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Endothelial-induced regulation of platelet signalling under flow: establishing the endothelial-controlled platelet phosphoproteome

1Dept of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands and Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences, University of Reading, Reading, United Kingdom, Reading, England, United Kingdom, 2Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V. Dortmund, Germany, Dortmund, Nordrhein-Westfalen, Germany, 3 Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands; Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg-University of Mainz, Germany, Maastricht, Limburg, Netherlands, 4School for Cardiovascular Diseases (CARIM), Maastricht University, 6200 MD Maastricht, The Netherlands, Maastricht, Limburg, Netherlands, 5Dept of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands, Maastricht, Limburg, Netherlands, 6Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University; Thrombosis Expertise Centre, Heart and Vascular Centre, Maastricht University Medical Centre, Maastricht, The Netherlands, Maastricht, Limburg, Netherlands, 7Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands; Thrombosis Expertise Center, Heart+ Vascular Center, Maastricht University Medical Center, Maastricht, The Netherlands, Maastricht, Limburg, Netherlands, 8Institute for Cardiovascular and Metabolic Research, School of Biological sciences, University of Reading, Reading, UK, Reading, England, United Kingdom, 9Department of Protein Dynamics, Leibniz Institute for Analytical Sciences - ISAS-e.V., Dortmund, Germany; Medizinische Fakultät, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany; Department of Chemistry, College of Physical Sciences, University of Aberdeen, United Kingdom., Dortmund, Nordrhein-Westfalen, Germany, 10Department of Biochemistry, CARIM, Maastricht University, Maastricht, the Netherlands; Synapse Research Institute Maastricht, The Netherlands, Maastricht, Limburg, Netherlands

Abstract Number: PB0401

Meeting: ISTH 2022 Congress

Theme: Platelets and Megakaryocytes » Platelet Signaling

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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