Regulating the Biosynthesis and Signaling of Thromboxane a2 in Hemostasis and Thrombosis
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Statement of the problem: TXA2 is a main contributor in hemostasis. TXA2 is mainly produced by the activated platelets, which can activate more platelets, induce platelet aggregation and facilitate the formation of platelet plug. The precursor of TXA2 is AA, which can be metabolized by COX-1 into the unstable intermediate PGH2. PGH2 could be further isomerized by TXA2 synthase (TXAS) to generate TXA2. However, other prostanoid synthase can compete with TXAS for the same substrate PGH2, to produce other prostanoids, such as PGI2 and PGE2, which are bleeding contributors. For the purpose of effective hemostasis, the production of TXA2 should be increased and the productions of other prostanoids should be constrained. But one obstacle is that all of the prostanoid synthases have the similar affinity to PGH2. Herein, we created a Single-Chain Hybrid Enzyme Complex (SCHEC), COX-1-10aa-TXAS, by linking the C-terminus of COX-1 to the N-terminus of TXAS through a 10 amino acid linker, to redirect the metabolism of AA toward the production of TXA2. On the other hand, the signaling mediated by TXA2 is highly involved in thrombosis. TXA2 can activate its receptor TP, mediate the downstream pro-thrombotic Gαq-calcium signaling, which can mediate platelet aggregation and vasoconstriction. The effects of TXA2 could be countered by PGI2, through triggering the anti-thrombotic Gαs-cAMP signaling. However, the patients with thrombotic diseases, always have a higher level of TXA2. The method to reverse the pro-thrombotic signaling activities of TXA2 has not been discovered yet. Therefore, this study was aimed to accomplish three aims. Specific aim 1: To Examine the biological functions of the novel hybrid enzyme, COX-1-10aa-TXAS. Specific aim 2: To investigate the application of COX-1-10aa-TXAS in hemostasis. Specific aim 3: To create a novel GPCR-G protein complex, which can reverse the pro-thrombotic signaling of TXA2, and investigate the functions of the novel fusion protein complexes in platelet aggregation. Procedure or methods: First, by expressing the hybrid enzyme in HEK 293 cells, the ability to redirect the metabolism of AA was studied. Considering the important role of TXA2 in hemostasis, we investigated the functions of this novel hybrid enzyme in vitro platelet aggregation assays and in vivo mouse bleeding models. Secondly, we created two Single-Chain (SC) GPCR-G protein complexes, SC-TP-Gαq and SC-TP-Gαs. By stably expressing the fusion protein complexes in HEK cells, the signaling mediated by the protein complexes with TXA2 stimulation was studied. In addition, through utilizing the platelet delivery system, the functions of SC-TP-Gαq and SC-TP-Gαs in vitro platelet aggregation were assessed. Results: The HEK 293 cells stably expressing the hybrid enzyme, COX-1-10aa-TXAS, redirected the metabolism of AA to be in favor of TXA2 production, and disfavor of other protanoids. Through being expressed in HEK cells, the hybrid enzyme indicated strong anti-bleeding functions both in vitro platelet aggregation assays and in vivo mouse tail-cut bleeding assays. The hybrid enzyme was also produced by using the S. cerevisiae yeast protein expression system, and purified by ultracentrifugation and chromatography methods. The purified hybrid enzyme indicated strong ability to stop bleeding in mouse bleeding models. In HEK cells stably expressing SC-TP-Gαq and SC-TP-Gαs, triggered by TXA2, the calcium signaling mediated by SC-TP-Gαq and the cAMP signaling mediated by SC-TP-Gαs were confirmed. Moreover, the platelets expressing SC-TP-Gαq could further promote platelet aggregation. While the platelets expressing SC-TP-Gαs could dramatically inhibit the aggregative activities of platelets. Conclusions: These findings suggested that the hybrid enzyme, COX-1-10aa-TXAS, has great potential to be developed into a novel biological reagent in dealing with various hemorrhagic emergencies. And the fusion protein complex, SC-TP-Gαs, through the platelet delivery system, could be used to treat various thrombotic diseases, or be used to prevent the stent thrombosis or in-stent restenosis for the patients with the placement of stent. Additionally, this fusion protein method could also be applied to other GPCRs, in studying or manipulating the downstream signaling by linking to different G proteins.