Platelet GPIIb/IIIa receptor occupancy studies using a novel fluoresceinated cyclic Arg-Gly-Asp peptide

Monitoring Platelet GP IIb/IIIa Antagonist Therapy

“…10 -12 The more rapid GP IIb/IIIa off-rates of many of the low-molecular-weight compounds compared with 7E3 have made it technically more difficult to directly assess GP IIb/IIIa receptor blockade with these agents, but binding studies have been reported that used fluorescent compounds in conjunction with flow cytometry, radiolabeled compounds, or the expression of ligand-induced binding sites on GP IIb/IIIa induced by the binding of the compounds. [13][14][15][16] The introduction of GP IIb/IIIa antagonists as a new class of therapeutic agents raises several important questions, especially because oral, and perhaps even transdermal and intranasal, agents may eventually be available for long-term therapy. Several questions must be addressed.…”

Comparative Antiplatelet Efficacy of a Novel, Nonpeptide GPIIb/IIIa Antagonist (XV454) and Abciximab (c7E3) in Flow Models of Thrombosis

“…Nonspecific XL086 binding was determined by the addition of a 5-fold excess of XV454 and accounted for Ͻ15% of the total binding. 28 Prior work has demonstrated that binding of fluoresceinated monoclonal antibodies (CD42b or abciximab) or cyclic RGD peptide (XL086) detected by flow cytometry and expressed as mean fluorescence intensity is highly correlated with that detected by a radiometric method. 28 Because treatment of blood specimens with XV454 or abciximab inhibits the subsequent binding of XL086-FITC or abciximab-FITC, respectively, to platelets, the decrease in FITC fluorescence values after drug incubation is correlated with the percentage of GPIIb/IIIa receptors occupied by the drug.…”

“…XL086-FITC was prepared by reaction of cyclic [D-Lys-N 2 -methyl-Larginylglycyl-L-aspartyl-3-(aminomethylbenzoic acid)] with FITC and purified by reverse-phase high-performance liquid chromatography. 28 XL086 has 1 fluorescein coupled at the D-Lys position. 28 CD42a (anti-GPIX)-FITC and CD42b (anti-GPIb)-FITC were purchased from Becton Dickinson and Pharmigen, respectively.…”

“…28 XL086 has 1 fluorescein coupled at the D-Lys position. 28 CD42a (anti-GPIX)-FITC and CD42b (anti-GPIb)-FITC were purchased from Becton Dickinson and Pharmigen, respectively.…”

“…Subsequently, specimens were diluted with 2 mL of 1% formaldehyde and analyzed in a FACScan (Becton-Dickinson) flow cytometer as described elsewhere in detail. 17,23,24 Binding of fluoresceinated CD42b (anti-GPIb), XL086 (an analogue of XV454), 28 or abciximab to platelets was calculated by measuring the mean FITC fluorescence intensity of at least 5000 platelets, which were identified on the basis of their characteristic forward-and side-scatter profiles in citrated blood specimens in the presence or absence of GPIIb/IIIa antagonists. At each experimental state, the FITC fluorescence values were normalized by using as a reference the pre-XV454/preabciximab incubation (control specimen) fluorescence levels.…”