Reprogramming Megakaryocytes for Controlled Release of Platelet-like Particles Carrying a Single-Chain Thromboxane A₂...

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Thromboxane A2 (TXA2) is a key regulator of human platelet function.
• The TXA2 receptor (TP) mediates platelet aggregation.
• Controlling platelet activity has significant therapeutic implications.

Purpose of the Study:

• To develop novel single-chain fusion proteins linking the TP to G-protein α-subunits q (Gαq) or s (Gαs).
• To utilize megakaryocyte-released platelet-linked particles (PLPs) as a delivery vehicle for these fusion proteins.
• To investigate the dual function of these fusion proteins in regulating opposite platelet activities via the same ligand, TXA2.

Main Methods:

• Stable expression of single-chain TP-Gαq and SC-TP-Gαs fusion proteins in megakaryocytes (MKs).
• Induction of mature MKs to release PLPs carrying the recombinant fusion proteins.
• Western blot and flow cytometry to confirm protein expression and biological activity.

Main Results:

• Successfully expressed and purified SC-TP-Gαq and SC-TP-Gαs within PLPs derived from MKs.
• PLPs carrying SC-TP-Gαq promoted platelet aggregation by activating Gαq signaling.
• PLPs carrying SC-TP-Gαs inhibited platelet aggregation by reversing Gαq to Gαs signaling.

Conclusions:

• Demonstrated successful bio-engineering of PLPs for targeted delivery of functional fusion proteins.
• These engineered PLPs can mediate opposing platelet aggregation responses (calcium and cAMP signaling) to TXA2.
• Nucleus-free PLPs represent a versatile platform for delivering recombinant membrane-bound GPCRs to modulate cellular activity.