Platelets and circulating extracellular vesicles in cancer therapy and thrombosis

Platelets are known for their role in haemostasis and thrombosis but are also key mediators of vascular communication. Tumour cells exploit platelet signalling to support survival, growth, immune evasion and metastasis. This process, termed Tumour Cell-Induced Platelet Activation (TCIPA), involves platelet receptor engagement, release of activation mediators, endothelial dysregulation, and coagulation cascade activation, creating a tightly interwoven relationship between platelets and cancer cells that elevates the prothrombotic potential of cancer patients. Activated platelets release extracellular vesicles (EVs) that influence cellular processes via endocytosis. Platelet-derived EVs enhance thrombotic and coagulant capacity, while tumour-derived EVs promote thrombosis and metastasis, acting synergistically in circulation. This interplay underpins fatal thrombotic events in cancer, highlighting the need to investigate platelet and EV roles in cancer therapies. This thesis focuses on two distinct but interrelated cancer therapies, investigating the platelets and EVs contributions to both mechanisms of action. The first aim evaluated EV and inflammatory profiles in active cancer patients presenting with venous thromboembolism (VTE) to the Mater Misericordiae University Hospital (MMUH). These patients were treated with the historic, Low Molecular Weight Heparin (LMWH) or the newly emerging drug group, Direct Oral Anticoagulants (DOACs). An embedded Quality Improvement (QI) Study successfully increased patient recruitment by implementing a clinical project sponsor, increasing interdisciplinary communication and efficient process monitoring. Characterisation of EVs using Nanoparticle Tracking Analysis (NTA), flow cytometry, and mass spectrometry revealed a non-significant trend of lower total and tissue factor-expressing EVs in DOAC-treated patients compared to LMWH. A similar numeric trend was observed in cytokine profiles, suggesting potential anti-inflammatory effects of DOACs. Although limited by sample size, these findings align with existing clinical data supporting the shift to DOACs for CAT treatment, highlighting the need for larger studies to confirm these promising observations. The second aim investigated the role of platelets and EVs in a leukapheresis treatment for Cutaneous T Cell Lymphoma, called Extracorporeal Photopheresis (ECP). In this procedure, leukocytes and platelets are extracted, exposed to UVA light and 8-methoxypsoralen, and reinfused into the patient. While ECP induces immunomodulation, 27% of patients show no clinical response, with the role of platelets in this process previously unknown. An ex vivo and in vitro study using NTA, flow cytometry, immunoassays, and platelet aggregometry revealed that platelet activation occurs the during density flow centrifugation step but not during UVA light/8-methoxypsoralen exposure. Contrary to previous assumptions, the irradiation step did not influence platelet activation, aggregation, or EV release. These results identify density flow centrifugation as the critical point for platelet activation, providing novel insights into ECP mechanisms. In summary, this comprehensive body of research highlights the subtle but successful initiatives to increase recruitment rates, uncovered intriguing trends indicating DOACs reduce tissue factor EVs and circulating cytokine levels compared to LMWH treatment of cancer associated thrombosis, potentially depicting anti-inflammatory pleotropic effects and begins to clarify the role of platelets in the ECP process, emphasizing the density flow centrifugation step as the critical point for platelet activation. These insights elucidate the diverse roles of platelets and EVs in immuno-thrombotic contributions to cancer therapies, revealing exciting avenues for future research and discovery.