Thrombosis is a fatal complication of major diseases, such as stroke, sepsis, and heart attack. In addition to aspirin, current medication for thrombosis prevention/treatment mainly focus on platelet P2Y12 receptor with plavix as an antagonist in routine clinical use. However, plavix has showed unstable performance, either failing to avoid thrombosis or causing bleeding. The long-term goal of this project is to establish non-platelet P2Y receptors, e.g. P2Y2 receptor (P2Y2R), as a new molecular target that controls inflammation-induced vascular thrombogenesis. Although it is well established that tissue factor (TF) is the initiator of thrombosis, very little is known on the contribution of P2Y receptors in regulation of various inflammatory stimuli-induced TF expressions. This is an important question because many pro-inflammatory stimuli promote cellular nucleotide release and inactivate ectonucleotidases, leading to accumulation of extracellular nucleotides, which in turn amplify original receptors? signaling via P2Y receptor co-activation. The PI reported previously that P2Y2R is up-regulated in stented coronary arteries and it is the predominant subtype of all P2Y receptors in human coronary artery endothelial cells (HCAEC). They also reported that activation of P2Y2R dramatically induces TF expression and activity in HCAEC. The PIs? preliminary studies showed that TF induction by P2Y2R is also applicable to human primary blood monocytes and mouse coronary endothelium in vivo, and it involves both transcriptional and post-transcriptional mechanisms. Further, LPS-induced TF induction is significantly decreased when extracellular nucleotides are removed by apyrase, and importantly, P2Y2R-null mice are protected from endotoxic death. Based on these original findings, the PI proposes the hypothesis: The non-platelet P2Y2R is a previously unrecognized key mediator in inflammation-induced thrombosis via TF induction. This hypothesis will be tested by the pursuit of three specific aims: 1) Determine the transcriptional mechanism underlying P2Y2R activation of the TF gene in vascular endothelial cells and blood monocytes; 2) Define the post-transcriptional mechanism by which P2Y2R activation leads to increased TF mRNA stability in vascular endothelial cells and blood monocytes; 3) Assess the role of P2Y2R/TF axis in mouse models of inflammation-induced thrombosis.
Aim 1 will determine the role of a new AP-1 binding site with new AP-1 components Fra-1/ATF2 in TF mRNA induction by P2Y2R.
Aim 2 will determine the roles of the AU-rich elements in TF 3?UTR along with their binding proteins and the miRNA mechanisms contribute to P2Y2R-mediated TF mRNA stabilization.
Aim 3 will verify if deletion of P2Y2R prevents LPS-induced disseminated intravascular coagulation and reduces atherothrombosis. The proposed study will be performed in primary cultured human cells and cells isolated from P2Y2R-null mice, followed by a series of in vivo studies. The proposed research is significant, because it is expected to advance and expand understanding of how inflammation leads to increased thrombogenicity of blood vessels via the new P2Y2R-TF axis. Ultimately, such knowledge has the potential to produce new strategies in combating thrombogenic diseases.
P2Y2 receptor is an important protein that acts as a sensor for both extracellular ATP and UTP. The applicants will investigate the functions of P2Y2 receptor in inflammation-induced blood clot formation. The results of the proposed studies may provide the foundation for novel therapy for the treatment of blood vessel occlusive diseases, such as angina, heart attack and stroke, which is relevant to public health and to the part of NIH?s mission ?developing fundamental knowledge that will help to reduce the burdens of human disability.
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