Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd ∼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (μM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.

Russell's viper venom factor V (FV) activator (RVV-V) is a thrombin-like proteinase that specifically cleaves the Arg1545-Ser1546 bond of FV. Here we present the crystal structure of RVV-V in complex with the FV14 peptide (residues 1533-1546 of human FV) determined at 1.8Å resolution. The structure reveals multiple interactions between RVV-V and the seven residues, Ile1539 (P(7))-Arg1545 (P(1)), of the cleaved substrate. Comparison with substrate-free structures reveals conformational changes of the RVV-V loops upon substrate binding, suggesting that the multiple interactions are mediated by an induced-fit mechanism. The results provide an explanation for the narrow specificity of RVV-V.

Coagulation factor V (fV) is the precursor of activated fV (fVa), an essential component of the prothrombinase complex required for the rapid activation of prothrombin in the penultimate step of the coagulation cascade. In addition, fV regulates the tissue factor pathway inhibitor α (TFPIα) and protein C pathways that inhibit the coagulation response. A recent cryogenic electron microscopy (cryo-EM) structure of fV has revealed the architecture of its A1-A2-B-A3-C1-C2 assembly but left the mechanism that keeps fV in its inactive state unresolved because of an intrinsic disorder in the B domain. A splice variant of fV, fV short, carries a large deletion of the B domain that produces constitutive fVa-like activity and unmasks epitopes for the binding of TFPIα. The cryo-EM structure of fV short was solved at 3.2 Å resolution and revealed the arrangement of the entire A1-A2-B-A3-C1-C2 assembly. The shorter B domain stretches across the entire width of the protein, making contacts with the A1, A2, and A3 domains but suspended over the C1 and C2 domains. In the portion distal to the splice site, several hydrophobic clusters and acidic residues provide a potential binding site for the basic C-terminal end of TFPIα. In fV, these epitopes may bind intramolecularly to the basic region of the B domain. The cryo-EM structure reported in this study advances our understanding of the mechanism that keeps fV in its inactive state, provides new targets for mutagenesis and facilitates future structural analysis of fV short in complex with TFPIα, protein S, and fXa.

Factor V is an essential clotting factor that plays a key role in the blood coagulation cascade on account of its procoagulant and anticoagulant activity. Eighty percent of circulating factor V is produced in the liver and the remaining 20% originates in the α-granules of platelets. In humans, the factor V gene is about 80 kb in size; it is located on chromosome 1q24.2, and its cDNA is 6914 bp in length. Furthermore, nearly 190 mutations have been reported in the gene. Factor V deficiency is an autosomal recessive coagulation disorder associated with mutations in the factor V gene. This hereditary coagulation disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft-tissue bleeds to potentially fatal hemorrhages. Current treatment of this condition consists in the administration of fresh frozen plasma and platelet concentrates. This article describes the cases of two patients with severe factor V deficiency, and of their parents. A high level of mutational heterogeneity of factor V gene was identified, nonsense mutations, frameshift mutations, missense changes, synonymous sequence variants and intronic changes. These findings prompted the identification of a new mutation in the human factor V gene, designated as Jaén-1, which is capable of altering the procoagulant function of factor V. In addition, an update is provided on the prospects for the treatment of factor V deficiency on the basis of yet-to-be-developed recombinant products or advanced gene and cell therapies that could potentially correct this hereditary disorder.

Numerous studies examined the association between factors FV, FVII, FXII, and FXIII-A gene polymorphisms and ischemic stroke, but conclusive evidence is yet to be obtained. Thus, this meta-analysis aimed to investigate the novel association of FV rs1800595, FVII rs5742910, FXII rs1801020, and FXIII-A rs5982 and rs3024477 polymorphisms with ischemic stroke risk. A systematic review was performed on articles retrieved before June 2018. Relevant data were extracted from eligible studies and meta-analyzed using RevMan version 5.3. The strength of association between studied polymorphisms and ischemic stroke risk was calculated as odds ratios and 95% confidence intervals, by applying both fixed- and random-effect models. A total of 25 studies involving 6100 ischemic stroke patients and 9249 healthy controls were incorporated in the final meta-analysis model. Specifically, rs1800595, rs5742910, rs1801020, rs5982, and rs3024477 consisted of 673, 3668, 922, 433, and 404 cases, as well as 995, 4331, 1285, 1321, and 1317 controls, respectively. The pooled analysis indicated that there was no significant association of FV rs1800595, FVII rs5742910, FXII rs1801020, FXIII-A rs5982, and FXIII-A rs3024477 polymorphisms with ischemic stroke risk, under any genetic models (dominant, recessive, over-dominant, and allelic). The present meta-analysis concluded that FV rs1800595, FVII rs5742910, FXII rs1801020, and FXIII-A rs5982 and rs3024477 polymorphisms are not associated with ischemic stroke risk.

FV-Short is a normal splice variant of Factor V (FV) having a short B domain, which exposes a high affinity-binding site for tissue factor pathway inhibitor α (TFPIα). FV-Short and TFPIα circulate in complex in plasma.

Plasma thrombin generation (TG) provides important information on coagulation status; however, current TG output parameters do not predict major bleeding of patients on anticoagulants. We recently reported that factor V (FV) activation by factor X (FX)a contributes importantly to the initiation phase of TG. Here we investigated how this pathway varies in the normal population and whether FXa-mediated activation of FV is associated with major bleeding in patients on anticoagulant therapy.

Factor V together with activated factor X forms the prothrombinase complex, which transforms prothrombin into thrombin. The Mus musculus species is characterized by very high levels of this factor and short clotting times, which hinders accurate measurements. For that reason, a detailed characterization of such parameters is indispensable. A method was designed as part of this study to provide an accurate determination and standardization of factor V levels, prothrombin time and activated partial thromboplastin time in Mus musculus. Those parameters were evaluated in a sample of 66 healthy animals using a semi-automated coagulometer and human diagnostic reagents in an attempt to determine the most appropriate time of day for the extractions. A mouse-based protocol was designed, capable of making corrections to the samples at dilutions of 1:100 for factor V and at of 1:3 for prothrombin time. The goal was to smoothen the calibration curves, which often present with steep slopes and narrow measurement ranges between one calibration point and another. It was found that the most stable period for blood sample extraction was that comprised between the first 6 h of light. No clinical differences were observed between the sexes and reference intervals were established for factor V (95.80% ± 18.14; 25.21 s ± 1.34), prothrombin time (104.31% ± 14.52; 16.85 s ± 1.32) and activated partial thromboplastin time (32.86 s ± 3.01). The results obtained are applicable to human or veterinary biomedical research, to transfusional medicine or to pathological models for diseases such as factor V deficiency.

We investigated interactions between genetically and autoimmune-mediated coagulopathies by inducing experimental antiphospholipid syndrome (eAPS) in mice carrying the factor V Leiden (FVL) mutation.

The role of Factor V Leiden (FVL) mutation in recurrent miscarriages has been disputed. It has been hypothesized that FVL mutation in patients with recurrent miscarriages is treatable. In this study, we evaluated 78 pregnant women for FVL mutations, among whom 50 had a history of recurrent miscarriages. Only 1 (2%) of the woman was positive for heterozygous FVL mutation. The incidence of FVL mutations in patients with recurrent pregnancy loss had an odds ratio of 1.72 (95% confidence interval, 0.0681-43.8257; P>0.05). However, the findings were not statistically significant. Thus, we suggest that FVL mutation study may not be included in the battery of tests for recurrent miscarriages in the Indian population.

Rare inherited coagulation disorders (RICDs) are congenital deficiencies of the plasma proteins that are involved in blood coagulation, which generally lead to lifelong bleeding manifestations. These diseases are generally qualitative and/or quantitative defects that are associated with monoallelic or biallelic mutations in the relevant gene. Among RICDs, factor V (FV) deficiency is one of the least characterized at the molecular level. Here, we investigated four unrelated patients with reduced plasma FV levels (three severe, one mild), which were associated with a moderately severe bleeding tendency. Sequence analysis of the FV gene identified seven different variants, five hitherto unknown (p.D1669G, c.5789-11C>A, c.5789-12C>A, c.5789-5T>G, and c.6528G>C), and two previously reported (c.158+1G>A and c.5789G>A). The possible pathogenic role of the newly identified missense variant was studied by in silico approaches. The remaining six genetic defects (all putative splicing mutations) were investigated for their possible effects on pre-mRNA splicing by transient transfection experiments in HeLa cells with plasmids expressing appropriate hybrid minigenes. The preparation of minigene constructs was instrumental to demonstrate that the two adjacent variants c.5789-11C>A and c.5789-12C>A are indeed present in cis in the analyzed FV-deficient patient (thus leading to the c.5789-11_12CC>AA mutation). Ex vivo experiments demonstrated that each variant causes either a skipping of the relevant exon or the activation of cryptic splice sites (exonic or intronic), eventually leading to the introduction of a premature termination codon.

Thrombophilia is a term used to define the conditions creating a tendency toward thrombosis. Factor V Leiden (FVL) is the most frequently observed genetic risk factor, and its frequency varies among societies and ethnicities. In this study, our aim is to identify the frequency of FVL mutation in patients with thrombosis, the frequency of FVL mutation for each thrombosis disease, whether there is any difference in the geographical distribution of FVL mutation in the Turkish population, correlation with age and gender, and correlation with arterial and venous thrombosis.

No abstract available

ATP6AP2 (also known as the [pro]renin receptor) is a type I transmembrane protein that can be cleaved into two fragments in the Golgi apparatus. While in Drosophila ATP6AP2 functions in the planar cell polarity (PCP) pathway, recent human genetic studies have suggested that ATP6AP2 could participate in the assembly of the V-ATPase in the endoplasmic reticulum (ER). Using a yeast model, we show here that the V-ATPase assembly factor Voa1 can functionally be replaced by Drosophila ATP6AP2. This rescue is even more efficient when coexpressing its binding partner ATP6AP1, indicating that these two proteins together fulfill Voa1 functions in higher organisms. Structure-function analyses in both yeast and Drosophila show that proteolytic cleavage is dispensable, while C-terminus-dependent ER retrieval is required for ATP6AP2 function. Accordingly, we demonstrate that both overexpression and lack of ATP6AP2 causes ER stress in Drosophila wing cells and that the induction of ER stress is sufficient to cause PCP phenotypes. In summary, our results suggest that full-length ATP6AP2 contributes to the assembly of the V-ATPase proton pore and that impairment of this function affects ER homeostasis and PCP signaling.

Factor VIII C-domains are believed to have specific functions in cofactor activity and in interactions with von Willebrand factor. We have previously shown that factor VIII is co-targeted with von Willebrand factor to the Weibel-Palade bodies in blood outgrowth endothelial cells, even when factor VIII carries mutations in the light chain that are associated with defective von Willebrand factor binding. In this study, we addressed the contribution of individual factor VIII C-domains in intracellular targeting, von Willebrand factor binding and cofactor activity by factor VIII/V C-domain swapping. Blood outgrowth endothelial cells were transduced with lentivirus encoding factor V, factor VIII or YFP-tagged C-domain chimeras, and examined by confocal microscopy. The same chimeras were produced in HEK293-cells for in vitro characterization and chemical foot-printing by mass spectrometry. In contrast to factor VIII, factor V did not target to Weibel-Palade bodies. The chimeras showed reduced Weibel-Palade body targeting, suggesting that this requires the factor VIII C1-C2 region. The factor VIII/V-C1 chimera did not bind von Willebrand factor and had reduced affinity for activated factor IX, whereas the factor VIII/V-C2 chimera showed a minor reduction in von Willebrand factor binding and normal interaction with activated factor IX. This suggests that mainly the C1-domain carries factor VIII-specific features in assembly with von Willebrand factor and activated factor IX. Foot-printing analysis of the chimeras revealed increased exposure of lysine residues in the A1/C2- and C1/C2-domain interface, suggesting increased C2-domain mobility and disruption of the natural C-domain tandem pair orientation. Apparently, this affects intracellular trafficking, but not extracellular function.

How individual protein subunits assemble into the higher order structure of a protein complex is not well understood. Four proteins dedicated to the assembly of the V(0) subcomplex of the V-adenosine triphosphatase (V-ATPase) in the endoplasmic reticulum (ER) have been identified in yeast, but their precise mode of molecular action remains to be identified. In contrast to the highly conserved subunits of the V-ATPase, orthologs of the yeast assembly factors are not easily identified based on sequence similarity. We show in this study that two ER-localized Arabidopsis proteins that share only 25% sequence identity with Vma21p can functionally replace this yeast assembly factor. Loss of AtVMA21a function in RNA interference seedlings caused impaired cell expansion and changes in Golgi morphology characteristic for plants with reduced V-ATPase activity, and we therefore conclude that AtVMA21a is the first V-ATPase assembly factor identified in a multicellular eukaryote. Moreover, VMA21p acts as a dedicated ER escort chaperone, a class of substrate-specific accessory proteins so far not identified in higher plants.

Factor V Leiden polymorphism is a well-recognized genetic factor in the etiology of preeclampsia. Considering that Ghana is recording high incidence of preeclampsia, we examined if factor V Leiden is a contributory factor to its development and pregnancy outcomes.

The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H(+)-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1 mutants exhibit a Vma(-) growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma(-) growth defect when combined with the rav1 mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma(-) growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1 mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1 and rav1vph1, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1 perturbed the normal localization of Stv1-green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma(-) phenotypes arise from defects in Vph1p-containing complexes caused by rav1, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.

We describe a mouse model of fetal loss in factor V Leiden (FvL) mothers in which fetal loss is triggered when the maternal prothrombotic state coincides with fetal gene defects that reduce activation of the protein C anticoagulant pathway within the placenta. Fetal loss is caused by disruption of placental morphogenesis at the stage of labyrinth layer formation and occurs in the absence of overt placental thrombosis, infarction, or perfusion defects. Platelet depletion or elimination of protease-activated receptor 4 (Par4) from the mother allows normal placentation and prevents fetal loss. These findings establish a cause-effect relationship for the observed epidemiologic association between maternal FvL status and fetal loss and identify fetal gene defects as risk modifiers of pregnancy failure in prothrombotic mothers. Pregnancy failure is mediated by Par4-dependent activation of maternal platelets at the fetomaternal interface and likely involves a pathogenic pathway independent of occlusive thrombosis. Our results further demonstrate that the interaction of two given thrombosis risk factors produces markedly disparate consequences on disease manifestation (i.e., thrombosis or pregnancy loss), depending on the vascular bed in which this interaction occurs.

Factor V Leiden G1691A (FVL) and Factor II prothrombin G20210A (PGM) mutations are the leading causes of thrombophilia. In this study, we have investigated the prevalence of the FVL G1691A and PGM G20210A single nucleotide polymorphisms (SNPs) among Libyan deep vein thrombosis (DVT) and myocardial infarction (MI) patients. SNP genotyping was performed using high-resolution melt analysis (HRM) and DNA sequencing. Biochemical parameters conducted on 112 males and 93 females showed no significant difference in means between the control group and the deep vein thrombosis and myocardial infarction groups. For Factor V Leiden, 40 samples were genotyped. Of the 40 samples, 6 (15.0%) of them were heterozygous and no one was homozygous. As for Factor II SNP, 59 samples were genotyped and only 2 (3.3%) were heterozygous. All the heterozygous samples showed 100% concordance between the HRM-PCR and DNA sequence analysis. Our study showed, for the first time, that both the FVL and PGM mutations are present among Libyan DVT and MI patients and that the FVL mutation is significantly associated with DVT but not with MI. However, our results do not support the association of PGM G20210A mutation with DVT or MI.