Background: The natural coagulation factor X (FX) substitution FXR386C, affecting the catalytic domain, provided us with a tool to investigate TF/FVIIa and FVIIIa/FIXa -dependent FX activation. Materials and methods: The natural (R386C) and designed (R386A and the inactive S379A-R386C) FX mutants were stably expressed in Human Embrionic Kidney cells and purified by ion exchange, immunoaffinity and hydroxyapatite chromatography. In vitro secretion and in vivo stability in mice of recombinant molecules were studied. Variants were characterized by coagulation tests (PT and APTT) and functional assays in plasma and in reconstituted systems. Results: Alignment of mammalian sequences of vitamin K-dependent coagulation factors showed that the R386 residue is not conserved among serine-proteases. Moreover, in the crystallographic structure of FXa it is exposed on the surface of the catalytic domain. Major alteration of FX biosynthesis or function of the 386C variant were excluded by its normal i) secretion in vitro (1008±504 ng/ml vs 813±190 ng/ml of rFXwt), ii) clearance in vivo iii) procoagulant activity in a PT-based assay (rFX386C 105,7±5,7%). PT activity in plasma of the rFX386A and rFX379A386C variants were 173.6±7.1% and 0±0%, respectively. Instead, significantly reduced APTT values (rFX386C 28.7±0.5%, rFX386A 52.3±0.6%, rFX379A386C 0±0%) suggested impaired intrinsic activation of mutants with substitution at the 386 position. Functional assays exploiting fluoro/chromogenic substrates were used to dissect activation and activity of FX variants. Extrinsic activation, amydolytic and thrombin generation activities were indistinguishable from those of rFXwt. Noteworthy, a significant variation in kinetic parameters was measured for the rFX386C and rFX386A mutants in FXa generation assay upon activation by purified FVIIIa/FIXa complex. Conclusions: The A386C substitution in the coagulation FX is compatible with normal protein biosynthesis and almost exclusively affects its intrinsic activation. The 386 residue is potentially included in a functional exosite involved in macromolecular interactions.
THE NATURAL ARG386CYS MUTATION IN THE COAGULATION FACTOR X, AS A TOOL TO INVESTIGATE DIFFERENT FX-REQUIREMENTS IN INITIATION AND PROPAGATION PHASES.
BARONI, Marcello;PINOTTI, Mirko;MONTI, Monia;BERNARDI, Francesco
2012
Abstract
Background: The natural coagulation factor X (FX) substitution FXR386C, affecting the catalytic domain, provided us with a tool to investigate TF/FVIIa and FVIIIa/FIXa -dependent FX activation. Materials and methods: The natural (R386C) and designed (R386A and the inactive S379A-R386C) FX mutants were stably expressed in Human Embrionic Kidney cells and purified by ion exchange, immunoaffinity and hydroxyapatite chromatography. In vitro secretion and in vivo stability in mice of recombinant molecules were studied. Variants were characterized by coagulation tests (PT and APTT) and functional assays in plasma and in reconstituted systems. Results: Alignment of mammalian sequences of vitamin K-dependent coagulation factors showed that the R386 residue is not conserved among serine-proteases. Moreover, in the crystallographic structure of FXa it is exposed on the surface of the catalytic domain. Major alteration of FX biosynthesis or function of the 386C variant were excluded by its normal i) secretion in vitro (1008±504 ng/ml vs 813±190 ng/ml of rFXwt), ii) clearance in vivo iii) procoagulant activity in a PT-based assay (rFX386C 105,7±5,7%). PT activity in plasma of the rFX386A and rFX379A386C variants were 173.6±7.1% and 0±0%, respectively. Instead, significantly reduced APTT values (rFX386C 28.7±0.5%, rFX386A 52.3±0.6%, rFX379A386C 0±0%) suggested impaired intrinsic activation of mutants with substitution at the 386 position. Functional assays exploiting fluoro/chromogenic substrates were used to dissect activation and activity of FX variants. Extrinsic activation, amydolytic and thrombin generation activities were indistinguishable from those of rFXwt. Noteworthy, a significant variation in kinetic parameters was measured for the rFX386C and rFX386A mutants in FXa generation assay upon activation by purified FVIIIa/FIXa complex. Conclusions: The A386C substitution in the coagulation FX is compatible with normal protein biosynthesis and almost exclusively affects its intrinsic activation. The 386 residue is potentially included in a functional exosite involved in macromolecular interactions.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
