Circulating platelet aggregates damage endothelial cells in culture

Abstract

BackgroundPresence of circulating endothelial cells (CECs) in systemic circulation may be an indicator of endothelial damage and/or denudation, and the body's response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt/denude the endothelium and contribute to the presence of CEC and EC-derived particles (ECDP). MethodsEndothelial cells were grown in glass tubes and tagged with/without 0.5 μm fluorescent beads. These glass tubes were connected to a mini-pump variable-flow system to study the effect of circulating AgPlts on the endothelium. ECs in glass tube were exposed to medium alone, nonaggregated platelets (NAgPlts), AgPlts, and 90 micron polystyrene beads at a flow rate of 20 mL/min for various intervals. Collected effluents were cultured for 72 h to analyze the growth potential of dislodged but intact ECs. Endothelial damage was assessed by real time polymerase chain reaction (RT-PCR) for inflammatory genes and Western blot analysis for von Willebrand factor. Results and conclusionNo ECs and ECDP were observed in effluents collected after injecting medium alone and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs, releasing ECs and ECDP in effluents as the time increased. Effluents collected when endothelial cell damage was seen showed increased presence of von Willebrand factor as compared to control effluents. Furthermore, we analyzed the presence of ECs and ECDPs in heart failure subjects, as well as animal plasma samples. Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelium damage.