Free Protease Activity Research Articles

Targeting Proteases in Cystic Fibrosis Lung Disease. Paradigms, Progress, and Potential.

Cystic fibrosis (CF) is the most common life-limiting hereditary condition of Caucasian populations and is characterised by chronic airways inflammation driving progressive structural lung damage. Despite tremendous advances in the treatment of CF and concomitant increased life expectancy for patients, chronic lung disease remains the main cause of morbidity and mortality among CF patients. While universal restoration of cystic fibrosis transmembrane conductance regulator activity remains a future hope, novel therapies aimed at reducing or preventing chronic airways inflammation and progressive structural lung damage are required. It is well-established that proteolytic enzymes are important in the CF lung beyond the basic turnover of proteins and intracellular degradation of pathogens. When secreted, these enzymes play key roles in extracellular substrate modification implicated in important biological processes such as matrix and airway remodelling, goblet cell metaplasia and mucus hypersecretion, immune cell recruitment and dysregulation of epithelial ion channels. Importantly, the burden of proteases in the CF lung is significantly elevated, overwhelming the endogenous antiprotease shield. Indeed, free protease activity has emerged as a major risk factor of the onset and progression of bronchiectasis and lung function decline in patients with CF. Recent research has highlighted the importance of new players such as cathepsin S and matrix metalloprotease-12, as well as the membrane-associated activity of key proteases such as neutrophil elastase on the surface of neutrophils. Here, we review the current knowledge and emerging concepts of the role of host proteases in the pathogenesis of CF lung disease and their potential as therapeutic targets.

A brain derived peptide preparation reduces the translation dependent loss of a cytoskeletal protein in primary cultured chicken neurons.

Neuronal cytoskeletal proteins like the microtubule associated protein 2 (MAP2) are objected to pathological proteolysis in case of Alzheimer's disease and brain ischemia. The neurotrophic peptidergic drug Cerebrolysin (EBEWE Arzneimittel, Austria, Europe) is produced by a standardized enzymatic break-down of lipid free porcine brain proteins. Cerebolysin protected MAP2 in primary neuronal cultures after a brief histotoxic hypoxia and in a rat model of acute brain ischemia. Furthermore the drug was shown to inhibit the proteases mu- and m-calpain dose dependently in several cell free protease activity assays. The question if the higher MAP2 levels are due to an alleviation of proteolysis, to a higher synthesis rate or both is addressed in the current investigation: Monitoring the MAP2 content of primary neuronal cell cultures over a period of eight days revealed MAP2 to reach a peak level on day six in vitro followed by a degradation phase. In other experiments the protein synthesis of Cerebrolysin treated and untreated cells was blocked with cycloheximide at that moment when all cells exhibited the same MAP2 content. After the following MAP2 degradation phase--i.e. after eight days in vitro--the MAP2 contents were determined by western blotting. Cerebrolysin treated cells contained more MAP2 than untreated controls proving that the drug protects MAP2 independently from de novo synthesis, although further work is in progress to investigate if the drug supplementary boosts this effect by increasing MAP2 synthesis.

Decreased Platelet Number and Function and Increased Fibrinolysis Contribute to Postoperative Bleeding in Cardiopulmonary Bypass Patients

We simultaneously evaluated platelet and fibrinolytic parameters to assess their individual and combined contributions to postoperative blood loss in cardiopulmonary (CP) bypass patients. Platelet count, platelet aggregability, hematocrit, plasminogen (PLG) concentration, alpha 2-antiplasmin (AP) concentration, free protease activity (fPA), and antithrombin-III (AT-III) were measured in nine patients undergoing surgery using cardiopulmonary bypass. Chest tube drainage was used as the measure of postoperative blood loss. Hematocrit, platelet count, PLG, AP, and AT-III all decreased during CP bypass, with PLG and AT-III decreasing much more than dilution. During CP bypass, platelet aggregability to ADP did not change significantly from pre-bypass, but aggregability to arachidonic acid (AA) decreased significantly. Following protamine administration there was a large increase (83%) in fPA, the platelet count showed a further drop (from 61% to 50% of pre-bypass levels), and platelet aggregability decreased significantly (from 95% to 34% of pre-bypass levels for ADP, and from 55% to 11.9% for AA). Chest tube drainage during the first four postoperative hours correlated positively (p less than 0.05) with the combination of increase in free protease activity and decrease in platelet count. The total chest tube drainage correlated significantly with the combination of decrease in platelet count and the decrease in platelet aggregability. These combinations of changes correlated significantly with postoperative blood loss whereas the individual changes did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced Free Protease Activity in the Lungs of Smokers

Enhanced Free Protease Activity in the Lungs of Smokers

Methods for studying fibrinolytic pathway components in human plasma

Methods have been developed to quantitatively measure the major plasma components of the human fibrinolytic system. Plasminogen is measured functionally with a 9M excess of streptokinase and immunochemically by rocket immunoelectrophoresis; the normal range was found to be 16.7–23.8 mg/dl and 17.4–21.6 mg/dl, respectively. α 2-plasmin inhibitor is measured functionally and immunochemically; the normal range for the major plasma plasmin inhibitor was found to be 5.30–6.60 mg/dl by both methods. Plasminogen activator concentrations, as well as, free, and complexed, protease activities are measured along with plasmin generation rates by spectrophotometric assays with chromogenic substrates. Both activator and free protease activities are zero in plasma samples from normal human subjects. Plasmin generation rates are 0.25–0.47% with urokinase and 5.30–9.70% with streptokinase; these values are the percentages of the respective initial velocities of activation in purified systems.