The Role of Endothelial Cells in Platelet Generation in vitro

Abstract

Platelets are derived from polypoid megakaryocytes (MKs). Platelet transfusion remains essential for treating thrombocytopenia and bleeding disorders, yet donor-derived platelets are limited by donor shortages, a short shelf life, and risk of contamination. Developing efficient in vitro systems for platelet generation is therefore a major biomedical goal. The lung is a primary site for platelet biogenesis in vivo, and our recent studies suggest the lung endothelium provides a key role for thrombopoiesis. This study investigates how endothelial components contribute to platelet generation using an established microfluidic system designed to mimic pulmonary capillary conditions.
To establish baseline conditions, microfluidic chambers were coated with different concentrations of bovine serum albumin (BSA) to determine the optimal concentration for platelet generation. Chambers were subsequently coated with whole endothelial cell (EC) lysate or subcellular EC fractions (cytosolic, soluble membrane, and insoluble membrane) to investigate their specific contributions to platelet yield and function. Generated platelets were quantified and characterised by flow cytometry. EC-derived proteins from the most effective fraction were analysed by mass spectrometry to identify potential molecular regulators.
A 1% BSA coating provided an optimal baseline. Coating with soluble EC membrane proteins significantly increased platelet generation, approximately doubling the yield compared to BSA or cytosolic fractions. However, these platelets displayed attenuated αIIbβ3 activation and reduced P-selectin surface expression following agonist stimulation, indicating partial pre-activation or functional exhaustion. Proteomic analysis of the soluble membrane fraction revealed enrichment of proteins—including LAMP-1, CD63, integrin β1, PECAM-1, and protein disulphide isomerases—implicated in adhesion, trafficking, and redox regulation.
In summary, specific endothelial membrane components can markedly enhance platelet biogenesis but may simultaneously impair the functionality of the generated platelets. These findings provide a mechanistic insight into the endothelial regulation of thrombopoiesis and inform the design of improved, endothelial-mimetic systems for efficient in vitro platelet production.

Date of Award	24 Feb 2026
Original language	English
Awarding Institution	University of Bristol
Supervisor	Alastair W Poole (Supervisor), Xiaojuan Zhao (Supervisor) & Ingeborg Hers (Supervisor)