PRODUCTION OF PLANT-BASED RECOMBINANT HUMAN THROMBIN

Human thrombin currently commercially used for wound hemostasis and other applications is entirely derived from donor plasma. This source is limited to a current capacity of less than 100 kg/year worldwide, requires virus and other pathogen inactivation steps to assure safety and thus is plagued by severe cost constraints that preclude widespread application of clotting factor-based wound adhesives. The objective of this proposed Phase I SBIR project is to demonstrate expression of correctly processed prothrombin in stable plant cell culture. The active thrombin form will then be obtained through correct cleavage of prothrombin by either factor Xa or snake venom Ecarin. We will use cloning and expression strategies that (1) encourage assembly of prothrombin in the lumen of the endoplasmic reticulum and (2) are easily portable to future efforts for commercial-scale prothrombin production in transgenic plants. Active thrombin derived from cell culture will be tested for its correct factor XIII activation function as well as in vitro clotting procedures using Factor XIII and fibrinogen. This project is a part of a larger effort at PhytaGenics for the production of blood clotting proteins. To date, we have completely demonstrated expression of Factor XIII in plants and its function in fibrinogen clotting and cross-linking, and have completed proof-of-concept experiments toward transient thrombin expression in plant protoplasts