease in ADP-induced platelet aggregation after the addition of eptifibatide in both groups. ADP-induced platelet aggregation was decreased to 6.4% ± 2.9% (citrate) and 10.3% ± 4.8% (UH) with eptifibatide at the concentration of 4 μg/mL (Fig. 1). There was a significant difference in % aggregation between citrate and UH samples at eptifibatide concentrations of 0.4, 0.8, and 1.6 μg/mL (P < 0.01).

Figure 1

Figure 1

The TEG® variables are shown in Table 1. The batroxobin TEG® showed a significantly shortened R time and a decreased α angle in comparison with the kaolin TEG® at all eptifibatide concentrations used. Dose-response curves of the percentage decrease in MA for both the kaolin and the batroxobin TEG® are shown in Figure 2. Changes in MA from baseline were detected only at a concentration of 24 μg/mL on kaolin TEG® (P < 0.01). Conversely, the batroxobin TEG® showed differences from baseline at concentrations of 0.8 μg/mL or larger (P < 0.01). Figure 3 shows the changes in kaolin- and batroxobin-activated TEG® tracings in response to increasing concentrations of eptifibatide.

Table 1

Table 1

Figure 2

Figure 2

Figure 3

Figure 3

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Discussion

Because of its close interaction with blood clotting, a specific assay of platelet function, typically aggregometry, is not possible without anticoagulation that prevents fibrin clotting mediated by thrombin, the most potent physiological platelet agonist. This allows clinicians to test platelet responses to various less potent platelet agonists, such as ADP. The platelet aggregometry reflects the early phase of platelet-mediated hemostatic processes (primary plugs), the importance of which is underscored by certain pathologic conditions associated with ADP secretion (10) or ADP receptor blockade (11). Conversely, TEG® is performed in the presence of calcium, and thrombin-mediated clot formation (viscoelasticity) is measured. The use of TEG® as a platelet function test has been controversial because the amount of fibrin, as well as platelet count and function, affects its clot strength (6,12). In this study, we evaluated two different modalities that are useful in monitoring platelet inhibition by eptifibatide, a commonly used GPIIb/IIIa antagonist: the Plateletworks® and the batroxobin-modified TEG®.

Plateletworks®, which measures platelet counts before and after the addition of ADP, showed a dose-dependent decrease in % aggregation with eptifibatide. The aggregation value was reduced to 20% at the concentration of 0.8 μg/mL. This is in close agreement with the PRIDE study (13), which reported that an eptifibatide plasma concentration of 1.65 μg/mL could achieve a GPIIb/IIIa receptor occupancy of >80%. When eptifibatide-induced platelet inhibition was compared in two types of anticoagulants, we found more pronounced platelet inhibition by eptifibatide in citrate than in UH (Fig. 1). Sodium citrate reduces the calcium ion concentration from a 1 mmol/L physiological level to approximately 40 to 50 μmol/L (14), which reduces the GPIIb/IIIa receptor binding affinity for fibrinogen (15). By comparing the antiplatelet efficacy (50% inhibitory concentration) of various GPIIb/IIIa (Class II) drugs, including eptifibatide, in blood samples collected in citrate versus heparin, Mousa et al. (1) have demonstrated not only that the ionized calcium concentration plays an important role in antiplatelet effects, but also that there is a significant differential calcium sensitivity for different drugs included in Class II antiplatelet drugs: this results in lower efficacy in heparin-collected samples as compared with citrate samples. Heparin inhibits coagulation via catalyzing antithrombin; therefore, the calcium ion level is unaffected. Thus, the use of anticoagulants that do not chelate calcium (e.g., heparin, hirudin, or d-Phe-Pro-Arg-chloromethyl ketone (PPACK)) may allow more accurate approximation of in vivo platelet aggregation, although all commonly used anticoagulants have some undesirable effects either on platelets or Ca2+-dependent functions (16).

In contrast to conventional aggregometry, platelet count-based assays with Plateletworks® offer the advantages of simplicity (no centrifugation), rapidity(less than a five-minute procedure time), and the ability to provide a complete cell count, which detects the thrombocytopenia that may occur during eptifibatide therapy (17,18).

Batroxobin, a thrombin-like enzyme derived from the Bothrops atrox snake, can cleave fibrinogen, even in the presence of heparin, but it affects only the amino terminus of the α-chain and lacks platelet activation; hence, limited fibrin cross-linking occurs (9,19). Alternatively, kaolin is one of the most potent contact activators for the TEG® assay, which allows thrombin generation in the presence of calcium. The thrombin cleaves both the α- and β-chains of fibrinogen and activates platelets and factor XIII, which leads to complex cross-linking of fibrin fibers. As shown in Table 1, the MA values of batroxobin-catalyzed clots were smaller than those of the corresponding thrombin-catalyzed clots, especially when a large concentration of eptifibatide was added. MA values of the batroxobin-modified TEG® were significantly reduced at clinically relevant plasma levels (plasma concentration, 1.6–2.5 μg/mL) (20). Minimal R times (1 to 2 minutes) were also noticed with the batroxobin method when compared with the conventional TEG® (5–5.5 minutes). Our data underscore the efficacy of thrombin as a clot activator, and important contribution of platelet activation for clot strength. Furthermore, batroxobin-modified TEG® allows us to use a specific platelet agonist by separating fibrin formation from platelet activation.

A prototypical GPIIb/IIIa antagonist, abciximab, causes a dose-dependent reduction of the TEG® amplitude (6). However, Waters et al. (7) have reported that TEG® was less sensitive than Sonoclot in detecting the antiplatelet effects of eptifibatide at a large concentration (4 μg/mL). This discordance can be, in part, attributed to different classes of GPIIb/IIIa antagonists. The Class I drugs (e.g., abciximab) are long-acting and have high affinity, whereas the Class II drugs (e.g., orbofiban and tirofiban) have a low affinity and are titratable. Mousa et al. (21) have reported that MA was not useful in evaluating the platelet inhibition induced by orbofiban (Class II) at up to 1000 nM concentration, although they have clearly shown the dose-dependent differences in MA by using another Class I drug, roxifiban.

Eptifibatide also belongs to Class II. Incomplete inhibition of amplitude (i.e., clot formation) on conventional TEG® can be explained by several mechanisms: 1) dissociation of eptifibatide from GPIIb/IIIa receptors (low affinity), 2) exocytosis of GPIIb/IIIa receptors by thrombin-induced platelet activation (22), and 3) non-GPIIb/IIIa receptor-mediated fibrin(ogen) binding (23).

In summary, the batroxobin-modified TEG® is a sensitive and fast method that detects eptifibatide-induced platelet inhibition. Its clinical utility and optimal platelet agonist dose need to be further tested in clinical settings. When used in conjunction with conventional TEG®, multiple phases of coagulation processes (i.e., primary platelet plug formation and clot formation) may be evaluated.

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