The role of autolysis loop in determining the specificity of coagulation proteases

Abstract

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX 143-154 ) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX 143-154 with AT, both the amidolytic and antifactor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp 143-154 ). Unlike an ~500-fold improvement in the reactivity of APC/fX [143][144][145][146][147][148][149][150][151][152][153][154] with AT in the presence of pentasaccharide, the reactivity of APC/Tryp 143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate. Correspondence

Autolysis loop restricts the specificity of activated protein C: Analysis by FRET and functional assays

“…2 indicate that this mutant has an APC-like activity toward fVa both in the absence and presence of protein S. These results suggest that the basic residues of the autolysis loop in APC-FX 143-154 are responsible for the protein S-independent improvement in the activity of the mutant, possibly by the basic residues making a productive interaction with fVa in the absence of protein S. Analysis of the activity of the autolysis loop mutants in the plasma-based assay indicated that the anticoagulant activities of APC derivatives (in particular APC-Tryp 143-154 ) have been dramatically impaired (data not shown, see Ref. [17]). Analysis of the inhibition profiles of APC derivatives with three different plasma inhibitors, PCI, AT and α 1 -antitrypsin have indicated that the reactivity of APC-Tryp 143-154 with all three inhibitors has been markedly improved, explaining a lack of anticoagulant activity for this mutant in plasma [17].…”

“…Expression, purification and preparation of wild-type protein C and the chimeric mutants in which the autolysis loop of the protein from residues 143-154 has been replaced with the corresponding loop of either factor X (PC-FX 143-154 ) or trypsin (PC-Tryp 143-154 ) have been described [16,17]. The expressed proteins were purified by a combination of immunoaffinity and ion exchange chromatography using the HPC4 monoclonal antibody immobilized on Affi-gel 10 and FPLC Mono Q column, respectively, as described [16].…”

“…Nevertheless, the substitution of this loop with the corresponding loop of trypsin (APC-Tryp 143-154 ) did not alter the activity of the mutant toward fVa in the purified system, however, it completely abolished the anticoagulant activity of the protease in a plasma-based assay system [17]. Further studies revealed that the APC-Tryp 143-154 mutant has adopted a trypsin-like specificity, thus becoming susceptible to inhibition by plasma inhibitors, accounting for its lack of activity in plasma [17]. Interestingly, the amidolytic activity of the APC-Tryp 143-154 was also markedly improved, as evidenced by its 3-4-fold decreased K m toward the cleavage of an APC-specific small synthetic chromogenic substrate [17].…”

“…In a recent study we also demonstrated that the substitution of the longer autolysis loop of APC from residues 143-154 (chymotrypsin numbering [15]) with the shorter loop of factor Xa (APC-FX 143-154 ) improved the anticoagulant activity of APC in a fVa degradation assay in the absence but not in the presence of protein S [16]. Nevertheless, the substitution of this loop with the corresponding loop of trypsin (APC-Tryp 143-154 ) did not alter the activity of the mutant toward fVa in the purified system, however, it completely abolished the anticoagulant activity of the protease in a plasma-based assay system [17]. Further studies revealed that the APC-Tryp 143-154 mutant has adopted a trypsin-like specificity, thus becoming susceptible to inhibition by plasma inhibitors, accounting for its lack of activity in plasma [17].…”

“…Further studies revealed that the APC-Tryp 143-154 mutant has adopted a trypsin-like specificity, thus becoming susceptible to inhibition by plasma inhibitors, accounting for its lack of activity in plasma [17]. Interestingly, the amidolytic activity of the APC-Tryp 143-154 was also markedly improved, as evidenced by its 3-4-fold decreased K m toward the cleavage of an APC-specific small synthetic chromogenic substrate [17]. The molecular basis for the overall improved catalytic activity of this mutant has not been investigated nor the effect of protein S on its proteolytic activity toward fVa in the purified system.…”