Circulating von Willebrand Factor Antigen as a Predictor of Short-term Prognosis in Pulmonary Hypertension: Blood Sampling and Inhibition of Proteolysis In Vitro
The diagnosis of primary’ pulmonary hypertension was established only in the absence of disorders that are known to be associated with pulmonary vascular disease, such as chronic airway obstruction or fibrotic lung disease, pulmonary thromboembolism, congenital heart disease, chronic liver disease, portal hypertension, collagen vascular disease, schistosomiasis, HIV infection, and antiphospholipid syndrome. After initial biochemical determinations, which were performed no longer than 15 days after hemodynamic measurements, patients were followed for 1 year. Flovent inhalers More info At the end of the follow-up period, the results of the initial determinations were compared in nonsurvivors and survivors to identify possible predictors of prognosis. Patients waiting for heart-lung transplantation were included if they were not subjected to any kind of surgical treatment during the period of the study. Patients or their parents were informed about the research purpose of blood sampling and gave their consent. The control group consisted of 20 healthy volunteers with an age range similar to that of the patients. The study protocol was approved by the Scientific Committee of the Heart Institute at the University’ of Sao Paulo.
Blood Sampling and Inhibition of Proteolysis In Vitro
Peripheral venous blood was collected in 1:10 volumes of 3.8% sodium citrate, in the presence of the following protease inhibitors: 5 mM edetic acid, 6 mM N-ethylmaleimide, 1 mM phenyl-methylsulfonyl fluoride, 0.25 mM of leupeptin, and 20 U/mL of aprotinin. Immediately after, plasma was separated by centrifugation at 3,000g for 20 min and was stored at — 70°C until use. Samples were thawed only once for use.
Plasma von Willebrand Factor Antigenic Activity
Plasma vWF:Ag was measured by electroimmunodiffusion using a kit obtained from Diagnostica Stago (Asnieres, France). Samples always were processed in duplicate. In patients with exceedingly high vWF:Ag, plasma was diluted 1:2 or 1:4 and was processed again. Results were compared with a standard curve and were expressed as a percentage of activity.