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The cessation of bleeding in mammals occurs due to a well-conserved sequence of protein activation known as the coagulation cascade. However, people who have a deficiency in one or more proteins in this cascade, whether due to genetics or blood loss, struggle to maintain hemostasis. In order to aid patients in the restoration of hemostasis, exogenous proteins are often administered in response to bleeding events. However, these proteins are limited and costly due to limited supply of donor blood available for the production of plasma-derived proteins and the high cost of mammalian cell bio-reactors required for the production of recombinant proteins. As an alternative to the two previously aforementioned methods, human recombinant coagulation proteins have also been produced in the mammary gland of mice, swine, and bovine. This technique offers high production of active coagulation protein at low scaled-up cost. The research in this dissertation details the preclinical trials and characterization of two such proteins, factor IX produced in swine (FIX) and fibrinogen produced in bovine (FI), as well as one traditional recombinant protein, factor XIII produced in Pichia pastoris (FXIII). FXIII and FI, in addition to thrombin, are the main components of fibrin sealant, which is typically used to seal a wound in the case of a catastrophic bleeding event. FIX is used in a completely different context: the treatment of hemophilia B, a disease where the body produces no active FIX. Pharmacokinetic analysis and characterization of FIX were performed both intravenously and buccally in mice and dogs. The analysis revealed that FIX produced in the mammary gland of swine exhibited enhanced endothelial binding while maintaining normal whole blood clotting times despite the reduced plasma retention times. FIX stored in the extravascular reservoir was shown to influence FIX retention times for samples infused over 24 hours after plasma levels had been depleted. FXIII produced in yeast was shown to be monomeric and containing an artificial activation peptide that enhanced the crosslinking of fibrinogen with a reduced activation time lag.
Adviser: William H. Velander