AID	Name	Type	Comment
2663	Inhibitors of Platelet Integrin alphallb-beta3: Summary	summary

NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLPCN Grant: 1 R03 MH083257-01
Assay Provider: Barry Coller, Rockefeller University

NCGC Assay Overview:

The alphaIIbbeta3 receptor plays a vital role in hemostasis and thrombosis, where receptor deficiency causes the hemorrhagic disorder, Glanzmann thrombasthenia, and uncontrolled receptor activation causes thrombosis and blood vessel occlusion. Small molecule inhibitors of alphaIIbbeta3, tirofiban and eptifibatide, have undesirable side effects thought to arise from conformational changes in alphaIIbbeta3. These changes transiently activate the receptor to cause spontaneous platelet aggregation and thrombus formation. The goal of this project is to identify new alphaIIbbeta3 antagonists that do not activate the receptor or initiate conformational changes that expose novel epitopes. This secondary assay measures the binding of human plates to fibrinogen, an extracellular matrix protein (Blue et al., 2008). Platelets were loaded with a fluorescent dye, calcein, and added to fibrinogen-coated plates. Following incubation, unbound platelets were removed by washing and bound platelets were detected by measuring fluorescence intensity. Inhibitors of alphaIIbbeta3 block platelet binding to fibrinogen and result in a decrease in fluorescence intensity.

NCGC Assay Protocol Summary:

Human fibrinogen (50 ug/mL) in Tris/saline (100 mM NaCl, 50 mM Tris/HCl, pH 7.4; American Diagnostica, Stamford, CT) was added to black, clear-bottom, untreated polystyrene, nonsterile 384-well microtiter plate wells (Corning no. 3711; Acton, MA) using a peristaltic microplate dispenser (WellMate; Matrix, Hudson, NH). After incubating at 22 C for 1 hour, plates were washed 4 times with Tris/saline using an automated plate washer (ELX405; Bio-Tek, Winooski, VT), and wells were then blocked with HBMT (138 mM NaCl, 12 mM NaHCO3, 10 mM HEPES, 2.7 mM KCl, 0.4 mM NaH2PO4, 0.1% glucose, 0.35% BSA, pH 7.4) for at least 1 hour. After 2 additional washes, 15 uL HBMT was allowed to remain in each well. Compounds were added to the wells with a liquid handling robot (MiniTrak V; PerkinElmer, Wellesley, MA) using a pin tool (VP Scientific, San Diego, CA) that added 0.1 uL compound in DMSO. Fifteen microliters 5E11/L calcein-labeled platelets were then added (2.5E11/L final concentration). After 1 hour of adhesion, wells were washed 4 times with HBMT-CaCl2/MgCl2 and the plates were read by an Envision (Perkin Elmer) to detect calcein fluorescence (490 nm excitation and 515 nm emission). Positive controls consisted of wells containing platelets without compounds. Negative controls were wells containing platelets and known inhibitors of alphaIIbbeta3, including mAbs 7E3 and 10E5, and EDTA.

Reference

Blue R, Murcia M, Karan C, Jirouskova M, and Coller BS. Application of high-throughput screening to identify a novel alphaIIb-specific small- molecule inhibitor of alphaIIbbeta3-mediated platelet interaction with fibrinogen. Blood. 2008, 111(3):1248-56

Compound Ranking:

Compounds with an IC50 value <20 uM were considered active and were assigned a score between 50 and 100 based on compound potency. Compounds with an IC50 value >20 uM or % inhibition at 30 uM <50% (when an IC50 was not determined) were considered inactive and assigned a score of 0. Other compounds were considered inconclusive and assigned a score of 25.

Grant Number: 1 R03 MH083257-01