Approach for Analysis of Intracellular Markers in Phosphatidylserine-Positive Platelets

Abstract

Phosphatidylserine- (PS-) positive platelets play an important role in thrombosis and hemostasis. They have high procoagulant activity, the ability to vesiculate, and can aggregate with activated PS-negative platelets. They are found in growing thrombus in vitro, but numerous mysteries remain regarding them. In particular, intracellular signaling and cytoskeletal reorganization in these platelets is poorly studied, since they have the ability to be destroyed by permeabilization, which is necessary for the penetration of antibodies to intracellular markers into the cell. In this work, we propose an approach that allows the analysis of intracellular signaling in calcium ionophore A23187-induced PS-positive platelets using flow cytometry or confocal microscopy. We used the mildest permeabilization of fixed PS-positive platelets using saponin and showed that such permeabilization allows us to significantly preserve PS-positive platelets. As an example, we analyzed the state of the polymerized form of actin in PS-positive platelets and showed that, despite the significant rearrangement of the cytoskeleton that occurs upon activation in such platelets, actin in them is partially presented in a polymerized form.