Hemogram cell counts over a 48 hour period.

<p>Individual cell counts from both control (●) and LPS (o) for neutrophils (A), monocytes (B), lymphocytes (C), platelets (D), red blood cells (E), and reticulocytes (F). Presented are means and standard deviation for five animals at each time point. * indicates a significant (P<0.05) difference between the control and LPS-treated groups.</p

Predicted Direct Relationships Among miRNA and Hemostatic or Adhesive Protein Targets.

<p>In response to LPS stimulation, previous studies have shown increased expression of miRNAs and interaction with target proteins. The relationships depicted are generated from Ingenuity Pathway Analyses (IPA, QIAGEN Redwood city, <a href="http://www.quiagen.com/ingenuity" target="_blank">www.quiagen.com/ingenuity</a>). Symbol definitions are included in the figure inset.</p

miRNA Changes in Serum from LPS-Treated Rats.

<p>miRNA Changes in Serum from LPS-Treated Rats.</p

Changes in hemostatic and adhesion proteins in plasma or serum over a 48 hour period.

<p>Mean protein concentrations and standard deviation were determined in both control (●) and LPS (o) for plasma PAI-1 (A), plasma D-Dimer (B), serum sICAM-1 (C), serum sE-selectin (D), and plasma fibrinogen (F) for five animals at each time point. * indicates a significant (P<0.05) difference between the control and LPS-treated groups</p

Endothelial alterations in a canine model of immune thrombocytopenia

<p>Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (<i>P</i> = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (<i>R</i> = 0.99103; <i>P</i> < 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.</p

Clinical observations in LPS-treated animals.

<p>Clinical observations in LPS-treated animals.</p

Histopathology, hematoxylin and eosin-stained sections.

<p>(A) Neutrophil sequestration (arrows) within pulmonary alveolar capillaries typical of 5/5 rats at hours 4, 8, 24, and 48 post-intraperitoneal LPS injection. (B) Normal pulmonary alveolar capillaries at 48 hours post-intraperitoneal saline injection. (C) Neutrophil sequestration (arrows) within hepatic sinusoids typical of 5/5 rats at hour 4 and 8 post-intraperitoneal LPS injection. (D) Normal hepatic sinusoids at 8 hours post-intraperitoneal saline injection. 400 X original magnification A, B, C, and D. (E) Single cell necrosis of lymphocytes (arrows) within splenic lymphoid follicles typical of 5/5 rats at hours 4 and 8 post-intraperitoneal LPS injection. (F) Normal spleen at 8 hours post-intraperitoneal saline injection. (G) Increased megakaryocytes (arrows) within spleen typical of 5/5 rats at hour 48 post-intraperitoneal LPS injection. 200 X original magnification E, F, and G. (H) Leukocyte accumulation in subepicardial myocardium of the right atrium typical of 5/5 rats at hour 48 post-intraperitoneal LPS injection. Inset 400X original magnification. (I) Normal right atrium at 48 hours post-intraperitoneal saline injection. 40 X original magnification H, I.</p

Experimental design and time-points analyzed.

<p>Experimental design and time-points analyzed.</p

Extracellular vesicle NTA counts.

<p>Population size distributions of extracellular vesicles in rat plasma. A. Fluorescence histograms of di-8 intensity (proportional to vesicle surface area) of EVs in rat plasma 1 hour and 24 hours after treatment with vehicle or LPS. Four replicate measurements are overlaid in each panel from a single representative sample. B. NTA diameter histograms of nanoparticles in rat plasma before and 24 hours after treatment with vehicle or LPS. Each panel shows the average histogram from a single representative sample. C. NTA population mean diameter (+/- SD) of nanoparticles in rat plasma as a function of time after treatment with vehicle or LPS.</p

Extracellular vesicle counts/concentrations in plasma over a 48 hour period.

<p>Total EV concentrations in control (●) and LPS (o) plasma as measured by VFC. (A) Total NP concentrations in control (●) and LPS (o) plasma as measured by NTA. (B) Marker positive EV concentrations in control and LPS plasma for annexin V (C), CD42d (D), CD54 (E), and CD106 (F). Presented are means and standard deviation for five animals at each time point. * indicate a significant (P<0.05) difference between the control and LPS-treated groups.</p