Journal of Invasive Cardiology

Platelet Function Studies
Glycoprotein IIb/IIIa receptor-binding assay. Blood samples were obtained in 25 patients receiving abciximab in standard fashion without pretreatment with eptifibatide, and in 25 patients receiving abciximab following eptifibatide pretreatment as outlined above. Samples were obtained prior to administration of abciximab, 10 minutes after administration of abciximab and 8 hours following administration of abciximab in all patients. We used the platelet GP IIb/IIIa occupancy kit (Biocytex, France) to determine MAB1 and MAB2 binding in our study groups. The method uses a flow cytometric assay to quantify binding of two monoclonal antibodies LYP18 (MAB1) and 4F8 (MAB2) to GP IIb/IIIa receptors.¹³ Binding of MAB1 to GP IIb/IIIa receptors has been shown to be inhibited by abciximab, whereas MAB2 binding is unaffected by it. In contrast, small molecular- weight antagonists inhibit MAB2, but not MAB1-binding.¹³ This kit employs whole blood diluted 1:4 in PBS BSA buffer, a negative isotypic control, MAB1 reagent, MAB2 reagent, a calibrated bead suspension and a polyclonal antimouse IgG-FITC. Samples were analyzed in a FACS Calibur (Becton-Dickenson, Franklin Lakes, New Jersey) and gated on log-forward versus log-side scatter to identify the platelet or bead populations. A calibration curve was plotted using the mean fluorescence intensity of the beads and their corresponding number of monoclonal antibody molecules. Results were expressed as the total number of binding sites/platelet for MAB1 and MAB2. Receptor occupancy was determined as (MAB2-MAB1)/MAB2 x 100% in the presence of abciximab, and as (MAB1-MAB2)/MAB1 x 100% in the presence of eptifibatide.
Ultegra RPFA. Determination of Ultegra RPFA (rapid platelet function assay) platelet activation units (PAU) was made with the same blood samples used to assess platelet receptor binding. Details of this method have been reported elsewhere.¹⁴ The RPFA is based on an interaction between platelet GP IIb/IIIa receptors and fibrinogen-coated beads, leading to the agglutination of the beads. Pharmacological blockade of GP IIb/IIIa receptors prevents this interaction and thereby diminishes agglutination in proportion to the degree of receptor blockade achieved. The light absorbance of the sample is measured as a function of time, and the rate of agglutination is quantified as PAU.
Outcomes evaluation. The procedural and hospital outcomes of all patients in the study were evaluated by dedicated research nurses for occurrence of complications and entered into a database following each procedure (CAOS, IBS, Winston-Salem, North Carolina).¹² As a supplement to the database query, an independent chart review of all patients was performed. CK measurements were performed prior to and every 8 hours and up to 24 hours after each coronary intervention or until discharge from the hospital, whichever came first. In the event of a myocardial infarction (MI) postprocedure, CK measurements were obtained until a peak was determined. Electrocardiography, blood and chemistry assays were performed on the morning of, 2 hours following the administration of abciximab and the day following each coronary intervention. Outcome measures conformed to the outcomes defined in the ACC-NCDR.¹⁵ Bleeding was defined according to TIMI classification.¹⁶ In-hospital and follow-up major adverse cardiac events (MACE) were defined as recurrent MI, any revascularization or death.