FVL Carriers Research Articles

Factor V Leiden but not the factor II 20210G>A mutation is a risk factor for premature coronary artery disease: a case-control study in Iran

BackgroundFactor V Leiden (FVL) and factor II c.∗97G>A (rs1799963) are genetic risk factors for venous thromboembolism. Their contribution to coronary artery disease (CAD) is less clear. ObjectivesThis study aimed to investigate the association between FVL, rs1799963, and premature CAD in Iranians. MethodsWe performed a genetic case-control study of 944 cases and 1081 controls from the premature CAD Milano-Iran study, including patients aged 18-55 (female) and 18-45 years (male) who underwent coronary angiography at the Tehran Heart Centre (Iran) in 2004-2011. Cases had luminal stenosis ≥50% in at least 1 main coronary artery or branch. Controls were age- and sex-matched with no CAD history. FVL and rs1799963 were genotyped using TaqMan SNP genotyping assays. Association was tested by logistic regression adjusted for matching factors and ethnicity. Effect modification by sex and cardiovascular risk factors (metabolic [obesity, hypertension, hyperlipidemia, and diabetes], and smoking) was assessed. ResultsThe risk of premature CAD was increased by 50% in FVL carriers (adjusted odds ratio [adjOR] 1.54 [95% CI, 0.95-2.48]) and slightly reduced in rs1799963 carriers (adjOR 0.71 [95% CI, 0.40-1.27]). These effects were more pronounced in women than men (FVL, adjOR 1.66 vs 1.25; rs1799963, adjOR 0.60 vs 1.07). The risk of premature CAD was substantially increased in carriers of FVL with at least 1 metabolic risk factor compared with noncarriers without metabolic risk factors (adjOR 25.14 [95% CI, 12.51-50.52]). ConclusionFVL but not FII rs1799963 was associated with an increased risk of CAD in young Iranians. This risk increased considerably when combined with metabolic cardiovascular risk factors.

Circulating Plasminogen Activator Inhibitor-1 (PAI-1) Is Reduced By In Vivo Thrombin Generation and Subsequent Formation of Activated Protein C (APC)

Background: APC has been suggested to contribute to a hyperfibrinolytic state in various acquired coagulation disorders, including coagulopathies induced by trauma and sepsis. However, in vivo evidence for the proposed underlying mechanism of proteolytic degradation of PAI-1 by APC remains inconclusive. Recently, we have shown increased APC generation in response to in vivo thrombin formation in carriers of the factor V Leiden mutation (FVL). In this approach of stimulated hemostasis activity pattern evaluation (SHAPE), in vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa). Aim of the present study was to investigate the resulting effects on the fibrinolytic system. Methods: The study population consisted of 30 FVL carriers (thereof 13 with a history of venous thromboembolism) and 15 healthy controls. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of APC were quantified using an oligonucleotide-based enzyme capture assay (OECA). Other monitored parameters included plasma levels of prothrombin activation fragment 1+2 (F1+2), tissue-type plasminogen (t-PA), PAI-1, plasminogen, α2-antiplasmin, plasmin-α2-antiplasmin complexes (PAP), soluble fibrin monomers, d-dimer, and thrombin-activatable fibrinolysis inhibitor (TAFI). Results: At baseline, FVL carriers showed higher median levels of APC in comparison to the controls (1.39 vs. 0.86 pmol/L, P=.001), higher PAI-1 levels (30.1 vs. 15.3 ng/mL, P=.002) and lower plasminogen levels (94.8 vs. 110.4%, P=6·10-4). The other baseline parameters did not differ significantly. In both cohorts a comparable increase of F1+2 was observed after administration of rFVIIa, whereas APC increased more (P=.004) in FVL carriers (by 6.40 pmol/L) than in healthy controls (by 2.14 pmol/L), Concurrently, median PAI-1 levels decreased more (P=.007) in FVL carriers (by 19.8 pmol/L) than in healthy controls (by 8.0 pmol/L) (Figure 1). TAFI levels decreased temporarily, from 104.8 to 94.4% (P=.007) in FVL carriers and from 105.0 to 92.1% (P=2·10-4) in healthy controls. PAP levels increased from 164 to 209 ng/mL (P<10-4) in FVL carriers and from 154 to 189 ng/mL (P=.023) in the control group. The extent of these changes did not differ between the two cohorts. D-dimer level increased only in FVL carriers, from 0.34 to 0.41 mg/L (P=.008). t-PA and the other parameters did not show significant changes after rFVIIa administration. Conclusion: Increased APC formation rates in FVL carriers were associated with a greater decline of PAI-1 levels in the absence of interfering changes in t-PA levels. These data provide further in vivo evidence that APC down-regulates PAI-1. Overall, the SHAPE approach utilized here does not induce a significant profibrinolytic response, even in patients with thrombophilia. Disclosures Reda: Grifols: Honoraria; Shire: Honoraria. Winterhagen:SOBI: Honoraria. Berens:Pfizer: Honoraria; Shire: Honoraria; Biotest: Honoraria; CSL Behring: Honoraria; Novo Nordisk: Honoraria; Baxter: Honoraria. Müller:NovoNordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Siemens: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Oldenburg:CSL Behring: Consultancy, Research Funding, Speakers Bureau; NovoNordisk: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Research Funding, Speakers Bureau; Swedish Orphan Biovitrum: Consultancy, Speakers Bureau; Grifols: Consultancy, Speakers Bureau; Takeda (Shire): Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Biotest: Consultancy, Research Funding, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau. Rühl:CSL Behring: Honoraria; Bayer: Honoraria; Shire: Honoraria; SOBI: Honoraria; Sanofi Genzyme: Honoraria; Grifols: Honoraria.

The Variable Clinical Expressivity of Factor V Leiden, but Not of Prothrombin G20210A, Correlates with the Extent of the APC Response to In Vivo Thrombin Formation

Background: The factor V Leiden mutation (FVL) and the prothrombin G20210A mutation (PRO) are the most frequent thrombophilic mutations among Caucasians. Both mutations show a highly variable expressivity, ranging from a lifelong asymptomatic course to severe venous thromboembolism (VTE). Recently, we have shown increased generation rates of thrombin and activated protein C (APC) in response to in vivo thrombin formation in asymptomatic FVL carriers (VTE-). In vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa). Aim of the present study was to extend this stress test approach to carriers of PRO and to FVL carriers with a history of VTE (VTE+).Methods: The study population consisted of 29 FVL carriers (thereof 25 heterozygotes: 13 VTE- and 12 VTE+) and 21 PRO carriers (thereof 19 heterozygotes: 12 VTE- and 7 VTE+). None of the subjects was under anticoagulant treatment at time of analysis. The control group consisted of 13 healthy volunteers tested negative for FVL and PRO. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of free thrombin and APC were quantified using oligonucleotide-based enzyme capture assays (OECAs). Prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and D-dimer were measured additionally.Results: Injections of rFVIIa were well-tolerated by all subjects and median D-dimer levels remained within the reference range in all groups. Compared with the controls, peak plasma levels of F1+2 and TAT were significantly higher after rFVIIa injection in FVL carriers (p=.009 and p=1.0∙10-4, respectively), and in PRO carriers (p=.045 and p=6.0·10-4, respectively), but they did not differ between the FVL and PRO cohorts, and between the VTE+ and VTE- subgroups. Median plasma concentrations of thrombin were below the limit of detection at baseline, and rFVIIa did not induce statistically significant changes of thrombin levels in any group. APC increased to peak levels 30 minutes after rFVIIa injection, from 0.63 (0.54-1.16) to 3.14 (2.55-3.55) pmol/L (p=.017) in the controls, and from 1.39 (1.14-1.86) to 7.86 (5.55-11.55) pmol/L (p<1.0∙10-4) in the FVL group. In PRO carriers, plasma levels of APC increased from 1.07 (0.86-1.23) pmol/L at baseline to a peak of 5.86 (4.44-6.41) pmol/L 1 hour after rFVIIa injection p=4.0·10-4). Peak plasma levels of APC were higher in both FVL carriers and PRO carriers in comparison to the controls (p<1.0∙10-4 and p=4.0∙10-4, respectively), and lower in PRO carriers compared with those in FVL carriers (p=.003). With peak levels of 8.11 (7.59-11.77) vs. 5.62 (4.94-6.49) pmol/L, APC was significantly higher in the VTE- subgroup than in the VTE+ subgroup (p=.004) in heterozygous FVL carriers, while in heterozygous PRO carriers, APC peak levels did not differ between the VTE- subgroup and the VTE+ subgroup (Figure 1).Conclusion: The APC response to in vivo thrombin formation is significantly lower in symptomatic than in asymptomatic FVL carriers, implying that higher APC levels protect FVL carriers from thrombosis development. This hypothesized effect appears to be specific for FVL as prothrombin G20210A carriers do not show differences in the APC response related to VTE history. Further studies are warranted to identify the factors that modulate the APC response in FVL carriers. [Display omitted] DisclosuresRühl:Swedish Orphan Biovitrum: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Shire: Research Funding; Grifols: Research Funding; CSL-Behring: Research Funding. Berens:Pfizer: Research Funding; Sanofi Genzyme: Research Funding; Shire: Research Funding; CSL-Behring: Research Funding; Biotest: Research Funding. Winterhagen:Swedish Orphan Biovitrum: Consultancy, Research Funding. Müller:Swedish Orphan Biovitrum: Consultancy, Research Funding. Oldenburg:Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Octapharma: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria; Grifols: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria; Biotest: Consultancy, Honoraria; Biogen Idec: Consultancy, Honoraria; Bayer: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Swedish Orphan Biovitrum: Consultancy, Honoraria.

Circulating microparticles in carriers of prothrombin G20210A mutation.

Factor V Leiden (FVL) and prothrombin gene mutation G20210A (PTM) are the two most common genetic polymorphisms known to predispose carriers to venous thromboembolism (VTE). A recent study in FVL carriers showed that circulating levels of microparticles (MP) may contribute to their thrombogenic profile. To further elucidate the prothrombotic state linked to genetic thrombophilia, we extended this study to carriers of PTM. The plasma level of annexin V-MP, endothelial-MP (EMP), platelet-MP (PMP), tissue factor-bearing MP (TF+) and the MP procoagulant activity (PPL) was measured in 124 carriers of PTM (105 heterozygous and 19 homozygous) and in 120 age- and gender-matched healthy individuals. Heterozygous and homozygous carriers of PTM showed significantly increased levels of annexin V-MP (2930 [1440-4646] MP/µl and 3064 [2412-4906] MP/µl, respectively) and significantly shorter PPL clotting time (54 [46-67] sec and 55 [46-64] sec) compared to controls (1728 [782-2122] MP/µl and 71 [61-75] sec, respectively; p<0.01). Similarly, heterozygous and homozygous subjects presented with significantly higher levels of EMP, PMP and TF+ than controls (p<0.05). PTM carriers with a VTE history had significantly higher MP numbers and activity than controls. No significant difference was seen between carriers with and without a VTE history. We conclude that the higher levels of circulating MP found in PTM carriers may play a role in the development of VTE possibly by increasing thrombin generation. Further studies are needed to better define the role of MP as triggering factors for the thrombotic complications characterizing mild genetic thrombophilic defects.

Fibrin clot properties in women heterozygous for factor V Leiden mutation: Effects of oral contraceptives

IntroductionOral contraceptives (OC) in the presence of factor V Leiden mutation (FVL) markedly increase the risk of venous thromboembolism (VTE). Little is known about the OC and FVL-related alterations in fibrin clot properties. Subjects and MethodsPlasma fibrin clot permeability (Ks) and efficiency of lysis, reflected by clot lysis time (CLT) and the rate of D-dimer release from clots (D-Drate) induced by recombinant tissue plasminogen activator (tPA) were determined in 25 women with a family history of VTE who were heterozygous for FVL [FVL(+/−) – twice, on third-generation OC and after their discontinuation. Female non-carriers of FVL, matched for demographics, using OC and after their discontinuation served as controls (n=25). All participants had no personal history of VTE. ResultsOC discontinuation in FVL(+/−) women resulted in shortened CLT (−9%), and increased Ks (+4%) and D-Drate (+1.4%; all p<0.01). Alterations in fibrin clot properties were associated with decreased prothrombin fragments 1+2 (F1+2) (−8%), plasminogen activator inhibitor-1 (PAI-1) antigen (−11%), and thrombin activatable fibrinolysis inhibitor (TAFI) activity (−20%; all p<0.01). During OC use FVL(+/−) carriers compared with non-carriers had higher platelet count, activity of PAI-1, TAFI, and tPA, as well as prolonged CLT and higher D-Dmax, along with lower D-Drate and Ks. Multiple regression analysis adjusted for fibrinogen and age, showed that PAI-1 antigen and TAFI activity independently predicted CLT in FVL(+/−) women on OC. ConclusionFVL(+/−) is associated with hypofibrinolysis in apparently healthy women and third-generation OC administration unfavorably alters plasma clot characteristics in female FVL(+/−) carriers with a family history of thrombotic events.

Usefulness of Thrombophilia Testing in Venous Thromboembolic Disease

Information on thrombophilia risk factors for patients with upper extremity deep vein thrombosis (UEDVT) is limited. The genetic, acquired, and coagulation risk factors of an acute episode of lower EDVT (LEDVT) or UEDVT, either isolated or associated with pulmonary embolism (PE), were studied. A total of 4503 patients participated in a thrombophilia study. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated. Mean age of the participants was 55 ± 19 years. The risk of LEDVT or UEDVT, isolated or associated with PE, was calculated according to thrombophilia factors. We found association between LEDVT and factor V Leiden ([FVL]; OR: 1.8; 95% CI 1.4-2.4) and resistance to activated protein C ([APC-R]; OR: 1.6; 95% CI 1.1-2.4). The LEDVT + PE presented an association with PTG20210A (OR: 1.5; 95% CI 1.1-2.1). No association was found between the thrombophilic defects studied and UEDVT or UEDVT + PE. Both FVL and APC-R carriers had the risk of developing LEDVT. The PTG20210A carriers had the risk of developing LEDVT + PE. No thrombophilic defects studied presented risk factors for UEDVT or UEDVT + PE.

Factor V Leiden Mutation and its Impact on Pregnancy Complications

The aim of this prospective study was to find the association between the factor V Leiden mutation and adverse pregnancy outcomes. This study is an analysis of a prospective observational study of the frequency of placenta-mediated complications of factor V Leiden mutation carriers. We compared pregnancy outcomes of 11 women with a heterozygous form of the factor V Leiden mutation with 41 women of a control group. All pregnancies ended with delivery of a living infant. None of the 52 pregnancies were complicated by venous thromboembolism. There were a few significant differences regarding placenta-mediated complications. The gestational age at delivery showed small significant differences. There was a significant difference in the birth weight deviation in percentage between FVL carriers and controls. The incidence of blood loss exceeding 1000 ml was higher in the control group. Carriership of the factor V Leiden mutation did not affect the incidence of preeclampsia. Adverse pregnancy outcomes such as placental abruption were rare. Eclampsia, intrauterine fetal death and venous thromboembolism did not occur. Our results provide evidence that the maternal heterozygous FVL mutation did not increase the risk of an adverse pregnancy outcome.

Increased Sperm Count Maintains High Population Frequency of Factor V Leiden.

Abstract 3994Poster Board III-930 IntroductionFactor V Leiden (FVL) increases the risk of venous thrombosis and pregnancy loss in carriers. Nevertheless, this relatively old mutation is still prevalent in Caucasion populations, which could be explained by positive selection pressure. Men with FVL have previously been found to have higher fecundity (the time between marriage and first pregnancy). Whether this is caused by increased fertility is unknown. We therefore aimed to assess whether men with FVL have increased fertility. Patients and methodsWe performed a pilot study in 19 men with FVL. For confirmation, we performed a cohort study among 961 consecutively included male partners of couples presenting at our university hospital for fertility workup between January 2000 and July 2007. We genotyped all participants and compared the outcome of the fertility workup between FVL carriers and non-carriers. ResultsThe pilot study showed increased sperm concentration and total sperm counts in the 19 FVL carriers as compared to men from the general population. In the cohort study, we identified 37 FVL carriers and 921 non-carriers. FVL carriers had higher total sperm counts and total motile sperm counts than non-carriers: 236 ×106 (95% CI 158-292 ×106) versus 163 ×106 (95% CI 147-178 ×106) and 81 ×106 (95% CI 54-105 ×106) versus 52 ×106 (95% CI 48-57 ×106), respectively. ConclusionsOur results provide a possible explanation for the high prevalence of FVL among Caucasians. To our knowledge, this is the first study that indicates that an increased prevalence of a genotype is controlled by increased sperm counts. Disclosures:No relevant conflicts of interest to declare.

Factor V Leiden Does Not Affect Bleeding in Aprotinin Recipients after Cardiopulmonary Bypass

Carriers of the factor V Leiden mutation (FVL) are resistant to activated protein C proteolysis. Therefore, they are at increased risk of thromboembolic events. Aprotinin is an unspecific proteinase inhibitor frequently used during cardiac surgery procedures to reduce bleeding. However, aprotinin may cause thromboembolic complications after cardiopulmonary bypass (CPB). The primary endpoint of this study was the amount of blood loss after CPB in aprotinin recipients, and secondary endpoints were thromboembolic complications. A total of 1,447 consecutive patients who underwent cardiac surgery with CPB were prospectively enrolled. All patients were screened for FVL by a fluorescence-based polymerase chain reaction method. Linear and logistic regression analyses were performed to assess associations of FVL on bleeding and thromboembolic complications. One hundred seven individuals (7.4%) were heterozygous FVL carriers. No difference was found between FVL carriers and noncarriers regarding age, sex, CPB, type of operation, EuroSCORE, antiplatelet treatment, and reoperation. FVL was not significantly associated with postoperative blood loss, whereas a significant influence was found for female sex (P < 0.0001), duration of CPB (P < 0.0001), reoperation (P = 0.001), and preoperative antiplatelet treatment (P < 0.002). Multiple linear regression analysis for total blood loss had an observed power of at least 99%. FVL carriers faced the same risk for postoperative transfusion (P = 0.391), reoperation (P = 0.675), myocardial infarction (P = 0.44), stroke (P = 0.701), and 30-day mortality (P = 0.4) as did noncarriers. These data suggest that FVL carriers do not have reduced blood loss compared with noncarriers. Furthermore, the combination of aprotinin and FVL does not enhance the risk for thromboembolic complications.

Mutations and Polymorphisms in Genes Affecting Hemostasis Proteins and Homocysteine Metabolism in Children with Arterial Ischemic Stroke

Background: The pathogenesis of thrombosis in childhood seems to be multifactorial implicating genetic and environmental factors. Aim: To compare the distributions of mutations/polymorphisms in genes affecting hemostasis (factor V Leiden – FVL, FV H1298R-FVR2, FII 20210A, b-Fib 455G>A, FXIII V34L, PAI-1 4G, HPA-1b) or homocysteine metabolism (MTHFR C677T, MTHFR A1298C) among 90 children with arterial ischemic stroke (AIS) and 103 controls, and to associate the carriage of these mutations/polymorphisms with their corresponding proteins in children with AIS. Results: AIS was more frequent in boys (p < 0.01). No studied mutation/polymorphism was found to be a risk factor for AIS, except for FVL [odds ratio 4.2 (95% CI 1.5–12.1)], the presence of which was even higher in 31 children with congenital AIS [odds ratio 6.82 (95% CI 2.0–22.8)]. FVL carriers had an odds ratio of 5.76 (95% CI 1.6–6.4) when FVR2 was absent. In thrombosed children, activated protein C resistance, prothrombin and fibrinogen levels were higher in the presence of FVL, FII20210A or b-Fib 455G→A, respectively. Double heterozygotes in both MTHFR C677T and A1298T or homozygotes in one had significantly elevated homocysteine levels. Conclusion: Except for FVL, no definite conclusion could be reached regarding the involvement of the studied mutations/polymorphisms in childhood AIS.

Gender-specific association of the factor V Leiden mutation with fertility and fecundity in a historic cohort. The Leiden 85-Plus Study

Factor V Leiden (FVL, Arg506Gln) mutation may facilitate embryo implantation and increase fertility and fecundity. This was studied in subjects who were of childbearing age in a time with minimal fertility control without modern contraceptive methods. From 1986 to 1999, 1502 inhabitants of Leiden, The Netherlands, reaching the age of 85 years were enrolled in the Leiden 85-Plus Study. Of 1176 subjects the FVL status was analysed, in 365 male and 811 female subjects. The FVL carrier rate was 4.3%. Fertility was not affected by FVL status. In male subjects, fecundity (interval between marriage and birth of first child) was significantly increased in FVL carriers; 67% of male FVL carriers had a child within 371 days of marriage (therefore conceived within 3 months of marriage), compared with 19% of male non-carriers [relative risk (RR), 3.5; 95% confidence interval (CI), 2.1-5.7; P < 0.001]. Within 6 months of marriage, 75% of male FVL carriers had conceived a child compared with 34% male non-carriers (RR, 2.2; 95% CI, 1.5-3.2; P = 0.01). In female subjects, fecundity was not influenced by FVL status. Fecundity is increased in male FVL carriers; in female subjects, no such association was observed.

Risk of Venous Thromboembolism in Relatives of Factor V Leiden and Factor II G20210A Carriers: A Combined Analysis of Retrospective and Prospective Studies.

There are scarce data on thrombotic risk in FVL and FII G20210A asymptomatic carriers, probably due to the requirement of a great number of observed people for a long period of time. This study evaluated retrospectively and prospectively the incidence of VTE in first-degree relatives of FVL and FII G20210A carriers and it performed combined analysis with previous similar observational studies. Retrospective combined analysis for FVL evaluated four previous studies and ours (Table 1). Of the 2016 included family members, 1065 subjects were FVL carries. 37464 and 33433 years of observation were recorded in the group of FVL carriers (car) and non-carriers (N-car), respectively. Annual incidence of VTE was 0.27% in FVL car and 0.08% in N-car (RR=3.4, CI95 2.22–5.19). For FII G20210A analysis, we added another study to the previously mentioned studies (Table 1). Of the 695 analysed family members, 210 subjects carried the mutation. 8312 and 18354 years of observation were recorded in the group of car and N-car, respectively. Annual incidence of VTE was 0.14% in car and 0.06% in N-car (RR=2.4, CI95 1.06–5.45). Prospective combined analysis for FVL evaluated two studies, including ours (Table 2). 653 family members were evaluated, being 357 FVL car. 1387 and 1145 years of observation were recorded in the group of car and N-car, respectively. Annual incidence of VTE was 0.79% in FVL car and 0.617% in N-car (RR=4.51, CI95 1–20.32). For FIIG20210A, the prospective analysis was performed only in our study. Included were 113 family members, being 64 FII G20210 carries. 194 and 151 years of observation were recorded in the group of car and N-car, respectively. Annual incidence of VTE was 1.55% in car and 0% in N-car (RR=∞). In conclusion, the incidence of VTE in asymptomatic carriers of the two most common prothrombotic mutations was found to be low in the retrospective and prospective analysis, not justifying a routine thrombophilia mutation screening in this population.Table 1 -Results of retrospective studies with relatives of FVL and FIIG20210A carriers.StudiesMutationNObserved yearsVTEAnnual Incidence rate VTE (%)CI 95FV LeidenMiddeldorp 1998present2366524290.45(0.28–0.61)absent201571660.10(0.02–0.19)FV LeidenSimioni 1999present2246114170.28(0.15–0.41)absent154440140.09(<0.01–0.18)FV LeidenLensen 2000present1225294180.34(0.2–0.5)absent111509050.10(0.01–0.2)FV LeidenMartinelli 2000present44718320350.19(0.13–0.25)absent43616720110.07(0.03–0.11)FV LeidenOur study 2005present36121240.33(0.10–0.88)absent49150610.07(<0.01–0.42)FII G20210AMartinelli 2000present158673890.13(0.06–0.24)absent43616720110.07(0.03–0.11)FII G20210AOur study 2005present36157430.19(0.04–0.59)absent49163400(0–0.023)Table 2 -Results of prospective studies with relatives of FVL carriersStudiesMutationNObserved yearsVTEAnnual incidence rate VTE (%)CI 95FV LeidenSimioni 2002present313125580.67(0.29–1.33)absent24898410.1(0.003–0.56)FV LeidenOur study 2005present4413232.27(0.49–6.81)absent4816110.62(<0.01–3.83)

Effects of Factor V Leiden mutation on postoperative blood loss under aprotinin: A prospective trial in 1500 cardiac surgical patients

Objectives: Carriers of the Factor V Leiden Mutation (FVL) have an inherited resistance to activated protein C (APC) proteoloysis. FVL is the main cause of thrombophilia, and FVL carriers are supposed to have lower postoperative blood loss after surgery. Aprotinin may inhibit APC. The aim of this study was to evaluate effects of the FVL mutation on postoperative blood loss and thrombotic complications in patients recieving aprotinin.

Improved Detection of FV Leiden Patients Using a Novel Snake-Venom-Based Activated Protein C Resistance Assay.

A new functional prothrombin-based activated protein C (APC) resistance (APC-R) test (Pefakit® APC-R Factor V Leiden, Pentapharm, Basel, Switzerland) is presented.Methods: The plasma sample is mixed with a reagent containing APC and snake venom specifically activating FV (RVV-V, Daboia russelli) and plasma that has been depleted of FV. During an incubation period of 180 sec the activated FV is inactivated by APC. Subsequently a reagent that contains a FV dependent prothrombin activator (Noscarin, Notechis scutatus) and EDTA is added. The clotting time is recorded. A second determination is performed under identical conditions, with the exception that no APC is added to the first reagent. A ratio between the two measurements is calculated. 703 samples of patients undergoing thrombophilia screening were analysed. Results were correlated to PCR based FVL testing, aPTT, PT, and to levels of Protein C, Protein S, Fibrinogen, FVIII and lupus anticoagulant index.Results: Using a predefined cut-off of a ratio of 2.5 a 100% sensitivity and specificity for the detection of a FVL mutation was found. Using a cut-off ratio of 1.2 a complete but narrow distinction of FVL heterozygous (n=192) and FVL homozygous samples (n=27) was determined. No interference by sample's INR and aPTT, PS, fibrinogen and FVIII levels and lupus anticoagulant ratio was detected.Conclusion: The new snake-venom-based APC-R assay provides an improved distinction of FV wild-type and FVL carriers compared to the data reported for the aPTT based methods. The use of a FV dependent prothrombin activator eliminates effects of FVIII concentration or lupus anticoagulants in the sample.

A Sensitized Genome-Wide ENU Mutagenesis Screen in the Mouse to Identify Genetic Modifiers of Thrombosis.

Factor V Leiden, (FVL) is the most common known inherited thrombotic risk factor and is present in approximately 5% of most Western populations and 25–50% of patients presenting with venous thrombosis. However, FVL is incompletely penetrant, with only approximately 10% of FVL carriers developing thrombosis in their lifetimes. Though interactions between FVL and other known prothrombotic mutations have been documented in a few cases, the genetic factors responsible for the incomplete penetrance of FVL remain largely unknown. We previously reported a remarkable synthetic lethality in mice carrying the FVL mutation and partial deficiency of a key coagulation component, tissue factor pathway inhibitor (TFPI). Complete TFPI deficiency in mice is embryonic lethal, whereas heterozygosity is compatible with normal survival. However, homozygosity for FVL (FvQ/Q) in the context of heterozygosity for TFPI (Tfpi+/−) is uniformly lethal due to disseminated perinatal thrombosis. In order to identify potential modifier genes contributing to FVL penetrance, we have utilized this lethal genetic interaction as a phenotyping tool for a sensitized ENU mutagenesis screen in laboratory mice. We hypothesize that dominant mutations in key components of the coagulation system will improve hemostatic balance and allow survival in mice carrying the lethal FvQ/Q Tfpi+/− genotype combination. As an example, we propose that loss of one tissue factor allele might compensate for reduced TFPI and rescue FvQ/Q Tfpi+/− . To test this hypothesis, we bred tissue factor heterozygous mice (Tf+/−) with FvQ/Q Tfpi+/− mice and observed complete rescue, with normal survival and the expected number (8 of 57) of FvQ/Q Tfpi+/− Tf+/− mice from a FvQ/+ Tfpi+/− Tf+/−x FvQ/Q cross. In order to identify candidate modifier genes, we performed a whole genome mutagenesis screen. In this screen, male FvQ/Q mice were mutagenized with ENU and bred to FvQ/+ Tfpi+/− double heterozygous females. DNAs from surviving offspring were PCR assayed to identify rescued mice with the FvQ/Q Tfpi+/− genotype. Analysis of 2250 offspring, corresponding to approximately half genome coverage, has identified 15 mice that survived to weaning. Heritability was demonstrated for the 5 mutant lines subjected to progeny testing to date. Genetic crosses are in progress to map the mutant genes in 3 of the 5 progeny tested lines. These preliminary results demonstrate the feasibility of this sensitized screen for the identification of dominant suppressors of thrombosis. Based on our data, we estimate that there are likely 10–20 mammalian genes for which a <50% reduction in expression could result in a major shift in hemostatic balance sufficient to rescue the lethal thrombosis associated with the FvQ/Q Tfpi+/− lethal genotype. Each of these loci represent a candidate for a human modifier gene in patients with FVL and other thrombophilic mutations. Finally, the biologic pathways uncovered by these studies should provide new insights into the overall regulation of hemostatic balance and identify potential new targets for therapeutic intervention.

Heterozygosity for the factor V Leiden (G1691A) mutation predisposes renal transplant recipients to thrombotic complications and graft loss.

Heterozygosity for a mutation in the coagulation factor V gene (factor V Leiden; FVL) leads to resistance to activated protein C and represents the most common cause of inherited thrombophilia. FVL is associated with a high risk for thromboembolic events and might be a risk factor for venous thrombosis and early graft loss in renal transplant recipients. We studied a cohort of 202 renal allograft recipients to assess the impact of the FVL mutation on thrombotic events and graft loss within 1 year after transplantation. We recorded the occurrence of deep venous thrombosis, pulmonary embolism, early graft perfusion defect, and graft loss. The occurrence of these events was then correlated with the presence or absence of heterozygosity for the FVL mutation. Heterozygosity for FVL was detected in 8 (4%) of 202 patients. The incidence of deep venous thrombosis or pulmonary embolism was higher in heterozygous compared with wild-type patients (25% vs. 5.7%, P=0.09). Furthermore, early graft perfusion defect (25% vs. 2.6%; P=0.03) and graft loss within 7 days after transplantation (2/8 vs. 1/194; P=0.004) were significantly more frequent among heterozygous carriers of FVL. All eight FVL carriers were negative for protein C or S deficiency and antiphospholipid and anticardiolipin antibodies, and were not carriers of the G20210A prothrombin mutation. Heterozygosity for the FVL mutation predisposes renal allograft recipients to venous thromboembolic complications, graft perfusion defects, and early transplant loss. Screening for the FVL mutation and appropriate peri- and postoperative anticoagulation after renal transplantation might prevent these thromboembolic complications.

Contribution of the glycoprotein Ia 807TT, methylene tetrahydrofolate reductase 677TT and prothrombin 20210GA genotypes to prothrombotic risk among factor V 1691GA (Leiden) carriers.

The cooperative effects of the GPIa 807TT, MTHFR 677TT and prothrombin 20210GA genotypes with the FV Leiden 1691GA (FVL) genotype were evaluated by comparing these genotype frequencies in 77 asymptomatic and 156 symptomatic heterozygous FVL carriers. The GPIa 807TT and MTHFR 677TT genotypes did not segregate within the symptomatic FVL carrier group and did not contribute to venous thrombotic risk in this patient cohort. There was no difference in the prothrombin 20210GA genotype frequency between asymptomatic FVL carriers and a random Caucasian control group; however, the prothrombin 20210GA genotype was nearly 5 times as prevalent (19/156 v 2/77; P < 0.02) in the symptomatic FVL carriers (odds ratio 5.21; 95% confidence interval 1.20-47.62), demonstrating that this important prothrombotic risk factor acts synergistically with FVL.