Approach for analysis of intracellular markers in phosphatidylserine-positive platelets

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 there remain a number of mysteries associated with them. In particular, intracellular signaling and cytoskeletal reorganization in these platelets is very poorly studied, because they are destroyed upon permeabilization, which is necessary for antibodies to intracellular markers to penetrate 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 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.