Analysis of procoagulant phosphatidylserine-exposing platelets by imaging flow cytometry

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 alpha II beta 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 (alpha II beta 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.