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Analysis of procoagulant phosphatidylserine-exposing platelets by imaging flow cytometry.


ABSTRACT:

Background

Upon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane.

Objective

To evaluate procoagulant PS-exposing platelets by imaging flow cytometry.

Methods

Platelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin ?IIb?3, CD62P, and PS exposure.

Results

A subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS-exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non-PS-exposing platelets. They expressed CD62P and ?IIb?3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS-exposing platelets possessing platelet-associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs.

Conclusions

Innovative imaging flow cytometry allowed detailed fluorescence-based, quantitative morphometric analysis of PS-exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical "balloons," almost devoid of their normal internal architecture. Almost all PS-exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet-associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

SUBMITTER: Reddy EC 

PROVIDER: S-EPMC6178738 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Publications

Analysis of procoagulant phosphatidylserine-exposing platelets by imaging flow cytometry.

Reddy Emily C EC   Wang Hong H   Christensen Hilary H   McMillan-Ward Eileen E   Israels Sara J SJ   Bang K W Annie KWA   Rand Margaret L ML  

Research and practice in thrombosis and haemostasis 20180823 4


<h4>Background</h4>Upon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane.<h4>Objective</h4>To evaluate procoagulant PS-exposing platelets by imaging flow cytometry.<h4>Methods</h4>Platelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets w  ...[more]

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