The frequency of factor V Leiden and concomitance of factor V Leiden with prothrombin G20210A mutation and methylene tetrahydrofolate reductase C677T gene mutation in healthy population of Denizli, Aegean region of Turkey.

Factor V Leiden causing activated protein C resistance is the most common inherited form of thrombophilia leading to thrombosis. Its frequency shows great ethnic and geographic variations. The aim of this study was to determine the frequency of FV Leiden and coinheritance of FV Leiden with two other frequent hereditary thrombophilia causes, namely, prothrombin G20210A and methylene-tetrahydrofolate reductase (MTHFR) C677T mutation in the Aegean region of Turkey. The study population consisted of 1030 (500 men and 530 women) apparently healthy subjects. Functional resistance to activated protein C (APC) was measured by using the test kit STA staclot APC-R ((Diagnostica Stago, Asnieres, France, Cat. No. 00721). In subjects with APC resistance, molecular analyses of FV Leiden and of prothrombin G20210A and MTHFR C677T mutation were performed by using FV-PTH-MTHFR StripA (Vienna Lab, Labordiagnostika GmbH, Austria) kit, which was based on hybridization of polymerase chain reaction (PCR) amplified DNA products with mutation-specific oligonucleotide probes. Functional APC resistance was present in 93 subjects (9%). FV Leiden mutation was found in 87 of 93 subjects with APC resistance by PCR method. The FV Leiden carrier frequency was found to be 8.4% (87/1030). Seventy-six individuals were heterozygous (7.3%), and 11 were homozygous (1.06%). Among the 87 subjects with FV Leiden mutation, 45 subjects had MTHFR C677T gene mutation (7 homozygous, 38 heterozygous) and 4 subjects had heterozygote prothrombin G20210A gene mutation. A combination of FV Leiden and prothrombin G20210A and MTHFR C677T gene mutation was detected in 3 subjects. The results indicate that FV Leiden prevalence is quite high and coexistence of FV Leiden with other hereditary causes of thrombosis such as prothrombin G20210A mutation and MTHFR enzyme defect is not rare in healthy population of Aegean region of Turkey.     

Pseudohomozygosity for activated protein C resistance is a risk factor for venous thrombosis

Pseudohomozygosity for activated protein C resistance (APC-r) is a rare condition due to the association of heterozygous FV Leiden mutation and partial type I FV deficiency. To assess the risk of venous thromboembolism in these subjects, seven families including 11 pseudohomozygotes and 45 relatives were examined. Among the relatives, 16 were heterozygous FV Leiden carriers, nine showed partial FV deficiency and 20 no abnormalities. Deep vein thrombosis occurred in 4/11 (36.3%) pseudohomozygous patients versus 6/16 (37. 4%) FV Leiden carriers and 1/20 (5%) normal relatives. Pseudohomozygotes and FV Leiden carriers had a significantly increased risk of venous thrombosis in comparison to normal relatives (RR 8.8 and 5.7, respectively). There was no difference between the thrombotic risk of pseudohomozygous subjects and of FV Leiden carriers (RR 1.6, 95% CI 0.43-5.7). Furthermore, there was no difference in thrombosis-free survival between pseudohomozygotes and 45 consecutive FV Leiden heterozygous outpatients, suggesting that a referral bias may explain the apparent younger age of thrombosis in the pseudohomozygotes in comparison to relatives with FV Leiden heterozygosity (27 years v 54 years; P = 0.01). Pseudohomozygosity for APC resistance carries a significantly higher risk for venous thromboembolism in comparison to normal subjects, but probably not in comparison to heterozygous FV Leiden carriers.     

The factor V (FV) gene ASP79HIS polymorphism modulates FV plasma levels and affects the activated protein C resistance phenotype in presence of the FV Leiden mutation

BACKGROUND AND OBJECTIVES: In carriers of the factor V (FV) Leiden mutation, different trans-acting gene variants (HR2 haplotype and FV Cambridge mutation) affect activated protein C (APC) sensitivity. Among a series of FV gene variants characterized, the Asp79His polymorphism appeared to be a good candidate for the modulation of FV activity. DESIGN AND METHODS: In a group of 150 apparently healthy subjects without the FV Leiden mutation and in 55 apparently healthy subjects with mutation, genotypes of the Asp79His polymorphism and of the HR haplotype were characterized and plasma levels of FV coagulant activity and APC ratios evaluated. RESULTS: In the group without the FV Leiden mutation, 16 subjects (10.7%) carried the His 79 allele and 15 subjects (10.0%) the HR2 haplotype. Two of them carried both gene variants. As compared to FV activity levels in non-carriers (106.4+18.5%), values were lower in subjects with the His79 allele (95.2+25.2%; p=0.025) and in those with the HR2 haplotype (93.7+16.2%; p =0.007). FV activity levels were further reduced in carriers of both FV gene variants (78.7+3.3%; p =0.009). APC values were similar among individuals carrying different FV genotypes. In the group with the FV Leiden mutation, APC ratios were lower in subjects carrying the His 79 allele (0.63; p =0.008) or the HR2 haplotype (0.63; p =0.026) than in subjects without (0.69) reflecting FV activity values. INTERPRETATION AND CONCLUSIONS: Present data suggest that carriership of the His79 allele modulate plasma levels of FV coagulant activity and, in subjects carrying the FV Leiden mutation, affects APC sensitivity.     

Association between adverse pregnancy outcomes and maternal factor V G1691A (Leiden) and prothrombin G20210A genotypes in women with a history of recurrent idiopathic miscarriages

Thrombophilia was implicated in the development of pregnancy complications, including recurrent idiopathic pregnancy loss, and is aggravated in women who are carriers of factor V G1691A (FV Leiden) and prothrombin (PRT) G20210A single-nucleotide polymorphisms (SNPs). Previous studies examined the role of FV-Leiden and PRT G20210A in recurrent pregnancy loss with conflicting results. Here we examined the prevalence of FV Leiden and PRT G20210A SNPs, in 200 women with 3 or more consecutive early (n = 87), late (n = 41), or early-late (n = 72) recurrent pregnancy losses, and 200 age-matched fertile parous control women. APC resistance (APCR) was detected functionally (measuring the activated clotting time triggered by activated factor X in presence of a fixed amount of purified APC), and FV-Leiden and PRT G20210A genotypes were assessed by PCR. The frequency of the mutant FV (0.1400 vs. 0.0276; P < 0.001) but not PRT 20210 (0.0100 vs. 0.0225; P = 0.159) allele was higher in patients than controls, respectively. APC resistance with factor V Leiden was seen in 27% of patients compared to 11.5% of controls, while APC resistance without factor V Leiden was seen in 12.5% of patients compared to 9.5% of controls. Regression analysis demonstrated that the significant predictors for early abortion was FV Leiden; those for late abortion were oral contraceptive, APCR, and FV Leiden; and predictors for early-late abortions were oral contraceptives, obesity, FV Leiden, and smoking. APC resistance and FV Leiden, as well as combination of both, are common thrombotic defects seen in women with idiopathic recurrent pregnancy loss, thus testing for these is recommended in women who have experienced recurrent miscarriages.     

Impaired APC cofactor activity of factor V plays a major role in the APC resistance associated with the factor V Leiden (R506Q) and R2 (H1299R) mutations

Activated protein C (APC) resistance is a major risk factor for venous thrombosis. Factor V (FV) gene mutations like FV(Leiden) (R506Q) and FV(R2) (H1299R) may cause APC resistance either by reducing the susceptibility of FVa to APC-mediated inactivation or by interfering with the cofactor activity of FV in APC-catalyzed FVIIIa inactivation. We quantified the APC cofactor activity expressed by FV(Leiden) and FV(R2) and determined the relative contributions of reduced susceptibility and impaired APC cofactor activity to the APC resistance associated with these mutations. Plasmas containing varying concentrations of normal FV, FV(Leiden), or FV(R2) were assayed with an APC resistance assay that specifically measures the APC cofactor activity of FV in FVIIIa inactivation, and with the activated partial thromboplastin time (aPTT)-based assay, which probes both the susceptibility and APC cofactor components. FV(R2) expressed 73% of the APC cofactor activity of normal FV, whereas FV(Leiden) exhibited no cofactor activity in FVIIIa inactivation. Poor susceptibility to APC and impaired APC cofactor activity contributed equally to FV(Leiden)-associated APC resistance, whereas FV(R2)-associated APC resistance was entirely due to the reduced APC cofactor activity of FV(R2). Thrombin generation assays confirmed the importance of the anticoagulant activity of FV and indicated that FV(Leiden) homozygotes are exposed to a higher thrombotic risk than heterozygotes because their plasma lacks normal FV acting as an anticoagulant protein.     

Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficient families

Heterozygous protein C deficiency is associated with an increased risk for thrombosis. This association is restricted to a minority of protein C-deficient families, which have been defined as clinically dominant protein C-deficient. In contrast, in the clinically recessive protein C- deficient families, only the homozygous family members are (severely) affected. One possible explanation for this difference in thrombotic risk between families may be the presence of a second hereditary risk factor. A good candidate for this second risk factor is the recently identified resistance to activated protein C (APC). APC resistance, which is associated with a mutation in the FV gene (FV Leiden), is a common and strong risk factor for thrombosis. We show here that the prevalence of the FV Leiden mutation is high among symptomatic protein C-deficient probands (19%). In 6 clinically dominant protein C- deficient families, the segregation of the FV Leiden mutation and the protein C gene mutation was studied. A thrombotic episode had been experienced by 73% of the family members having both the protein C gene mutation and the FV Leiden mutation. In contrast, respectively, 31% and 13% of the family members having either the protein C gene mutation or the FV Leiden mutation had experienced a thrombotic episode. Moreover, the result of a two locus linkage analysis support the assumption that the FV gene and the protein C gene are the two trait loci responsible for the thrombophilia. These results indicate that carriers of both gene defects have an increased risk for thrombosis compared with related carriers of the single defect.     

Increased incidence of venous thrombosis in patients with shortened activated partial thromboplastin times and low ratios for activated protein C resistance

A cohort of 69 hospital patients with shortened activated partial thromboplastin time (APTT) were prospectively identified and were further investigated for resistance to activated protein C (APC). This was quantified by APTT-based and Russel viper venom time (RVVT)-based methods. The prevalence of objectively confirmed venous thromboembolism (VTE) in this cohort was 19% (13/69). Of these 69 patients, 28 also had low APC resistance ratios and the incidence of VTE among these patients (group 1) was 36% (10/28). This was significantly higher (P=0.003) than that in the remaining 41 patients (group 2) with shortened APTT and normal APC resistance (7%, 3/41). DNA analysis confirmed 13 of the group 1 patients were FV Leiden positive. The incidence of VTE in the FV Leiden group (group 1a, n=13) was 38% (5/13) and in the group whose abnormal resistance to APC was independent of FV Leiden (group 1b, n=15) was 33% (5/15). These results suggest that a shortened APTT, coexisting with a low APC resistance ratio, regardless of FV Leiden carriership status, is a marker for VTE. Increased resistance to the anticoagulant activity of APC is multifactorial as reflected by evidence of abnormal resistance differing in the two assays.     

Coinheritance of Factor V (FV) Leiden enhances thrombin formation and is associated with a mild bleeding phenotype in patients homozygous for the FVII 9726+5G>A (FVII Lazio) mutation

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.     

Analysis of phenotype resistance to activated protein C (APC resistance)

The laboratory diagnosis of resistance to activated C protein (APC-resistance) involves examination of the phenotype and genotype of this thrombophilia. For examination of the phenotype coagulation and chromogenic tests are used. Their essence is examination in the presence and absence of exogenous APC. While the result of the original coagulation examination of APC-resistance which uses the APTT principle is influenced by a number of factors, the sensitivity and specificity of the modification of this examination (dilution of the examined plasma sample by FV deficient plasma before making the test) in relation to detection of FV Leiden is almost 100% and eliminates the majority of limitations of the original examination. The chromogenic assessment of APC-resistance has similar advantages, however, it cannot differentiate between the heterozygous and homozygous form of FV Leiden. During examination of the genotype of subjects with APC-resistance the mutation of FV Leiden is detected in as many as 90%. The group of subjects with the phenotype of APC-resistance comprises in particular subjects with acquired APC-resistance caused by conditions which lead to a disbalance between procoagulation and anticoagulation proteins of haemostasis which influence the reactions of the applied laboratory examinations. The acquired phenotype of APC-resistance can be also associated with an increased risk of thrombosis and the clinical manifestations of this thrombophilia resemble the classical, FV Leiden conditioned APC resistance. Rarely also congenital causes of the phenotype of APC-resistance are encountered caused by another mutation than the Leiden mutation of gene FV. The concurrent examination of the patient's plasma with the original and modified coagulation test makes it possible to assess the inborn cause of APC-resistance (positive finding also in modified examination). The presence of FV Leiden is then confirmed by examination of the genotype by the polymerase chain reaction.     

Diagnosis strategies in activated protein C resistance: is genotyping still necessary?

Resistance to activated protein C (APC) is due, in most cases, to a G to A mutation at nucleotide 1691 of factor V (FV) gene (the Leiden mutation). This inherited abnormality is now considered to be the major hereditary cause associated with an elevated risk of thrombosis. For this reason, laboratories are faced with an increasing number of samples referred for APC resistance diagnosis. This could have serious economic consequences and a comprehensive laboratory screening strategy for APC resistance is necessary. An original DNA assay based on denaturing gradient gel electrophoresis (DGGE) was designed in our laboratory. During a first period we systematically performed DNA analysis and compared the results with phenotypic assays. Using the modified functional test with a 1 : 5 predilution of plasmas, the cut-off value for APC resistance ratio was 2.6 in our sample. Among 94 consecutive patients referred to our laboratory we found a clear cut-off between the APC resistance ratio obtained for normal and abnormal individuals. The modified test had a predictive value of 1.0 found by a cut-off ≤2.6 for the heterozygote FV Leiden. This obviates the necessity of genotyping subjects with a normal phenotype. Among patients with an abnormal phenotype we were able to fully discriminate between homozygous and heterozygous patients using a cut-off value of 1.5. Nevertheless, our results demonstrate that, because of false-positive results such as lupus anticoagulant, genotyping is still indicated for patients with an abnormal ratio determined with the modified APC resistance test. The strategy described here allows us to safely lower the number of samples analysed by DGGE.     

Determination of activated protein C resistance in anticoagulated and lupus positive patients.

Clotting-based activated protein C (APC) assays have limitations when testing patients on oral anticoagulant (OA) therapy or with a lupus anticoagulant (LA). Predilution in factor V (FV)-deficient plasma and testing with phospholipid-rich Russell Viper venom (RVV)-based methods have been shown to be the most suitable methods when testing these patient groups, respectively. We evaluated a modified RVV based clotting test (Gradileiden V test; Gradipore, Sydney, Australia) in a large patient cohort and determined its sensitivity to the FV Leiden mutation. We also examined whether normal plasma can be used to dilute plasma from warfarinized patients without compromising sensitivity to the FV Leiden mutation. A total of 1,956 plasmas were studied including congenital protein C (five plasmas), and protein S deficiency (five plasmas), LA (29 plasmas), FV Leiden heterozygote (102 plasmas), and homozygote (five plasmas), warfarin (54 plasmas), standard heparin therapy (37 plasmas) and normal healthy controls (21 plasmas). Molecular analysis was performed on all samples. The effect of FV Leiden concentration on the APC ratio was examined by determining the APC resistance of a homozygous plasma serially diluted in six sources of normal plasma (NP). The relationship was non-linear and dependent on the initial APC ratio of the chosen source of NP. APC resistance was demonstrated in the varying sources of NP in dilutions of 1/4 (25% FV Leiden) to 1/32 (3% FV Leiden). A 1/2 dilution in pooled NP is recommended for patients on OA therapy because the test remains sensitive at levels of 25% FV Leiden and this is the dilution routinely used for other applications in a coagulation laboratory. The effect of a LA on the APC ratio was similarly studied by determining the APC resistance of a homozygous plasma serially diluted in two sources of LA-positive plasma. This relationship was also non-linear and dependent on the initial APC ratio of the LA-positive plasma. APC resistance was demonstrated in dilutions of 1/16 (6% FV Leiden) to 1/64 (1.5% FV Leiden) demonstrating the sensitivity of the test to APC resistance in the presence of a LA. Our results show the modified RVV-based test clearly predicts the presence of factor V Leiden in a large cohort of patients. The method offers advantages when testing patients with a LA and patients receiving warfarin providing a 1/2 predilution step in pooled NP is performed. Pooled NP does not affect the sensitivity of the test to the mutation, is routinely used in coagulation laboratories, and is considerably less expensive than FV-deficient plasma.     

Congenital and acquired activated protein C resistance

Resistance to the anticoagulant action of activated protein C, APC resistance, is a highly prevalent risk factor for venous thrombosis among individuals of Caucasian origin. In most cases, APC resistance is associated with a single missense mutation in the gene for coagulation factor V (FV (Leiden)), which predicts the replacement of Arg (506) with a Gln at one of the cleavage sites for APC in factor V. Factor V is a Janus-faced protein with dual functions, serving as an essential nonenzymatic cofactor in both pro- and anticoagulant pathways. Procoagulant factor Va, generated after proteolysis by thrombin or factor Xa, is a cofactor to factor Xa in the activation of prothrombin, whereas anticoagulant factor V, generated after proteolysis by APC, functions as a cofactor in the APC-mediated degradation of FVIIIa. The FV (Leiden) mutation affects the anticoagulant response to APC at two distinct levels of the coagulation pathway, as it impairs degradation of both activated factor V and activated factor VIII, the latter effect inasmuch as FVLeiden is a poor APC cofactor. Several other genetic traits, some of them quite common, are known to affect the anticoagulant response to APC, but none of them cause the same severe APC-resistance phenotype as FV (Leiden) and their importance as risk factors for thrombosis is unclear. A poor APC response may also result from acquired conditions, some of which are clearly involved in the pathogenesis of venous thrombosis. Venous thrombosis is a typical multifactorial disease, the pathogenesis of which involves multiple gene-gene and gene-environment interactions. In many patients with severe thrombophilia, APC resistance is found as a contributing risk factor.     

The activated protein C (APC)-resistant phenotype of APC cleavage site mutants of recombinant factor V in a reconstituted plasma model.

Recently, new missense mutations in the activated protein C (APC) cleavage sites of human factor V (FV) distinct from the R506Q (FV Leiden) mutation have been reported. These mutations affect the APC cleavage site at arginine (Arg) 306 in the heavy chain of activated FV. Whether these mutations result in APC resistance and are associated with a risk of thrombosis is not clear. The main objective of the present study was to identify the APC-resistant phenotype of FV molecules with different mutations in APC cleavage sites. To study this, recombinant FV mutants were reconstituted in FV-deficient plasma, after which normalized APC-sensitivity ratios (n-APC-SRs) were measured in activated partial thromboplastin time-based and Russell's Viper Venom time-based APC-resistance tests. The mutations introduced in FV were R306G, R306T, R506Q, R679A and combinations of these mutations. Based on the APC-sensitivity ratios, we conclude that the naturally occurring mutations at Arg306 (i.e. FV HongKong and FV Cambridge) result in a mildly reduced sensitivity for APC (n-APC-SR, 0.74-0.87), whereas much lower values (n-APC-SR, 0.41-0.51) are obtained for the mutation at Arg506 (FV Leiden). No effect on the n-APC-SR was observed for the recombinant FV mutant containing the single Ala679 mutation. Because reduced sensitivity for APC, not due to FV Leiden, is a risk factor for venous thrombosis, these data suggest that mutations at Arg306 might be associated with a mild risk of venous thrombosis.     

The factor VR506Q mutation causing APC resistance is highly prevalent amongst unselected outpatients with clinically suspected deep venous thrombosis

Objective. Resistance to activated protein C (APC resistance), caused by a single point mutation in the factor V gene (FV:R506Q), is a major risk factor for venous thrombosis. As the significance of this mutation among unselected outpatients with deep-vein thrombosis (DVT) is not established, we have studied its prevalence among consecutive outpatients attending the emergency room due to a clinically suspected DVT.
Design, setting and subjects. The FV:R506Q mutation was determined in 223 consecutive Swedish outpatients with clinically suspected DVT, and in 288 healthy controls. Using phlebography, the patients were classified as DVT-positive or DVT-negative.
Main outcome measure. The prevalence of FV:R506Q mutation.
Results. The prevalence of the FV:R506Q mutation was 28% (28/99) in the DVT-positive subgroup (relative risk: 3.1; 95% CI: 1.7–5.5), and 23% (28/124) in the DVT negative subgroup (relative risk: 2.0; 95% CI: 1.1–3.6), as compared to 11% (32/288) in the control group. In the DVT-positive subgroup, the FV:R506Q mutation was most common among younger patients with primary thrombosis (47%) and least common among older patients with secondary thrombosis (19%). The high prevalence of FV:R506Q mutation among DVT-negative patients was associated with a high frequency of previous venous thrombosis. Thus, 46% (13/28) of the DVT-negative FV:R506Q carriers had a history of thrombosis, compared with only 22% (21/96) of the DVT-negative patients lacking the mutation ( P =0.01).
Conclusion. To sum up, the FV:R506Q mutation is present in more than a quarter of Swedish DVT-positive outpatients with clinically suspected DVT, indicating that APC-resistance is a major thrombotic risk factor contributing to the high incidence of venous thrombosis in Sweden.     

A comparison between two activated protein C resistance methods as routine diagnostic tests for factor V Leiden mutation

The most common commercially available test measuring activated protein C (APC) resistance relies on the the anticoagulant response to added APC in an activated partial thromboplastin time (APTT) based method. Another method is a Russell Viper venom time (RVVT) based system. To improve the specificity for factor V Leiden of the APTT based method, pre-dilution of test plasma in FV-deficient plasma has recently been recommended. In this study we tested the relative suitabilities of the APTT-based system, the RVVT-based system and their corresponding assays modified by pre-dilution in FV-deficient plasma, for screening asymptomatic subjects, a group of thrombophilic patients (in particular those with low APC ratios), patients on oral anticoagulants, and patients with lupus anticoagulant (LAC). We found the RVVT-based assay to be superior to the APTT-based method in the separation of normals from those with FV Leiden mutation both in asymptomatic subjects and in the thrombophilic patient group. Both modified assays demonstrated a sensitivity and specificity of 100% for FV Leiden, as verified by genotyping in asymptomatic subjects, thrombophilic patients and patients on oral anticoagulants, with the modified RVVT-based assay giving better separation between normals and FV Leiden. Inhibition of phospholipid-dependent coagulation by LAC antibodies rendered the APTT-based system less suitable than the phospholipid-rich RVVT-based one, and as nine of the 20 LAC-positive patients were on warfarin, we showed only the modified RVVT assay to be a reliable predictor of factor V Leiden in this patient group.     

Phenotypic homozygous activated protein C resistance associated with compound heterozygosity for Arg506Gln (factor V Leiden) and His1299Arg substitutions in factor V

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.     

High prevalence of a mutation in the factor V gene within the U.K. population: relationship to activated protein C resistance and familial thrombosis

Summary. Recent findings have indicated the importance of factor V (FV) in causing resistance to activated protein C (APC) in a high proportion of patients with venous thrombosis. This prompted us to investigate whether resistance could be due to defective inactivation of FVa by APC. Consequently, we amplified a 3.2 kb fragment of the FV gene sequence encoding the heavy chain APC cleavage site. DNA analysis showed a guanine to adenine transition at nucleotide 1691 in all affected members of two families with inherited APC resistance associated with thrombosis and confirmed suspected homozygosity in two individuals. The mutation, in heterozygous form, was also found in ˜3.5% of our normal population (n = 144) and correlated with low APC resistance. The high prevalence of this mutation suggests that it may be a major contributory factor in early thrombosis.     

Prevalence of factor V gene mutation amongst myocardial infarction patients and healthy controls is higher in Sweden than in other countries

Objective. Haemostatic imbalance may be an aetiological factor in the development of acute coronary syndromes. Inherited resistance to activated protein C (APC) is a common disorder associated with hypercoagulability and lifelong risk of venous thrombosis. APC resistance is due to a single mutation in the gene coding for coagulation factor V (FV:Q⁵⁰⁶). To test the importance of the FV:Q⁵⁰⁶ mutation in premature myocardial infarction (MI), its prevalence was investigated in Swedish patients with MI before the age of 50 years.
Design, setting and subjects. In a retrospective case-control study, the FV:Q⁵⁰⁶ mutation was investigated in 101 survivors of MI (79 men, 22 women) and in 101 healthy sex- and age-matched controls.
Main outcome measure. The prevalence of FV:Q⁵⁰⁶ mutation.
Results. The FV:Q⁵⁰⁶ mutation was found in 18% of patients versus 11% of controls ( P =0.16). The mutation was significantly more frequent amongst male patients than amongst controls (23 vs. 10%; P =0.03), the calculated odds ratio being 2.6 (95% CI, 1.1–6.4).
Conclusion. The high prevalence of the FV:Q⁵⁰⁶ mutation found amongst Swedish MI patients, especially amongst men, is noteworthy, and calls for further studies on the outcome of MI in APC-resistant patients. The prevalence of the FV:Q⁵⁰⁶ mutation in controls is higher than figures reported from other countries, suggesting that at least 10% of the Swedish population are carriers of a congenital prothrombotic disorder.     

Factor V Leiden: a disorder of factor V anticoagulant function

PURPOSE OF REVIEW: Activated protein C (APC) resistance, which is often associated with the factor V R506Q (FV Leiden) mutation, is a common risk factor for venous thrombosis. Study of the mechanism of APC resistance has revealed that coagulation FV stimulates the APC-catalysed inactivation of FVIIIa, and that this anticoagulant function of FV is impaired in FV Leiden. The present review covers the discovery, the physiological significance and the structural requirements of the APC-cofactor activity of FV. RECENT FINDINGS: Recent in vitro and in vivo experiments indicate that the anticoagulant activity of FV is physiologically relevant and that FV plays a major role in the maintenance of the haemostatic balance. Quantitative and functional defects of the APC-cofactor activity of FV lead to increased thrombin generation and are associated with a prothrombotic state. Although the structural requirements for the expression of the APC-cofactor activity of FV are now beginning to be unravelled, the underlying molecular mechanism remains elusive. SUMMARY: The APC-cofactor activity of FV and its impairment in FV Leiden can explain the different thrombosis risks associated with heterozygosity, homozygosity and pseudo-homozygosity for FV Leiden. Elucidation of the molecular mechanism of the anticoagulant function of factor V may provide novel targets for the design of antithrombotic drugs.     

Functional characterization of recombinant FV Hong Kong and FV Cambridge

In factor V (FV) Cambridge (Arg306Thr) and Hong Kong (Arg306Gly), a cleavage site for anticoagulant activated protein C (APC), which is crucial for the inactivation of FVa, is lost. Although patients carrying FV Hong Kong have a normal APC response, those with FV Cambridge were reported to be APC resistant. To elucidate the molecular characteristics of the 2 FV mutants, we recreated them in a recombinant system and evaluated their functional properties. The 2 FV variants yielded identical APC resistance patterns, with APC responses being intermediate to those of wild-type FV and FV Leiden (Arg506Gln), which is known to be associated with the APC resistance phenotype. In the absence of protein S, APC mediated FVa inactivation curves obtained with the 2 variants were identical, resulting in partial FVa inactivation. In the presence of protein S, both FVa variants were almost completely inactivated because of protein S stimulation of the cleavage at Arg679. In a FVIIIa degradation system, both FV variants demonstrated slightly impaired APC cofactor activity. The ability of APC to cleave at Arg506 and at Arg679 in FVa Cambridge and Hong Kong and the slight decrease in APC cofactor activity of the 2 FV variants may explain the low thrombotic risk associated with these Arg306 mutations. In conclusion, we demonstrate that recombinant FV Cambridge and Hong Kong behave identically in in vitro assays and provide a mechanism for the low thrombotic risk associated with these FV mutations.