scholarly journals Quantitative measurement of APC-Resistant factor V clotting activity (FV-Leiden) and potential graduation of the associated thrombo-embolic risk

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
PEYRAFITTE MARIE ◽  
VISSAC MARIE ◽  
AMIRAL JEAN
Keyword(s):  
Blood Coagulation ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Protein S ◽  
Coagulation Cascade ◽  
Factor V ◽  
Thrombotic Risk

Coagulation Factor V (FV) is a key factor for regulating blood coagulation cascade, and it acts at the crossroads of the intrinsic and extrinsic pathways. It shows a dual activity as the procoagulant cofactor for Factor Xa in the prothrombinase complex, but it also supports an anticoagulant activity in combination with TFPI and Protein S. Its rapid cleavage by Activated Protein C (APC) complexed with Free Protein S (FPS), in presence of phospholipids and calcium, inhibits its activity and limits the propagation of blood coagulation, keeping it to where it is beneficial. Rapid inactivation of active FV by APC-FPS is essential for preventing the risk of thrombosis development. In 1993, Dahlbäck and coworkers reported an inherited disorder characterized by activated protein C resistance (APC-R) and associated to an increased occurrence of thromboembolic events in affected families. In 1994 Bertina demonstrated that this diathesis resulted from a Factor V mutation (R506Q), rendering this factor resistant to inactivation by APC. This mutated Factor V was called Factor V Leiden (FV-L). APTT based assays and molecular biology methods for detecting the mutation were developed, but these methods are only qualitative and classify tested individuals as normals, heterozygous or homozygous for the coagulation defect. Our group developed a quantitative assay for FV-L, which is described in this report, along with its performances. This assay allows to quantitate specifically FV-L coagulant activity, and to graduate its amount in heterozygous or homozygous patients. FV-L is absent in normal individuals and present in homozygous or heterozygous patients, accounting respectively for 100 % or 50 % of blood FV. Its amount is compared with FV clotting activity or antigenic concentration. Measured FV-L activities overlap between heterozygous patients with high FV and homozygous ones with low FV levels. This assay allows to better discriminate for the FV-L associated thrombotic risk, which depends on the effective FV-L concentration rather than on patients’ genetic status. This expectation is supported by literature review, which shows that FV-L concentrations correlate with presence of platelet released microparticles in patients carrying that mutation.

The APC-independent anticoagulant activity of protein S in plasma is decreased by elevated prothrombin levels due to the prothrombin G20210A mutation

Blood ◽  
2003 ◽  
Vol 102 (5) ◽  
pp. 1686-1692 ◽  
Author(s):  
Rory R. Koenen ◽  
Guido Tans ◽  
René van Oerle ◽  
Karly Hamulyák ◽  
Jan Rosing ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Factor V Leiden ◽  
Protein S ◽  
Prothrombin G20210a ◽  
Healthy Population ◽  
Factor V ◽  
Thrombotic Risk ◽  
G20210a Mutation

AbstractProtein S exhibits anticoagulant activity independent of activated protein C (APC). An automated factor Xa–based one-stage clotting assay was developed that enables quantification of the APC-independent activity of protein S in plasma from the ratio of clotting times (protein S ratio [pSR]) determined in the absence and presence of neutralizing antibodies against protein S. The pSR was 1.62 ± 0.16 (mean ± SD) in a healthy population (n = 60), independent of plasma levels of factors V, VIII, IX, and X; protein C; and antithrombin, and not affected by the presence of factor V Leiden. The pSR strongly correlates with the plasma level of protein S and is modulated by the plasma prothrombin concentration. In a group of 16 heterozygous protein S–deficient patients, the observed mean pSR (1.31 ± 0.09) was significantly lower than the mean pSR of the healthy population, as was the pSR of plasma from carriers of the prothrombin G20210A mutation (1.47 ± 0.21; n = 46). We propose that the decreased APC-independent anticoagulant activity of protein S in plasma with elevated prothrombin levels may contribute to the thrombotic risk associated with the prothrombin G20210A mutation.

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

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4173-4179 ◽  
Author(s):  
Elisabetta Castoldi ◽  
Jeroen M. Brugge ◽  
Gerry A. F. Nicolaes ◽  
Domenico Girelli ◽  
Guido Tans ◽  
...  
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Gene Mutations ◽  
Factor V Leiden ◽  
Activated Partial Thromboplastin Time ◽  
Factor V ◽  
Thrombotic Risk ◽  
Apc Resistance ◽  
Cofactor Activity

Abstract Activated protein C (APC) resistance is a major risk factor for venous thrombosis. Factor V (FV) gene mutations like FVLeiden (R506Q) and FVR2 (H1299R) may cause APC resistance either by reducing the susceptibility of FVa to APC-mediated inactivation or by interfering with the cofactor activity of FV in APC-catalyzed FVIIIa inactivation. We quantified the APC cofactor activity expressed by FVLeiden and FVR2 and determined the relative contributions of reduced susceptibility and impaired APC cofactor activity to the APC resistance associated with these mutations. Plasmas containing varying concentrations of normal FV, FVLeiden, or FVR2 were assayed with an APC resistance assay that specifically measures the APC cofactor activity of FV in FVIIIa inactivation, and with the activated partial thromboplastin time (aPTT)-based assay, which probes both the susceptibility and APC cofactor components. FVR2 expressed 73% of the APC cofactor activity of normal FV, whereas FVLeiden exhibited no cofactor activity in FVIIIa inactivation. Poor susceptibility to APC and impaired APC cofactor activity contributed equally to FVLeiden-associated APC resistance, whereas FVR2-associated APC resistance was entirely due to the reduced APC cofactor activity of FVR2. Thrombin generation assays confirmed the importance of the anticoagulant activity of FV and indicated that FVLeiden homozygotes are exposed to a higher thrombotic risk than heterozygotes because their plasma lacks normal FV acting as an anticoagulant protein.

Comparison of Activated Protein C Resistance With Factor V Leiden Testing by Molecular Assay

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S5-S5
Author(s):  
William R Perry ◽  
Steven W Pipe ◽  
Shih-Hon Li
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Protein S ◽  
European Ancestry ◽  
Variant Form ◽  
Factor V ◽  
Low Protein ◽  
Protein C Activity

Abstract Factor V Leiden (FVL) is a variant form of coagulation factor V that is the most common inherited risk factor for venous thromboembolism in people of European ancestry. FVL is associated with the missense mutation, p.R506Q, which encodes a FV protein resistant to cleavage by activated protein C (APC). Laboratory testing for FVL includes both phenotypic assays that assess APC resistance (APCR) and molecular assays that evaluate FVL genotype (FVLM) directly. Although APCR results are typically highly concordant with FVL genotype, discrepancies are known to occur and may result in inference of an incorrect FV genotype if only APCR testing is used. The objective of this study was to compare the results of these two testing methodologies and to identify potential explanations for discrepancies in results. Data were obtained by searching the electronic medical record of a large academic hospital for patients who underwent both APCR and FVLM testing from 2013 to 2018. APCR was evaluated using the ratio between two dilute Russell Viper Venom Time (DRVVT) tests, one preincubated with a protein C activator derived from A contortrix contortrix venom and the other with vehicle; the validated normal APCR ratio is ≥1.7. FVLM was performed by invader analysis utilizing fluorescence resonance energy transfer (FRET). In total, 424 patients underwent testing with both assays. Of 21 patients who had APCR assay clot times that exceeded the measurement range, all were FVLM negative, and 15 were anticoagulated during APCR testing. Of the 403 patients with reportable results for both tests, 385 (95.5%) patients had normal APCR and were FVLM negative. Of the 18 (4.5%) patients with discrepant results, 15 (3.7%) had an abnormally low APCR but were FVLM negative, and 3 (0.8%) had a normal APCR but were FVLM heterozygous. Among the 15 FVLM-negative patients with abnormal APCR <1.7, 11 (73.3%) were on warfarin with/out enoxaparin and had low protein S activity and/or low protein C activity. Of the 3 FVLM heterozygous patients with normal APCR, 1 was on apixaban. Our results demonstrated a high concordance between APCR phenotype and FVLM genotype. The concordance of APCR and FVLM may be limited in patients with low protein S or low protein C activity and those on a range of anticoagulant therapy.

scholarly journals Coagulation Factor V Mediates Inhibition of Tissue Factor Signaling By Activated Protein C

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 216-216
Author(s):  
Hartmut Weiler ◽  
Hai-Po Liang ◽  
Edward J Kerschen ◽  
Alireza Rezaie ◽  
Jose A. Fernandez ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Protein S ◽  
Survival Advantage ◽  
Factor V ◽  
Apc Resistance

Abstract BACKGROUND: The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. The naturally occurring R506Q Leiden polymorphism in fV largely abrogates the anticoagulant functions of aPC by rendering fVa partially refractory to aPC proteolysis, but also by preventing the formation of the anticoagulant cofactor form of fV. Among patients enrolled in the placebo arm of the PROWESS sepsis trial, heterozygous fV Leiden carriers showed significantly reduced mortality 1, and a similar survival advantage of heterozygous Leiden carriers was documented in mice harboring the fV R504Q mutation (equivalent to the human R506Q mutation) that were challenged with endotoxin1, gram-positive (S.aureus), or gram-negative infection (Y.pestis)2. The objective of the current study was to examine how aPC-resistance of fV Leiden modulates responsiveness to sepsis therapy with aPC in mice. RESULTS: In murine sepsis models of S.aureus-induced septic peritonitis, aPC-resistance of endogenous fV R504Q prevents marked disease stage-specific deleterious effects associated with aPC's anticoagulant activity, but also abrogated the mortality-reducing benefits of therapy with the signaling-selective 5A-aPC variant that only exerts minimal anticoagulant activity towards activated fVa. In mice homozygous for the R504Q mutation (fVQQ mice), 5A-aPC failed to suppress inflammatory gene expression in the presence of fVR504Q. This finding was reproduced in an in vitro culture model of murine RAW cells and bone marrow-derived dendritic cells, in which thrombosis and thrombin generation play no role. Gene expression analyses and functional in vitro studies of LPS-induced inflammatory cell signaling showed that fV, as well as protein S were required for the aPC-mediated suppression of inflammatory tissue factor-PAR2 signaling3. Structure-function analyses of recombinant variants of aPC and fV showed that this anti-inflammatory cofactor function of protein S and fV involved the same structural features that underlie their accessory role for aPC's anticoagulant function, but did not involve the degradation of activated fVa or fVIIIa. CONCLUSION: These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection. This cofactor function is structurally related, but mechanistically distinct from the anticoagulant cofactor activities of protein S and fV. APC-resistance of fV thus emerges as a response modifier of the endogenous host response to infection, as well as the outcome of sepsis therapy with normal APC and signaling-selective variants thereof. REFERENCES 1. Kerlin BA, Yan SB, Isermann BH, et al. Survival advantage associated with heterozygous factor V Leiden mutation in patients with severe sepsis and in mouse endotoxemia. Blood. 2003;102(9):3085-3092. 2. Kerschen E, Hernandez I, Zogg M, Maas M, Weiler H. Survival advantage of heterozygous factor V Leiden carriers in murine sepsis. J Thromb Haemost. 2015;13(6):1073-1080. 3. Liang HP, Kerschen EJ, Hernandez I, et al. EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice. Blood. 2015. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Coagulation Factor V Leiden Mutation Was Detected in the Patients with Activated Protein C Resistance in Thailand

1998 ◽  
Vol 80 (08) ◽  
pp. 344-345 ◽  
Author(s):  
Pasra Arnutti ◽  
Motofumi Hiyoshi ◽  
Wichai Prayoonwiwat ◽  
Oytip Nathalang ◽  
Chamaiporn Suwanasophon ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Factor V Leiden Mutation ◽  
Coagulation Factor V

“Pseudo Homozygous” Activated Protein C Resistance due to Double Heterozygous Factor V Defects (Factor V Leiden Mutation and Type I Quantitative Factor V Defect) Associated with Thrombosis: Report of Two Cases Belonging to Two Unrelated Kindreds

1996 ◽  
Vol 75 (03) ◽  
pp. 422-426 ◽  
Author(s):  
Paolo Simioni ◽  
Alberta Scudeller ◽  
Paolo Radossi ◽  
Sabrina Gavasso ◽  
Bruno Girolami ◽  
...  
Keyword(s):  
Protein C ◽  
Dna Analysis ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Type I ◽  
Factor V ◽  
Factor V Leiden Mutation ◽  
Thrombotic Risk ◽  
Apc Resistance ◽  
Quantitative Factor

SummaryTwo unrelated patients belonging to two Italian kindreds with a history of thrombotic manifestations were found to have a double heterozygous defect of factor V (F. V), namely type I quantitative F. V defect and F. V Leiden mutation. Although DNA analysis confirmed the presence of a heterozygous F. V Leiden mutation, the measurement of the responsiveness of patients plasma to addition of activated protein C (APC) gave results similar to those found in homozygous defects. It has been recently reported in a preliminary form that the coinheritance of heterozygous F. V Leiden mutation and type I quantitative F. V deficiency in three individuals belonging to the same family resulted in the so-called pseudo homozygous APC resistance with APC sensitivity ratio (APC-SR) typical of homozygous F. V Leiden mutation. In this study we report two new cases of pseudo homozygous APC resistance. Both patients experienced thrombotic manifestations. It is likely that the absence of normal F. V, instead of protecting from thrombotic risk due to heterozygous F. V Leiden mutation, increased the predisposition to thrombosis since the patients became, in fact, pseudo-homozygotes for APC resistance. DNA-analysis is the only way to genotype a patient and is strongly recommended to confirm a diagnosis of homozygous F. V Leiden mutation also in patients with the lowest values of APC-SR. It is to be hoped that no patient gets a diagnosis of homozygous F. V Leiden mutation based on the APC-resi-stance test, especially when the basal clotting tests, i.e., PT and aPTT; are borderline or slightly prolonged.

scholarly journals Cryo-EM structures of human coagulation factors V and Va

Blood ◽  
2021 ◽  
Author(s):  
Eliza A Ruben ◽  
Michael J Rau ◽  
James Fitzpatrick ◽  
Enrico Di Cera
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Proteolytic Processing ◽  
Coagulation Cascade ◽  
Factor V ◽  
Prothrombinase Complex ◽  
A2 Domain

Coagulation factor V is the precursor of factor Va that, together with factor Xa, Ca2+ and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. Here we present cryo-EM structures of human factors V and Va at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding factor Xa and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain responsible for prothrombin binding. Ordering of this region and full exposure of the factor Xa epitope emerge as a necessary step for the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of factors V and Va and pioneer the analysis of coagulation factors by cryo-EM.

scholarly journals High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance) [see comments]

Blood ◽  
1995 ◽  
Vol 85 (6) ◽  
pp. 1504-1508 ◽  
Author(s):  
FR Rosendaal ◽  
T Koster ◽  
JP Vandenbroucke ◽  
PH Reitsma
Keyword(s):  
Venous Thrombosis ◽  
Protein C ◽  
Absolute Risk ◽  
Thrombotic Event ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Factor V ◽  
Increased Risk ◽  
The Absolute

Resistance to activated protein C (APC) is a common inherited risk factor for venous thrombosis, which is associated with a mutation in coagulation factor V (factor V Leiden). We investigated the risk of venous thrombosis in individuals homozygous for this abnormality. We determined the factor V Leiden genotype in 471 consecutive patients aged less than 70 years with a first objectively confirmed deep-vein thrombosis and in 474 healthy controls. We found 85 heterozygous and seven homozygous individuals among the cases with thrombosis and 14 heterozygous individuals among the control subjects. The expected number of homozygous individuals among the controls was calculated from Hardy-Weinberg equilibrium and estimated at 0.107 (allele frequency, 1.5%). Whereas the relative risk was increased sevenfold for heterozygous individuals, it was increased 80-fold for homozygous individuals. These patients experienced their thrombosis at a much younger age (31 v 44 years). The homozygous individuals were predominantly women, most likely due to the effect of oral contraceptives. Because of the increased risk of thrombosis with age, the absolute risk becomes most pronounced in older patients, both for heterozygous and homozygous individuals. For the homozygous individuals, the absolute risk may become several percentage points per year. This implies that most individuals homozygous for factor V Leiden will experience at least one thrombotic event in their lifetime.

Evaluation of a New Assay Based on a FV-Dependent Prothrombinase Activator for the Detection of Activated Protein C Resistance Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3994-3994
Author(s):  
Jogin Wu ◽  
Peter Quehenberger ◽  
Katherine Foltyn ◽  
Patricia Dillard
Keyword(s):  
Protein C ◽  
Tertiary Care ◽  
False Negative ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Reference Method ◽  
Coagulation Cascade ◽  
Factor V ◽  
Resistance Phenotype ◽  
Apc Resistance

Abstract Activated protein C (APC) resistance is the most frequent hereditary defect associated with deep venous thrombosis. Major cause of APC resistance phenotype is due to a point mutation of factor V (Factor V Leiden). A new clotting assay, Pefakit® APC-R Factor V Leiden (Pentapharm Ltd., Switzerland) for the detection of APC resistance phenotype was evaluated at two tertiary care hospitals, Duke University Medical Center, USA (Duke) and University of Vienna, Austria (Vienna). Samples of 242 subjects from Duke and 187 subjects from Vienna were included in the study among patients who were subjects for thormbophilia screening. The Pefakit® method is based on clotting time measurement triggered by a prothrombin activator added to a mixture of patient plasma diluted with factor V deficient plasma with and without APC. Robustness and specificity of the assay is enhanced by elimination of possible disturbing influence by factors upstream the coagulation cascade and heparin interference is precluded up to heparin level of 2 IU/ml by heparin inhibitor added to the regent. A similar, FDA approved commercial APC-R kit (COATEST of IL) was used for method comparison. Patients with elevated factor VIII (n=11), Coumadin (n=23), Lupus Anticoagulant (n-14), protein C deficient (n=7), protein S deficient (n=9), AT III (n=6) and women with pregnancy (n=9) were included in Duke study and no interference were found in phenotype. Using PCR/FRET DNA method as reference method the Pefakit® method provided 100 % sensitivity and 100 % specificity for the Vienna study and 99.0 % sensitivity and 98.6 % specificity for the Duke study and the COATEST provided 97.1 % specificity and 93.2 % sensitivity with the Duke study. Using two levels of genotype controls both studies showed similar intra and inter-assay precision (less than 6 % for the Vienna study and 9 % for the Duke study) as compared with the gold standard IL APC-R COATEST kit (less than 5 % CV). Of great interest one false positive sample from the Duke study is under investigation due to that the functional detection of the assay is supposed to detect other FV mutations leading to APC-R phenotype as well. Reasons that cause the other two false negative results for the Duke study are still unknown and under investigation. Both studies showed that the Pefakit® is simple and rebust assay. Both wild type and heterozygous groups have much higher ratio as compared with the reference method in differentiating them from homozygous phenotype. Figure Figure

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