Characterization of the intracellular signalling capacity of natural FXa mutants with reduced pro-coagulant activity.
BACKGROUND
Factor X (FX) is a serine-protease playing a crucial role in the blood coagulation pathway and triggering intracellular signalling in a variety of cells via protease-activated receptors (PARs). By exploiting naturally occurring variants (V342A and G381D, catalytic domain; E19A, gamma-carboxyglutamic acid (GLA)-rich domain), we investigated the relationship between the pro-coagulant activity and the signal transduction capacity of FX.


METHODS
Recombinant FX (rFX) variants were expressed in Human Embryonic Kidney cells and purified by immunoaffinity chromatography. Activated rFX (rFXa) variants were characterized for pro-coagulant, amidolytic and thrombin generation activity. rFXa signalling was assessed through evaluation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in C2C12 myoblasts.


CONCLUSIONS
rFX variants showed reduced (rFX-342A, 29%; rFX-19A, 12%) or not detectable (rFX-381D) amidolytic activity. Thrombin generation activity in a plasma system was also decreased either upon activation by Russell's viper venom (rFX-342A, 38%; rFX-19A, 7%; rFX-381D, not detectable) or by the extrinsic pathway (rFX-342A, 36%; rFX-19A, rFX-381D, not detectable). The rFXa-381D mutant displayed little or no enzymatic activity, and did not induce any appreciable signal transduction capacity. The rFXa-342A mutant induced a dose-dependent signalling with a 50% reduced signalling capacity. At the highest concentration (174 nM), signalling progressed with a time course similar to that of rFXa-wt. Zymogen rFX-19A showed defective and incomplete activation resulting in strongly reduced enzymatic activity and signalling. Taken together our data are consistent with a close correlation between pro-coagulant activity and intracellular signalling capacity.
Monti
Monia
M
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy. mntmno@unife.it
Borensztajn
Keren S
KS
Pinotti
Mirko
M
Canella
Alessandro
A
Branchini
Alessio
A
Marchetti
Giovanna
G
Reitsma
Pieter H
PH
Bernardi
Francesco
F
Spek
C Arnold
CA
D000818:Animals; D002460:Cell Line; D003029:Coagulants / Q000378:metabolism; D015951:Factor Xa / Q000235:genetics* / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011516:Prothrombin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity
D003029:Coagulants; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D011516:Prothrombin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D015951:Factor Xa