PB0401 - Endothelial-induced regulation of platelet signalling under flow: establishing the endothelial-controlled platelet phosphoproteome

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Isabella Provenzale

PhD Student
Dept 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

Co-Author(s)

FS

Fiorella A. Solari

Research Associate
Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V. Dortmund, Germany
Dortmund, Nordrhein-Westfalen, Germany
Claudia Schönichen, MSc

PhD Student
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
Delia I. Fernández de la Fuente, MSc

PhD Student
School for Cardiovascular Diseases (CARIM), Maastricht University, 6200 MD Maastricht, The Netherlands
Maastricht, Limburg, Netherlands
SB

Sanne L.N. Brouns

PhD Student
Dept of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands
MK

Marijke Kuijpers

Associate professor
Maastricht University
Maastricht, Limburg, Netherlands
Paola van der Meijden, PhD

Assistant Professor
Department of Biochemistry, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands
Maastricht, Limburg, Netherlands
JG

Jonathan M. Gibbins, PhD

Professor
Institute for Cardiovascular and Metabolic Research, School of Biological sciences, University of Reading, Reading, UK
Reading, England, United Kingdom
AS

Albert Sickmann

Professor
Department 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
CJ

Chris Jones

Associate Professor
Reading School of Biological Sciences, University of Reading, Whiteknights, Reading, United Kingdom
Reading, England, United Kingdom
JW

Johan W. M. Heemskerk

Emeritus Professor
Synapse research insitute
MAASTRICHT, Limburg, Netherlands

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.