Enhanced clot formation and altered platelet subpopulations in mesothelioma characterized by a high-dimensional analytical platform

Abstract Background: Mesothelioma is a rare and aggressive lung cancer primarily caused by asbestos exposure. Thrombotic complications are common in mesothelioma and represent a significant contributor to morbidity and mortality. Platelets play a central role in thrombosis by initiating clot formation, supporting thrombin generation, and stabilizing the developing clot. However, the specific alterations in platelet behavior and function in mesothelioma remain poorly understood. Understanding these changes is critical to uncovering the mechanisms underlying the hypercoagulable state observed in these patients and may offer insight into disease progression and therapeutic opportunities.Methods: Peripheral blood samples were collected from patients with malignant mesothelioma (n=36) and healthy controls (n=17). To assess coagulation dynamics, thromboelastography (TEG) was performed on citrated whole blood, evaluating parameters related to clot initiation, kinetics, and clot strength. To study platelet phenotypes, we developed a 13-color spectral flow cytometry panel along with a high-dimensional analysis pipeline, termed PlateletProfiler. This platform was designed to categorize platelets into different subpopulations based on the expression of activation receptors and the exposure of phosphatidylserine. In addition, GPVI levels were measured in both platelet lysates and plasma using ELISA to evaluate differences in expression and potential shedding between mesothelioma patients and healthy controls.Results: Mesothelioma patients exhibited a hypercoagulable profile compared to healthy controls. TEG showed significantly elevated platelet-dependent parameters, including maximum amplitude, MA (p=0.002), clot strength, G (p=0.0003), and elasticity, E (p=0.0002), reflecting increased clot firmness in mesothelioma patients as measured by parameters known to be influenced by platelet activity.Using PlateletProfiler, we identified different platelet subpopulations based on surface marker expression patterns associated with activation and procoagulant function. Mesothelioma blood samples demonstrated a significantly higher proportion of total procoagulant platelets (p=0.001) relative to controls. Analysis of surface marker expression showed increased expression of CD62P (p=0.01), TLT-1 (p=0.052), and annexin-V-binding (p=0.004). Patients with increased platelet activation also displayed elevated platelet-driven TEG parameters, supporting a functional link between surface phenotype and clot strength. In addition, surface GPVI expression was significantly reduced (p=0.007) on mesothelioma platelets, and these platelets exhibited reduced responsiveness to collagen-related peptide A (CRP-A). Reticulated platelets, which represent immature and metabolically active platelets, were significantly elevated in mesothelioma patients compared to controls. These reticulated platelets showed increased annexin V binding (p=0.002) and elevated expression of CD62P (p=0.052) and PD-L1 (p=0.01), indicating both a procoagulant and immune-modulatory profile. ELISA revealed reduced levels of soluble GPVI in mesothelioma patients compared to controls, both in plasma (p=0.01) and in platelet lysates (p=0.05), supporting a potential mechanism of GPVI shedding.Conclusions:This study demonstrates enhanced clot formation in mesothelioma patients, accompanied by shifts in platelet subpopulations, including increased levels of procoagulant and reticulated platelets, along with decreased GPVI expression. Since procoagulant platelets have been associated with thrombotic risk, their presence may serve as a marker for identifying individuals at increased risk of thrombosis. Additionally, reduced levels of soluble GPVI in plasma may be used as a liquid biomarker to monitor platelet activation and thrombotic potential in mesothelioma.Keywords:Mesothelioma, Procoagulant Platelet, Reticulated Platelet, GPVI