Reprogramming Megakaryocytes for Controlled Release of Platelet-like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential

dc.contributor.authorLu, Renzhong
dc.contributor.authorLi, Yan
dc.contributor.authorXu, Anna
dc.contributor.authorKing, Bridgette
dc.contributor.authorRuan, Ke-He
dc.date.accessioned2023-12-22T13:45:22Z
dc.date.available2023-12-22T13:45:22Z
dc.date.issued2023-12-06
dc.date.updated2023-12-22T13:45:23Z
dc.description.abstractIn this study, we reported that novel single-chain fusion proteins linking thromboxane A<sub>2</sub> (TXA<sub>2</sub>) receptor (TP) to a selected G-protein &alpha;-subunit q (SC-TP-G&alpha;q) or to &alpha;-subunit s (SC-TP-G&alpha;s) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-G&alpha;q or the SC-TP-G&alpha;s to regulate human platelet function. To understand how the single-chain TP-G&alpha; fusion proteins could regulate opposite platelet activities by an identical ligand TXA<sub>2</sub>, we tested their dual functions&mdash;binding to ligands and directly linking to different signaling pathways within a single polypeptide chain&mdash;using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-G&alpha;q and SC-TP-G&alpha;s, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-G&alpha;q or SC-TP-G&alpha;s (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-G&alpha;q or SC-TP-G&alpha;s. Flow cytometry analysis showed that the PLPs carrying SC-TP-G&alpha;q were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-G&alpha;s reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-G&alpha;q and SC-TP-G&alpha;s were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA<sub>2</sub> with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.
dc.identifierdoi: 10.3390/cells12242775
dc.identifier.citationCells 12 (24): 2775 (2023)
dc.identifier.urihttps://hdl.handle.net/10657/15680
dc.titleReprogramming Megakaryocytes for Controlled Release of Platelet-like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential

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