TY - JOUR
T1 - Effects of Platelet Agonists and Priming on the Formation of Platelet Populations
AU - Veninga, A.
AU - Baaten, C.C.F.M.J.
AU - De Simone, I.
AU - Tullemans, B.M.E.
AU - Kuijpers, M.J.E.
AU - Heemskerk, J.W.M.
AU - van der Meijden, P.E.J.
N1 - Funding Information:
This work is supported by the Landsteiner Foundation for Blood Transfusion Research Grant No. 1711. C.C.F.M.J.B. received funding from The Dutch Heart Foundation (2020T020) and the START-Program of the Faculty of Medicine at the RWTH Aachen University (105/20). I.D. S. is supported by a joint PhD scholarship of Maastricht and Reading Universities, and by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 766118.
Publisher Copyright:
© 2022 Georg Thieme Verlag. All rights reserved.
PY - 2022/5
Y1 - 2022/5
N2 - 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 alpha (IIb) beta (3) (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 (alpha- and dense-granules; CD62P+, TLT1+, CD63+), aggregating plus alpha -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 alpha -granule secreting), which was hardly affected by the stimulus strength or priming substances.
AB - 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 alpha (IIb) beta (3) (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 (alpha- and dense-granules; CD62P+, TLT1+, CD63+), aggregating plus alpha -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 alpha -granule secreting), which was hardly affected by the stimulus strength or priming substances.
KW - platelets
KW - populations
KW - priming
KW - activation markers
KW - flow cytometric analysis
KW - COATED-PLATELETS
KW - FLOW-CYTOMETRY
KW - THROMBUS FORMATION
KW - ADENOSINE
KW - SUCCINATE
KW - STROKE
KW - ACTIVATION
KW - SECRETION
U2 - 10.1055/s-0041-1735972
DO - 10.1055/s-0041-1735972
M3 - Article
C2 - 34689320
SN - 0340-6245
VL - 122
SP - 726
EP - 738
JO - Thrombosis and Haemostasis
JF - Thrombosis and Haemostasis
IS - 05
ER -