Resistenza alla Proteina C Attivata
Alterazioni nel gene del Fattore V determinano la sintesi di una forma di Fattore V resistente alla degradazione da parte di un fattore plasmatico anticoagulante, la Proteina C Attivata (APC). L'alterazione più frequente è il Fattore V Leiden (10% della popolazione europea) che aumenta il rischio trombotico di circa 7 volte nei portatori eterozigoti e 80 volte negli omozigoti.
Blood. 2005 Oct 1;106(7):2363-5. Epub 2005 Jun 16.
An underestimated combination of opposites resulting in enhanced thrombotic tendency.
Simioni P, Castoldi E, Lunghi B, Tormene D, Rosing J, Bernardi F.
Department of Medical and Surgical Sciences, Second Chair of Internal Medicine, University of Padua Medical School, Padua, Italy. paolo.simioni@unipd.it
Heterozygous carriers of factor V (FV) Leiden who also carry FV deficiency often develop venous thromboembolism, but the thrombosis risk associated with this rare condition (pseudohomozygous activated protein C resistance) is still unclear. The thrombosis risk of genetically characterized pseudohomozygotes (n = 6) was compared with that of FV Leiden heterozygotes (n = 683) and homozygotes (n = 50) recruited within a large cohort study on familial thrombophilia. Both thrombin generation and Kaplan-Meier thrombosis-free survival analyses were performed in different FV genotype groups. FV Leiden pseudohomozygotes showed significantly higher thrombosis risk than heterozygotes. The thrombin generation test in pseudohomozygotes showed a pattern similar to homozygotes. Accordingly, early thrombotic manifestations occurred in pseudohomozygotes at a similar rate as in homozygotes. Thus, failure to recognize FV deficiency in FV Leiden heterozygotes may result in an underestimate of the thrombosis risk and inadequate management of affected patients.
PMID: 15961511 [PubMed - indexed for MEDLINE]
Thromb Haemost. 2003 Jun;89(6):983-9.
A FV multiallelic marker detects genetic components of APC resistance contributing to venous thromboembolism in FV Leiden carriers.
Mingozzi F, Legnani C, Lunghi B, Scanavini D, Castoldi E, Palareti G, Marchetti G, Bernardi F.
Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Italy.
Activated protein C resistance (APCR) is a major risk factor for venous thromboembolism (VTE). Although the factor V (FV) Leiden mutation accounts for the vast majority of APCR cases, other polymorphisms may contribute to the APCR phenotype. Genetic components of APCR and thrombophilia were investigated by two dinucleotide repeats, characterized in introns 2 and 11 of the FV gene. Only the intron 11 marker was genetically stable and thus suitable for further analysis. Its allelic frequencies were found to differ significantly (P=0.003) between subjects selected for increased APCR in the absence of the FV R506Q mutation (n=70, normalized ratios </=0.80), and for increased APC sensitivity (n=98, normalized ratios >/=1.31). Genotype differences were also found (P=0.017) between FV R506Q heterozygotes (n=100) who had experienced previous VTE and those (n=100), who were still asymptomatic for VTE. Significance was mostly driven by the relative over-representation of the 12R allele and to a minor extent by the under-representation of the 15R allele among the symptomatic versus the asymptomatic FV Leiden carriers. Two SNPs (4070A/G and 2391A/G) were found to underlie the 12R and 15R alleles respectively, and marked extended haplo-types, previously (HR2) or newly (HT2) identified. Only the FV HR2 differed (P=0.002) in frequency between the two groups of FV R506Q heterozygotes, suggesting that it represents the most relevant FV genetic component of APCR or VTE detectable by this experimental and clinical approach. Our analysis indicates that frequent FV genetic components might contribute to shape the risk for VTE in FV Leiden carriers.
PMID: 12783110 [PubMed - indexed for MEDLINE]
Arterioscler Thromb Vasc Biol. 1999 Feb;19(2):336-42.
Phenotype and genotype expression in pseudohomozygous factor VLEIDEN : the need for phenotype analysis.
Kalafatis M, Bernardi F, Simioni P, Lunghi B, Girolami A, Mann KG.
Department of Biochemistry, College of Medicine, University of Vermont, Burlington, USA. m.kalafatis@popmail.csuohio.edu
The presence of a DNA mutation is frequently used to define a disease or a risk state. Because DNA typing has become easy and convenient in contrast to protein characterization, it is generally assumed that a mutation if present (or not) at the DNA level will be also present (or not) in the corresponding protein. However, discrepancies between phenotype and genotype can occur. A point mutation in the coagulation factor V gene (G1691-->A, resulting in an Arg506-->Gln amino acid substitution in the factor V molecule [factor VLEIDEN], leading to activated protein C resistance) is the most common genetic risk factor for familial thrombophilia. A pseudohomozygous factor VLEIDEN phenotype would occur if a heterozygous individual for factor VLEIDEN also did not express the "normal" (non-Leiden) factor V allele. However, to date, no data have been available to confirm the presence of only the factor VLEIDEN form in the plasma of these individuals. Platelet mRNA from 2 presumed pseudohomozygous patients and their family members was isolated, the amplified partial cDNAs were sequenced or restricted, and the allelic bands were quantified. Both patients were found to be heterozygous for the G1691-->A substitution at both the DNA and mRNA levels. The presence of either the normal or mutated form of factor V in the patients' plasmas was investigated using a monoclonal antibody to factor V that recognizes an epitope located between residues 307 and 506 of the factor Va heavy chain. No normal factor V could be detected in the plasmas of the 2 propositi. The present data demonstrate absence of a correlation between genotype at position 1691 (at the DNA and mRNA levels) and the corresponding phenotype data found in the plasmas of patients with pseudohomozygous factor VLEIDEN. Overall, these data suggest the existence of heterogeneous genetic "lesions," which interfere with factor V expression, processing, secretion, and/or stability. Because the presence of the factor VLEIDEN molecule in plasma is directly related to pathology, identification and quantification of the circulating forms of factor V in plasma may be required for the diagnosis of individuals with activated protein C resistance.
PMID: 9974416 [PubMed - indexed for MEDLINE]
Thromb Haemost. 1998 Sep;80(3):403-6.
Molecular bases of pseudo-homozygous APC resistance: the compound heterozygosity for FV R506Q and a FV null mutation results in the exclusive presence of FV Leiden molecules in plasma.
Castoldi E, Kalafatis M, Lunghi B, Simioni P, Ioannou PA, Petio M, Girolami A, Mann KG, Bernardi F.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy.
Pseudo-homozygous APC resistance, the condition resulting from compound heterozygosity for FV R506Q (FV Leiden) and quantitative FV deficiency, provides a natural model to study the interaction between procoagulant and anticoagulant defects. This paper reports a complete FV characterization of a pseudo-homozygous APC resistant thrombotic patient. The expression of the patient's non-Leiden gene was found to be severely impaired both at the mRNA and protein levels. In particular, only FV Leiden molecules were detected in the patient's plasma by immunoblotting, which accounts for the observed marked APC resistance. Analysis of the FV cDNA obtained by reverse transcription of platelet RNA revealed that the mRNA of the non-Leiden gene was extremely reduced in amount. A PAC clone containing the whole FV gene was used to design primers for a complete FV exon scanning. A 2-bp insertion at nucleotide 3706 in the large exon 13 of the non-Leiden gene, predicting a frame-shift and premature termination of protein synthesis, was identified as responsible for the FV defect. Failure to find any case of pseudo-homozygous APC resistance in a large sample (6,804) of blood donors suggests that this condition is extremely rare among normal controls and that its detection is favoured by the thrombotic risk that it may confer.
PMID: 9759618 [PubMed - indexed for MEDLINE]
Br J Haematol. 1997 Nov;99(2):257-61.
Phenotypic homozygous activated protein C resistance associated with compound heterozygosity for Arg506Gln (factor V Leiden) and His1299Arg substitutions in factor V.
Castaman G, Lunghi B, Missiaglia E, Bernardi F, Rodeghiero F.
Department of Haematology and Haemophilia and Thrombosis Centre, San Bortolo Hospital, Vicenza, Italy.
Two patients from two unrelated families with a history of thrombosis showed severe plasma activated protein C (APC) resistance. However, genotypic analysis demonstrated that the patients were heterozygous for factor V (FV) Leiden mutation. Coagulation studies revealed that FV clotting activity and antigen were similarly reduced at about 50% of normal in the patients. One brother of propositus A also showed the same abnormalities. Genetic analysis showed that, in addition to FV Leiden mutation in exon 10 of the FV gene (G1691A), these patients had a transition in exon 13 of the FV gene (A4070G; R2 allele) predicting His1299Arg substitution in the mature FV. Study by RT-PCR of platelet FV mRNA indicated that the mRNA produced by the FV gene, marked by the R2 allele, was reduced in amount in both pseudohomozygous patients of family A. The R2 allele has previously been demonstrated to be significantly associated with plasma FV deficiency in the Italian population. The presence of FV deficiency did not protect the propositi from thrombosis. These data confirm that genotypic analysis is mandatory in patients with phenotypic severe APC resistance before these patients are definitely classified as homozygotes for FV Leiden and that further genotypic analysis is advisable.
PMID: 9375735 [PubMed - indexed for MEDLINE]
Blood. 1997 Aug 15;90(4):1552-7.
A factor V genetic component differing from factor V R506Q contributes to the activated protein C resistance phenotype.
Bernardi F, Faioni EM, Castoldi E, Lunghi B, Castaman G, Sacchi E, Mannucci PM.
Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Italy.
Factor V gene polymorphisms were investigated to detect components that may contribute to the activated protein C (APC) resistance phenotype in patients with venous thromboembolism. A specific factor V gene haplotype (HR2) was defined by six polymorphisms and its frequency was found to be similar in normal subjects coming from Italy (0.08), India (0.1), and Somalia (0.08), indicating that it was originated by ancestral mutational events. The relationship between the distribution of normalized APC ratios obtained with the functional assay and haplotype frequency was analyzed in patients heterozygous for factor V R506Q (factor V Leiden). The HR2 haplotype was significantly more frequent in patients with ratios below the 15th percentile than in those with higher ratios or in normal controls. Moreover, the study of 10 patients with APC resistance in the absence of the factor V R506Q mutation showed a 50-fold higher frequency of HR2 homozygotes. The HR2 haplotype was associated with significantly lower APC ratios both in patients with venous thromboembolism and in age- and sex-matched controls. However, the two groups showed similar HR2 haplotype frequencies. Plasma mixing experiments showed that an artificially created double heterozygote for the factor V R506Q mutation and the HR2 haplotype had an APC ratio lower than that expected for a simple R506Q heterozygote. Time-course experiments evaluating the decay of factor V in plasma showed the normal stability of the molecule encoded by the factor V gene marked by the HR2 haplotype, which ruled out the presence of a pseudo-homozygous APC resistance mechanism. Our results provide new insights into the presence of factor V genetic components other than the factor V R506Q that are able to contribute to the APC resistance phenotype in patients with venous thromboembolism.
PMID: 9269773 [PubMed - indexed for MEDLINE
Contraception. 1996 Sep;54(3):149-52.
Resistance to activated protein C, associated with oral contraceptives use; effect of formulations, duration of assumption, and doses of oestro-progestins.
Olivieri O, Friso S, Manzato F, Grazioli S, Bernardi F, Lunghi B, Girelli D, Azzini M, Brocco G, Russo C, Corrocher R.
Institute of Medical Pathology, University of Verona, Italy.
Resistance to activated protein C (APC-R) is at present considered the most frequent laboratory abnormality in patients with deep vein thrombosis. An increased risk for venous thrombosis is associated with the use of oral contraceptives (OCs). We recently described a statistically significant association between APC-R status and oral contraceptives use in a healthy group of women. We re-evaluated 50 healthy women taking low-dose combination OCs in order to consider a possible correlation between the APC sensitivity ratio (APC-SR) and different oral contraceptive formulations. Seven women showed an APC ratio < or = 2 (APC-resistant). Only one of the seven women was found to be heterozygous for Leiden factor V mutation. We observed no significant differences between normally sensitive and APC-resistant women in terms of duration of OC use, amount of estrogenic or progestogenic dose, or type of formulation. We conclude that APC-resistance associated with oral contraceptives use seems to occur only in predisposed subjects (in our results, about 12% of the healthy population).
PMID: 8899255 [PubMed - indexed for MEDLINE]
Br J Haematol. 1996 Jun;93(3):694-9.
Activated protein C resistance: a comparison between two clotting assays and their relationship to the presence of the factor V Leiden mutation.
Legnani C, Palareti G, Biagi R, Coccheri S, Bernardi F, Rosendaal FR, Reitsma PH, de Ronde H, Bertina RM.
Department of Angiology and Blood Coagulation, University Hospital S. Orsola, Bologna, Italy.
Resistance to the anticoagulant effect of activated protein C (APC resistance), a frequent abnormality in patients with a history of venous thrombosis, is known to be due, in the large majority of cases, to the presence of an abnormal factor V: the factor V Leiden. It is reasonable to surmise that screening for this abnormality should be performed with a clotting method for APC resistance, before submitting the patients with abnormal results to DNA analysis. The present study was performed on 216 individuals enrolled at the Bologna centre, of which 189 were unrelated patients with a history of juvenile venous thromboembolism and 27 were relatives with or without thrombosis. APC resistance was first measured in Bologna by a standard commercial method and then, in Leiden, by an in-house method: DNA analysis was performed in those cases in which at least one of the clotting methods was abnormal. The data obtained confirm the good performance and the optimal positive predictive value for the Leiden mutation (100%) of the Leiden in-house clotting method. Performance of the commercial method was less satisfactory but markedly improved by expressing the data in relation to the values simultaneously obtained with a normal plasma pool. Even with optimal data expression, however, the positive predictive value of the commercial method, versus DNA analysis, did not exceed 88%. It is concluded that further standardization of the commercial method here evaluated is necessary before it can be widely adopted for the screening of APC resistance and prediction of the presence of factor V Leiden.
PMID: 8652396 [PubMed - indexed for MEDLINE]
Br J Haematol. 1995 Oct;91(2):465-70.
Resistance to activated protein C in healthy women taking oral contraceptives.
Olivieri O, Friso S, Manzato F, Guella A, Bernardi F, Lunghi B, Girelli D, Azzini M, Brocco G, Russo C, et al.
Institute of Medical Pathology, University of Verona, Italy.
Resistance to activated protein C (APC) is at present considered the most frequent laboratory abnormality in patients with deep-vein thrombosis. An increased risk for venous thrombosis is associated to the use of oral contraceptives (OC). We studied APC sensitivity in 50 healthy women taking OC and in 50 healthy controls, matched for age, smoking habit, educational and social levels, and the main biochemical routinary parameters. Subjects with a personal or familial history of thrombosis and also with chronic or acute diseases were excluded. Protein C, protein S, antithrombin III and lupus anticoagulant activity (LAC) were also evaluated. Increased fibrinogen and protein C levels, decreased protein S. and shortened PT and APTT were also observed in women taking OC. APC sensitivity ratio (APC-SR) was significantly lower in the OC group than in a control group (2.6 +/- 0.38 v 2.81 +/- 0.35, P < 0.01). Seven of eight women with APC ratio < or = 2 (APC resistant) were OC users: the difference of prevalence was statistically significant (chi-squared test, P < 0.05). Only two out of eight women were found heterozygous for the Leiden factor V mutation. Two APC-resistant women without the Leiden mutation subsequently discontinued OC and both then normalized their APC-SR. We conclude that acquired factors, i.e. oral contraceptives, may play an important role in determining plasma APC resistance.
PIP: During April-June 1994, at Borgo Roma Polyclinic in Verona, Italy, clinical researchers compared data on 50 healthy women 18-41 who used low-dose combined oral contraceptives (OCs) with data on 50 healthy women matched for age, smoking, education, social class, and biochemical routinary parameters. Almost all the subjects were medical students or medical staff working in the hospital where the study occurred. The researchers aimed to examine the prevalence of resistance to activated protein C (APC) in both groups. They also evaluated protein C, protein S, antithrombin III, and lupus anticoagulant activity. The APC-sensitivity ratio (APC-SR) was much lower in OC users than nonusers (2.6 vs. 2.81; p 0.01). Seven of the eight women with an APC-SR of no greater than 2 (i.e., demonstration of APC resistance) used OCs (p 0.05). Prevalence of APC resistance was higher among OC users than nonusers (14% vs. 2%; p 0.05). Among the eight women with APC resistance, two were heterozygous for the Leiden factor V mutation. One of these women used OCs and the other did not. Two APC resistant women who did not have the Leiden factor V stopped using OCs and their APC-SR subsequently normalized. OC users had higher fibrinogen and protein C levels, a lower protein S level, and shorter prothrombin and activated partial thromboplastin times than nonusers. These findings suggest that OCs may contribute to plasma APC resistance, which in turn increases the risk of venous thrombosis.
PMID: 8547095 [PubMed - indexed for MEDLINE]
Thromb Haemost. 1993 Dec 20;70(6):1067-71.
Resistance to activated protein C in nine thrombophilic families: interference in a protein S functional assay.
Faioni EM, Franchi F, Asti D, Sacchi E, Bernardi F, Mannucci PM.
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Maggiore Hospital, Milamo, Italy.
Nine thrombophilic patients who had had previous diagnoses of functional protein S deficiency were reinvestigated. The functional protein S assays gave dose-response curves that were not parallel to those of the reference plasma. The same pattern was true for approximately half of the first-degree relatives of the propositi. When protein S was extracted from the plasma of the patients by immunoabsorption, it had a normal ratio of functional activity to immunologic concentration. Restriction fragment length polymorphism analysis, informative in one family, showed no linkage between the protein S gene marker and the abnormal behavior of the protein S functional assay. All the propositi and 23/36 first-degree relatives were resistant to the prolongation of activated partial thromboplastin time induced by activated protein C. Furthermore, there was striking concordance in all patients and relatives between the abnormal pattern of the protein S functional assay and resistance to activated protein C. We conclude that a plasma-based functional protein S assay is sensitive to activated protein C resistance and this may lead to spuriously low results in the assay. In agreement with the results of others, this study indicates that resistance to activated protein C is a frequent hemostatic defect in selected thrombophilic populations.
PMID: 8165605 [PubMed - indexed for MEDLINE]
Arterioscler Thromb Vasc Biol. 1999 Feb;19(2):336-42.
Phenotype and genotype expression in pseudohomozygous factor VLEIDEN : the need for phenotype analysis.
Kalafatis M, Bernardi F, Simioni P, Lunghi B, Girolami A, Mann KG.
Department of Biochemistry, College of Medicine, University of Vermont, Burlington, USA. m.kalafatis@popmail.csuohio.edu
The presence of a DNA mutation is frequently used to define a disease or a risk state. Because DNA typing has become easy and convenient in contrast to protein characterization, it is generally assumed that a mutation if present (or not) at the DNA level will be also present (or not) in the corresponding protein. However, discrepancies between phenotype and genotype can occur. A point mutation in the coagulation factor V gene (G1691-->A, resulting in an Arg506-->Gln amino acid substitution in the factor V molecule [factor VLEIDEN], leading to activated protein C resistance) is the most common genetic risk factor for familial thrombophilia. A pseudohomozygous factor VLEIDEN phenotype would occur if a heterozygous individual for factor VLEIDEN also did not express the "normal" (non-Leiden) factor V allele. However, to date, no data have been available to confirm the presence of only the factor VLEIDEN form in the plasma of these individuals. Platelet mRNA from 2 presumed pseudohomozygous patients and their family members was isolated, the amplified partial cDNAs were sequenced or restricted, and the allelic bands were quantified. Both patients were found to be heterozygous for the G1691-->A substitution at both the DNA and mRNA levels. The presence of either the normal or mutated form of factor V in the patients' plasmas was investigated using a monoclonal antibody to factor V that recognizes an epitope located between residues 307 and 506 of the factor Va heavy chain. No normal factor V could be detected in the plasmas of the 2 propositi. The present data demonstrate absence of a correlation between genotype at position 1691 (at the DNA and mRNA levels) and the corresponding phenotype data found in the plasmas of patients with pseudohomozygous factor VLEIDEN. Overall, these data suggest the existence of heterogeneous genetic "lesions," which interfere with factor V expression, processing, secretion, and/or stability. Because the presence of the factor VLEIDEN molecule in plasma is directly related to pathology, identification and quantification of the circulating forms of factor V in plasma may be required for the diagnosis of individuals with activated protein C resistance.
PMID: 9974416 [PubMed - indexed for MEDLINE]
J Thromb Haemost. 2005 Dec;3(12):2695-702.
Expression of the normal factor V allele modulates the APC resistance phenotype in heterozygous carriers of the factor V Leiden mutation.
Brugge JM, Simioni P, Bernardi F, Tormene D, Lunghi B, Tans G, Pagnan A, Rosing J, Castoldi E.
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, Netherlands.
BACKGROUND: Functional defects of the protein C pathway, detectable in plasma as activated protein C (APC) resistance, are a prevalent risk factor for venous thrombosis. The factor V (FV) Leiden mutation causes APC resistance by interfering with the APC-mediated inactivation of both FVa and FVIIIa. Co-inheritance of FV Leiden and quantitative FV deficiency on different alleles, a rare condition known as pseudo-homozygous APC resistance, is associated with pronounced APC resistance and 50% reduced FV levels, because of non-expression of the non-Leiden FV allele. OBJECTIVES: The role of normal FV in modulating the APC resistance phenotype in carriers of FV Leiden was investigated in patients with pseudo-homozygous APC resistance and in model systems. PATIENTS/METHODS: Four functional plasma assays probing both components of APC resistance (susceptibility of FVa to APC and cofactor activity of FV in FVIIIa inactivation) were employed to compare seven clinically and genetically characterized FV Leiden pseudo-homozygotes to 30 relatives with different FV genotypes (including 12 FV Leiden heterozygotes and seven carriers of FV deficiency) and to 32 unrelated FV Leiden homozygotes. RESULTS AND CONCLUSIONS: All assays consistently indicated that FV Leiden pseudo-homozygotes are significantly more APC-resistant than heterozygotes and indistinguishable from homozygotes. Thrombin generation measurements in FV-deficient plasma reconstituted with purified normal FV and FV Leiden confirmed these observations and showed that the expression of the normal FV allele is an important modulator of APC resistance in FV Leiden heterozygotes. These findings provide an explanation for the higher thrombotic risk of FV Leiden pseudo-homozygotes when compared with heterozygotes.
PMID: 16359508 [PubMed - indexed for MEDLINE]
Brugge JM, Simioni P, Bernardi F, Tormene D, Lunghi B, Tans G, Pagnan A, Rosing J, Castoldi E.
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, Netherlands.
BACKGROUND: Functional defects of the protein C pathway, detectable in plasma as activated protein C (APC) resistance, are a prevalent risk factor for venous thrombosis. The factor V (FV) Leiden mutation causes APC resistance by interfering with the APC-mediated inactivation of both FVa and FVIIIa. Co-inheritance of FV Leiden and quantitative FV deficiency on different alleles, a rare condition known as pseudo-homozygous APC resistance, is associated with pronounced APC resistance and 50% reduced FV levels, because of non-expression of the non-Leiden FV allele. OBJECTIVES: The role of normal FV in modulating the APC resistance phenotype in carriers of FV Leiden was investigated in patients with pseudo-homozygous APC resistance and in model systems. PATIENTS/METHODS: Four functional plasma assays probing both components of APC resistance (susceptibility of FVa to APC and cofactor activity of FV in FVIIIa inactivation) were employed to compare seven clinically and genetically characterized FV Leiden pseudo-homozygotes to 30 relatives with different FV genotypes (including 12 FV Leiden heterozygotes and seven carriers of FV deficiency) and to 32 unrelated FV Leiden homozygotes. RESULTS AND CONCLUSIONS: All assays consistently indicated that FV Leiden pseudo-homozygotes are significantly more APC-resistant than heterozygotes and indistinguishable from homozygotes. Thrombin generation measurements in FV-deficient plasma reconstituted with purified normal FV and FV Leiden confirmed these observations and showed that the expression of the normal FV allele is an important modulator of APC resistance in FV Leiden heterozygotes. These findings provide an explanation for the higher thrombotic risk of FV Leiden pseudo-homozygotes when compared with heterozygotes.
PMID: 16359508 [PubMed - indexed for MEDLINE]
