. The goal of this proposal is to develop a prothrombin time (PT) reagent using purified, reconstituted recombinant human tissue factor (rTF). The availability at Corvas of purified recombinant human tissue factor makes this feasible. A human tissue factor based PT reagent will allow development of ultra-sensitive and specific monitoring of oral anticoagulant therapy and deficiencies in the extrinsic pathway of coagulation. In Phase I, we will examine the reconstitution of tissue factor into phospholipid vesicles. A Chinese hamster ovarian cell transformant expressing high levels of tissue factor (rTF) has already been selected by Corvas. In preliminary studies, reconstitution of the inactive, detergent solubilized protein with crude mixed brain phospholipids of animal origin resulted in the formation of active protein capable of initiating the coagulation cascade. Parameters to be investigated include: 1. Phospholipid requirements for reconstitution. 2. Methods for vesicle formation and protein insertion. 3. Physical stability of the optimally formulated reagent. The result of successful Phase I research will be a PT reagent suitable for standardization, clinical evaluation and commercialization. In Phase II, we will manufacture rTF, perform clinical trials and obtain regulatory approval to market product.
