15. DETECTION OF THROMBIN AND OTHER PROTEASE ACTIVITIES DIRECTLY IN WHOLE BLOOD SAMPLES

Department: Bioengineering
Faculty Advisor(s): Michael Heller

Primary Student
Name: Augusta Esmeralda Modestino
Email: amodesti@ucsd.edu
Phone: 857-998-0560
Grad Year: 2015

Student Collaborators
Johnson Yu, jcyu@ucsd.edu | Mrudul Bhine, mrudulb@gmail.com

Abstract
A novel assay technique has now been developed which allows thrombin and other proteases to be detected and measured directly in a few microliters of whole blood. Many indigenous proteases like thrombin are important for blood clotting activities and related cardiac diseases (DVT, atherosclerosis, etc.). Additionally, the appearance of certain non-indigenous proteases like trypsin, chymotyrpisn, and MMP?s are frequently biomarkers for shock, cancer and other diseases. Thus, the ability to measure protease activity in whole blood will allow researchers to further elucidate the relationship between circulating protease levels and many important diseases. We have now developed a number of synthetic charge-changing fluorescent peptide substrates which are specific for trypsin, chymotrypsin, MMP2/9, elastase and thrombin. These unique substrates allow one to directly detect protease activity in whole blood and essentially eliminate the need for any sample preparation. The substrates are added to a few microliters of blood sample for reaction, and the cleaved products are then rapidly separated in a simple electrophoretic microgel format. In the past year, using our new thrombin substrate we demonstrated the monitoring of thrombin enzyme activity in untreated whole blood, which directly correlates with blood clotting. The technique provides an unprecedented quantification approach to measure coagulation kinetics and moreover, to monitor thrombin levels in blood. We are also in the process of developing a point-of-care device that will allow for the rapid measurement of thrombin activity in whole blood without the need for sample preparation.

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