Proteases are enzymes that cleave proteins by hydrolyzing their peptide bonds. The level of proteases have been shown to be elevated in many diseases including shock, diabetes, several types of cancers, and coagulation disorders. Thus the ability to measure protease activity directly in whole blood, and other complex samples will allow for better diagnostics, better understanding of the disease progression, and the development of better therapeutics. Current techniques used to measure protease activity required considerable amounts of sample preparation, which is 1) time-consuming 2) costly and 3) alters the sample, making the reads less accurate. In this dissertation we have further developed a novel assay, which allows rapid measurements of different proteases activities directly in whole blood, and other complex samples, requiring no sample preparation. The technology used by the charge-changing fluorescent substrates assays consists of a simple electrophoretic format. In the past proteases have not been fully studied in many conditions, because sample preparation lead to un-accurate measurements of the proteases in most cases. In this dissertation we present the measurement of protease activity in the lymph fluid of rats, after physiological shock. An elevation of the metalloproteases-2, and -9, trypsin, and chymotrypsin activity after shock was observed. We also were able to measure the protease activity in whole blood samples of type 2 diabetics during a meal; where we found an elevation of metalloproteases -2, and -9, and elastase activity. Metalloproteases-2, and -9 activities were measured in whole untreated blood samples of CLL patients, where they were elevated, and it was observed that this activity came from B-cells. Because of the simple format of our assay, we have design and tested a prototype for a point of care system, which detected thrombin activity in whole untreated blood in only 27- minutes. We have then demonstrated, that our charge- changing fluorescent substrate assay can be used with real complex clinical samples and no sample preparation for the detection of protease activities to : 1) gain a better understanding of the disease progression, 2) develop better diagnostics, and 3) develop new therapeutics for the different diseases, which will ultimately help patients managing their conditions

The small intestine plays a key role in the pathogenesis of multiple organ failure following circulatory shock. Current results show that reduced perfusion of the small intestine compromises the mucosal epithelial barrier, and the intestinal contents (including pancreatic digestive enzymes and partially digested food) can enter the intestinal wall and transport through the circulation or mesenteric lymph to other organs such as the lung. The extent to which the luminal contents of the small intestine mediate tissue damage in the intestine and lung is poorly understood in shock. Therefore, rats were assigned to three groups: No-hemorrhagic shock (HS) control and HS with or without a flushed intestine. HS was induced by reducing the mean arterial pressure (30 mmHg; 90 min) followed by return of shed blood and observation (3 h). The small intestine and lung were analyzed for hemorrhage, neutrophil accumulation, and cellular membrane protein degradation. After HS, animals with luminal contents had increased neutrophil accumulation, bleeding, and destruction of E-cadherin in the intestine. Serine protease activity was elevated in mesenteric lymph fluid collected from a separate group of animals subjected to intestinal ischemia/reperfusion. Serine protease activity was elevated in the plasma after HS but was detected in lungs only in animals with nonflushed lumens. Despite removal of the luminal contents, lung injury occurred in both groups as determined by elevated neutrophil accumulation, permeability, and lung protein destruction. In conclusion, luminal contents significantly increase intestinal damage during experimental HS, suggesting transport of luminal contents across the intestinal wall should be minimized.

Objective: To examine resting and postprandial peripheral protease activity in healthy controls and individuals with type 2 diabetes mellitus (T2DM) and pre-T2DM. Methods: Individuals with T2DM or pre-T2DM and healthy controls (mean age 55.8 years) were studied before and for a span of 300 minutes following a single high-calorie McDonald's breakfast. Metalloproteases-2/-9 (MMP-2/-9), elastase, and trypsin activities were assessed in whole blood before and following the meal using a novel high-precision electrophoretic platform. Also assessed were circulating levels of inflammatory biomarkers and insulin receptor density on peripheral blood mononuclear cells (PBMCs) in relationship to protease activity. Results: Premeal MMP-2/-9 and elastase activity levels in T2DM and in pre-T2DM participants were significantly elevated as compared to controls. The T2DM group showed a significant increase in elastase activity 15 minutes after the meal; elastase activity continued to increase to the 30-minute time point (p < 0.01). In control participants, MMP-2/-9, elastase, and trypsin were significantly increased at 15 minutes after the meal (p < 0.05) and returned to premeal values within a period of approximately 30 to 60 minutes post meal. PBMCs incubated for 1 hour with plasma from T2DM and pre-T2DM participants had significantly lower levels of insulin receptor density compared to those incubated with plasma from control participants (p < 0.001). Conclusions: The results of this study suggest that individuals with T2DM and pre-T2DM have higher resting systemic protease activity than nonsymptomatic controls. A single high-calorie/high-carbohydrate meal results in further elevations of protease activity in the systemic circulation of T2DM and pre-T2DM, as well as in healthy controls. The protease activity in turn can lead to a downregulation of insulin receptor density, potentially supporting a state of insulin resistance.