Abstract
AbstractAbstract 3195Antiplatelet therapy is a key component of the treatment regimen for acute coronary syndrome and stroke. A major obstacle in developing antiplatelet agents is for agents to be effective without causing bleeding complications. Protease activated receptor 4 (PAR4) is a potential attractive target for antiplatelet therapies because of its requirement for stable clot formation and limited tissue distribution. The purpose of the current study is to determine how PAR4 interacts with other proteins on the surface of platelets and how these interactions influence activation and signaling of PAR4. We have demonstrated that PAR4 forms homodimers using two techniques, bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC). These interactions are specific, as PAR4 does not interact with two unrelated G-protein coupled receptors (GPCRs) rhodopsin and dopamine D2 receptor. In addition to homodimers, PAR4 forms heterodimers with PAR1. The heterodimers are also specific since PAR1 also does not interact with other GPCRs. PAR1 does not interact with rhodopsin; therefore we generated a series of rhodopsin-PAR4 chimeras to determine the region on PAR4 that interacts with PAR1. We have mapped the region on PAR4 required for heterodimerization to transmembrane helix 4 (TM4). When the rhodopsin-PAR4 junction is at the beginning of TM2, TM3 or TM4, the chimera interacts with PAR1 in a manner indistinguishable from PAR4-wt. However, when the junction is moved to the beginning of TM5, TM6 or TM7 the chimera does not interact with PAR1-wt. Molecular modeling of PAR1 and PAR4 has identified residues within TM4 that are exposed and are directed away from the other TM helices creating a potential interaction interface. Co-expression of PAR1 with PAR4 increased the efficiency of PAR4 activation by thrombin ∼10-fold indicating a functional significance for PAR1-PAR4 interactions. The results from these studies provide detailed map of the interactions between two GPCRs on the cell surface and may provide insights for developing PAR antagonists. The current strategy of developing PAR antagonists is to target the individual receptors. However our data show that the functional unit is the PAR1-4 heterodimer and, as such, this may the relevant target to disrupt thrombin activation of platelets. Antagonists to PAR1 or PAR4 need to developed and studied in the context of these interactions, not in isolation. Disclosures:No relevant conflicts of interest to declare.
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