Effects of Platelet Agonists and Priming on the Formation of Platelet Populations

Alicia Veninga; Constance C. F. M. J. Baaten; Ilaria De Simone; Bibian M. E. Tullemans; Marijke J. E. Kuijpers; Johan W. M. Heemskerk; Paola E. J. van der Meijden

doi:10.1055/s-0041-1735972

Thrombosis and Haemostasis, Table of Contents

CC BY-NC-ND 4.0 · Thromb Haemost 2022; 122(05): 726-738
DOI: 10.1055/s-0041-1735972

Cellular Haemostasis and Platelets

Effects of Platelet Agonists and Priming on the Formation of Platelet Populations

Authors

Alicia Veninga

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
Constance C. F. M. J. Baaten

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands

²Institute for Molecular Cardiovascular Research, University Hospital Aachen, RWTH Aachen University, Germany
Ilaria De Simone

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands

³Institute for Cardiovascular and Metabolic Research, University of Reading, Reading, United Kingdom
Bibian M. E. Tullemans

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
Marijke J. E. Kuijpers

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands

⁴Thrombosis Expertise Center, Heart and Vascular Center, Maastricht University Medical Center, Maastricht, The Netherlands
Johan W. M. Heemskerk

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
Paola E. J. van der Meijden

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands

⁴Thrombosis Expertise Center, Heart and Vascular Center, Maastricht University Medical Center, Maastricht, The Netherlands

Abstract

Platelets from healthy donors display heterogeneity in responsiveness to agonists. The response thresholds of platelets are controlled by multiple bioactive molecules, acting as negatively or positively priming substances. Higher circulating levels of priming substances adenosine and succinate, as well as the occurrence of hypercoagulability, have been described for patients with ischaemic heart disease. Here, we present an improved methodology of flow cytometric analyses of platelet activation and the characterisation of platelet populations following activation and priming by automated clustering analysis.

Platelets were treated with adenosine, succinate, or coagulated plasma before stimulation with CRP-XL, 2-MeSADP, or TRAP6 and labelled for activated integrin α_IIbβ₃ (PAC1), CD62P, TLT1, CD63, and GPIX. The Super-Enhanced Dmax subtraction algorithm and 2% marker (quadrant) setting were applied to identify populations, which were further defined by state-of-the-art clustering techniques (tSNE, FlowSOM).

Following activation, five platelet populations were identified: resting, aggregating (PAC1 + ), secreting (α- and dense-granules; CD62P + , TLT1 + , CD63 + ), aggregating plus α-granule secreting (PAC1 + , CD62P + , TLT1 + ), and fully active platelet populations. The type of agonist determined the distribution of platelet populations. Adenosine in a dose-dependent way suppressed the fraction of fully activated platelets (TRAP6 > 2-MeSADP > CRP-XL), whereas succinate and coagulated plasma increased this fraction (CRP-XL > TRAP6 > 2-MeSADP). Interestingly, a subset of platelets showed a constant response (aggregating, secreting, or aggregating plus α-granule secreting), which was hardly affected by the stimulus strength or priming substances.

Keywords

platelets - populations - priming - activation markers - flow cytometric analysis

Full Text

References

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