Autori: Scanavini D, Girelli D, Lunghi
B, Martinelli N, Legnani C, Pinotti M, Palareti G, Bernardi F.
Rivista: Arterioscler Thromb Vasc Biol. Volume:
24 Anno: 2004
Abstract: OBJECTIVE: Functional polymorphisms contributing
to coagulation factor levels are preferential markers for association studies
aimed at identifying prothrombic genetic components. METHODS AND RESULTS:
Factor V (FV) microsatellite genotypes were found to be associated with
FV levels (P=0.003). Single nucleotide polymorphisms analysis and sequencing
of the promoter and of coding regions identified two polymorphisms (Met2120Thr,
Asp2194Gly) present in 20% of the population (n=1013) that are responsible
for genotype-phenotype associations. The effect of the Met2120Thr polymorphism,
both in plasma (mean reduction of FV level in the heterozygous condition:
25%) and in recombinant FV studies (34% reduction), was comparable to that
of the Asp2194Gly change (20% and 34%, respectively). The study of 10 subjects
with a rare genotype indicated that the Asp2194Gly substitution is the functional
determinant of the reduced FV levels associated with the FVHR2 haplotype.
Among Leiden carriers, the doubly heterozygous condition for FV2120Thr was
found to be associated with a significantly increased activated protein-C
resistance (APCR) (P<0.05), and the doubly heterozygous condition for
FV2194Gly was found to be more frequent (P=0.009) in symptomatic than in
asymptomatic subjects. CONCLUSIONS: Extensive analysis of FV polymorphisms
indicated that changes in the C2 domain modulate FV levels and might increase
APCR and thrombotic risk in FV Leiden carriers through a pseudohomozygous
mechanism.
Breve commento esplicativo: L’analisi di diversi polimorfismi localizzati nel dominio C2 del fattore V (FV) della coagulazione ha mostrato che questi possono modulare i livelli plasmatici del FV stesso ed incrementare la resistensa alla proteina C attivata. La conseguenza è l’aumento del rischio di trombosi nei soggeti già portatori della mutazione Leiden. Questi polimorfismi funzionali possono essere utilizzati come marcatori genetici di rischio trombotico.
Autori: Castoldi E, Govers-Riemslag JW,
Pinotti M, Bindini D, Tans G, Berrettini M, Mazzucconi MG, Bernardi F, Rosing
J.
Rivista: Blood Volume: 102 Anno:
2003
Abstract: We investigated the role of thrombophilic mutations
as possible modifiers of the clinical phenotype in severe factor VII (FVII)
deficiency. Among 7 patients homozygous for a cross-reacting material-negative
(CRM-) FVII defect (9726+5G>A, FVII Lazio), the only asymptomatic individual
carried FV Leiden. Differential modulation of FVII levels by intragenic
polymorphisms was excluded by a FVII to factor X (FX) gene haplotype analysis.
The coagulation efficiency in the FV Leiden carrier and a noncarrier was
evaluated by measuring FXa, FVa, and thrombin generation after extrinsic
activation of plasma in the absence and presence of activated protein C
(APC). In both patients coagulation factor activation was much slower and
resulted in significantly lower amounts of FXa and thrombin than in a normal
control. However, more FXa and thrombin were formed in the plasma of the
patient carrying FV Leiden than in the noncarrier, especially in the presence
of APC. These results were confirmed in FV-FVII doubly deficient plasma
reconstituted with purified normal FV or FV Leiden. The difference in thrombin
generation between plasmas reconstituted with normal FV or FV Leiden gradually
decreased at increasing FVII concentration. We conclude that coinheritance
of FV Leiden increases thrombin formation and can improve the clinical phenotype
in patients with severe FVII deficiency.
Breve commento esplicativo: In questo studio si è mostrato come la presenza del fenotipo Leiden aumenta la produzione di trombina migliorando il quadro clinico di pazienti aventi difetto di fattore VII (FVII). Ciò evidenzia come una mutazione che normalmente aumenta il rischio di trombosi possa, in alcuni casi, compensare la carenza di un fattore procoagulante.
Autori: Pinotti M, Marchetti G, Baroni
M, Cinotti F, Morfini M, Bernardi F.
Rivista: Thrombosis and haemostasis Volume:
88 Anno: 2002
Abstract: 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.
Breve commento esplicativo: Sono stati caratterizzati pazienti con difetto di fattore X (FX) della coagulazione dovuto ad una mutazione che ne coinvolge il dominio ricco in gamma-carbossiglutammico (Gla). Il dato più ecclatante di questo studio riguarda il fatto che la completa attivazione del FX mutato richiede una quantità di fattore VIIa/fattore tissutale 30 volte superiore alla forma normale, mentre l’attivazione tramite fattore IXa/fattore VIIIa è confrontabile tra le due diverse forme. Ciò può essere un valido suggerimento per studiare le interazioni tra il FX e gli altri fattori della coagulazione che coinvolgono il dominio Gla.
Autori: Castoldi E, Simioni P, Kalafatis
M, Lunghi B, Tormene D, Girelli D, Girolami A, Bernardi F.
Rivista: Blood Volume: 96 Anno:
2000
Abstract: The study of the molecular bases of thrombophilia
in a large family with 4 symptomatic members is reported. Three thrombophilic
genetic components (FV R506Q, FV H1299R, and PT 20210G/A), all affecting
the activity of the prothrombinase complex, were detected alone and in combination
in various family members. In addition, a newly identified missense mutation
(factor V [FV] Y1702C), causing FV deficiency, was also present in the family
and appeared to enhance activated protein C (APC) resistance in carriers
of FV R506Q or FV H1299R by abolishing the expression of the counterpart
FV allele. The relationships between complex genotypes, coagulation laboratory
findings, and clinical phenotypes were analyzed in the family. All symptomatic
family members were carriers of combined defects and showed APC resistance
and elevated F1 + 2 values. Evidence for the causative role of the FV Y1702C
mutation, which affects a residue absolutely conserved in all 3 A domains
of FV, factor VIII, and ceruloplasmin, relies on (1) the absolute cosegregation
between the mutation and FV deficiency, both in the family and in the general
population; (2) FV antigen and immunoblot studies indicating the absence
of Y1702C FV molecules in plasma of carriers of the mutation, despite normal
levels of the FV Y1702C messenger RNA; and (3) molecular modeling data that
support a crucial role of the mutated residue in the A domain structure.
These findings help to interpret the variable penetrance of thrombosis in
thrombophilic families and to define the molecular bases of FV deficiency.
Breve commento esplicativo: In questo sono state identificate
mutazioni che determinano cali di attività del complesso protrombinasico
o riduzione dei livelli di fattore V (FV). Quando queste compaiono in associazione
ad altre mutazioni del FV possono determinare una maggiore resistenza alla
proteina C attivata. Ciò suggerisce di estendere lo screenig a tutte
queste varianti per avere un quadro più dettagliato delle possibili
cause genetiche della patologia.
Autori: Pinotti M, Bertolucci C, Portaluppi
F, Colognesi I, Frigato E, Foa A, Bernardi F.
Rivista: Arterioscler Thromb Vasc Biol. Volume:
16 Anno: 2004
Abstract: OBJECTIVE: Diurnal variations in levels of factor
VII (FVII), FVIII, proteins C and S, antithrombin, plasminogen activator
inhibitor-1, prothrombin fragment F1+2, and D-dimers in healthy humans point
to the existence of circadian rhythms of coagulation factors. We sought
for temporal fluctuations of tissue factor pathway inhibitor (TFPI) activity
in human and mouse plasma. METHODS AND RESULTS: TFPI activity showed significant
daily variations with highest levels in the morning in healthy men (+11%)
and in mice at the light-to-dark transition (+63%), the beginning of the
physically active period. Variations in FVII activity paralleled those in
TFPI. In mice, the feeding schedule had a strong impact on these rhythms.
Although restricted feeding and fasting shifted the peak of TFPI, the FVII
peak disappeared. Investigation of temporal fluctuations in constant darkness
indicated the existence of daily rhythms for TFPI and of true circadian
rhythms for FVII. CONCLUSIONS: For the first time, we report, both in humans
and mice, temporal variations in TFPI activity. The coherent variations
in FVII and TFPI activity could interplay to maintain the coagulation equilibrium.
The chronobiological patterns should be considered to analyze activity levels
of these factors. Moreover, the mouse model could be exploited to investigate
modifiers of coagulation rhythms potentially associated to morning peaks
of cardiovascular events.
Breve commento esplicativo: In questo
studio abbiamo mostrato la variazione secondo ritmi circadiani dell’inibitore
della via del fattore tissutale (TFPI) e del fattore VII (FVII) della coagulazione,
sia in modello murino che nell’uomo. Il passo successivo potrebbe
riguardare l’analisi di altri fattori coinvolti nel processo emostatico
per comprendere la relazione che hanno con il picco di eventi cardiovascolari
che si verifica preferenzialmente al mattino.