OC 75.4 - Investigating the health and function of murine platelets generated using a new ex-vivo lung system

Background: Producing fully functional platelets outside of the body has been challenging, with notably low platelet yields per megakaryocyte. We have recently designed a new murine lung-based ex vivo vascular system that generates physiological levels of platelets or up to 3000 per megakaryocyte (https://www.biorxiv.org/content/10.1101/2021.11.01.466743v1).

Aims: This study builds on the functional analysis of these generated platelets conducted in the original report by looking specifically at their procoagulant features and mitochondrial health and function.

Methods: Cultured mouse megakaryocytes were passaged through the vasculature of the new mouse lung ex vivo system until platelets were generated. Using flow cytometry, tetramethylrhodamine methyl ester (TMRM) fluorescence was measured in resting and collagen-related peptide + thrombin stimulated platelets (n=8) to assess their mitochondrial depolarisation compared to control platelets isolated directly from mouse whole blood. Surface phosphatidylserine (PS) was assessed by annexin V binding to determine their procoagulant activity (n=5).

Results: Surface activation of αIIbβ3 integrin and P-Selectin exposure upon single agonist stimulation was normal. In their resting state, the generated platelets showed normal TMRM mean fluorescent intensity but significantly more of them (12% of the whole sample) exposed PS compared to control platelets (1.5%) (p < 0.005, Student's t-test). Upon dual agonist stimulation, significantly fewer generated platelets start presenting procoagulant features compared to controls (p < 0.05). Notably, 19% have depolarised mitochondria and 20% expose PS compared to 38% and 35% of controls respectively.

Conclusion(s): Under basal conditions, the platelets generated in the mouse lung vasculature have healthy mitochondria but increased surface PS likely due to some degree of pre-activation by exposure to tissue injury while in the ex vivo system. Once activated, they have reduced procoagulant activity compared to control platelets, possibly a mechanism that ensures young platelets remain quiescent until they are established in the blood system. Their functionality should now be assessed over time as they age in vitro.