Extracellular vesicles from activated platelets possess a phospholipid-rich biomolecular profile and enhance prothrombinase activity

Background: Extracellular vesicles (EVs), in particular those derived from activated platelets, are associated with a risk of future venous thromboembolism.

Objectives: To study the biomolecular profile and function characteristics of EVs from control (unstimulated) and activated platelets.

Methods: Biomolecular profiling of single or very few (1-4) platelet-EVs (control/stimulated) was performed by Raman tweezers microspectroscopy. The effects of such EVs on the coagulation system were comprehensively studied.

Results: Raman tweezers microspectroscopy of platelet-EVs followed by biomolecular component analysis revealed for the first time 3 subsets of EVs: (i) protein rich, (ii) protein/lipid rich, and (iii) lipid rich. EVs from control platelets presented a heterogeneous biomolecular profile, with protein-rich EVs being the main subset (58.7% ± 3.5%). Notably, the protein-rich subset may contain a minor contribution from other extracellular particles, including protein aggregates. In contrast, EVs from activated platelets were more homogeneous, dominated by the protein/lipid-rich subset (>85%), and enriched in phospholipids. Functionally, EVs from activated platelets increased thrombin generation by 52.4% and shortened plasma coagulation time by 34.6% ± 10.0% compared with 18.6% ± 13.9% mediated by EVs from control platelets (P = .015). The increased procoagulant activity was predominantly mediated by phosphatidylserine. Detailed investigation showed that EVs from activated platelets increased the activity of the prothrombinase complex (factor Va:FXa:FII) by more than 6-fold.

Conclusion: Our study reports a novel quantitative biomolecular characterization of platelet-EVs possessing a homogenous and phospholipid-enriched profile in response to platelet activation. Such characteristics are accompanied with an increased phosphatidylserine-dependent procoagulant activity. Further investigation of a possible role of platelet-EVs in the pathogenesis of venous thromboembolism is warranted.