Lu, R.; Li, Y.; Xu, A.; King, B.; Ruan, K.-H. Reprogramming Megakaryocytes for Controlled Release of Platelet-Like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential. Cells2023, 12, 2775.
Lu, R.; Li, Y.; Xu, A.; King, B.; Ruan, K.-H. Reprogramming Megakaryocytes for Controlled Release of Platelet-Like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential. Cells 2023, 12, 2775.
Lu, R.; Li, Y.; Xu, A.; King, B.; Ruan, K.-H. Reprogramming Megakaryocytes for Controlled Release of Platelet-Like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential. Cells2023, 12, 2775.
Lu, R.; Li, Y.; Xu, A.; King, B.; Ruan, K.-H. Reprogramming Megakaryocytes for Controlled Release of Platelet-Like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential. Cells 2023, 12, 2775.
Abstract
In this study, we reported that novel single-chain fusion-proteins linked thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq), or s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MK). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the over-expressed SC-TP-Gαq), or s (SC-TP-Gαs) to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we used dual-binding domains linked to different signaling pathways within a single polypeptide chain using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (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-Gαq or SC-TP-Gαs. Flow cytometry analysis showed the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding. This bio-engineering led to the formation of two sets of biologically active PLP forms, which mediate calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as required for therapeutic process.The results also demonstrated the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.
Keywords
platelet linked particles; megakaryocyte; G protein coupled recepto
Subject
Medicine and Pharmacology, Medicine and Pharmacology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
