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Platelet subpopulations remain despite strong dual agonist stimulation and can be characterised using a novel six-colour flow cytometry protocol
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Orebro Univ, Sweden.ORCID iD: 0000-0002-1920-3962
2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 1441Article in journal (Refereed) Published
Abstract [en]

It is recognised that platelets respond differently to activation, where a subpopulation of platelets adopt a procoagulant phenotype while others are aggregatory. However, it has not been thoroughly tested whether these subpopulations will remain in maximally activated samples, or if they are merely a result of different platelet sensitivities to agonist activation. Here platelets were activated with gradually increasing concentrations of thrombin and/or the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Platelet activation was investigated using a novel six-colour flow cytometry protocol evaluating exposure of phosphatidylserine, active conformation of the fibrinogen receptor alpha(IIb)beta(3), alpha-granule and lysosomal release (P-selectin and LAMP-1 exposure), mitochondrial membrane integrity and platelet fragmentation. Upon activation by CRP-XL or thrombin+CRP-XL, platelets formed three differently sized subpopulations. Normal-sized platelets showed high exposure of aggregatory active alpha(IIb)beta(3) and intact mitochondria, while the smaller platelets and platelet fragments showed high exposure of procoagulant phosphatidylserine. The distribution of platelets between the differently sized subpopulations remained stable despite high agonist concentrations. All three were still present after 30 and 60 min of activation, showing that all platelets will not have the same characteristics even after maximal stimulation. This suggests that platelet subpopulations with distinct activation patterns exist within the total platelet population.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP , 2018. Vol. 8, article id 1441
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:liu:diva-145133DOI: 10.1038/s41598-017-19126-8ISI: 000423045400014PubMedID: 29362366OAI: oai:DiVA.org:liu-145133DiVA, id: diva2:1182455
Note

Funding Agencies|LiU Fund of U and Linkoping University

Available from: 2018-02-13 Created: 2018-02-13 Last updated: 2018-10-25
In thesis
1. Formation and Relevance of Platelet Subpopulations
Open this publication in new window or tab >>Formation and Relevance of Platelet Subpopulations
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Platelets are important players in the hemostatic system, acting as guardians of vessel integrity. When they come across a breach in the vessel wall, they quickly adhere to the damaged surface, secrete activating and adhesive compounds from their secretory granules, recruit additional platelets into a growing platelet plug and support the action of the coagulation system. In the past decades, it has become clear that platelets form functionally different platelet subpopulations. The aggregatory platelets build the platelet plug, whereas the procoagulant subpopulation support and direct the actions of the coagulation system. The aim of this thesis was to examine the formation and features of the different platelet subpopulations, and elucidate their respective roles in hemostasis.

Platelet lysosomal secretion is not well characterized. In Paper I, we found that lysosomal secretion, detected as LAMP-1 surface exposure, occur upon potent platelet stimulation including secondary activation by ADP. This is regulated by the endothelial platelet inhibitors nitric oxide and prostacyclin. As observed in Paper II, lysosomal secretion might also be of clinical relevance as a quality indicator for platelet concentrates used for transfusion, an area were quality control may become increasingly important in the future. Among several evaluated platelet activation markers, platelet LAMP-1 exposure and the ability to form procoagulant platelets may be useful as novel indicators of platelet responsiveness. Moreover, the ability to form procoagulant platelets varies extensively between individuals, something we established in Paper III. Here we also present a novel flow cytometry protocol enabling the simultaneous investigation of 6 different platelet activation markers. Using this protocol we investigate the formation of procoagulant platelets and reveal that only a subpopulation of platelets may become procoagulant. Further we show that this is dependent on the agonist stimulation applied. Finally in Paper IV, we explore the influence of the procoagulant platelet subpopulation on different aspects of hemostasis. While platelet aggregation was not affected, the fraction of procoagulant platelets was found to strongly correlate to peak thrombin generation, and to be associated with plasma cholesterol levels.

In conclusion, this thesis presents evidence for the use of LAMP-1 surface exposure and the formation of a procoagulant platelet subpopulation as potential indicators of platelet activation potential. The formation of procoagulant platelets varies extensively between individuals, influence hemostasis and is associated with the known risk factor cholesterol. Thus, the formation of a procoagulant platelet subpopulation may be a candidate biomarker for cardiovascular disease, to be explored in the future.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. p. 61
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1642
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:liu:diva-152280 (URN)10.3384/diss.diva-152280 (DOI)9789176852156 (ISBN)
Public defence
2018-11-02, Berzeliussalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2018-10-25 Created: 2018-10-25 Last updated: 2018-10-25Bibliographically approved

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