DREAM, a Transcription Repressor, Is Critical for Calcium Signaling and Platelet Thrombus Formation Independently of Its Transcriptional Activity

DREAM (Downstream Regulatory Element Antagonistic Modulator) is a neuronal calcium sensor and acts as a transcriptional repressor for pain modulation. Recent studies showed that endothelial cell DREAM transcriptionally regulates NF-κB signaling during inflammation (Tiruppathi et al. Nat Immunol. 2014). However, it is unknown whether DREAM plays a role during thrombosis and hemostasis. Using real-time fluorescence intravital microscopy with DREAM knockout (KO) mice, we demonstrated that intravascular cell DREAM is required for platelet thrombus formation and fibrin generation at the site of laser-induced cremaster arteriolar injury. Studies of bone marrow chimeric mice further revealed that both hematopoietic and endothelial cell DREAM regulate thrombus formation following arteriolar injury. Using in vitro flow chamber assays, we found that DREAM is important for platelet thrombus formation on collagen-coated surfaces under shear conditions. Further, tail bleeding time and blood loss significantly increased in DREAM KO mice, compared with wild-type (WT) mice, suggesting the importance of DREAM for hemostasis as well as thrombus formation in vivo. We observed that DREAM KO platelets show markedly decreased aggregation, ATP secretion, P-selectin exposure, and αIIbβ3 integrin activation induced by thrombin, collagen-related peptide, ADP, and A23187 (calcium ionophore). Interestingly, DREAM deletion did not affect phorbol 12-myristate 13-acetate-induced platelet functions. Consistently, DREAM KO platelets exhibited decreased phosphorylation of phosphoinositide-3-kinase, AKT, and ERK, but not protein kinase C (PKC) isoforms during cell activation, suggesting that DREAM does not regulate protein kinase C signaling pathway. Electron microscopic analysis demonstrated that ultrastructural features of DREAM KO platelets are indistinguishable from those of WT platelets. Since DREAM is a transcription factor that binds to calcium in four EF-hand motifs, we investigated whether platelet DREAM regulates calcium signaling. Indeed, DREAM deletion in platelets resulted in significant reduction in both intracellular calcium release and store-operated calcium entry (SOCE) following agonist stimulation. When we transiently knocked down DREAM in a human megakaryoblastic leukemia cell line (MEG-01) using siRNA, we found that A23187-induced calcium release and SOCE are impaired by DREAM knockdown in MEG-01 cells without altering the expression of calcium signaling molecules, such as inositol trisphosphate receptor, stromal interaction molecule 1, and ORAI1. These defects were rescued by overexpression of siRNA-resistant WT but not EF-hand domain mutant DREAM. Therefore, our results provide novel evidence that platelet DREAM regulates calcium signaling independently of its transcriptional factor activity, thereby playing a critical role during thrombus formation and hemostasis in vivo.