UC San Diego

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