An Assay of Measuring Platelet Reactivity Using Monoclonal Antibody against Activated Platelet Glycoprotein IIb/IIIa in Patients Taking Clopidogrel

Abstract

Background and ObjectivesResidual platelet reactivity in patients who are taking clopidogrel is commonly measured with VerifyNow assay, which is based on the principle of light transmission aggregometry. However, to evaluate the residual platelet reactivity, it would be more accurate if the reactivity of platelet glycoprotein (GP) IIb/IIIa is directly monitored. In this study, PAC1, a monoclonal antibody against activated platelet GP IIb/IIIa, was used to measure the residual platelet reactivity.Subjects and MethodsTwenty seven patients with coronary artery disease taking clopidogrel were enrolled. Platelets in whole blood were stained with fluorescein isothiocyanate (FITC)-conjugated PAC1. Mean fluorescence intensity (MFI) and % positive platelets (PP) were measured with flow cytometry, and the binding index (BI; MFI × %PP/100) was calculated. P2Y12 reaction unit (PRU) and % inhibition of VerifyNow assay were also measured in the usual manner.ResultsPRU of VerifyNow assay correlated significantly with MFI, %PP, and BI at 10 µM (r=0.59, 0.73, and 0.60, respectively, all p<0.005) and 20 µM of adenosine diphosphate (ADP; r=0.61, 0.75, and 0.63, respectively, all p<0.005). The % inhibition also correlated significantly with MFI, %PP, and BI at 10 µM (r=-0.60, -0.69, and -0.59, respectively, all p<0.005) and 20 µM of ADP (r=-0.63, -0.71, and -0.62, respectively, all p<0.005).ConclusionDirect measurements of the reactivity of platelet GP IIb/IIIa were feasible using PAC1 and flow cytometry in patients taking clopidogrel. Further clinical studies are required to determine the cut-off values which would define high residual platelet reactivity in patients on this treatment protocol.

Anti-platelet effects of epigallocatechin-3-gallate in addition to the concomitant aspirin, clopidogrel or ticagrelor treatment

“…Various agonists can activate platelets and induce their aggregation [ 20 ]. Signaling process of platelet activation and aggregation can be classified into three steps: (1) agonist-dependent early platelet activation, (2) the intermediate common signaling events, and (3) integrin-mediated final signaling pathway [ 21 ].…”

Detection of Sepsis in Platelets Using MicroRNAs and Membrane Antigens

“…Platelet-rich plasma samples were pre-incubated for 15 min at room temperature with 5 µL of one of the following monoclonal antibodies: P2Y12 Platelet ADP Receptor antibody (Sigma-Aldrich, Saint Louis, MO, USA), CD62P/P-Selectin Antibody APC conjugate (Thermo Fisher Scientific ® , Waltham, MA, USA), Integrin alpha-2b/CD41 Antibody FITC conjugate (Thermo Fisher Scientific ® , Waltham, MA, USA), and Integrin beta-3/CD61 Antibody APC conjugate (Thermo Fisher Scientific, Waltham, MA, USA), which have specificity for the platelet antigens P2Y12, P-Selectin (CD62P), GpIIb (CD41), and GpIIIa (CD61), respectively. We characterized the basal state of platelet activation, measured by the mean fluorescence intensity (MFI) of the plasma, which means the higher the fluorescence, the greater the labeling of antibodies against the plasma membrane [ 12 ]. For each sample submitted to flow cytometry, 10,000 events were analyzed using Attune NxT Software (Thermo Fisher Scientific, Waltham, MA, USA).…”

Opposite effects of Agrimonia pilosa Ledeb aqueous extracts on blood coagulation function

“…Platelets are critical for hemostasis both for the formation of blood clots, as platelet aggregates are an essential constituent of the arterial thrombus, and as a platform for activation of coagulation proteins (4). The most important characteristic of GP IIb/IIIa is its affinity modulation of binding to fibrinogen involved in platelet aggregation (26,27). Binding of fibrinogen to the platelet surface can mediate platelet aggregation, and GP IIb-IIIa is an activation dependent receptor for fibrinogen (28).…”