Homozygous Factor V Leiden Research Articles

Prevalence of Factor V Leiden Mutation in Healthy Females of Sindh, Pakistan and Comparison of Three Detection Methods in Resource-Limited Settings

Factor V Leiden (FVL) mutation is related to venous thromboembolism and pregnancy complications. It is highly prevalent among Caucasians; however, few studies have been conducted on the Asian population. Objective: To find the prevalence, genotype, and allelic frequency of FVL mutation in females of Sindh, Pakistan, and to develop a valid and economical method for the detection of FVL mutation in resource-limited settings. Methods: Hundred (n=100), unrelated healthy females of Sindh, Pakistan, were recruited. FVL was detected using three methods, i.e., tetra primer amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and PCR-sequencing. Prevalence, genotype, and allele frequencies were calculated. We compared the three mutation detection methods to find a suitable protocol for FVL detection in developing countries. Results: The prevalence of FVL mutation was 3% in healthy females of Sindh, Pakistan. No homozygous FVL mutation was found. The results from tetra primer ARMS-PCR, PCR-RFLP, and PCR-sequencing were 100% concordant. Tetra primer ARMS-PCR was a valid, reliable, faster, economical genotyping and screening method. Conclusions: In conclusion, FVL mutation was found in our population, and further studies should be conducted to find its role in thromboembolic and obstetrical complications. In addition, we have suggested tetra primer ARMS-PCR as an appropriate method for FVL detection in resource-limited settings.

Factor V Leiden homozygous mutation: Recurrent venous thromboembolism during periods of discontinuation of anticoagulant therapy

Venous thromboembolism (VTE), also known as deep vein thrombosis and pulmonary thromboembolism, is a medical condition that has a high incidence and a multifactorial pathogenesis. One of the causes is a mutation of the Factor V Leiden (FVL), which is the most common inherited thrombotic disorder. A mutation inherited from both parents is found in about 0.05-0.5% of cases, making our case more medically interesting. We presented the case of a young male patient with recurrent VTE and few risk factors who was admitted to the hospital 4 times with VTE symptoms over a 5-year period. In the background, there was a homozygous FVL mutation and improper use of anticoagulant therapy. We examined the medical data, diagnostics, therapy, and precautions that were and are required.

Evaluation of the Thrombophilic Response to rFVIIa-Induced Hemostasis Activation in Factor V Leiden Carriers By Monitoring of Activated Protein C (APC) In Vivo

Background: The majority of patients with unprovoked venous thromboembolism (VTE) does not exhibit laboratory risk factors. Monitoring a limited and standardized activation of the hemostatic system in these patients might help to identify prothrombotic phenotypes in VTE. Promising biomarkers to monitor subclinical changes of the hemostatic activation status are plasma levels of active enzymes including thrombin and APC. Aim of this study was to characterize the prothrombotic phenotype in factor V (FV) Leiden carriers as a prerequisite for the identification of novel prothrombotic mechanisms.Methods: Subclinical activation of extrinsic hemostasis was induced by i.v. injection of recombinant activated factor VII (rFVIIa, 15 µg/kg) into 14 asymptomatic FV Leiden carriers (thereof 12 heterozygous, five males) and 12 healthy FV wild type (WT) carriers (six males). rFVIIa-induced hemostatic activation was monitored by measuring plasma levels of free thrombin and APC at baseline and repeatedly during an eight-hour follow-up period using oligonucleotide-based enzyme capture assays (OECA). In addition, prothrombin fragment F1+2 (F1+2), thrombin-antithrombin complex, plasmin-α2-antiplasmin complex, soluble fibrin monomer, and D-dimer were determined.Results: Administration of rFVIIa was well tolerated in all subjects. Its pharmacokinetics showed an expected course with no differences between FV Leiden carriers and WT carriers (Figure 1A). At baseline, median (interquartile range, IQR) APC plasma levels were 0.081 (0.068-0.117) ng/ml in heterozygous FV Leiden carriers and 0.035 (0.028-0.067) ng/ml in WT carriers. Following rFVIIa injection, a significant (p<0.05) elevation of APC levels from baseline was observed between t=10 min and t=5 h (3 h for WT carriers). Peaks of 0.509 (0.431-0.721) ng/ml (p=10-4) in heterozygous FV Leiden carriers and of 0.164 (0.139-0.194) ng/ml (p=3·10-4) in WT carriers were observed at t=1 h (Figure 1B). In addition to higher peaks (p=2·10-4) in heterozygous FV Leiden carriers than in WT carriers, the area under the APC generation curve (AUC) was greater (p=4·10-6) with 2.40 (1.84-2.84) vs. 0.77 (0.63-0.97) ng/ml·h. Interestingly, the AUC of both homozygous FV Leiden carriers (2.82 and 2.67 ng/ml·h) and their APC peak levels (0.717 and 1.123 ng/ml) lay within the ranges of the heterozygous cohort. Among the other biomarkers, only F1+2 showed an increase from baseline (0.178, 0.122-0.197 nmol/l in heterozygous FV Leiden carriers; 0.125, 0.087-0.188 nmol/l in WT carriers) that became statistically significant at t=2 h (0.229, 0.187-0.264 nmol/l, p=0.001), t=3 h (0.230, 0.190-0.281 nmol/l, p=0.006), and t=5 h (0.230, 0.172-0.274 nmol/l, p=0.02) in heterozygous FV Leiden carriers, and at t=2 h (0.179, 0.111-0.200 ng/ml, p=0.03) in WT carriers. All other biomarkers remained unchanged, and no statistically significant differences were observed between FV carriers and WT carriers.Conclusion: Increased thrombin generation rates are counterbalanced by increased APC generation rates in clinically asymptomatic FV Leiden carriers after subclinical activation of extrinsic coagulation. Furthermore, the data presented demonstrate that subclinical activation of the hemostatic system by rFVIIa combined with serial APC testing is a new diagnostic tool to assess the functionality of the anticoagulant APC pathway in vivo. [Display omitted] DisclosuresRühl:Grifols: Research Funding; Sobi: Consultancy. Winterhagen:Sobi: Consultancy.

Assessment and Reduction of Unnecessary Thrombophilia Testing in Patients 18-60 Years with Acute Cryptogenic Ischemic Stroke

Patients &lt; 60 years with acute ischemic stroke (AIS) are commonly tested for thrombophilia. Underlying this practice is the perception that the stroke may be the result of a hypercoagulable state, especially when an alternative cause is not evident. Although certain conditions, such as antiphospholipid antibody syndrome (APS), can be associated with AIS and require anticoagulation, inherited thrombophilia, largely, is not a risk factor for AIS even in young adults with cryptogenic stroke, as supported by case-control and prospective observational studies. Moreover, testing in the acute phase of AIS may give erroneous results (i.e., increase of Factor 8 as an acute phase reactant), and increase health care costs. To assess our institutional performance on this matter, we conducted a retrospective chart review of 104 patients, age 18-60 years, who were admitted for AIS to Memorial Hermann Hospitals at the Texas Medical Center between 11/2016 and 7/2018, and whose stroke was considered cryptogenic or of undetermined origin (i.e., more than one possible causes) after initial diagnostic evaluation. Mean age at the time of event was 43 (range 20-58) years. There were 61 (59%) males. Fifty nine (57%) patients were White, 42 (40%) Black, 18 (17%) Hispanic, 2 (2%) Asian, and one (1%) of unknown race. Risk factors for AIS were: hypertension in 64 (61%) patients, diabetes in 26 (25%), smoking in 27 (26%), dyslipidemia in 32 (31%), prior AIS in 17 (16%), coronary artery disease in 4 (4%), and obesity in 10 (10%). None had known atrial fibrillation or other cardiac arrhythmia. Thrombophilia testing (at least one test) was performed in 82 (79%) of the patients. In the vast majority (70 patients, 85%) of cases, such testing was done within 1-3 days of admission as part of the initial stroke evaluation. A total of 752 tests were performed on the 82 patients, 56 (7%) of which were positive. Increased Factor 8 (F8) activity (i.e., ≥200%) was the commonest abnormality observed (31/58 patients tested, or 53%), followed by low protein S (PS) activity (13/77, 17%), Factor V Leiden (FVL) mutation (3/71, 4%), low antithrombin (AT) activity (3/73, 4%), elevated homocysteine (2/79, 3%), low protein C (PC) activity (1/76, 1%), and positive APL (1/251, 1%), while no prothrombin gene mutation was seen. All but one of F8, PS, and AT tests were performed in the acute phase of the stroke. The estimated annual cost for testing was $65000. Forty-two (51%) of the 82 patients tested had at least one abnormal test. In 2/42 (5%) of these patients, repeat testing (i.e., PS activity, F8) was done at least a month later, which, however, did not confirm the abnormality. Forty-seven (45%) patients were seen in the Outpatient Neurology Clinic after their stroke, for a median follow up of 5 (0.2 - 24) months. In 54 (52%) patients the cause of stroke remained undetermined even after thorough evaluation. Sixteen (15%) patients had a patent foramen ovale (PFO). Of all patients tested, thrombophilia was confirmed in three, i.e., two patients with heterozygous FVL mutation and one with known homozygous FVL mutation and a significant personal history of venous thromboses. Only in one patient (1/82, 1%) management was changed taking into account the thrombophilia testing results: a 57 year old male with low PS activity (tested 3 months after the stroke) and a PFO not amenable to closure, for whom anticoagulation was started despite the lack of venous thromboembolism. Based on these results, and due to concerns of indiscriminate testing, we created a practice guideline supported by current literature, as a reference for clinicians (see Figure). Briefly, we eliminated inherited thrombophilia testing in the acute phase, and rendered it optional in the chronic phase at the discretion of the treating physician, when anticoagulation is considered (i.e., some cases of PFO or recurrent stroke). Accordingly, we removed these tests from the admission order panel in the Electronic Medical Record. After internal audit and feedback from health care providers across disciplines, we submitted the new guideline for multidisciplinary review and system-wide adoption (i.e., multiple hospitals). We conclude that routine thrombophilia testing is not an effective tool for secondary stroke prevention even in young patients with cryptogenic AIS. Institutional practice guidelines are helpful in tailoring such testing, hence avoiding misdiagnosis of a thrombophilic state and unnecessary costs. Figure Disclosures No relevant conflicts of interest to declare.

High risk of thrombosis recurrence in patients with homozygous and compound heterozygous factor V R506Q (Factor V Leiden) and prothrombin G20210A

ObjectiveHeterozygous Factor V R506Q [Factor V Leiden (FVL)] and prothrombin G20210A (PGM), the most common inherited thrombotic disorders in the Caucasian population, confer a low-moderate risk for first venous thromboembolic (VTE) event. We investigated the thrombotic complications of rare homozygous and compound heterozygous FVL and PGM. MethodsA cohort of patients with homozygous and compound heterozygous FVL and PGM were evaluated at a major referral center in Central Pennsylvania, USA between June 2001 and March 2019. Data including incidence of first and recurrent thrombosis, associated risk factors, family history and demographics were collected. ResultsSeventy-five patients were eligible for analysis: 47 had homozygous FVL, three had homozygous PGM, 19 had compound heterozygous FVL and PGM, five had compound homozygous FVL and heterozygous PGM, and one had compound heterozygous FVL and homozygous PGM. Fifty-nine patients experienced 111 thromboembolic events. Forty-seven percent of first thrombotic events occurred in patients without clinical or surgical conditions predisposing to thrombosis. The rate of recurrent thromboembolism was 59%. The mean time to recurrence was 8.5 years. Ninety percent of recurrent events occurred during times when patients were not treated with anticoagulation. ConclusionPersons with homozygous and compound heterozygous FVL and PGM are at a significantly increased risk of first unprovoked and recurrent VTE. Patients with first thromboembolic events should be considered for long-term anticoagulation.

Compound Homozygous Factor V Leiden and Heterozygous Prothrombin Gene Mutation: The First Report in a Pregnant African with Extensive Thrombosis

Background: Factor V Leiden (FVL) mutation and Protein gene G20210A mutation (PGM) are the most common inherited thrombophilias in the world. (Limdi NA et.al, Blood Cells Mol Dis. 2006 Sep-Oct;37(2):100-6) Both are inherited in an autosomal recessive fashion with individuals who are homozygous having higher risk of thrombosis compared to those who are heterozygous.(Rodger MA et.al, PLoS Med. 2010 Jun 15;7(6):e1000292.) The global prevalence of FVL and PGM is variable with Caucasians carrying the highest prevalence and Africans living in Africa, Asians and Native Americans having the lowest rate of these mutations; it is zero in West and Southern African countries, 2.4%-3.9% in North African countries including Morocco, Tunisia, and Algeria. (Limdi NA et.al, Blood Cells Mol Dis. 2006 Sep-Oct;37(2):100-6, Dziadosz M et. al, Blood Coagul Fibrinolysis. 2016 Jul;27(5):481-9)Pregnancy increases the risk of developing venous thromboembolism (VTE) by 0.05-1.8 %, (Eldor A, Thromb Haemost. 2001 12.12.2017;86(07):104-11) which is 4 to 5 fold greater than in non-pregnant female.(Croles FN et.al, BMJ. 2017;359, Greer IA, N Engl J Med. 2015 Aug 6;373(6):540-7) Inheritance of FVL and PGM increases the risk for development of VTE in pregnancy, the risk is higher with homozygous than heterozygous mutations.(Rodger MA et.al, PLoS Med. 2010 Jun 15;7(6):e1000292.) Concurrent presentation of FVL and PGM in pregnancy presenting as thrombosis is not common.Aim: The aim of this case report is to present a patient of African descent with concurrent FVL and PGM mutation who had thrombosis during pregnancy.The Case: A 32-year-old Nigerian female who was 28 weeks pregnant presented to the gynecologist with a swollen left leg. Physical examination and the Wells score were very suggestive of a deep vein thrombosis (DVT). The Duplex Doppler performed confirmed a diagnosis of bilateral lower limb DVT. The patient was referred to a physician, who together with the haematologist performed a thrombophilia screen including FVL, PGM, Protein C, Protein S and Anticardiolipin antibody tests. The D-DIMERS were raised and the viral markers including HIV, HBV, and HCV were negative. The PT, APTT and full blood counts were normal. The thrombophilia tests revealed that the patient was homozygous for FVL and also heterozygous for the PGM mutations. The rest of the thrombophilia screen including Protein C, Protein S and Antithrombin tests were all negative. She has no family history of thrombosis; no past history of hormone based contraceptives. She was counselled on the need for using anticoagulation, low molecular weight heparin (LMWH) during the rest of her pregnancy period, and therapeutic anticoagulation with a LMWH at a dose of 1mg/kg twice a day until onset of labour was started. During labour LMWH was discontinued, delivery was per vaginal and was uncomplicated. Postpartum LMWH was continued at therapeutic doses for six weeks with no bleeding or thrombotic events. After six weeks, the patient was started on lifelong warfarin.Results (See Table)Discussion: To our knowledge this is a first case of a patient born and living in Africa presenting with thrombosis and found to have homozygous FVL and heterozygous PGM during pregnancy, the one report that is similar was in a second generation South African woman of German, Dutch and French ancestry. (Wilson J et. al, Medical Technology SA. [Case Report]. December 2011;25(2):4) Most reports have shown ethnic and regional affectation. (Limdi NA et.al, Blood Cells Mol Dis. 2006 Sep-Oct;37(2):100-6, Bavikatty NR, et.al, Am J Clin Pathol. 2000 Aug;114(2):272-5)There is no single agreed reason as to why the mutation is rare in Africa though founder effect is being studied to define how the mutation came about, it could have taken place before divergence of race. (Jadaon MM. [Thrombosis, Familiar Thrombophilia]. 2011 2011-11-28;3(1)We could not do family studies to help define the origin of this mutation whether it is denovo for Nigeria; this has to do with sentiments about genetic inheritance in Africa.Conclusion: Concurrent inheritance of both FVL and PGM is possible in Africa. This rare case has open opportunities to revisit the prevalence of thrombophilia in Nigeria and other African countries. DisclosuresMahlangu:Alnylam: Consultancy, Research Funding, Speakers Bureau; Biomarin: Research Funding, Speakers Bureau; Catalyst Biosciences: Consultancy, Research Funding; Chugai: Consultancy; Amgen: Consultancy; Bayer: Research Funding; Biogen: Research Funding, Speakers Bureau; CSL Behring: Consultancy, Research Funding, Speakers Bureau; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; LFB: Consultancy; Roche: Consultancy, Research Funding, Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau; Spark: Consultancy, Research Funding.

Detection of high-risk thrombophilia with an automated, global test: the Coagulation Inhibitor Potential assay.

: The diagnosis of thrombophilia is a cost-consuming and time-consuming process, as each defect should be separately investigated. The Coagulation Inhibitor Potential (CIP) assay is a promising new global test, sensitive for most of the hereditary thrombophilias, developed for manual methodology. We adapt the original method to an optical coagulation analyser. By this automation, the test will be easier, faster and more precise, and it also allows carrying out 18 measurements simultaneously. The CIP assay was performed in 126 healthy subjects and 193 patients with different types of hereditary thrombophilia conditions. Detected with conventional laboratory tests high-risk thrombophilia was present in 70 patients: deficiencies of antithrombin (AT) (n = 12), protein C (PC) (n = 14), protein S (PS) (n = 6), homozygous factor V Leiden (FVL) mutation (n = 9) and combined types (n = 29). Low-risk thrombophilia was present in 123 patients: heterozygous FVL (n = 115) and FII G2010A mutation (n = 8). Significantly lower median CIP values were found for AT-,PC-, PS deficiencies, homozygous and heterozygous FVL mutations and combined thrombophilias (P < 0.01) as compared with healthy controls. There was no significant difference between the heterozygous FIIG20210A (P = 0.669) thrombophilia group and the healthy controls. The best performance of the test was achieved at the cut-off value of 90.0 U (area: 0.981) with 96% sensitivity and 92% specificity in the high-risk thrombophilia group estimated by receiver operating characteristic analysis. The new method seems to be appropriate and reliable for the detection of AT-, PC- and PS deficiencies, homozygous FVL mutation and also for combined deficiencies. The automated CIP test is insensitive to FII G2010A mutation.

Factor V Leiden paradox in a middle-aged Swedish population: A prospective study.

Few prospective studies have examined the factor V paradox: factor V Leiden (FVL) is a stronger risk factor for deep venous thrombosis (DVT) than for pulmonary embolism (PE). The present study, to the best of our knowledge, is the first population-based study aimed to examine the relationship between FVL and incidence of venous thromboembolism (VTE), DVT and PE in a prospective cohort study of middle-aged Swedish individuals. FVL was determined in 4890 subjects (aged 46-68 years, 57% women) from the general population without previous VTE or cancer, who participated in the Malmö Diet and Cancer study between 1991 and 1994. Incident cases of VTE were identified from the Swedish patient register during a mean follow-up of 15.6 years. Of 4890 subjects with determination of FVL (10.2% carriers), 220 had VTE during follow-up (113 DVT, 78 PE, 29 both). Incidence of VTE was significantly higher in subjects with heterozygous and homozygous FVL: adjusted hazard ratios (HR) were 1.8 (95% CI 1.3-2.6, p=0.001) and 6.5 (2.1-21, p=0.001), respectively. The population attributable fraction was 8.7% for FVL. Adjusted HRs for DVT were 2.2 (1.4-3.3, p<0.001) for heterozygotes and 3.3 (0.5-24, p=0.233) for homozygotes. Adjusted HRs for PE were 1.2 (0.65-2.2, p=0.582) for heterozygotes and 8.7 (2.1-36, p=0.003) for homozygotes. The FVL paradox was confirmed for heterozygotes for FVL. However, homozygotes for FVL had a high risk for PE, suggesting that the FVL paradox is related to the carriership of one wild type and one mutated factor V allele.

Lack of rivaroxaban influence on a prothrombinase-based assay for the detection of activated C protein resistance: an Italian ex vivo and in vitro study in normal subjects and factor V Leiden carriers.

Activated protein C resistance (APCr) leads to hypercoagulability and is due, often but not exclusively, to Factor V Leiden (FVL). The aim of this study was to assess the ex vivo and in vitro interference of the direct factor Xa inhibitor rivaroxaban (RIV) on a prothrombinase-based assay for APCr detection. An ex vivo study was performed on fresh plasma samples obtained from 44 subjects with FV wild-type and seven with FVL heterozygous, all treated with RIV. An in vitro study was performed on 15 plasma samples (six from normal subjects, six from heterozygous, and three from homozygous FVL carriers, all frozen specimens) spiked with RIV. RIV concentration was evaluated using a chromogenic assay, and APCr was evaluated by a prothrombinase-based assay. No significant interference of RIV on APCr results obtained by a prothrombinase-based assay was observed for drug concentrations up to 400 ng/mL in FV wild-type and FVL carriers (homozygous and heterozygous). These results were confirmed both ex vivo and in vitro. RIV did not significantly interfere with the prothrombinase-based assay used for the assessment of APCr, and this was observed to occur independently of FV status. However, only concentrations up to 400 ng/mL were tested and, therefore, what occurs in the presence of higher doses remains to be investigated.

The Association between Prothrombin Gene G20210A and Factor V Leiden Mutation in Women with Complications of Pregnancy in Baghdad Province

Factor V Leiden (FVL) (G1691A) and prothrombin gene (G20210A) mutations are the most common inherited forms of thrombophilia. The main objective of this study is to analyze the association between inherited thrombophilia FVL mutation and prothrombin G20210A mutation and recurrent pregnancy loss (RPL) among women suffering from complications of pregnancy. The study included 40 buccal swab samples collected from women at the reproductive age complications; abruption placenta 12.5%, dead fetal 37.5%, and recurrent spontaneous abortions (RSA) 50% in comparison with 30 women who had one or more normal pregnancies from Elwiyah Obstetric teaching hospital through the period from mid of September 2014 to the mid of March 2015. The median age of patients was 32 years (range: 19–42 years) while for the control group, it was 28 years (range: 17–41 years). Out of 40 women, 65% had one pregnancy loss and 35% for more than two previous pregnancy losses. According to the time of pregnancy losses, 22(55%) women had early pregnancy loss (EPL), and 18 (45%) women had Late Pregnancy Loss (LPL). For FVL mutation detection the restriction fragment length polymorphism (RFLP) was used. DNA fragment of interest was amplified by PCR and then subjected to digestion by MnlI specific restriction enzyme. For prothrombin G20210A mutation detection different PCR products were generated using a set of primers in A/A, G/G, A/G alleles. Out of the 70 samples tested for FVL mutation no homozygous FVL mutation was detected and Prothrombin gene mutation G20210A was totally absent among patient and control.

Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.

Thrombophilic risk of individuals with rare compound factor V Leiden and prothrombin G20210A polymorphisms: an international case series of 100 individuals.

The risk of thrombosis in individuals with rare compound thrombophilias, homozygous factor V Leiden (FVL) plus heterozygous prothrombin G20210A (PTM), homozygous PTM plus heterozygous FVL, and homozygous FVL plus homozygous PTM, is unknown. We identified, worldwide, individuals with these compound thrombophilias, predominantly through mailing members of the International Society on Thrombosis and Haemostasis. Physicians were sent a clinical questionnaire. Confirmatory copies of the genetic results were obtained. One hundred individuals were enrolled; 58% were female. Seventy-one individuals had a venous thrombosis (includes superficial and deep vein thrombosis, and pulmonary embolism), 4 had an arterial thrombosis and 6 had both. Nineteen individuals had never had a thrombotic event. Thrombosis-free survival curves demonstrated that 50% of individuals had experienced a thrombotic event by 35 yrs of age, while 50% had a first venous thromboembolic event (VTE; includes all venous thrombosis except superficial thrombosis) by 41 yrs of age; 38.2% of first VTEs were unprovoked. 37% of patients had at least one VTE recurrence. Seventy percent of first pregnancies carried to term and not treated with anticoagulation were thrombosis-free. In conclusion, patients with these rare compound thrombophilias are not exceedingly thrombogenic, even though they have a substantial risk for VTE.

TFPIα Has Reduced Ability to Inhibit FV Leiden

Introduction: Tissue factor pathway inhibitor (TFPI)α dampens the initiation phase of coagulation by inhibiting the tissue factor/factor VIIa complex and early thrombin generation. Activated protein C (APC) dampens the propagation phase of coagulation by degrading factor Va (FVa) and factor VIIIa. Kinetic assays using purified TFPIα, protein C, and thrombomodulin have shown that TFPIα and APC act synergistically to inhibit thrombin production. In these assays, thrombin generation is markedly altered by factor V Leiden (FVL), a prothrombotic mutation that reduces the ability of APC to degrade FVa. A 50% reduction in TFPIα (2.5nM to 1.25nM) completely abolishes the anticoagulant effect of APC in reactions performed with FVL, but has no effect in reactions performed with FV. The physiological importance of these in vitro results was subsequently supported by the severe perinatal thrombosis observed in mice with homozygous FVL and heterozygous TFPI deficiency. TFPIα inhibits thrombin generation via a high affinity exosite interaction between its basic C-terminus and an acidic region in early forms of FVa. We hypothesized that the Leiden mutation alters this interaction in a manner that reduces TFPIα inhibitory activity during the initiation of coagulation.Methods: Plasma and platelet-rich plasma (PRP) were collected from homozygous FVL, heterozygous FVL, and control donors. Thrombin generation assays were performed in the presence or absence of recombinant TFPIα, a peptide mimicking the TFPIα basic C-terminus (LIKTKRKRKK), or anti-TFPI antibodies. Purified prothrombinase assays were performed using FV810, a B-domainless form of FV(a) that binds and is inhibited by TFPIα, and FVL810.Results: Thrombin generation assays were initiated with limiting FXa to determine an activation threshold. The threshold for thrombin generation was between 10 and 20 pM FXa in all 10 control plasmas, but was reduced in homozygous FVL plasmas, with 4 of 10 responding to only 5 pM FXa. Additional thrombin generation assays were performed using 100 pM FXa to determine the effect of FVL on TFPI activity, as there were no baseline differences between control and FVL plasmas in these conditions. When substances that alter TFPI function were added, the lag time for initiation of thrombin generation was primarily affected. Addition of 1 nM recombinant TFPIα and 1 μM LIKTKRKRKK prolonged the lag time in control plasma by 58% and 28%, respectively, but had progressively less effect in FVL heterozygous (45% and 19%) and homozygous (39% and 9.6%) plasmas (p<0.03 for all comparisons except addition of TFPIα to heterozygous plasma). Conversely, anti-TFPI antibodies directed against the second (anti-K2) and third (anti-K3) Kunitz domains decreased the lag time 32% and 23% in control plasmas, respectively, but only 25% and 17% in FVL homozygous plasma (p=0.03 for both). Thrombin generation assays were repeated using PRP. There were minimal and equivalent effects of recombinant TFPIα or LIKTKRKRKK in control and FVL PRP, possibly due to the presence of additional exogenous TFPIα in platelets. However, the anti-TFPI antibodies decreased the lag time more in control plasma than in FVL homozygous plasma, with anti-K2 decreasing the lag time of control PRP by 28% and FVL homozygous PRP by 20% (p=0.009). In purified prothrombinase assays, TFPIα was a weaker inhibitor of prothrombinase containing FV810 (IC50 = 4.8nM; C.I.= 3.8-6.1nM) compared to FVL810 (IC50 = 7.3nM; C.I. = 6.9-7.9nM). A similar effect of FVL was observed when inhibiting with LIKTKRKRKK.Conclusion: The biochemical mechanism underlying the procoagulant phenotype of FVL is well-described as a resistance to APC, which dampens the propagation phase of clot formation. The data described here suggest a potential second biochemical mechanism underlying this procoagulant phenotype, a reduced ability of TFPIα to inhibit FVL. This effect is evidenced by alterations in the threshold for initiation of thrombin generation in FVL homozygous plasma and PRP, as well as the reduced IC50 for inhibition of thrombin production by prothrombinase assembled with FVL. This effect is enhanced by reducing TFPIα concentration, offering an explanation for the dramatic procoagulant effect of 50% reduced TFPI in assays using human proteins and mice with FVL. The findings could have clinical relevance in patients with FVL who use oral contraceptives that can reduce the plasma TFPIα concentration. DisclosuresCamire:Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Research Funding; Pfizer: Consultancy, Patents & Royalties, Research Funding. Mast:Novo Nordisk: Honoraria, Research Funding.

Abstract 35: Identification of a Mouse Strain Modifier Locus for Factor V Leiden/Tissue Factor Pathway Inhibitor Dependent Thrombosis

Factor V Leiden (FVL) is the most common genetic risk factor for venous thromboembolism (VTE). FVL is incompletely penetrant as only 10% of FVL heterozygotes develop VTE. Previously, nearly uniform perinatal lethality was observed in C57BL/6J (B6) mice hemizygous for tissue factor pathway inhibitor (Tfpi+/-) on a homozygous FVL background (FVQ/Q). However, inbred mouse strains exhibit significant genetic variation and can exert a profound influence on phenotype. We tested the lethality of FVQ/Q Tfpi+/- on the DBA/2J inbred strain background by crossing FVQ/+ Tfpi+/- to DBA/2J followed by a double heterozygous (FVQ/+ Tfpi+/-) x FVQ/Q cross. Surprisingly, FVQ/Q Tfpi+/- mice on the DBA/2J genetic background are born at expected Mendelian frequencies (N=62, nexp=15, nobs=15, N.S.) and display no overt thrombosis. This demonstrates the existence of dominant antithrombotic DBA/2J strain-specific thrombosis modifier gene(s). Analysis of progeny from a DBA/2J-B6 F1 FVQ/Q Tfpi+/- x B6 FVQ/Q cross yielded equal proportions of FVQ/Q Tfpi+/- and FVQ/Q Tfpi+/+ (N=130, nexp=43, nobs=63, p&lt;0.001) which suggested that multiple modifier genes were contributing to the phenotype. To isolate individual dominant modifier loci, we adopted a serial backcrossing strategy in which F1 FVQ/Q Tfpi+/- mice were serially backcrossed to B6 FVQ/Q mice for 17 generations. A standard mouse positional cloning strategy was used on the 110 surviving FVQ/Q Tfpi+/- mice from these crosses which identified a chromosome 2 modifier locus (148-169 Mb) under genetic selection. Currently there are 16 genes in this locus with known annotated non-synonymous coding variants. Ongoing studies are focused on identifying the responsible genetic DBA/2J variant within this locus. Identification of this modifier gene will represent a significant advance for the genetics of thrombotic disease. Not only will it give insight into the pathways leading to thrombosis, it may also reveal previously unexplored therapeutic targets for the prevention of thrombotic events.

Low Molecular Weight Heparin Modulates Maternal Immune Response in Pregnant Women and Mice with Thrombophilia

Thrombophilia is associated with pregnancy complications. Treatment with low molecular weight heparin (LMWH) improves pregnancy outcome, but the underlying mechanisms are not clear. We analyzed Treg frequency in blood from thrombophilic pregnancies treated with LMWH (n = 32) or untreated (n = 33) and from healthy pregnancies (n = 39) at all trimesters. Additionally, we treated pregnant wild-type, heterozygous and homozygous factor-V-Leiden (FVL) mice with LMWH or PBS and determined Treg frequency, pro-/anti-inflammatory cytokine levels and Caspase-3-activity in placenta and decidua. Treg frequencies were increased in second and third trimester in LMWH-treated thrombophilic pregnancies compared to controls. Treg levels were comparable to those of normal pregnancies. Homozygous FVL mice had decreased decidual Tregs compared to wild-type mice. LMWH treatment normalized Tregs and was associated with increased decidual IL-10 mRNA. LMWH diminished Caspase-3-activity in mice of all genotypes. We demonstrated anti-apoptotic and anti-inflammatory effects of LMWH in pregnant FVL mice. LMWH increased Treg levels in mice and humans, which suggests benefits of LMWH treatment for thrombophilic women during pregnancy.

Influence of proband's characteristics on the risk for venous thromboembolism in relatives with factor V Leiden or prothrombin G20210A polymorphisms

AbstractIn family studies, the risk for venous thromboembolism (VTE) in relatives with factor V Leiden (FVL) or G20210A prothrombin (PT20210A) gene polymorphisms may differ according to genotype and clinical presentation of the proband. To address this hypothesis, a retrospective cohort family study was carried out on 192 kindreds with at least one member with homozygous FVL or PT20210A, for a total of 886 relatives. The proband of the family was heterozygous in 68 and homozygous or with both polymorphisms in 124 kindreds. Twenty-three probands were asymptomatic, 11 had had arterial thrombosis, 7 obstetrical complications, and 151 venous thrombosis (122 VTE and 29 superficial vein thrombosis). The incidence of VTE (per 1000 patient-years) in relatives was higher when the proband had heterozygous rather than homozygous polymorphism (1.25 [95% confidence interval (CI), 0.73-1.91] vs 0.44 [0.19-0.78]) and when the proband had had VTE instead of other or no clinical manifestations (0.95 [0.57-1.42] vs 0.50 [0.19-0.96]). Compared with relatives belonging to kindreds with homozygous probands without VTE, the adjusted hazard ratio of VTE for relatives selected from kindreds with heterozygous probands with VTE was 4.14 (95% CI, 1.17-14.71). The genotype and clinical presentation of the proband influence the risk for VTE in relatives with FVL or PT20210A.

Coagulopathy triggered autoimmunity: experimental antiphospholipid syndrome in factor V Leiden mice

BackgroundWe investigated interactions between genetically and autoimmune-mediated coagulopathies by inducing experimental antiphospholipid syndrome (eAPS) in mice carrying the factor V Leiden (FVL) mutation.MethodseAPS was induced in heterozygous and homozygous FVL transgenic mice (C57BL/6 background) by immunization with β2-glycoprotein I (β2-GPI). Autoantibody levels were measured at 1 and 5 months post-immunization. Mice were tested at 4 months post-immunization for behavior and cognitive function in the staircase, elevated plus-maze, and swim T-maze tests. Brains were removed and analyzed by immunohistochemistry for inflammatory markers and neurodegenerative processes.ResultsA single immunization with β2-GPI induced significantly higher and longer-lasting immune responses, and this was dependent on the number of FVL alleles. At 1 and 5 months post-immunization, levels of antibodies rose from 1.17 ± 0.07 to 1.62 ± 0.17 (optical density units; ODU) in homozygous FVL mice, compared with stable levels of 0.59 ± 0.17 and 0.48 ± 0.16 ODU in heterozygous FVL mice and a drop from 1.62 ± 0.21 to 0.61 ± 0.13 ODU in wild-type mice. Behavioral and cognitive clinical features of eAPS were also correlated with FVL allele load, as assessed by the elevated plus-maze (altered anxiety), staircase (hyperactivity and higher exploration), and swim T-maze (impaired learning) tests. Histological studies identified significant neurodegenerative changes in both grey and white matter in the eAPS-FVL brains. In spite of the potential interaction of two prothrombotic disease states, there were no ischemic lesions seen in this group.ConclusionsThe results indicate that genetically mediated coagulopathies increase the risk of developing coagulation-targeted autoimmune responses, and suggest the importance of antibody-mediated neurodegenerative processes in the brain in APS.

Homozygous factor V Leiden and double heterozygosity for factor V Leiden and prothrombin mutation

The most common forms of familial thrombophilia are factor V Leiden (FVL) and prothrombin mutation (PTM). Homozygous FVL and PTM have long been feared conditions thought to cause high rates of morbidity and mortality. To analyse clinical features in patients with homozygous FVL and PTM, as well as patients with double heterozygosity for FVL and PTM. All patients with homozygous FVL, PTM or double heterozygosity in the MATS database of 1465 consecutive unselected patients were analysed regarding age at inclusion venous thromboembolism (VTE), age at first thrombosis, recurrence, clinical course and acquired risk factors. We found 36 patients homozygous for FVL. Patients homozygous for FVL were younger than controls at group level (56±18 vs. 63±17, p<0.02). Homozygous women were younger than female controls (50±19 vs. 63±18, p<0.002). No difference was observed when comparing male subjects. Women were younger than men at inclusion thrombosis (50±19 vs. 65±14, p<0.02) and at first thrombosis (47±19 vs. 64±14, p<0.01). Deep venous thrombosis (DVT) was seen in 33 patients (92%), 6 (17%) had pulmonary embolism (PE) and 3 (8%) had combined DVT and PE. PE was less frequent in homozygous FVL women compared to female controls (p<0.03). VTE recurred in 3 subjects during the duration of the study. Odds ratio for VTE in homozygous FVL patients compared to controls was 13.9 (95% CI 9.9-19.7). We found no subjects with homozygous PTM. Double heterozygosity for FVL and PTM was seen in 12 subjects. There was no difference in age at inclusion VTE between double heterozygotes and controls (59±16 vs. 63±17, ns.). DVT was seen in 92% at inclusion, 8% had PE. Mean age at first VTE was 52±17 (27-82). Consecutive homozygous FVL patients had a higher age at first thrombosis than previously described. Homozygous females are affected at an earlier age than homozygous men and female controls. It seems that thrombi in homozygous FVL have a different pattern compared to controls i.e. more prone for thrombosis in the lower extremity. The odds ratio for thrombosis among homozygous FVL seems to be lower than previously described.

Factor V Leiden: to test or not to test, that is the debate.

The first reference to factor V Leiden (FVL) is in a letter to the journal Nature by Bertina and colleagues1 from the University of Leiden in the Netherlands. This is now a citation classic, having been cited a remarkable 3,279 times (Google Scholar - accessed 14 May 2012). The paper followed the key discovery by Dahlback of an autosomal dominantly inherited form of activated protein C resistance which was associated with a predisposition to venous thrombosis2. A point mutation in the factor V gene that causes a missense mutation in the protein (Arg506Gln, factor V Leiden) renders the molecule less susceptible to inactivation by activated protein C. The mutation is found in 3–7% of the healthy Caucasian population and is associated with a sevenfold increase in the risk of thrombosis3. The presence of the FVL defect is associated with venous thromboembolism as well as with recurrent miscarriage and other pregnancy complications such as severe preeclampsia, growth retardation and stillbirth. Testing for FVL is fairly straightforward because in practise it is usually associated with a single mutation3. Just because a test is easy to perform however, does not mean that there is value in performing it, unless knowledge of the result alters the management of the affected individual. It is sometimes assumed that identification of a positive result is always going to be helpful but positive patients frequently suffer from psychological anxiety as a result of knowing they have a genetic disease, may suffer higher insurance premiums, and there is an actual monetary cost associated with performance of the test. In this issue of Blood Transfusion, Franchini4 and, Favaloro and McDonald5 debate the utility of FVL testing. The clinical utility of a test is its ability to influence or alter a clinical outcome. Franchini describes the background to the development of the FVL test and supports the recommendations of the American College of Medical Genetics in selecting categories where testing for FVL is justified, but also argues for the addition of blood donors to these categories4,6. With the aim of improving blood donor safety, the Italian authorities recommend avoidance of donors who are homozygous for FVL or who have combined thrombophilia but the reference is in Italian and we do not know the precise reasoning behind this recommendation. Even if it is accepted that these thrombophilic individuals are at some risk, it must be questionable whether it is justified to screen blood donors to identify these individuals especially since the prevalence of homozygous FVL in the population would be expected to be around 1 in 1,000. In discussing the futility of FVL testing, Favaloro and McDonald take the opposite view5. The authors observed a dramatic year on year increase in the number of requests for FVL testing in their laboratory service which was accompanied by a concomitant reduction in the number of positive tests, suggesting that tests were being requested inappropriately. Thus, despite the presence of recommendations on who to test for FVL, clinicians are not following these and continue to request the test unselectively. The authors conclude that in the majority of cases FVL testing will actually have a negative impact on identified individuals and their relatives, and clinicians should be discouraged from testing on the basis of theoretical benefits. The issue of thrombophilia testing in general has been tackled by a number of publications including recently the British Committee for Standards in Haematology7. A common finding of many guidelines is, that although testing will identify cases, the evidence that knowledge that an individual is a carrier of a thrombophilic defect rarely influences management. Ultimately the decision of whether to test or not comes from the definition of the term “utility”, if knowing the result will affect your management you should test, but unfortunately this is rarely followed when referring to FVL.

Determination of Factor V Leiden Mutation and R2 Polymorphism in Cis Position

FVA4070G (R2 polymorphism) influences plasma factor V (FV) concentration and was associated with mild activated protein C resistance. This polymorphism was reported to have a trans inheritance with FV Leiden (FVL) mutation. The aim of this study is to investigate the inheritance of R2 polymorphism in the homozygous FVL carriers. In this study, 99 patients with thrombosis and 7 individuals without a history of thrombosis all of which homozygous for FVL were included. Of 99 patients, 1 was heterozygous for FV A4070G. Additionally, 6 polymorphisms in the FV gene were analyzed for the heterozygous R2 patient and her family. When the allelic distribution was classified, 8 different haplotypes were obtained. In contrast to the literature, it was shown that R2 polymorphism could be inherited in cis position with FVL and also the family members could have co-inheritance of the FVL and R2 on the same chromosome as proband.