Protein Engineering Thrombin for Optimal Specificity and Potency of Anticoagulant Activity in Vivo

Abstract

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

Functional Requirements for Inhibition of Thrombin by Antithrombin III in the Presence and Absence of Heparin

“…Mutant prothrombin in conditioned medium from transiently transfected COS-7 cells was concentrated by ultrafiltration, processed to thrombin by Echis carinatus venom, and quantitated by enzyme-linked immunosorbent assay. All these methods have been described in detail previously (32,34). a Residues numbered consecutively from the start of the thrombin B chain.…”

“…This mixture was added to a spectrophotometric cuvette containing 10 l of 300 nM mutant thrombin (final concentration of 5 nM), and hydrolysis of S-2238 was immediately measured for 2 min. The pseudo-first order rate constant of inhibition kЈ was determined by curve fitting as described previously (32). The second-order rate constant, k 2 , was determined from kЈ after correcting for substrate competition using the K m value of S-2238 hydrolysis (32).…”

“…The pseudo-first order rate constant of inhibition kЈ was determined by curve fitting as described previously (32). The second-order rate constant, k 2 , was determined from kЈ after correcting for substrate competition using the K m value of S-2238 hydrolysis (32).…”

“…Inhibition by ATIII is the primary mechanism for the clearance of active thrombin in vivo (11) and we have previously shown that mutations in thrombin that confer resistance to ATIII in vitro may correlate with a prolonged half-life in plasma and decreased clearance rate in vivo (31,32). By analogy with naturally occurring mutations in ATIII (10,12), mutations in thrombin that modulate inhibition by ATIII, such as those described in this study, may predispose individuals to an increased risk of thrombosis.…”

“…In contrast, the mutations identified in this study involving residues that are implicated in direct interactions with ATIII (Trp 50 , Glu 229 , Arg 233 ) would not be expected to be prothrombotic. Because of their proximity to the active site, the mutation of these residues also modulates the recognition of other substrates and alters the specificity of thrombin to favor the anticoagulant substrate protein C (31,32). Thus naturally occurring mutations in this class may have a net anticoagulant effect and may be protective of thrombosis.…”