Abstract Number: PB0401
Meeting: ISTH 2022 Congress
Background: Whole blood based microfluidic assays represent new tools to model thrombosis and haemostasis in vitro. Yet these assays often lack key vascular elements that regulate platelet activation and control thrombus formation.
Aims: Development of an endothelialised microfluidic system to reveal mechanisms of endothelial control of platelet and coagulant processes. Use of a phosphoproteomic approach to delineate endothelial effects on platelets.
Methods: Human umbilical vein endothelial cells (HUVEC), cultured in microfluidic channels coated with collagen and tissue factor, were used for whole blood perfusion (shear rate 1000/s). Fluo4-loaded platelets and labelled fibrinogen were added to measure platelet activation and fibrin formation. Calcium responses were also measured in isolated platelets exposed to HUVEC, and post-activated with thrombin or collagen-related peptide (CRP-XL). Guided by functional assays, (un)stimulated platelets exposed to HUVEC (n=3 donors, 11 conditions) were used for label-free phosphoproteomic analysis. Peptides were generated with a bottom-up proteomic approach, phopsphopeptides were enriched using Fe(III)-IMAC based workflow and analysed via LC-MS/MS.
Results: Whole blood perfusion over sub-confluent HUVEC resulted in reduced platelet adhesion with surface area coverage (SAC) of 4.6±3.4%, when compared to absence of HUVEC (40.4±6.9 %SAC, p < 0.001). Residual thrombi were restricted to areas between HUVEC, and the platelets had low Ca2+ responses. Under coagulant condition, HUVEC strongly delayed the fibrin formation (p < 0.01). Under stasis, pre-incubation with HUVEC suppressed the platelet Ca2+ responses to thrombin or CRP-XL. In mass-spectrometric analyses, 5,463 phospho-peptides were identified (77.5% Ser, 15.1% Thr, 7.4% Tyr) with good correlation between biological replicates (Pearson 0.86). Multiple regulated proteins were identified with consensus PKA and PKG phosphorylation motives, pointing to activation control via prostacyclin, nitric oxide and other endothelial-derived biomolecules, being antagonised by platelet post-stimulation.
Conclusion(s): Endothelial cells negatively regulate platelet responses (under flow) through phosphorylation changes affecting collagen and thrombin receptor-induced activation involving multiple pathways.
To cite this abstract in AMA style:Provenzale I, Solari F, Schönichen C, Fernández de la Fuente D, Brouns S, Kuijpers M, van der meijden P, Gibbins J, Sickmann A, Jones C, Heemskerk J. Endothelial-induced regulation of platelet signalling under flow: establishing the endothelial-controlled platelet phosphoproteome [abstract]. https://abstracts.isth.org/abstract/endothelial-induced-regulation-of-platelet-signalling-under-flow-establishing-the-endothelial-controlled-platelet-phosphoproteome/. Accessed November 30, 2023.
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