Background: In vitro studies have shown that endothelial-cell derived microparticles (EMP) can bind and activate leukocytes. It is unknown whether EMP binding to leukocytes regulates leukocyte activation in vivo. In this study, we examined the correlation between EMP binding to neutrophils (EMP-neutrophil conjugates) and neutrophil activation in various clinical conditions.

Methods: We studied a total of 251 venous blood samples from 221 subjects with several prothrombotic and inflammatory conditions, including acute venous thromboembolism (n=25), atrial fibrillation (n=48), metabolic syndrome (n=37), congestive heart failure (n=44), early sepsis (n=35), late sepsis (n=30), as well as normal controls (n=32). Using flow cytometry, we measured leukocyte expression of activation marker CD11b and 2 different populations of EMP-neutrophil conjugates. Bitmapping by forward- and side-scatter gating was used to identify neutrophils; EMP62E+-neutrophil conjugates and EMP54+-neutrophil conjugates were measured based on the detection of E-selectin (CD62E) or CD54, respectively, co-expressed with CD45 in neutrophils. Neutrophil nitric oxide (NO) levels were measured by flow cytometry after loading neutrophils with the membrane permeable NO-selective fluorescent indicator DAF-DA.

Results: Levels of EMP62E+-neutrophil conjugates consistently and significantly correlated with CD11b expression. This finding was present in patients with acute venous thromboembolism (r=0.46; p=0.02), atrial fibrillation (r=0.42; p=0.003), metabolic syndrome (r=0.56; p<0.0001), congestive heart failure (r=0.70; p<0.0001), early sepsis (r=0.50; p= 0.003), late sepsis (r=0.55;p=0.002), as well as normal controls (r=0.86; p<0.0001). In contrast, a correlation between EMP54+-neutrophil conjugates and neutrophil activation was not found in any of the studied populations (all p>0.05). EMP62E+-neutrophil conjugates correlated with NO levels in neutrophils in patients with congestive heart failure (r=0.48; p=0.001) and atrial fibrillation (r=0.33; p=0.02), but no correlation was seen in other disease states.

Conclusions: EMP62E+-neutrophil conjugates strongly correlate with neutrophil activation in normal adults, as well as in multiple pro-inflammatory and pro-thrombotic disease states. EMP62E+-neutrophil conjugates may serve as a marker of prothrombotic and inflammatory states. Our results combined with prior in vitro studies suggest that EMP62E+ binding to neutrophils is an important and “universal” determining factor for neutrophil activation in vivo in humans. In contrast, binding of EMP54+ to neutrophils does not seem to regulate neutrophil activation. These findings also support the concept that different species of endothelial microparticles have different biologic functions. EMP binding to neutrophils seems to affect NO production in only some disease states.

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