Identification and Characterization of Platelet Surface-membrane-derived Microparticles before and after Total-body Irradiation

Conventionally, the microparticles that are derived from the platelet surface membrane in the size range of 0.1 to 1.0 µm in diameter, as detected by flow cytometry with platelet-specific marker/s, are collectively categorized as PMPs, despite their structural heterogeneity. We aim to determine the detailed structural classification of this heterogeneous population based on their scatter properties using flow cytometry, both before and several months after total-body irradiation (TBI), an exposure that may occur during nuclear attacks or accidents, or as part of myeloablative conditioning prior to allogeneic stem cell transplantation for leukemia, in wild-type C57BL/6J (WT) mice and in a relatively radiation-sensitive strain deficient in platelet glycoprotein (GP) Ibα (KO), including both sexes and with or without agonist treatments. We exposed WT and KO mice to a single dose of TBI. We collected blood 233 days after 7.8 Gy from male mice and 254 days after 8.0 Gy from female mice, prepared platelet-rich plasma (PRP), activated PRP with 3 different platelet agonists [adenosine 5'-diphosphate (ADP), collagen and thrombin], and characterized the structural heterogeneity of PMPs. Additionally, we quantified the total number of microparticles using Nanoparticle Tracking Analysis (NTA) in WT and KO mice of both sexes. We then cocultured either RAW264.7 cells or splenic CD19+ B cells with microparticles from sham-irradiated and irradiated female mice of both strains to measure reactive oxygen species (ROS) generation and immunoglobulin production, respectively, to assess strain- and TBI-dependent effects of PMPs on immune cell function. We identified 3 distinct PMP subpopulations based on flow cytometry scatter profiles. The percentage of PMP subpopulations altered depending on irradiation status, GPIbα expression, platelet agonist used, and biological sex. Depending on sex, mouse strain, and TBI status, total microparticle count and microparticles-mediated ROS generation and immunoglobin production were altered in RAW264.7 cells and splenic B cells, respectively. These findings offer important insights into a poorly understood area of platelet biology and highlight the potential significance of PMP subpopulations in the context of radiation exposure.