PB0776 - Using a multi-omics approach to understand endothelial dysregulation of platelet activation in inflammatory conditions

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

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

Co-Author(s)

IP

Isabella Provenzale

PhD Student
Department for Biochemistry, CARIM School for Cardiovascular Diseases, Maastricht University, The Netherlands; Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences, University of Reading, Reading (UK)
Maastricht, Limburg, Netherlands
AL

Annekathrin Ludt

Scientific Staff
Institute of Medical Biostatistics, Epidemiology and Informatics (IMBEI), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
Mainz, Rheinland-Pfalz, Germany
FS

Fiorella A. Solari

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

Federico Marini

Head of the Bioinformatics Group - Genomic Statistics and Bioinformatics
Institute of Medical Biostatistics, Epidemiology and Informatics (IMBEI), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany; Virchow Fellow, Center for Thrombosis and Hemostasis Mainz (CTH), Germany
Mainz, Rheinland-Pfalz, Germany
AC

Audrey Cleuren

Principle Investigator
Oklahoma Medical Research Foundation, Oklahoma City, OK, USA
Oklahoma City, Oklahoma, United States
KJ

Kerstin Jurk

Principle Investigator
Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg-University of Mainz, Germany
Mainz, Rheinland-Pfalz, Germany
MS

Monika Stoll

Professor
Departments of Biochemistry and Genetic Epidemiology and Statistical Genetics, CARIM School for Cardiovascular Diseases, Maastricht University, The Netherlands
Maastricht, Limburg, Netherlands
AS

Albert Sickmann

President
ISAS
Dortmund, Nordrhein-Westfalen, Germany
MK

Marijke Kuijpers

Associate professor
Maastricht University
Maastricht, Limburg, Netherlands
JW

Johan W. M. Heemskerk

Emeritus Professor
Synapse research insitute
MAASTRICHT, Limburg, Netherlands

Background: Endothelial cells (EC) suppress platelet activation and coagulation to prevent blood clotting. In inflammatory conditions, this negative regulation can be hampered and lead to thrombotic complications, but the mechanism is largely unknown.

Aims: Unravel the pathways by which (inflammatory) EC influence platelet signaling using vessel-on-a-chip and multi-omics approaches.

Methods: The transcriptomes of control-, TNFalpha- or LPS-treated Human Umbilical Venous EC (HUVEC) were characterized by RNA-sequencing. Platelet activation and thrombus formation on sub-confluent EC were monitored in whole-blood and high shear conditions. Changes in phosphorylation levels of platelets exposed to (treated) EC were assessed by TMT phosphoproteomic analysis. Platelet integrin activation (PAC1 mAb) in these conditions were measured by flow cytometry.

Results: Out of >18k robustly detectable genes, the HUVEC transcriptome showed ~0.7k and ~6.5k expression changes (LPS or TNF-treatment vs. Control respectively, adjusted p-value >0.01), in particular of inflammatory pathways and secretory mediators, after TNFalpha >> LPS treatment. Pathway analysis via ClusterProfiler and topGO revealed distinct inflammatory footprints of the treated EC. In high shear flow conditions on a collagen/tissue factor surface, the presence of sub-confluent EC potently suppressed platelet adhesion, thrombus and fibrin formation. When pre-treated with TNFalpha, the thrombo-protective and anticoagulant effect of the EC was significantly reverted. Flow cytometry revealed a ~80% reduction in agonist-induced integrin activation of platelets exposed to the control EC. This effect was half as strong after TNFalpha treatment, but not after LPS treatment. Key changes in the phosphoproteome (3.0k phospho-sites) in EC-exposed platelets are currently validated to uncover most relevant signaling alterations due to TNFalpha or LPS treatment of the cells.

Conclusion(s): The combined use of OMICs and microfluidic approaches reveals a high complexity of inflammation-modulated interactions between EC and platelets.
This project is funded from the EU Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 813409 (TICARDIO).