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Chemical Genetic Analysis of Platelet Granule Secretion-Probe 3

Publication TypeJournal Article
Year of Publication2012
AuthorsVerPlank, L, Dockendorff, C, Negri, J, Perez, JR, Dilks, J, Macpherson, L, Palmer, M, Flaumenhaft, R, Schreiber, SL
Date Published2012/01/09

Platelet activation occurs in response to vascular injury, which triggers an array of cascading signaling events that potentiate the formation of thrombi to control bleeding. One downstream effect of activation is the secretion of granules, which plays a role in the formation of thrombi. Platelet activation inhibitors that target various surface receptors and cytosolic pathways within platelets are well validated for inhibiting thrombosis, clinically preventing adverse cardiovascular events such as intermittent claudication and stroke, and reducing mortality and morbidity in acute myocardial infarction. These inhibitors have provided a greater understanding of the mechanisms of platelet biology. Some inhibitors are currently in use in the clinical setting or in development for the treatment of thrombotic conditions. Unfortunately, many of the characterized platelet inhibitors can cause undesired side effects in patients. None of the characterized inhibitors are known to target the granule secretion machinery. We report the outcome of a high-throughput chemical library screen and a series of secondary conformation assays to identify novel, small-molecule inhibitors of platelet activation, specifically fitting into three possible attribute classes: 1) inhibitors of the pathways required for dense granule secretion, 2) inhibitors of pathways required for granule secretion, and 3) inhibitors of upstream G protein-coupled receptor (GPCR) signaling. A primary assay was developed to measure the release of granules from platelets activated through the thrombin GPCR PAR1. Of 302,457 screened compounds, 28 prioritized compounds were selected for additional characterization in secondary specificity assays, which yielded three probe candidates. These three probe candidates met the criteria as possible modulators of GPCR signaling in platelet activation. Of these, a diaminophenyl scaffold (CID 1048267/ML161) showed acceptable potency (av. 11.2 μM ± 1.3) and inhibited P-selectin expression in a concentration-dependent manner (EC50 0.3 μM), as well as inhibiting SFLLRN-induced thrombus formation. This new GPCR-specific probe will be useful in future cell-based investigations of the complex mechanisms of platelet activation and in vivo studies as an antithrombotic agent in murine models.