Department of Anesthesiology, Emory University School of Medicine, Division of Cardiothoracic Anesthesiology and Surgery, Emory Healthcare, Atlanta, Georgia, USA
* Corresponding author: Department of Anesthesiology, Emory University Hospital, 1364 Clifton Road, NE, Atlanta, Georgia 30322, USA. E-mail: kenichi_tanaka{at}emoryhealthcare.org
Accepted for publication February 11, 2004.
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Abstract |
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Methods. Blood samples were obtained from healthy subjects (n=8) and cardiac surgical patients (n=34). Thrombin formation was measured in platelet-rich plasma with a Thrombogram®-Ascent fluorescent plate reader system. Platelet inhibition by tirofiban was evaluated with Plateletworks®, and the interaction of tirofiban and heparin (>1.5 U ml1) on clot formation was evaluated with Sonoclot Analyzer® or kaolin activated clotting times (ACTs).
Results. Addition of tirofiban (70280 ng ml1) progressively delayed onset of thrombin generation triggered by adenosine diphosphate (ADP). Plateletworks showed platelet inhibition with tirofiban (>35 ng ml1), whereas heparin per se failed to produce platelet inhibition at 7 U ml1. Heparin (1.5 U ml1) slowed the onset and rate of fibrin formation on Sonoclot analyses, and this was further slowed after addition of tirofiban (70 ng ml1) to heparin-containing blood samples. Significant increases in ACT at all heparin concentrations were observed with the addition of tirofiban (70 ng ml1). The addition of antithrombin (0.2 units/ml) to heparinized blood samples further prolonged ACTs, but the difference was not statistically significant when compared with heparin alone.
Conclusion. Tirofiban delays platelet activation-mediated thrombin generation and prolongs ACT in heparinized blood.
Keywords: blood, antithrombin ; blood, platelet inhibitors, tirofiban ; complications, heparin resistance
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Introduction |
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Materials and methods |
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For the preparation of Fluca buffer, HEPES buffer 1.750 ml (pH 7.35, HEPES 20 mM, BSA 60 mg ml1) was added to CaCl2 1 M 0.2 ml (Sigma) in a glass test tube, mixed, and warmed for a few minutes at 37°C. Just before use, fluorogenic substrate 50 µl (Z-GGR-AMC, Bachem Switzerland) DMSO 100 mM (Sigma) was added to the HEPESCaCl2 solution and mixed to dissolve. This buffer now contained substrate 2.5 mM and CaCl2 100 mM. Tirofiban (Aggrastat®; Merck, Whitehouse Station, NJ, USA), AT (Thrombate IIITM; Bayer, Elkhart, IN, USA) and porcine sodium heparin (Elkins-Sinn, Cherry Hill, NJ, USA) were diluted with saline and then added to platelet-rich (PRP) or whole blood samples to obtain appropriate drug concentrations. The volumes of drugs added to PRP or whole blood resulted in <1% dilution.
Thrombin generation and platelet aggregation
The study was performed after institutional approval and with informed consent.
Blood samples were collected from eight volunteers in 3.2% sodium citrate tubes. Plasma was centrifuged (5 min at 800 g) to obtain PRP, and platelet-poor plasma (PPP) (15 min at 2000 g). PPP was used to adjust the platelet count of PRP to 200 000 µl1. Thrombin generation was measured on the basis of the development of the fluorescent signal over time,9 by using a slow fluorogenic thrombin substrate and a thrombin calibrator. Briefly, for the thrombin generation experiments, PRP 80 µl and the thrombin generation trigger (20 µl of ADP or tissue factor) were added to wells of 96-well solid black microtitre plates (Microfluor 2; ThermoLabsystems, Franklin, MA, USA) followed by substratecalcium chloride buffer 20 µl. The reaction was monitored using microplate fluorometer (Fluoroskan Ascent; ThermoLabsystems, Franklin, MA, USA) set at 390 nm (excitation) and 460 nm (emission). Fluorescence was recorded every 20 s for 60 min and the acquired data were processed for thrombin generation parameters: lag time and endogenous thrombin potential (ETP) (Synapse, Maastricht, The Netherlands). The area under the curve (the ETP) represents the amount of thrombin enzymatic action that can potentially be triggered in a given plasma sample before its inhibition by physiological antithrombins (AT, 2-macroglobulin).10 11
The contribution of platelets to thrombin generation was assessed by adding different concentrations of tirofiban to PRP (plasma tirofiban 0, 100, 200 and 400 ng ml1; final concentration 0, 70, 140 and 280 ng ml1 respectively) or porcine heparin (plasma heparin 0, 0.1, 0.2 and 0.3 U ml1; final concentration 0, 0.07, 014 and 0.21 U ml1 respectively). For aggregation studies, blood was collected from eight normal volunteers in 3.2% sodium citrate (9:1 vol/vol) or sodium heparin (final concentration 7 U ml1). Two different anticoagulants were used to verify that neither anticoagulant on its own had any aggregation effects. All the measurements were obtained within 40 min after blood sampling. For each sample, platelet inhibition by tirofiban was tested before and after in vitro addition of tirofiban (final concentration 0140 ng ml1), using Plateletworks (Helena, Beaumont, TX, USA) in ADP test tubes (20 µM). The instrument works as a cell counter. In the presence of an agonist (i.e. ADP) platelets aggregate, thus exceeding the threshold limitations for platelet size, and are therefore excluded from being counted as individual platelets. The differential platelet count between two samples (baseline and ADP) is used to calculate the percentage of aggregation.
Whole blood clot formation
We also evaluated the antithrombotic effects of tirofiban alone and in combination with heparin, and compared these with the effects of AT, which is known to augment heparin anticoagulation, using an in vitro whole blood clot protocol. Blood was collected from eight normal volunteers in 3.2% sodium citrate (9:1 vol/vol). Baseline Sonoclot analysis (Sienco, Morrison, CO, USA) was performed with a recalcified blood sample, which was taken from a 1 ml-volume aliquot containing celite 1.5 mg. Analyses were repeated with a cuvette containing (final concentrations) heparin 1.5 U ml1 plus either normal saline, AT 0.2 U ml1 or tirofiban 70 ng ml1. The addition of heparin and other drugs resulted in less than 1% dilution as compared to baseline samples. The following variables were measured: (i) clotting time (s); and (ii) clot rate (the gradient of the primary slope). Clotting time reflects the onset of fibrin formation, and clot rate reflects the rate of fibrin formation.
Blood samples were also obtained from patients undergoing elective cardiac surgery with concurrent heparin therapy (n=34). The durations of both of i.v. heparin therapy and concomitant drug administration were recorded. Patients with pre-existing haemostatic disorder, hepatic or renal disease, and concurrent anticoagulant therapy (GPIIb/IIIa antagonist, warfarin, or tissue plasminogen activator) were excluded. Baseline platelet count (x103 µl1), prothrombin time as the international normalized ratio (INR), and activated partial thromboplastin time (APTT, s) were recorded. Blood samples were obtained after induction of general anaesthesia but before full heparinization (400 U kg1). Blood (0.4 ml) was placed in kaolin-ACT cartridges (Medtronic, Parker, CO, USA) to determine baseline ACT, and into cartridges containing porcine intestinal heparin at final concentration of 1.5, 2.5, and 4.1 U ml1. Blood was also added to cartridges that contained the same amount of heparin plus either AT (final concentration 0.2 U ml1), or tirofiban (final concentration 70 ng ml1), and ACTs were recorded. All tests were performed in duplicate. For determination of AT activity, blood samples from patients were collected into plastic tubes containing sodium citrate 3.2% (9:1 vol/vol), and centrifuged at room temperature for 15 min at 3000 g. Plasma was separated and stored at 80°C until analysis. AT activity was determined using a commercially available kit (Coamatic Antithrombin; Chromogenix, Mondal, Sweden) with a coefficient of variation <8.0%.
Statistical analysis
All data are expressed as mean (SD). Differences between groups were analysed using analysis of variance with the Bonferroni correction. A P value 0.05 was considered significant.
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Results |
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Discussion |
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Tirofiban prolonged lag time when ADP was used to trigger thrombin formation. ADP-induced thrombin generation depends on platelets, because ADP does not induce thrombin in PPP. Although tirofiban did not reduce the total amount of thrombin generated (ETP), this does not exclude its potential anticoagulant effects because other clinically useful anticoagulants are known to cause a similar delay without reducing ETP.13 When tissue thromboplastin (Innovin) was used as a trigger, tirofiban did not effectively suppress thrombin generation. This can be explained by the direct activation of clotting factors seen in PPP. Our results are in agreement with Wegert and colleagues, who reported that abciximab inhibited platelet agonist-induced but not Innovin-induced thrombin generation.9 Heparin concentration-dependently delayed and reduced thrombin formation against ADP- or thromboplastin-induced thrombin generation in our assay system, but persistent thrombin generation and platelet activation seems to occur during CPB even when heparin concentrations and ACTs are adequately maintained.14 15
Platelets are activated when only a small amount of thrombin is generated,16 and support prothrombinase (factor Vafactor Xa) complexes by providing a catalytic surface for thrombin generation. In the presence of heparin, free plasma thrombin and factor Xa are rapidly inhibited by AT in plasma. However, clot-bound thrombin is not inhibited by ATheparin complex,16 and factor Xa also becomes resistant to inhibition when it is incorporated into the prothrombinase complex on the platelet surface.17 Additionally, factor IXa cannot be inhibited by AT when factor VIIIa and platelets are present.18
Platelet aggregation is not inhibited with heparin anticoagulation per se (Fig. 3). Tirofiban inhibited ADP-induced platelet aggregation in citrate- and heparin-anticoagulated blood in a concentration-dependent manner. Moreover, platelet function seemed to be less inhibited with tirofiban at 35 ng ml1 in our study when heparin anticoagulation was used (Fig. 3). This difference presumably resulted from the overestimation of platelet inhibition due to the calcium-chelating citrate.19
By inhibiting platelet aggregation, GPIIb/IIIa antagonists are effective in reducing propagation of procoagulant events. Reverter and colleagues showed that in vitro thrombin generation was delayed and reduced in the presence of abciximab.20 Tirofiban is a non-peptide tyrosine derivative that selectively and reversibly binds to platelet GPIIb/IIIa receptors. Plasma half-life of tirofiban is approximately 2 h; therefore, it is less likely to cause postoperative bleeding such as that seen in patients with abciximab therapy. In the second part of the study, we used clot-based methods because thrombin generation assay and platelet aggregation tests were not suitable for the evaluation of potentially additive effects of tirofiban and high-dose heparin. The in vitro tirofiban dose of 70 ng ml1 was chosen on the basis of a rough approximation of administering a bolus dose of 510 µg kg1 drug into a 70-kg adult.21
The first part of the clot-based study was performed with blood from healthy volunteers, using the Sonoclot analyser. The Sonoclot analyser detects clot gel formation as oscillatory changes, generating a continuous signal curve (signature), and subsequently parameters are calculated using proprietary computer algorithms. We used two calculated parameters of Sonoclot, clot formation time (onset) and clot rate. The former is comparable to activated clotting time, but the latter reflects the rate of fibrin polymerization, which is unique to Sonoclot. Both of these numerical parameters are calculated and then displayed on the instrument.
Sonoclot analyses showed that the onset of clot formation was further delayed with addition of AT or tirofiban in comparison with anticoagulation with heparin only (Table 1 and Fig. 4). The rate of subsequent clot formation also showed a trend of reduction with addition of tirofiban or AT. We expanded the experiments with a MedtronicACT system to study the effects of AT or tirofiban at varied heparin concentrations in blood samples from cardiac surgical patients treated before surgery with heparin. Although the Sonoclot analyser and the MedtronicACT system do not measure thrombin formation directly, the changes in blood viscosity that are detected with these monitors reflect thrombin-induced fibrin formation and platelet activation.22 23 In patient blood samples, tirofiban seemed to be more effective than AT in increasing ACT. Smaller increases in ACTs induced by addition of AT 0.2 U ml1 may be related to the heparin concentrations used in this study (1.54.1 U ml1). In a previous study from our group, AT supplementation caused statistically significant ACT prolongations over non-AT-supplemented samples only at higher heparin concentrations (5.46.8 U ml1) in heparin-treated patients.24 Statistically significant ACT prolongations over non-supplemented samples were found with tirofiban at all heparin concentrations, suggesting that tirofiban prolongation of ACTs may be due to a mechanism other than heparin-AT mediated thrombin inhibition (Fig. 5).
Extensive platelet activation occurs during CPB, and platelets undergo morphological changes to form aggregates. Heparin anticoagulation is also susceptible to neutralization by platelet factor 4,25 which is released upon platelet activation. Addition of GPIIb/IIIa inhibitor to heparin has significantly improved the outcome of patients undergoing coronary interventions. Enhanced ACT responses to heparin in patients treated with abciximab, a classical GPIIb/IIIa inhibitor, have been reported.26 Prolongation of ACTs with abciximab could be attributed to other integrin receptors, such as vß3 (vitronectin receptor), but our study shows that specific blockade of GPIIb/IIIa (
2bß3) receptors with tirofiban can effectively prolong ACTs. Platelet impairment induced by prostacyclin is also known to prolong ACTs up to 60%.27
Lack of platelet inhibition is one of the critical disadvantages of this current regimen of CPB anticoagulation. Strategies to supplement high-dose heparin with AT concentrate or recombinant AT may be effective, but the level at which AT activity should be supplemented to reduce thrombotic complications after CABG is not well defined.1 24 28 The mean value of AT activity in blood samples from cardiac patients was 78.5%. In the subgroup of patients who had AT activity <80%, the mean value was 64.1%, which is consistent with an AT concentration that is associated with clinical heparin resistance.1 2 Although tirofiban does not replete AT concentrations, this platelet inhibitor therapy may be useful in augmenting heparin anticoagulation when AT levels are moderately decreased. During CPB there is extensive activation of haemostatic and inflammatory systems, resulting in increased thrombin generation, which is only partially suppressed by the use of high doses of heparin.29 Suppression of platelets with tirofiban may potentially reduce thrombin formation on the activated platelet surface, and ultimately reduce platelet activation and clot formation on extracorporeal circuits. Preservation of platelet count and function by high-dose tirofiban has been reported in an experimental CPB in baboons.30 Experiences from the use of tirofiban immediately before7 or during CPB 30 also provide evidence that tirofiban addition to heparin more effectively suppresses haemostatic activation on CPB.6 30 Bizzari and colleagues noted that the incidence of thrombocytopenia and haemostatic product use was less in 20 patients who received preoperative tirofiban plus aspirin than in 68 patients who received only heparin plus aspirin.7
In summary, we have shown that a clinical dose of tirofiban can effectively delay platelet activation-mediated thrombin generation and prolong the ACTs of heparinized blood. Suppression of platelet function with tirofiban may be useful in preserving coagulatory function for cardiac surgical patients who present with clinical heparin resistance with moderately decreased AT concentrations.
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Acknowledgments |
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Footnotes |
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References |
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