Abstract

Phosphatidylserine-positive (PS+) platelets represent a critical subpopulation in hemostasis and thrombosis, providing the necessary catalytic surface for efficient thrombin generation. While their procoagulant function is well-established, a comprehensive understanding of the intracellular signaling pathways and molecular effectors that drive their formation and function remains challenging due to the difficulty in isolating and analyzing this fragile subset. This study addresses this limitation by developing a novel, robust approach for the simultaneous analysis of intracellular markers within PS+ platelets. The method integrates a specific PS-binding probe, such as Annexin V, with optimized fixation and permeabilization protocols to preserve intracellular epitopes for subsequent immunolabeling and flow cytometric analysis. We demonstrate that this approach successfully minimizes cell loss and non-specific staining, enabling the precise quantification of key signaling molecules—such as activated kinases or cytoskeletal components—specifically in the PS+ population. The key finding is the successful application of this method to reveal distinct activation profiles in PS+ platelets compared to their PS-negative counterparts. This new analytical tool provides a powerful platform for dissecting the molecular mechanisms underlying platelet procoagulant transformation, offering new insights into the pathophysiology of bleeding and thrombotic disorders.