The highly variable levels of fetal hemoglobin (HbF) reactivation among patients with sickle cell disease is not well understood. We previously determined that pancellular reversal of gamma-globin gene silencing is achievable through the activation of specific signal transduction pathways using stem cell factor (SCF) and transforming growth factor beta (TGF-B). Based upon the reliability of this culture assay for HbF reactivation, the culture system is additionally being utilized to screen for inhibitors of HbF reactivation. As an initial screening strategy, published reports, high-throughput sequencing efforts, and array-based transcription profiles were examined to identify erythroblast-expressed receptors with known ligands that may serve a signaling role in the regulation of globin expression. A dose-escalation strategy was used to screen seventeen ligands that bind to a subset of G-protein coupled receptors (GPCR). Among those ligands, oleoyl-lysophosphatidic acid (OLPA) and neurokinin A (NKA) were identified as potential inhibitors of HbF reactivation. Lysophosphatidic acid is a lipid metabolite that is released after tissue injury and may play a role in vascular remodeling. Neurokinin A is a small neuropeptide mainly involved in pain signaling. OLPA inhibited HbF reactivation in a dose-dependent manner (HbF/HbF+HbA ratios: EPO: 0.7±0.01%; EPO+SCF+TGF-B (EST): 36.3±0.1%; EST+10nM OLPA: 36.5±0.1%; EST+100nM OLPA: 31.6±0.1%; EST+1uM OLPA: 12.7±0.1%, p=1.9E-03). NKA also inhibited HbF reactivation, but the magnitude of the NKA effect was donor-specific. Cells from one donor demonstrated a robust response (HbF/HbF+HbA ratios: EPO: 1.7%; EST: 26.7%; EST+10nM NKA: 24.5%; EST+100nM NKA: 8.8%; EST+1uM NKA: 7.8%). Since OLPA and NKA may both act through adenylate cyclase, the role of this enzyme was examined further using the activator, forskolin (0.8 uM-20uM) and inhibitor, SQ22536 (40uM – 600uM). While we were unable to demonstrate a significant increase in HbF among cultures grown in forskolin, the addition of SQ22536 resulted in a profound, dose-dependent inhibitory effect on the expression of HbF (EPO: 1.3±1.2%; EST: 36.7±11.3%; EST+40uM SQ: 30.3±10.2%; EST+200 uM SQ : 14.5±4.5%, p=1.9E-02; EST+600uM SQ: 2.3±2.7%, p=4.1E-03). Flow cytometry revealed that the inhibitory effects of SQ22536 on HbF were pancellular. Importantly, inhibition of the main downstream target of adenylate cyclase (protein kinase A) with H89 (10nM-1.0 uM) did not result in a significant reduction in HbF. However, phosphorylation studies demonstrated that SQ22536 altered the kinetics of MEK activation. MEK is a kinase that helps mediate the HbF-activating effect of SCF [Blood 2004, 1;103(5):1929–33]. These data suggest that inhibitory effects upon HbF reactivation in adult erythroblasts can be mediated by endogenous signaling molecules. Based upon these results, we propose that patient-specific responses to tissue injury and pain may play a negative role in HbF reactivation in sickle-cell disease.