Engineering the proteolytic specificity of activated protein C improves its pharmacological properties

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 prot...

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Vydáno v:Proceedings of the National Academy of Sciences - PNAS Ročník 100; číslo 8; s. 4423
Hlavní autoři: Berg, David T, Gerlitz, Bruce, Shang, Jing, Smith, Tommy, Santa, Paula, Richardson, Mark A, Kurz, Ken D, Grinnell, Brian W, Mace, Ken, Jones, Bryan E
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States 15.04.2003
Témata:
alpha 1-Antitrypsin - pharmacology
Amino Acid Sequence
Amino Acid Substitution
Animals
Anticoagulants - chemistry
Anticoagulants - metabolism
Anticoagulants - pharmacology
Genetic Variation
Humans
In Vitro Techniques
Kinetics
Macaca fascicularis
Models, Molecular
Protein C - chemistry
Protein C - genetics
Protein C - metabolism
Protein C - pharmacology
Protein C Inhibitor - pharmacology
Protein Engineering
Rabbits
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Recombinant Proteins - pharmacology
Serpins - pharmacology
Substrate Specificity
ISSN:0027-8424
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Shrnutí: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.
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ISSN:0027-8424
DOI:10.1073/pnas.0736918100