High-throughput elucidation of thrombus formation reveals sources of platelet function variability
Johanna P. van Geffen,
Sanne L.N. Brouns,
Joana Batista,
Harriet McKinney,
Carly Kempster,
Magdolna Nagy,
Suthesh Sivapalaratnam,
Constance C.F.M.J. Baaten,
Nikki Bourry,
Mattia Frontini,
Kerstin Jurk,
Manuela Krause,
Daniele Pillitteri,
Frauke Swieringa,
Remco Verdoold,
Rachel Cavill,
Marijke J. E. Kuijpers,
Willem H. Ouwehand,
Kate Downes,
Johan W.M. Heemskerk
Affiliations
Johanna P. van Geffen
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
Sanne L.N. Brouns
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
Joana Batista
Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, UK;National Health Service Blood and Transplant (NHSBT), Cambridge Biomedical Campus, UK
Harriet McKinney
Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, UK;National Health Service Blood and Transplant (NHSBT), Cambridge Biomedical Campus, UK
Carly Kempster
Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, UK;National Health Service Blood and Transplant (NHSBT), Cambridge Biomedical Campus, UK
Magdolna Nagy
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
Suthesh Sivapalaratnam
Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, UK;The Royal London Haemophilia Centre, London, UK
Constance C.F.M.J. Baaten
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
Nikki Bourry
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
Mattia Frontini
Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, UK;National Health Service Blood and Transplant (NHSBT), Cambridge Biomedical Campus, UK;BHF Centre of Excellence, Division of Cardiovascular Medicine, Cambridge University Hospitals, Cambridge Biomedical Campus, UK
Kerstin Jurk
Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Germany
Manuela Krause
DKD Helios Klinik Wiesbaden, Germany
Daniele Pillitteri
DKD Helios Klinik Wiesbaden, Germany
Frauke Swieringa
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
Remco Verdoold
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
Rachel Cavill
Department of Data Science & Knowledge Engineering, Faculty of Humanities and Sciences, Maastricht University, the Netherlands
Marijke J. E. Kuijpers
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
Willem H. Ouwehand
Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, UK;National Health Service Blood and Transplant (NHSBT), Cambridge Biomedical Campus, UK;BHF Centre of Excellence, Division of Cardiovascular Medicine, Cambridge University Hospitals, Cambridge Biomedical Campus, UK;NIHR BioResource, University of Cambridge, Cambridge Biomedical Campus, UK;Department of Human Genetics, The Wellcome Sanger Institute, Hinxton, Cambridge, UK
Kate Downes
Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, UK;National Health Service Blood and Transplant (NHSBT), Cambridge Biomedical Campus, UK;NIHR BioResource, University of Cambridge, Cambridge Biomedical Campus, UK
Johan W.M. Heemskerk
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands
In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter- and intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: i.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced αIIbβ3 activation and secretion. Common sequence variation of GP6 and FCER1G, associated with GPVI-induced αIIbβ3 activation and secretion, affected parameters of GPVI-and CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.
