Information is scarce on human responses to high pressure exposures out of water, such as related to tunnel construction workers. We hypothesized that differences in the longer durations of exposures for tunnel workers versus underwater divers results in greater inflammatory responses linked to the pathophysiology of decompression sickness (DCS). Blood was analyzed from 15 tunnel workers (36.1 ± 10.5 (SD) years old, 6 women) exposed to 142-156 kPa pressure for 4.1-4.9 h compared to 8 SCUBA divers (39.3 ± 13.3 (SD) years old, 6 women) exposed to 149 kPa for 0.61 hours. Despite differences in pressure duration between groups, elevations were the same for blood microparticles (MPs) (128 ± 28% MPs/μl) and intra-MPs interleukin (IL-1β) (376 ± 212% pg/million MPs), and for decreases of plasma gelsolin (pGSN, 31 ± 27% μg/mL). The number of circulating CD66b + neutrophils and evidence of cell activation, insignificant for divers, increased in tunnel workers. Across 3 exposures, the mean neutrophil count increased 150 ± 11%. Neutrophil activation increased by 1 to 2% of cells expressing cell surface CD18, myeloperoxidase, platelet-specific CD41, and decrease of cell bound pGSN. We conclude that MPs elevations occur rapidly in humans and reach steady state in minutes with pressure exposures and neutrophil activation requires significantly longer exposure times.
© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.

Coronavirus disease (COVID-19) induces inflammation, coagulopathy following platelet and monocyte activation, and fibrinolysis, resulting in elevated D-dimer levels. Activated platelets and monocytes produce microvesicles (MVs). We analyzed the differences in platelet and monocyte MV counts in mild, moderate, and severe COVID-19, as well as their correlation with D-dimer levels.
In this cross-sectional study, blood specimens were collected from 90 COVID-19 patients and analyzed for D-dimers using SYSMEX CS-2500. Platelet MVs (PMVs; PMVCD42b+ and PMVCD41a+), monocyte MVs (MMVs; MMVCD14+), and phosphatidylserine-binding annexin V (PS, AnnV+) were analyzed using a BD FACSCalibur instrument.
PMV and MMV counts were significantly increased in COVID-19 patients. AnnV+ PMVCD42b+ and AnnV+ PMVCD41a+ cell counts were higher in patients with severe COVID-19 than in those with moderate clinical symptoms. The median (range) of AnnV+ PMVCD42b+ (MV/μL) in mild, moderate, and severe COVID-19 was 1,118.3 (328.1-1,910.5), 937.4 (311.4-2,909.5), and 1,298.8 (458.2-9,703.5), respectively (P =0.009). The median (range) for AnnV+ PMVCD41a+ (MV/μL) in mild, moderate, and severe disease was 885.5 (346.3-1,682.7), 663.5 (233.8-2,081.5), and 1,146.3 (333.3-10,296.6), respectively (P =0.007). D-dimer levels (ng/mL) weak correlated with AnnV+ PMVCD41a+ (P =0.047, r=0.258).
PMV PMVCD42b+ and PMVCD41a+ counts were significantly increased in patients with severe clinical symptoms, and PMVCD41a+ counts correlated with D-dimer levels. Therefore, MV counts can be used as a potential biomarker of COVID-19 severity.

Donor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, ∼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)-based production system for an induced pluripotent stem cell-derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB.
© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

: Mycophenolate mofetil (MMF) raises platelet counts in patients with primary immune thrombocytopenia. However, studies indicate that MMF inhibits collagen-induced platelet aggregation, potentially increasing bleeding risk following MMF therapy. The study evaluates the in-vitro effect of MMF on platelet function. Blood samples (n = 6) from healthy donors were incubated with vehicle, MMF or mycophenolic acid (MPA) at clinically relevant concentrations. Platelet aggregation was measured with flow cytometry and 96-well light transmission aggregometry (LTA). Using flow cytometry, we measured the expression of platelet CD49b, CD42b, CD42a, CD61 and CD41. Platelet activation was measured as the expression of P-selectin and the active form of the GPIIb/IIIa receptor following agonist stimulation. Agonists were: ADP, thrombin receptor-activating peptide, collagen, collagen-related peptide and U46619. The Platelet Function Analyzer-200 was used to measure global platelet function. MMF and MPA did not change platelet aggregation regardless of the agonist used. An exception was a significant, but minor decrease in collagen-induced platelet aggregation in samples with MMF (6 ± 3%, P = 0.02) and MPA (8 ± 4%, P = 0.01) compared with vehicle (22 ± 11%). However, this was not observed using the lesser sensitive LTA method. Compared with vehicle, MPA led to a significantly lower relative disposition of the surface collagen-receptor GPVI (7.8 ± 1.8 versus 8.8 ± 2.1 mean fluorescence intensity, P < 0.001). In all other platelet-related tests, neither MMF nor MPA showed any effect. In conclusion, MMF and MPA only had a minor effect on collagen-induced platelet aggregation, with MPA reducing the relative disposition of surface GPVI receptors.

Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large numbers of platelets in whole blood. In the following chapter, we provide a number of flow cytometry-based protocols broadly aimed at (1) assessment of constitutively expressed platelet membrane receptors to diagnose inherited platelet bleeding disorders and (2) investigation of basal and agonist-induced platelet functional responses including generation of platelet-leukocyte aggregates, alpha and dense granule release, calcium flux, and phosphatidylserine exposure.