Increased Concentrations of Tirofiban in Blood and Their Correlation With Inhibition of Platelet Aggregation After Greater Bolus Doses of Tirofiban David J. Schneider, MD, Howard C. Herrmann, MD, Nasser Lakkis, MD, Frank Aguirre, MD, Man-Wai Lo, PhD, Kuo-Chang Yin, MA, Atul Aggarwal, MD, Samer S. Kabbani, MD, and Peter M. DiBattiste, MD P atients in the do Tirofiban And Reopro Give sim- ilar Efficacy outcomes Trial (TARGET) treated with abciximab compared with tirofiban had a lower 30-day incidence of the combined end point of death, myocardial infarction, and target vessel revasculariza- tion that reflected, in large part, a decrease in the incidence of periprocedural myocardial infarction. 1 We have found that the extent of platelet aggregation inhibition was greater from 15 to 60 minutes after abciximab than that after the TARGET study dosage of tirofiban (10 g/kg bolus, followed by a 0.15 g/ kg/min infusion). 2–4 To identify a bolus associated with maximal inhibition of platelet aggregation, we used flow cytometry to characterize the inhibitory effects of tirofiban in blood from patients with acute coronary syndromes. In subsequent studies, the aver- age extent of inhibition of turbidometric aggregation in response to 20 M of adenosine diphosphate (ADP) was found to be 90% during the first hour when a bolus of 25 g/kg was combined with the same infu- sion (0.15 g/kg/min for 18 to 24 hours) employed in the TARGET study. 5 In this report, we describe the concentrations of tirofiban in blood and their correla- tion with the inhibition of turbidometric aggregation in response to 20 M of ADP. ••• Protocols were approved by the institutional review boards of the participating institutions. All patients pro- vided written informed consent. Eligible patients in- cluded those with an acute coronary syndrome (ischemic symptoms plus either 0.5 mm of ST-segment depression on electrocardiogram or increased troponin or creatine kinase-MB) in whom a percutaneous coronary interven- tion was clinically mandated and performed. Exclusion criteria included treatment with antiplatelet agents other than aspirin in the previous 14 days, thrombolytic ther- apy within 24 hours, renal insufficiency (creatinine 2.5 mg/dl), and contraindication to treatment with a glyco- protein IIb/IIIa inhibitor. To perform in vitro studies, blood was obtained from patients with acute coronary syndromes before coronary intervention or treatment with an antiplatelet agent other than aspirin. After anticoagulation with corn trypsin inhibitor, a specific inhibitor of coagula- tion factor XIIa, 6 the blood was spiked with 0, 50, 100, or 150 ng/ml of tirofiban. After incubation for 15 minutes at room temperature, the capacity to bind fibrinogen in response to 1 M of ADP was deter- mined with the use of flow cytometry. The concentration of tirofiban in blood and its associated inhibition of platelet function were as- sessed in a preliminary study in which patients were treated with a 10 g/kg bolus 2 and in a subsequent study in which patients were treated in a sequential, open-label, nonrandomized fashion with a 20 or 25 g/kg bolus. 5 Tirofiban was administered before the start of the coronary intervention, and each bolus was combined with an 18- to 24-hour infusion of 0.15 g/kg/min. Patients were treated with aspirin (325 mg before the procedure and daily) and unfractionated heparin (target activated clotting time 250 seconds). Clopidogrel (300 mg initially and then 75 mg daily) was begun after the coronary intervention (1 hour). The concentration of tirofiban was determined by radioimmunoassay after 5, 15, 30, 45, 60, 120, and 360 minutes. 7 Platelet function was assessed by light transmission aggregometry (Bio Data Corp., Helena Laboratories, Horsham, Pennsylvania), and flow cy- tometry before treatment and after 15, 30, 45, 60, and 120 minutes, and an additional assessment was made after 5 minutes with flow cytometry. Blood for aggre- gometry was anticoagulated with D-Phe-Pro-Arg- chloromethyl ketone (PPACK, 38 M) to avoid ef- fects of citrate on inhibitory properties of tirofiban. 8 Blood for flow cytometry was anticoagulated with corn trypsin inhibitor. Maximal turbidometric ex vivo aggregation after 4 minutes was assessed in platelet- rich plasma in response to 20 M of ADP (Chronolog, Havertown, Pennsylvania). Assessment of the capac- ity of platelets to bind fibrinogen was performed as previously described in detail. 9 Assay and fixation were performed at each site for flow cytometry. Sam- ples were analyzed at the University of Vermont within 30 hours. As previously described, 5 the primary objective was to identify a bolus dose of tirofiban that achieved a mean inhibition of platelet aggregation of 90% plus a lower bound of the 95% confidence interval of inhibition of 85% from 15 to 45 minutes after onset of treatment. The slope of the association between selected concentrations of tirofiban and the extent of From the University of Vermont, Burlington, Vermont; University of Pennsylvania Medical Center, Philadelphia, Pennsylvania; Baylor Col- lege of Medicine, Houston, Texas; Prairie Cardiovascular, Spring- field, Illinois; and Merck & Company, Inc., West Point, Pennsylvania. This report was supported by Merck & Company, Inc., West Point, Pennsylvania. Dr. Schneider’s address is: University of Vermont, 208 South Park Drive, Suite 2, Colchester, Vermont 05446. E-mail: djschnei@zoo.uvm.edu. Manuscript received August 7, 2002; re- vised manuscript received and accepted September 24, 2002. 334 ©2003 by Excerpta Medica, Inc. All rights reserved. 0002-9149/03/$–see front matter The American Journal of Cardiology Vol. 91 February 1, 2003 doi:10.1016/S0002-9149(02)03163-6