Laboratory Testing for Activated Protein C Resistance (APCR): An Update.

Activated protein C resistance (APCR) reflects a hemostatic state defined by a reduced ability of activated protein C (APC) to affect an anticoagulant response. This state of hemostatic imbalance is characterized by a heightened risk of venous thromboembolism. Protein C is an endogenous anticoagulant that is produced by the hepatocytes and undergoes proteolysis-mediated activation to APC. APC in turn degrades activated Factors V and VIII. APCR describes a state of resistance by activated Factors V and VIII to APC-mediated cleavage of these factors, thereby promoting amplified thrombin production and a potentially procoagulant state. This resistance of APC may be inherited or acquired. Mutations in Factor V are responsible for the most frequent form hereditary APCR. The predominant mutation, a G1691A missense mutation at Arginine 506, the so-called Factor V Leiden [FVL], causes a deletion of an APC-targeted cleavage site in Factor Va, thereby rendering it resistant to inactivation by APC. There are a variety of laboratory assays for APCR, but this chapter focuses on a procedure using a commercially available clotting assay that utilizes a snake venom and ACL TOP analyzers.

Factor V Leiden-independent activated protein C resistance: Communication from the plasma coagulation inhibitors subcommittee of the International Society on Thrombosis and Haemostasis Scientific and Standardisation Committee.

Activated protein C resistance (APC-R) due to the single-nucleotide polymorphism factor V Leiden (FVL) is the most common cause of hereditary thrombophilia. It is found predominantly in Caucasians and is uncommon or absent in other populations. Although FVL is responsible for >90% of cases of hereditary APC-R, a number of other F5 variants that also confer various degrees of APC-R and thrombotic risk have been described. Acquired APC-R due to increased levels of coagulation factors, reduced levels of inhibitors, or the presence of autoantibodies occurs in a variety of conditions and is an independent risk factor for thrombosis. It is common for thrombophilia screening protocols to restrict assessment for APC-R to demonstrating the presence or absence of FVL. The aim of this Scientific and Standardisation Committee communication is to detail the causes of FVL-independent APC-R to widen the diagnostic net, particularly in situations in which in vitro APC-R is encountered in the absence of FVL. Predilution clotting assays are not FVL specific and are used to detect clinically significant F5 variants conferring APC-R, whereas different forms of acquired APC-R are preferentially detected using the classical activated partial thromboplastin time-based APC-R assay without predilution and/or endogenous thrombin potential APC-R assays. Resource-specific recommendations are given to guide the detection of FVL-independent APC-R.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Activated protein C resistance impact on Syrian candidates for in vitro fertilisation and the benefit of anticoagulation therapy: a retrospective cohort study.

Activated protein C resistance (APCR) is a common thrombophilia, caused mainly by a mutation. The impact of APCR on the efficacy of In Vitro Fertilization (IVF) are still unclear, and no solid recommendations for its management were published. To investigate the effect of APCR on IVF outcomes and assess the efficacy of our management protocol, we retrospectively scanned the medical records of women who were tested with APCR assay in 2019 at our fertility centre. The 66 women (12%) positive for APCR had lower odds of reaching clinical pregnancies after IVF 0.18 [95% CI: 0.07-0.47] and fewer live births. The administration of low-molecular-weight heparin and aspirin associated with more implantation in treated compared to untreated APCR-positive women with an odds ratio of 43.2 [7.51-248.6]. In conclusion, APCR negatively affects the number of clinical pregnancies after IVF, but anticoagulation therapy can mitigate this effect and significantly increase clinical pregnancies.Impact StatementWhat is already known on this subject? The evidence about the impact of APCR on IVF outcomes is still inconclusive. According to the Canadian guideline, routine screening for thrombophilia in patients with recurrent pregnancy loss is not recommended. No clear recommendations regarding the management of APCR in the planning for IVF are yet available.What do the results of this study add? APCR significantly increases implantation failure among infertile women who conduct IVF. Management of APCR using LMWH and aspirin was effective in mitigating this effect and increasing successful implantation.What are the implications of these findings for clinical practice and/or further research? Our findings can support the recommendation to include APCR assay in the routine tests for infertile women conducting IVF, and suggest the combination between LMWH and aspirin as an effective therapy to increase successful implantation in APCR positive candidates. However, more controlled clinical trials are still needed to confirm our results.

Main Factors Predicting Nonresponders to Autologous Cell Therapy for Critical Limb Ischemia in Patients With Diabetic Foot.

Autologous cell therapy (ACT) is a new treatment for patients with no-option critical limb ischemia (NO-CLI). We evaluated the factors involved in the nonresponse to ACT in patients with CLI and diabetic foot. Diabetic patients (n = 72) with NO-CLI treated using ACT in our foot clinic over a period of 8 years were divided into responders (n = 57) and nonresponders (n = 15). Nonresponder was defined as an insufficient increase in transcutaneous oxygen pressure by <5 mm Hg, 3 months after ACT. Patient demographics, diabetes duration and treatment, and comorbidities as well as a cellular response to ACT, limb-related factors, and the presence of inherited thrombotic disorders were compared between the 2 groups. The main independent predictors for an impaired response to ACT were heterozygote Leiden mutation (OR 10.5; 95% CI, 1.72-4) and homozygote methylenetetrahydrofolate reductase (MTHFR 677) mutation (OR 3.36; 95% CI, 1.0-14.3) in stepwise logistic regression. Univariate analysis showed that lower mean protein C levels (P = .041) were present in nonresponders compared with responders. In conclusion, the significant predictors of an impaired response to ACT in diabetic patients with NO-CLI were inherited thrombotic disorders.

Prevalence of the Factor V Leiden Mutation Arg534Gln in Western Region of Saudi Arabia: Functional Alteration and Association Study With Different Populations.

The rare Gln534 (Factor V Leiden; FVL) allele (1:169,519,049 T>C) is associated with an increased risk of venous thrombosis. The purpose of this study was to measure the prevalence of Factor V Leiden mutation in thrombophilia patients with deep vein thrombosis. Also, we investigated the functional and structural characteristics of this mutation p.(Arg534Gln) to be examined the cumulative impact on venous thrombosis risk as well correlated with different populations by Genome Wide Association Studies (GWAS). A total of 108 patients with idiopathic deep vein thrombosis were examined for Factor V Leiden gene mutation. Our preliminary data show that about 10% of patients were detected with the heterozygous and homozygous form of the Factor V Leiden mutation. An association analysis confirmed that the Factor V SNP variant (rs6025) was highly associated (P-value 4.91 x10-^ -39) with an increased risk of venous thrombosis. Also, we found that the recognized SNP was important among HapMap populations. Our results indicated that among the 3 populations (Asian, African, and American) studied, this association was highest in the African population based on the r(2) significant threshold (P-value 5e-190). In addition, this mutation was located at the domain F5/8 type A 2, which can disturb this domain and abolish its function. Because of aspartic acid nearby wild type position as form in the salt bridge due to this discharge will disturb the ionic interaction made by the wild type residue Arg534. This residue was not found to be in contact with other domains of which the function was known. However, contact with other molecules or domains (THPH2: MIM: 188055) were still possible and might be affected by this mutation that may cause thrombophilia due to activated protein C resistance.

Pulmonary embolism in Behcet's disease: a case report.

Behcet's disease (BD) is a vasculitis of unknown etiology. It is often correlated with thrombophilic factors such as V Leiden. Pulmonary involvement is reported in 1-10% of patients. The most common manifestations are pulmonary aneurysms while pulmonary embolism is a rare complication. A 41-year old man with BD and V Leiden heterozygosity complained of pleurodynia and fever. Pleurodynia deteriorated in the following days and PE was confirmed by CT angiography, without the presence of aneurysms. After the exclusion of the antiphospholipid syndrome, a therapeutic dose of apixaban was initiated. Two weeks later, pleurodynia relapsed in combination with pleural effusion unilaterally. These findings were attributed to disease exacerbation. For this reason, we decided to enhance the immunosuppressive therapy. Six months later, CTPA showed complete remission of the clots. Vasculitis predisposes to thrombosis with or without coexisting thrombophilia. Clinicians should include them in their differential diagnosis and provide personalized treatment, based on immunosuppressants.

Interaction of thrombophilic SNPs in patients with unexplained infertility-multifactor dimensionality reduction (MDR) model analysis.

PURPOSE: Our aim was to evaluate the frequency and SNP-SNP interactions between factor V Leiden (FVL) G1691A, prothrombin G20210A mutation, and C677T MTHFR and PAI-1 4G/5G gene polymorphisms in female IVF patients with unexplained infertility (UI) by using a multifactor dimensionality reduction (MDR) model analysis. METHODS: A total of 225 subjects were enrolled in the study. There were 105 females in UI group and 120 healthy controls. Designated SNPs were determined by using allele-specific PCR methods. The difference in thrombophilia prevalence was assessed by a chi-square test and logistic regression analysis. Four-locus SNP interaction model was tested using the MDR approach. A ten-fold cross-validation consistency (CVC) and permutation testing were performed. RESULTS: There was a significant difference of MTHFR C677T polymorphism frequency between the groups. Significantly less UI patients had MTHFR CC genotype (p = 0.005), while the risk allele T was more frequent (OR = 1.83, p = 0.0018). Logistic regression determined a significant association only for MTHFR C677T in our patients (TT genotype OR = 2.99). The MDR analysis confirmed the significance of a single-locus model for MTHFR C677T polymorphism (p = 0.015; OR = 2.93). However, the best, significant predictive model was the two-locus model comprising MTHFR C677T and FVL (CVC = 10/10, testing accuracy = 60.95%, p = 0.013; OR = 3.02). CONCLUSION: The MTHFR C677T polymorphism was significantly associated with UI, with minor allele T being more frequent. Additionally, there was a significantly increased presence of MTHFR C677T with FVL mutation in these patients. Therefore, MTHFR and its interaction with FVL should be recognized as contributing factors in the pathogenesis of infertility.

Comparative analysis of "APTT vs RVVT" based activated protein C resistance assay in the diagnosis of Factor V Leiden mutation.

BACKGROUND: Thrombophilia is a hypercoagulable state characterized by increased venous thrombosis. The most common cause of heritable thrombophilia is Factor V Leiden (FVR506Q) homozygous state, with a relative risk of 10-80 times as compared to normal individuals and Lupus anticoagulant is the most common cause of acquired thrombophilia. The main objective of this study is to compare the sensitivity of activated partial thromboplastin time (APTT) vs dilute Russell viper venom test (DRVVT) based APCR assays with predilution in Factor V-deficient plasma for diagnosis of Factor V Leiden mutation. MATERIALS AND METHODS: The coagulometer used for APCR test was Sysmex CS-5100. APTT reagent used is Pathrombin SL supplied by Siemens. All data were expressed as mean ± SD. Statistical analysis was done using unpaired students t-test and a P value <0.05 was considered as statistical significance. RESULTS: A total of 300 cases of APCR (200 cases of Factor V Leiden mutation was confirmed by PCR and 100 acquired) were studied. The sensitivity of screening APTT-based APCR for detection of Factor V Leiden mutation is 67% and for the noncarrier state, it is 62%. The sensitivity of modified APTT and DRVVT with predilution in FV-deficient plasma for detection of Factor V Leiden mutation is 82% and 84%, respectively and for acquired causes, it is 48% and 86%, respectively. CONCLUSION: Screening APTT test has increased in activated protein C resistance (APCR) due to Factor V Leiden mutation as well as acquired causes such as patients on direct-acting oral anticoagulants, warfarin, lupus anticoagulants, and oral contraceptive pills which are independent risk factors of venous thrombosis. Modified DRVVT with predilution in FV-deficient plasma is more sensitive than screening and modified APTT-based APCR test in the diagnosis of Factor V Leiden mutation and the former test can distinguish homozygous and heterozygous states from normal individuals.

Inherited Thrombophilia in a Lebanese Family of Four Generations: A Case Report of Recurrent Miscarriage.

INTRODUCTION: Factor V Leiden (G1691A), prothrombin (G20210A) and MTHFR (C677T) gene mutations were investigated in many studies for their association with Deep Venous Thrombosis. CASE PRESENTATION: A North Lebanese family has been examined, from an index case, a 40-year-old woman, who had a history of venous thrombosis with unexplained recurrent miscarriage. The index case was found to be heterozygous for factor V Leiden G1691A, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T gene variants. Her family members were heterozygous for at least two of the three-point mutations, and multiple risk factors associated with thrombophilia were identified. CONCLUSION: Our findings emphasize the need for clarifying the utility and futility of thrombophilia testing in the era of molecular diagnostics.

Apixaban Does Not Interfere With Protein S or Activated Protein C Resistance (Factor V Leiden) Testing Using aPTT-Based Methods.

CONTEXT.­: Apixaban causes a false increase in activated protein C resistance (APCR) ratios and possibly protein S activity. OBJECTIVE.­: To investigate whether this increase can mask a diagnosis of factor V Leiden (FVL) or protein S deficiency in an actual population of patients undergoing apixaban treatment and hypercoagulation testing. DESIGN.­: During a 4.5-year period involving 58 patients, we compared the following 4 groups: heterozygous for FVL (FVL-HET)/taking apixaban, wild-type/taking apixaban, heterozygous for FVL/no apixaban, and normal APCR/no apixaban. Patients taking apixaban were also tested for protein S functional activity and free antigen (n = 40). RESULTS.­: FVL-HET patients taking apixaban had lower APCR ratios than wild-type patients (P < .001). Activated protein C resistance in FVL-HET patients taking apixaban fell more than 3 SD below the cutoff of 2.2 at which the laboratory reflexes FVL DNA testing. No cases of FVL were missed despite apixaban. In contrast to rivaroxaban, apixaban did not interfere with the assessment of protein S activity (mean activity 93.9 IU/dL, free antigen 93.1 IU/dL, P = .39). A total of 3 of 40 patients (8%) had low free protein S antigen (30, 55, and 57 IU/dL), with correspondingly similar activity results (27, 59, and 52 IU/dL, respectively). Apixaban did not cause a missed diagnosis of protein S deficiency. CONCLUSIONS.­: Despite apixaban treatment, APCR testing can distinguish FVL-HET from healthy patients, rendering indiscriminate FVL DNA testing of all patients on apixaban unnecessary. Apixaban did not affect protein S activity.

[Retinal artery occlusion and anterior ischemic optic neuropathy associated with factor V Leiden mutation: A case report]. / Occlusion de l'artère centrale de la rétine associée à une neuropathie optique ischémique antérieure secondaire à une mutation du facteur V Leiden : à propos d'un cas.

Factor V is a pro-coagulant cofactor required for the transformation of prothrombin into thrombin. Thrombin activates factor V, which is then deactivated by protein C. A mutation in factor V is responsible for the formation of factor V Leiden, resistant to activated protein C. The association of this mutation with venous thromboses has been established. Its association with arterial occlusions is still controversial. We report the case of a central retinal artery occlusion associated with a non-arteritic anterior optic neuropathy associated with a Leiden mutation of factor V (FVL). The presence of FVL has been associated with lack of reperfusion and rapid progression to neovascularization. It seems that FVL intervenes mainly during the reperfusion phase after the occurrence of arterial thrombosis.

DOAC-Remove abolishes the effect of direct oral anticoagulants on activated protein C resistance testing in real-life venous thromboembolism patients.

Background Direct oral anticoagulants (DOACs) may cause false results of activated protein C resistance (APC-R) ratio. DOAC-Remove, a new reagent based on activated carbon, has been designed to eliminate the interference of DOACs on coagulation assays. The aim of the study was to investigate whether the use of DOAC-Remove enables to determine APC-R in patients treated with DOACs. Methods We assessed 74 venous thromboembolism (VTE) patients, including 25 on rivaroxaban, 25 on apixaban and 24 taking dabigatran. APC-R was determined using the Russell Viper Venom Time (RVVT)-based clotting test. APC-R and DOAC concentrations were tested at baseline and following DOAC-Remove. Thrombophilia, including factor V Leiden (FVL) mutation was tested. Results FVL mutation was found in 20 (27%) patients. The APC-R ratio at baseline was measurable in 43 patients (58.1%), including 20 (80%) on rivaroxaban, 19 (76%) on apixaban and four (16.7%) on dabigatran. In patients with measurable APC-R at baseline, the ratio >2.9 was found in 23 patients (53.5%). In 16 (37.2%) subjects APC-R ratio <1.8 suggested FVL mutation which was genetically confirmed. Four (9.3%) FVL carriers on dabigatran showed negative/equivocal APC-R results. In 11 (14.9%) patients taking rivaroxaban or apixaban, in whom blood was collected 2-5 h since the last dose, we observed unmeasurable APC-R. DOAC-Remove almost completely eliminated all plasma DOACs. After addition of DOAC-Remove all APC-R ratios were measurable. In four FVL carriers on dabigatran with false negative APC-R, DOAC-Remove resulted in APC-R ratios <1.8. Conclusions DOAC-Remove effectively reduces DOACs concentration in plasma, which enables FVL testing using APC-R.

Evaluation of Factor V Leiden and prothrombin G20210A mutations in Sudanese women with severe preeclampsia.

BACKGROUND: Preeclampsia (PE) is a common pregnancy complication and one of the main causes of maternal and fetal morbidity and mortality, worldwide. While the pathogenesis of PE is unclear, it has been suggested that hypercoagulability due to Factor V Leiden (FVL) and prothrombin gene mutation (FII G20210A) play a role in its progression. PURPOSE: This study aimed to determine if there is an association between FVL and FII G20210A mutations and severe PE. PATIENTS AND METHODS: This case-control study enrolled 50 women with severe PE and 50 healthy pregnant women as the control, at Khartoum North Teaching Hospital, in Khartoum State, Sudan, from January 2017 to June 2017. The presence of point mutations in FVL and FII G20210A were determined for each of the participants. Deoxyribonucleic acid (DNA) was extracted, and then an allele-specific polymerase chain reaction (PCR) was used to detect the point mutations in FVL and FII G20210A. RESULTS: The results revealed a significant difference between the subjects in the severe PE group and the control group for the means of parity, gestational age/ week and hemoglobin concentration (P < 0.05). No statistically significant body mass index (BMI) differences were found between the groups (P > 0.05). Women with severe PE were found to have a significant difference in FVL (16%; P value = 0.0058; OR: 20.20; 95%CI: 1.132-360.5) and FII G20210A (14%; P value = 0.0125; OR: 17.41; 95%CI: 0.9659-314.0) in comparison to the women in the control group (0%). CONCLUSION: Our findings intensely indicate that there is a statistically proven significant association between FVL, FII G20210A mutations and the development of severe preeclampsia in Sudanese pregnant women.

Antiphospholipid syndrome's genetic and epigenetic aspects.

Studies on last genetic and epigenetic predisposition to APS are summarized. It is well known that genetic predisposition is in HLA system (DR4 and DRw53) and that lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) are both associated with the same HLA antigens. Other genes, outside the MHC, give their contribution to the development of this autoimmune syndrome, such as IRF5, STAT4 and those related to inherited thrombophilia - factor V Leiden and G20210A prothrombin polymorphisms. Finally, post-transcriptional modifications of anti-beta2GPI antibodies could be implicated too. The most important discovery of last years is that altered microRNAs' expression is linked to autoimmunity, thrombosis, early atherosclerosis, and oxidative stress in APS.

Comparison of Phenotypic Activated Protein C Resistance Testing With a Genetic Assay for Factor V Leiden.

Objectives: To compare the accuracy and reliability of phenotypic activated protein C resistance (aPC-R) assays with a genotypic assay for the factor V Leiden F5 p.R506Q (FVL) mutation. Methods: Data were obtained from an electronic data warehouse for FVL testing performed at an academic institution with a large referral laboratory service. In total, 1,596 patients were identified who had undergone both phenotypic aPC-R and genotypic FVL mutation testing. Results: Phenotypic testing showed a high level of sensitivity, specificity, and other biostatistical values compared with genotypic testing. Improvements in technology decreased the amount of equivocal phenotypic results. Conclusions: Phenotypic assays had close to total concordance with genotypic assays over 16 years of testing. Changing ordering practices could result in up to an 80% reduction in testing costs.

Placenta-mediated pregnancy complications are not associated with fetal or paternal factor V Leiden mutation.

OBJECTIVE: Maternal thrombophilia is a risk factor for adverse pregnancy outcomes. The aim of this study was to elucidate the controversial role of fetal and paternal thrombophilia in the development of severe placenta-mediated pregnancy complications. STUDY DESIGN: The study group comprised 126 mothers, 72 fetuses and 58 fathers. 111 mothers, 50 fetuses and 91 fathers acted as controls. 106 couples were selected to study the thrombophilias of paternal inheritance, 58 from the study group and 48 from the control group. The prevalence of factor V Leiden mutation, prothrombin G20210 A mutation and homozygous 10-methylenetetrahydrofolate reductase C677 T mutations were compared between the study and control groups to study whether maternal, fetal or paternal thrombophilias increase the risk of severe preeclampsia, intrauterine growth restriction, placental abruption and stillbirth. RESULTS: The total prevalence of fetal thrombophilic mutations was 8.3% in the study group and 14.0% in the control group. Paternal prevalence of thrombophilic mutations was 6.8% and 4.3%, respectively. There were no statistical differences between fetal or paternal thrombophilic mutations between the study and control groups. CONCLUSION: Fetal or paternal factor V Leiden mutation is not associated with severe placenta-mediated pregnancy complications.