title: Engineering the proteolytic specificity of activated protein C improves its pharmacological properties.
abstract: Human activated protein C (APC) is an antithrombotic, antiinflammatory serine protease that plays a central role in vascular homeostasis, and activated recombinant protein C, drotrecogin alfa (activated), has been shown to reduce mortality in patients with severe sepsis. Similar to other serine proteases, functional APC levels are regulated by the serine protease inhibitor family of proteins including alpha(1)-antitrypsin and protein C inhibitor. Using APC-substrate modeling, we designed and produced a number of derivatives with the goal of altering the proteolytic specificity of APC such that the variants exhibited resistance to inactivation by protein C inhibitor and alpha(1)-antitrypsin yet maintained their primary anticoagulant activity. Substitutions at Leu-194 were of particular interest, because they exhibited 4- to 6-fold reductions in the rate of inactivation in human plasma and substantially increased pharmacokinetic profiles compared with wild-type APC. This was achieved with minimal impairment of the anticoagulant/antithrombotic activity of APC. These data demonstrate the ability to selectively modulate substrate specificity and subsequently affect in vivo performance and suggest therapeutic opportunities for the use of protein C derivatives in disease states with elevated serine protease inhibitor levels.
authors.affiliation: Division of BioResearch Technologies and Proteins, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
authors.forename: David T
authors.initials: DT
authors.lastname: Berg
authors.forename: Bruce
authors.initials: B
authors.lastname: Gerlitz
authors.forename: Jing
authors.initials: J
authors.lastname: Shang
authors.forename: Tommy
authors.initials: T
authors.lastname: Smith
authors.forename: Paula
authors.initials: P
authors.lastname: Santa
authors.forename: Mark A
authors.initials: MA
authors.lastname: Richardson
authors.forename: Ken D
authors.initials: KD
authors.lastname: Kurz
authors.forename: Brian W
authors.initials: BW
authors.lastname: Grinnell
authors.forename: Ken
authors.initials: K
authors.lastname: Mace
authors.forename: Bryan E
authors.initials: BE
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014644:Genetic Variation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008252:Macaca fascicularis; D008958:Models, Molecular; D011486:Protein C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018046:Protein C Inhibitor / Q000494:pharmacology; D015202:Protein Engineering; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015843:Serpins / Q000494:pharmacology; D013379:Substrate Specificity; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
chemical_list: D000925:Anticoagulants; D011486:Protein C; D018046:Protein C Inhibitor; D011994:Recombinant Proteins; D015843:Serpins; D000515:alpha 1-Antitrypsin