Non-canonical Platelet activation by oral squamous cell carcinoma extracellular vesicles through cooperative thrombin...

Coagulation contributes to cancer mortality, and tumor cells activate platelets to release growth factors. However, this activation is non-canonical, employs unknown agonists and, uniquely, is not immediate. Platelets are activated by human cell lines derived from Oral Squamous Cell Carcinomas (OSCC) with this atypical delay. The stimulatory activity was not a soluble factor, but rather was completely recovered in spontaneously shed extracellular vesicles (EV). EV did not contain established platelet agonists. Instead, chemical and pharmacologic inhibitors show OSCC EV interacted with quiescent platelets to assemble extrinsic and common coagulation complexes that generated thrombin after a significant delay. Newly generated thrombin then stimulated platelets through their PAR1 thrombin receptor. Translocation of intracellular phosphatidylserine to the platelet surface, enabled by its enzymatic oxidation, is essential for de novo formation of coagulation complexes. The delay in OSCC EV-induced platelet aggregation correlated to the delay in phosphatidylserine surface expression. Platelets express the oxidant-generating prorenin receptor (p)RR, while OSCC-EV contained its prorenin ligand. (p)RR non-proteolytically activates the prorenin zymogen and blockade of either this interaction by the decoy PRO20 peptide or inhibition of renin activity suppressed phosphatidylserine translocation and platelet activation. OSCC EV induced delayed peroxidation of platelet membrane lipids, while the intra-membranous radical trap Liproxstatin-1 suppressed phospholipid oxidation, phosphatidylserine display, and platelet activation. We conclude hysteresis characteristic of non-canonical, tumor cell-induced platelet activation reflects a novel time-dependent process of pro-thrombin activation. The rate-limiting step is membrane oxidation that enables phosphatidylserine translocation to the platelet surface and subsequent formation of the premier platelet agonist, thrombin.