thromboplastin and Familial-Primary-Pulmonary-Hypertension

To develop and evaluate a multicolour flow cytometry method for analysis of microparticles (MPs) in fresh whole blood without any centrifugation steps or freezing/thawing procedure.. Flow cytometry was performed using a FC500 MPL cytometer. The compensation in the protocol was performed based on the platelet population. Polystyrene microspheres 0.50-1.27 μm were used for size position, and the MP gate was set as particles 0.5-1.0 μm. Whole blood was incubated with annexin V and antibodies to tissue factor (TF), platelets (CD41 and CD62P), monocyte (CD14) and endothelial cells (CD144). For comparison, MPs from platelet free supernatant was used. The TF activity was evaluated by Calibrated Automated Thrombogram.. Annexin V was used to distinguish true events from background noise. For standardization, each analysis included 10,000 events in the gate of platelets. There were 622(462-1001) MP(annV+)/10,000 platelets and of these, 66 (49-82)/10,000 platelets expressed TF. After correction for the individual platelet counts, the amount of circulating MP(annV+) was 17.1 (12.1-24.9) × 10(9)/L in whole blood, and of these, 10% (6-12%) expressed TF. The majority of the MPs expressed CD41, and 5.6% (2.2-6.9%) of these co-expressed TF. The amount of CD41 + MP(annV+) tended to correlate to the TF activity in whole blood. There was no correlation between the MP(annV+) in whole blood and MPs derived from platelet free supernatant. Patients with pulmonary arterial hypertension and stable coronary artery disease had increased concentrations of CD41 + MP(annV+) in whole blood.. This multicolour flow cytometry assay in whole blood mimics the in vivo situation by avoiding several procedure steps interfering with the MP count. By standardized quantification of MPs a reference interval of MPs can be created.

Topics: Annexin A5; Antibodies, Monoclonal; Antigens, CD; Blood Platelets; Calibration; Cell-Derived Microparticles; Coronary Artery Disease; Endothelial Cells; Familial Primary Pulmonary Hypertension; Female; Flow Cytometry; Humans; Hypertension, Pulmonary; Male; Microspheres; Middle Aged; Monocytes; Particle Size; Polystyrenes; Reference Values; Thromboplastin

Inflammation, endothelial dysfunction, and thrombosis contribute to the pathogenesis and development of human pulmonary arterial hypertension (PAH). The aim of this study was to investigate the effects of ruscogenin, a natural anti-inflammatory and anti-thrombotic agent, on the development of monocrotaline (MCT)-induced PAH in rats. Our results revealed that ruscogenin had favorable effects on hemodynamics and pulmonary vascular remodeling, preventing the development of PAH 3 weeks after MCT. In addition, ruscogenin resulted in markedly reduced expression of inflammatory cytokine and leukocyte infiltration via the inhibition of nuclear factor (NF)-κB activity in rat lungs. Ruscogenin also attenuated MCT-induced endothelial cell apoptosis in the remodeled pulmonary arterioles and rescued destruction of endothelial cell membrane proteins such as eNOS, caveolin-1, and CD31. Our findings suggest that ruscogenin might have therapeutic benefits for PAH patients.

Topics: Animals; Anti-Inflammatory Agents; Antihypertensive Agents; Arterial Pressure; Caveolin 1; Endothelium, Vascular; Familial Primary Pulmonary Hypertension; Hypertension, Pulmonary; Interleukin-1beta; Male; Monocrotaline; NF-kappa B; Nitric Oxide Synthase Type III; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Sprague-Dawley; Spirostans; Thromboplastin