Fattore X
Thromb Res. 2009 Apr;123(6):914-8. Epub 2008 Dec 23.
Characterization of the intracellular signalling capacity of natural FXa mutants with reduced pro-coagulant activity.
Monti M, Borensztajn KS, Pinotti M, Canella A, Branchini A, Marchetti G, Reitsma PH, Bernardi F, Spek CA.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy. mntmno@unife.it
Abstract
INTRODUCTION: 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.
MATERIALS AND 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.
RESULTS AND 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.
PMID: 19108874 [PubMed - indexed for MEDLINE]
Characterization of anti-coagulant properties of prenylated coumarin ferulenol.
Monti M, Pinotti M, Appendino G, Dallocchio F, Bellini T, Antognoni F, Poli F, Bernardi F.
Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Italy.
We investigated the mechanisms underlying severe bleeding occurring upon consumption of Ferula communis. The prenylated coumarin ferulenol extracted from this plant did not directly affect blood coagulation but showed hepatocyte cytotoxicity and, at non-cytotoxic concentrations (<100 nM), impaired factor X biosynthesis (40% reduction). Studies with ferulenol derivatives indicated the prenyl residue as major determinant of ferulenol activity.
PMID: 17693024 [PubMed - indexed for MEDLINE]
Molecular characterization of factor X deficiency associated with borderline plasma factor X level.
Pinotti M, Monti M, Baroni M, Marchetti G, Bernardi F.
Borderline plasma factor X (FX) levels might complicate the diagnosis of FX deficiency. An asymptomatic individual with 73% FX activity was identified to be heterozygous for the Val342Ala mutation. Expression studies suggested that this substitution is responsible for a CRM+ FX variant with normal activation but modestly reduced catalytic function.
PMID: 15075089 [PubMed - indexed for MEDLINE]
Pinotti M, Camire RM, Baroni M, Rajab A, Marchetti G, Bernardi F.
Department of Biochemistry and Molecular Biology, University of Ferara, Ferrara, Italy.
We investigated three members of a large Omani family affected by severe factor X (FX) deficiency (coagulant activity <1%) and showing marked differences in the onset of severe hemorrhagic symptoms. All patients were homozygous for a novel FX mutation (Gly381Asp) in the structurally conserved region of the serine protease active site. Expression levels of recombinant 381D-FX were similar to those of wt-FX, indicating the presence of a severe CRM+ FX deficiency, a poorly investigated condition. The 381D-FX was normally activated and did not show a detectable amidolytic activity. Instead, we observed a residual activity in a prothrombin-time based assay (1%) and in prothrombinase assays both in plasma (1%) and in purified systems (3%). Comparison with FX variants characterized by reduced activation suggests that mutations affecting FX activity might result in a more pronounced impairment of coagulation and thus in severe hemorrhagic phenotype. In addition, this study indicates that the hemorrhagic heterogeneity observed in FX deficiencies is only partially explained by molecular analysis of FX gene.
PMID: 12574802 [PubMed - indexed for MEDLINE]
Pinotti M, Marchetti G, Baroni M, Cinotti F, Morfini M, Bernardi F.
Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Ferrara, Italy.
We characterized a symptomatic CRMred factor X (FX) deficiency produced by the Glu19Ala mutation in the gamma-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19Ala-FX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation. The study of this FX deficiency provides an "in vivo" and "in vitro" model for the investigation of Gla domain interactions.
PMID: 12195695 [PubMed - indexed for MEDLINE]
Molecular bases of CRM+ factor X deficiency: a frequent mutation (Ser334Pro) in the catalytic domain and a substitution (Glu102Lys) in the second EGF-like domain.
Marchetti G, Castaman G, Pinotti M, Lunghi B, Di Iasio MG, Ruggieri M, Rodeghiero F, Bernardi F.
Centro Studi Biochimici Patologie del Genoma Umano-Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Italy.
The presence of gene lesions in coagulation factor X (FX, Stuart factor) was investigated in asymptomatic subjects with FX deficiency characterized by the presence of dysfunctional molecules in plasma, as demonstrated by the discrepancy between clotting activity and antigen level. A missense mutation (Ser334Pro) in the catalytic domain was found in three unrelated families in both the homozygous and the heterozygous conditions, and also in the compound heterozygous form with the substitution of Lys for 102 Glu. None of the mutations was detected in 40 unrelated subjects from the same geographic area. The Ser334Pro mutation affects a serine protease region characterized by extensive variation in the coagulation factors but conserved in mammalian factor X molecules. The Glu102Lys mutation affects a residue of the second EGF-like module also conserved in protein C. Both mutated residues are surface-exposed and found in protein regions suggested to be involved in macromolecular interactions which are impaired in the dysfunctional molecules.
PMID: 7669671 [PubMed - indexed for MEDLINE]
Partial gene deletion in a family with factor X deficiency.
Bernardi F, Marchetti G, Patracchini P, Volinia S, Gemmati D, Simioni P, Girolami A.
Centro Studi Biochimici Patologie del Genoma Umano, Università di Ferrara, Italy.
The presence of gene lesions in coagulation factor X (FX, Stuart factor) was investigated in patients with FX deficiency or an FX abnormality (FX Friuli). The proposita had a heterozygous partial deletion of the FX gene with severe deficiency of FX activity and antigen. The lesion, which was inherited from her mother, removes the 3' portion of the gene coding for the catalytic domain of the factor. In this family, two differently affected FX genes are present, leading to double heterozygosity of the proposita and thus excluding consanguinity of parents. An apparently normal gene structure was observed in the other patient with FX abnormality, suggesting the presence of a small gene lesion.
PMID: 2567188 [PubMed - indexed for MEDLINE]
