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 ␣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 ␣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 anticoagulant͞ 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.

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

“…The importance of residues within the putative S2 pocket of proteases to the inhibition by serpins has been confirmed by a mutagenesis study of APC [37]. Substitution of residues within the active-site cleft of APC resulted in variants exhibiting resistance to inhibition by protein C inhibitor and α 1 -antitrypsin, yet maintained anticoagulant activity.…”

“…When Thr 254 (c99) was substituted with a serine residue, the t 1/2 in human plasma was prolonged 2-fold due to reduced rates of inactivation by circulating serpins found in plasma. The effect was more pronounced when the T254S (c99) mutation was combined with mutations of residue Leu 194 (c40), yielding up to 11-fold prolongation of the plasma t 1/2 [37]. Thus it appears that residue c99 in homologous enzymes such as FVIIa and APC influences the inhibitor specificity towards different serpins.…”

“…The present study of FVIIa and the one on APC variants described by Berg et al [37] shows how it is possible to engineer the primary specificity of serine proteases without compromising their biological function in the propagation and regulation of the coagulation cascade. These interesting findings reinforce the notion that the in vivo specificity of proteases towards their macromolecular substrates is, to a large degree, dictated by exosite interactions.…”

Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3

“…Because APC is not restricted by highaffinity binding to the PAR3 hirudin-like sequence, cleavage at Arg41 is not surprising considering that the PAR3 P5-P59 (IKTFR/ GAPPN) specificity subsite is very similar to that of protein C inhibitor (IFTFR/SARLN), with 80% identity in the P5-P1 residues and 40% in the P19-P59 residues. 37 The PAR3 P5-P59 region has been highly conserved (. 80%) between various species, except for rodents (supplemental Table 1).…”

Engineering activated protein C to maximize therapeutic efficacy

“…APC variants have been generated that fail to be efficiently inhibited by plasma inhibitors α1-anti-trypsin and protein C inhibitor (PCI) [37]. Mutations at the Leu 194 site on APC reduced interaction with both serpins and mediated a 6-fold increased plasma half-life compared with wild-type APC that was reflected in improved pharmacokinetic profiles for Leu 194 variants in rabbits and cynomolgus monkeys.…”