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

In this study, we reported that novel single-chain fusion proteins linking thromboxane A2 (TXA2) 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 TXA2, we tested their dual functions-binding to ligands and directly linking 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-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 TXA2 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.