Characterization of the Procoagulant Activity of a Novel Thromboplastin Reagent
In order to effectively monitor patients receiving oral anticoagulation therapies, physicians use the Prothrombin Time clotting assay. The main ingredient in these Prothrombin Time clotting assays is the thromboplastin reagent containing tissue factor, phospholipids and Calcium Chloride. This research will focus on characterizing a novel thromboplastin reagent that is efficient and easy to create. Additionally we will need to determine the International Sensitivity Index (ISI) of our thromboplastin regent. This is a necessary value assigned to each thromboplastin reagent in order to standardize it amongst the many commercially available today. We have determined that the optimal phospholipid concentration for our new thromboplastin reagent is 100uM phospholipid vesicles containing 8% Ni-NTA lipid, 30% PE, and between 5-15% PS with the balance being PC. This reagent also contains 1 ug/mL soluble tissue factor with a hexahistidine tag at the carboxy-terminus. The initial ISI of our reagent was 0.51 and in order to get an ISI closer to 1.00, we added more PS to increase the affinity of our thromboplastin reagent to the vitamin-k-dependent factors found in human plasma. However, with a sufficient PS concentration to yield an ISI near 1.00 our clot times with pooled normal plasma were too long for a typical thromboplastin reagent.
We have developed a novel thromboplastin reagent with soluble tissue factor that has similar clotting activity to that of membrane-bound tissue factor. In addition, we have determined that it is very sensitive to the presence of vitamin-k-dependent clotting factors. Future research will focus on decreasing the sensitivity of this reagent and achieving an ISI of approximately 1.00.
We have developed a novel thromboplastin reagent with soluble tissue factor that has similar clotting activity to that of membrane-bound tissue factor. In addition, we have determined that it is very sensitive to the presence of vitamin-k-dependent clotting factors. Future research will focus on decreasing the sensitivity of this reagent and achieving an ISI of approximately 1.00.
School:
University of Illinois at Urbana-Champaign
Department:
David A. Barounis
Research Advisor:
James Morrissey
Department of Research Advisor:
Department of Biochemistry
Year of Publication:
2005