A novel factor V compound heterozygous mutation associated with thrombosis (Y1961C; FV-Kanazawa, together with 1982_1983del).

BACKGROUND: Factor (F)V is pivotal in both procoagulant and anticoagulant mechanisms. The present report describes a novel F5 mutation in a FV-deficient patient (FV activity, 6 IU/dL; FV antigen, 32 IU/dL) complicated by recurrent deep vein thrombosis. The patient demonstrated activated protein C resistance (APCR) with compound heterozygous mutations consisting of FV-Y1961C (FVKanazawa) and FV-1982_1983del. OBJECTIVES: To clarify thrombotic mechanisms associated with this FV abnormality. METHODS AND RESULTS: Levels of FV-1982_1983del were below the detection sensitivity in our expression experiments using human embryonic kidney 293T cells, and analyses were targeted, therefore, on the FV-Y1961C mutation. Activated partial thromboplastin time-based clotting assays demonstrated that FV-Y1961C exhibited APCR and that the reduced activated protein C (APC) susceptibility in FVa-Y1961C resulted in a marked depression of APC-catalyzed inactivation with delayed cleavage at Arg506 and little cleavage at Arg306 with or without protein S. The APC cofactor activity of FV-Y1961C in APC-catalyzed FVIIIa inactivation promoted by Arg336 cleavage in FVIII was impaired. The binding affinity of FVa-Y1961C to phospholipid membranes was reduced in reactions involving APC/protein S-catalyzed inactivation and in prothrombinase activity. Furthermore, the addition of FVa-Y1961C to plasma failed to inhibit tissue factor-induced procoagulant function. These characteristics were similar to those of FV-W1920R (FVNara) and FV-A2086D (FVBesançon). CONCLUSION: We identified a compound heterozygous FV-Y1961C mutation in the C1 domain representing a novel FV mutation (FVKanazawa) resulting in not only APCR due to impaired FVa susceptibility and FV cofactor activity for APC function but also impaired inhibition of tissue factor-induced procoagulant function. These defects in anticoagulant function associated with FV in FV-Y1961C contributed to a prothrombotic state.

Coagulation factor V in breast cancer: a p53-regulated tumor suppressor and predictive marker for treatment response to chemotherapy.

BACKGROUND: Patients with cancer are at an increased risk of developing coagulation complications, and chemotherapy treatment increases the risk. Tumor progression is closely linked to the hemostatic system. Breast cancer tumors express coagulation factor V (FV), an essential factor in blood coagulation. The functional role of FV during treatment with chemotherapy is poorly understood and was explored in this study. OBJECTIVES: We aimed to investigate the role of FV in breast cancer progression by exploring associations with treatment response, gene regulation, and the functional effects of FV. METHODS: The receiver operating characteristic plotter was used to explore the predictive value of FV mRNA (F5) expression for treatment with FEC (5-fluorouracil, anthracycline, and cyclophosphamide). Breast cancer cohorts were analyzed to study treatment response to FEC. The effect of chemotherapy on F5 expression, the regulation of F5, and the functional effects of FV dependent and independent of chemotherapy were studied in breast cancer cell lines. RESULTS: F5 tumor expression was significantly higher in responders to FEC than in nonresponders. In vitro experiments revealed that anthracycline treatment increased the expression of F5. Inhibition and knockdown of p53 reduced the anthracycline-induced F5 expression. Mutation of a p53 half-site (c.158+1541/158+1564) in a luciferase plasmid reduced luciferase activity, suggesting that p53 plays a role in regulating F5. FV overexpression increased apoptosis and reduced proliferation slightly during anthracycline treatment. CONCLUSION: Our study identified F5 as a p53-regulated tumor suppressor candidate and a promising marker for response to chemotherapy. FV may have functional effects that are therapeutically relevant in breast cancer.

KLF5 inhibits the migration and invasion in cervical cancer cell lines by regulating SNAI1.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is an important biological process by which malignant tumor cells to acquire migration and invasion abilities. This study explored the role of KLF5 in the EMT process of in cervical cancer cell lines. OBJECTIVE: Krüpple-like factor 5 (KLF5) is a basic transcriptional factor that plays a key role in cell-cycle arrest and inhibition of apoptosis. However, the molecular mechanism by which KLF5 mediates the biological functions of cervical cancer cell lines has not been elucidated. Here, we focus on the potential function of ELF5 in regulating the EMT process in in vitro model of cervical cancer cell lines. METHOD: Western-blot and real-time quantitative PCR were used to detect the expression of EMT-related genes in HeLa cells. MTT assays, cell scratch and Transwell assays were used to assess HeLa cells proliferation and invasion capability. Using the bioinformatics tool JASPAR, we identified a high-scoring KLF5-like binding sequence in the SNAI1 gene promoter. Luciferase reporter assays was used to detect transcriptional activity for different SNAI1 promoter truncates. RESULT: After overexpressing the KLF5 gene in HeLa cells, KLF5 not only significantly inhibited the invasion and migration of HeLa cells, but also increased the expression of E-cadherin and decreased the expression of N-cadherin and MMP9. In addition, the mRNA expression of upstream regulators of E-cadherin, such as SNAI1, SLUG, ZEB1/2 and TWIST1 was also decreased. Furthermore, KLF5 inhibiting the expression of the SNAI1 gene via binding its promoter region, and the EMT of Hela cells was promoted after overexpression of the SNAI1 gene. CONCLUSION: These results indicate that KLF5 can downregulate the EMT process of HeLa cells by decreasing the expression of the SNAI1 gene, thereby inhibiting the migration and invasion of HeLa cervical cancer cells.

Structural architecture of the acidic region of the B domain of coagulation factor V.

BACKGROUND: Coagulation factor (F)V features an A1-A2-B-A3-C1-C2 domain organization and functions as the inactive precursor of FVa, a component of the prothrombinase complex required for rapid thrombin generation in the penultimate step of the coagulation cascade. An intramolecular interaction within the large B domain (residues 710-1545) involves the basic region (BR, residues 963-1008) and acidic region (AR, residues 1493-1537) and locks FV in its inactive state. However, structural information on this important regulatory interaction or on the separate architecture of the AR and BR remains elusive due to conformational disorder of the B domain. OBJECTIVES: To reveal the structure of the BR-AR interaction or of its separate components. METHODS: The structure of FV is solved by cryogenic electron microscopy. RESULTS: A new 3.05 Å resolution cryogenic electron microscopy structure of FV confirms the overall organization of the A and C domains but resolves the segment 1507 to 1545 within a largely disordered B domain. The segment contains most of the AR and is organized as recently reported in FV short, a spliced variant of FV with a significantly shorter and less disordered B domain. CONCLUSION: The similar architecture of the AR in FV and FV short provides structural context for physiologically important interactions of this region with the BR in FV and with the basic C-terminal end of tissue factor pathway inhibitor α in FV short.

In vitro and ex vivo rescue of a nonsense mutation responsible for severe coagulation factor V deficiency.

BACKGROUND: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents. OBJECTIVES: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy. METHODS: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation. RESULTS: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 µM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo. CONCLUSION: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation.

Developmental adcyap1b loss leads to hemorrhage, disrupted hemostasis, and a blood coagulation cascade in zebrafish.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide is a neuropeptide with diverse roles in biological processes. Its involvement in the blood coagulation cascade is unclear. OBJECTIVES: This study unraveled adcyap1b's role in blood coagulation using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 in zebrafish. Effects were validated via adcyap1b knockdown. Gene expression changes in adcyap1b mutants were explored, linking them to clotting disorders. An analysis of proca gene splicing illuminated its role in adcyap1b-related anticoagulation deficiencies. METHODS: Zebrafish were genetically modified using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to induce adcyap1b knockout. Morpholino-mediated gene knockdown was employed for validation. Expression levels of coagulation factors, anticoagulant proteins, and fibrinolytic system genes were assessed in adcyap1b mutant zebrafish. Alternative splicing of proca gene was analyzed. RESULTS: Adcyap1b mutant zebrafish exhibited severe hemorrhage, clotting disorders, and disrupted blood coagulation. Morpholino-mediated knockdown replicated observed phenotypes. Downregulation in transcripts related to coagulation factors V and IX, anticoagulation protein C, and plasminogen was observed. Abnormal alternative splicing of the proca gene was identified, providing a mechanistic explanation for anticoagulation system deficiencies. CONCLUSION: Adcyap1b plays a crucial role in maintaining zebrafish blood coagulation and hemostasis. Its influence extends to the regulation of procoagulant and anticoagulant pathways, with abnormal alternative splicing contributing to observed deficiencies. These findings unveil a novel aspect of adcyap1b function, offering potential insights into similar processes in mammalian systems.

KLF5 promotes esophageal squamous cell cancer through the transcriptional activation of FGFBP1.

Krüpple-like factor 5 (KLF5) is a zinc-finger-containing transcription factor implicated in several human malignancies, but its potential regulatory mechanisms implicated in esophageal squamous cell carcinoma (ESCC) remain elusive. Here, we show that KLF5 is upregulated in ESCC, where its level was significantly associated with tumor differentiation and lymph node metastasis status. Upregulated KLF5 expression promoted the proliferation, migration, and invasion of ESCC cells. Reduced KLF5 showed the opposite effects. Mechanistically, KLF5 exerts its tumor promotion effect by up-regulating fibroblast growth factor binding protein 1 (FGF-BP1) and snail family transcriptional repressor 2 (SNAIL2). KLF5 binds to the promoter regions of FGF-BP1 and transcriptionally activates its expression. Our study indicated that KLF5 could promote esophageal squamous cell cancer proliferation, migration, and invasion by upregulating FGF-BP1/SNAIL2 signaling. Our work suggests that KLF5 might be a proto-oncogene in ESCC and implicated in ESCC metastasis.

Bromodomain Factor 5 as a Target for Antileishmanial Drug Discovery.

Leishmaniases are a collection of neglected tropical diseases caused by kinetoplastid parasites in the genus Leishmania. Current chemotherapies are severely limited, and the need for new antileishmanials is of pressing international importance. Bromodomains are epigenetic reader domains that have shown promising therapeutic potential for cancer therapy and may also present an attractive target to treat parasitic diseases. Here, we investigate Leishmania donovani bromodomain factor 5 (LdBDF5) as a target for antileishmanial drug discovery. LdBDF5 contains a pair of bromodomains (BD5.1 and BD5.2) in an N-terminal tandem repeat. We purified recombinant bromodomains of L. donovani BDF5 and determined the structure of BD5.2 by X-ray crystallography. Using a histone peptide microarray and fluorescence polarization assay, we identified binding interactions of LdBDF5 bromodomains with acetylated peptides derived from histones H2B and H4. In orthogonal biophysical assays including thermal shift assays, fluorescence polarization, and NMR, we showed that BDF5 bromodomains bind to human bromodomain inhibitors SGC-CBP30, bromosporine, and I-BRD9; moreover, SGC-CBP30 exhibited activity against Leishmania promastigotes in cell viability assays. These findings exemplify the potential BDF5 holds as a possible drug target in Leishmania and provide a foundation for the future development of optimized antileishmanial compounds targeting this epigenetic reader protein.

Activated protein C, protein S, and tissue factor pathway inhibitor cooperate to inhibit thrombin activation.

INTRODUCTION: Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation. MATERIALS AND METHODS: We hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems. RESULTS: In plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS. CONCLUSIONS: We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot.

Lactadherin's Multistate Binding Predicts Stable Membrane-Bound Conformations of Factors V and VIII's C Domains.

Protein binding to negatively charged lipids is essential for maintaining numerous vital cellular processes where its dysfunction can lead to various diseases. One such protein that plays a crucial role in this process is lactadherin, which competes with coagulation factors for membrane binding sites to regulate blood clotting. Despite identifying key binding regions of these proteins through structural and biochemical studies, models incorporating membrane dynamics are still lacking. In this study, we report on the multimodal binding of lactadherin and use it to gain insight into the binding mechanisms of its C domain homologs, factor V and factor VIII. Molecular dynamics simulations enhanced with the highly mobile mimetic model enabled the determination of lactadherin's multimodal binding on membranes that revealed critical interacting residues consistent with prior NMR and mutagenesis data. The binding occurred primarily via two dynamic structural ensembles: an inserted state and an unreported, highly conserved side-lying state driven by a cationic patch. We utilized these findings to analyze the membrane binding domains of coagulation factors V and VIII and identified their preferred membrane-bound conformations. Specifically, factor V's C domains maintained an inserted state, while factor VIII preferred a tilted, side-lying state that permitted antibody binding. Insight into lactadherin's atomistically resolved membrane interactions from a multistate perspective can guide new therapeutic opportunities in treating diseases related to blood coagulation.

The TFPIα C-terminal tail is essential for TFPIα-FV-short-protein S complex formation and synergistic enhancement of TFPIα.

BACKGROUND: For maximal TFPIα functionality, 2 synergistic cofactors, protein S and FV-short, are required. Both interact with TFPIα, protein S through Kunitz 3 residues Arg199/Glu226 and FV-short with the C-terminus. How these interactions impact the synergistic enhancement remains unclear. OBJECTIVES: To determine the importance of the TFPIα-protein S and TFPIα-FV-short interactions for TFPIα enhancement. METHODS: TFPIα variants unable to bind protein S (K3m [R199Q/E226Q]) or FV-short (ΔCT [aa 1-249]) were generated. TFPIα-FV-short binding was studied by plate-binding and co-immunoprecipitation assays; functional TFPIα enhancement by FXa inhibition and prothrombin activation. RESULTS: While WT TFPIα and TFPIα K3m bound FV-short with high affinity (Kd∼2nM), TFPIα ΔCT did not. K3m, in contrast to WT, did not incorporate protein S in a TFPIα-FV-short-protein S complex while TFPIα ΔCT bound neither FV-short nor protein S. Protein S enhanced WT TFPIα-mediated FXa inhibition, but not K3m, in the absence of FV-short. However, once FV-short was present, protein S efficiently enhanced TFPIα K3m (EC50: 4.7nM vs 2.0nM for WT). FXa inhibition by ΔCT was not enhanced by protein S alone or combined with FV-short. In FXa-catalyzed prothrombin activation assays, FV-short enhanced TFPIα K3m function in the presence of protein S (5.5 vs 10.4-fold enhancement of WT) whereas ΔCT showed reduced or lack of enhancement by FV-short and protein S, respectively. CONCLUSION: Full TFPIα function requires the presence of both cofactors. While synergistic enhancement can be achieved in the absence of TFPIα-protein S interaction, only TFPIα with an intact C-terminus can be synergistically enhanced by protein S and FV-short.

Anti-ß2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies interfere with cleavage of factor V(a) by activated protein C.

BACKGROUND: The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against ß2-glycoprotein I (ß2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against ß2GPI and prothrombin cause APC resistance is currently unclear. OBJECTIVES: To investigate how anti-ß2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance. METHODS: The effects of anti-ß2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies. RESULTS: APC resistance was observed in LA-positive patients with anti-ß2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-ß2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-ß2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-ß2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306. CONCLUSION: Anti-ß2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage.

Mutant IAA21 genes from Dendrocalamus sinicus Chia et J. L. Sun inhibit stem and root growth in transgenic tobacco by interacting with ARF5.

Woody bamboos are important resource of industrial fibres. Auxin signaling plays a key role in multiple plant developmental processes, as yet the role of auxin/indole acetic acid (Aux/IAA) in culm development of woody bamboos has not been previously characterized. Dendrocalamus sinicus Chia et J. L. Sun is the largest woody bamboo documented in the world. Here, we identified two alleles of DsIAA21 gene (sIAA21 and bIAA21) from the straight- and bent-culm variants of D. sinicus, respectively, and studied how the domains I, i, and II of DsIAA21 affect the gene transcriptional repression. The results showed that bIAA21 expression was rapidly induced by exogenous auxin in D. sinicus. In transgenic tobacco, sIAA21 and bIAA21 mutated in domains i, and II significantly regulated plant architecture and root development. Stem cross sections revealed that parenchyma cells were smaller in transgenic plants than that in wild type plants. Domain i mutation changed the leucine and proline at position 45 to proline and leucine (siaa21L45P and biaa21P45L) strongly repressed cell expansion and root elongation by reducing the gravitropic response. Substitution of isoleucine with valine in domain II of the full length DsIAA21 resulted in dwarf stature in transgenic tobacco plants. Furthermore, the DsIAA21 interacted with auxin response factor 5 (ARF5) in transgenic tobacco plants, suggesting that DsIAA21 might inhibit stem and root elongation via interacting with ARF5. Taken together, our data indicated that DsIAA21 was a negative regulator of plant development and suggested that amino acid differences in domain i of sIAA21 versus bIAA21 affected their response to auxin, and might play a key role in the formation of the bent culm variant in D. sinicus. Our results not only shed a light on the morphogenetic mechanism in D. sinicus, but also provided new insights into versatile function of Aux/IAAs in plants.

The Magic of Proteases: From a Procoagulant and Anticoagulant Factor V to an Equitable Treatment of Its Inherited Deficiency.

Proteostasis, i.e., the homeostasis of proteins, responsible for ensuring protein turnover, is regulated by proteases, which also participate in the etiopathogenesis of multiple conditions. The magic of proteases is such that, in blood coagulation, one same molecule, such as coagulation factor V, for example, can perform both a procoagulant and an anticoagulant function as a result of the activity of proteases. However, this magic has an insidious side to it, as it may also prevent the completion of the clinical value chain of factor V deficiency. This value chain encompasses the discovery of knowledge, the transfer of this knowledge, and its translation to clinical practice. In the case of rare and ultra-rare diseases like factor V deficiency, this value chain has not been completed as the knowledge acquisition phase has dragged out over time, holding up the transfer of knowledge to clinical practice. The reason for this is related to the small number of patients afflicted with these conditions. As a result, new indications must be found to make the therapies cost-effective. In the case of factor V, significant research efforts have been directed at developing a recombinant factor V capable of resisting the action of the proteases capable of inactivating this factor. This is where bioethics and health equity considerations come into the equation.

Cryo-EM structure of coagulation factor V short.

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.

[The role of methylation of placental glucocorticoid response gene in the association between pregnancy-related anxiety in the third trimester and birth outcomes].

Objective: To investigate the role of methylation of placental glucocorticoid response gene in the association between pregnancy-related anxiety in the third trimester and birth outcomes. Methods: Based on a prospective cohort study, singleton live births and their mothers from the Ma'anshan Birth Cohort Study (MABC) were included as participants in this study. The maternal pregnancy-related anxiety symptoms in the third trimester of pregnancy were evaluated by using the Pregnancy-related Anxiety Questionnaire. The neonatal birth outcomes were collected from medical records. The placental tissues from 300 pregnant women with pregnancy-related anxiety and 300 without pregnancy-related anxiety were collected to detect the methylation of FKBP5, NR3C1 and HSD11B2 genes using the Methyl Target approach. The methylation factors were extracted by exploratory factor analysis. Linear regression or logistic regression models were used to analyze the association between pregnancy-related anxiety in the third trimester, methylation factor scores, and birth outcomes. The mediating role of methylation factors in the association between pregnancy-related anxiety in the third trimester and birth outcomes was analyzed by using the Process procedure. Results: The mean age of 2 833 pregnant women was (26.60±3.60) years old. After adjusting for confounding factors, pregnancy-related anxiety in the third trimester increased the risk of small-for-gestational-age (OR=1.32, 95%CI:1.00-1.74). A total of 5 methylation factors were extracted, and the factor 5 was loaded with FKBP5 CpGs 18-21. Pregnancy-related anxiety in the third trimester was negatively correlated with the factor 5 (ß=-0.24,95%CI:-0.44--0.05). The factor 5 was positively correlated with the gestational age (ß=0.17, 95%CI:0.06-0.27). In addition, the factor 2 (ß=0.02,95%CI:0.00-0.04) and factor 3 (ß=0.03,95%CI:0.01-0.05) were positively correlated with 5-min Apgar score after delivery. However, this study did not found the mediating role of the scores of the factor characterized by FKBP5 in the relationship between pregnancy-related anxiety and birth outcomes. Conclusion: Pregnancy-related anxiety in the third trimester may reduce the methylation level of FKBP5 CpGs 18-21 in placental tissues and is associated with the risk of small-for-gestational-age.

Natural anticoagulant discovery, the gift that keeps on giving: finding FV-Short.

The complex reactions of blood coagulation are balanced by several natural anticoagulants resulting in tuned hemostasis. During several decades, the knowledge base of the natural anticoagulants has greatly increased and we have also learned about antiinflammatory and cytoprotective activities expressed by antithrombin and activated protein C (APC). Some coagulation proteins have also been found to function as anticoagulants; e.g., thrombin when bound to thrombomodulin activates protein C. Another example is factor V (FV), which in addition to being a procofactor to FVa has emerged as an anticoagulant. The discovery of APC resistance, caused by FVLeiden, as a thrombosis risk factor resulted in the identification of FV as an APC cofactor working in synergy with protein S in the regulation of FVIIIa in the Xase complex. More recently, a natural anticoagulant FV splice isoform (FV-Short) was discovered when investigating the East Texas bleeding disorder. In FV-Short, the truncated B domain exposes a high-affinity binding site for tissue factor pathway inhibitor alpha (TFPIα), and together with protein S a high-affinity trimolecular complex is generated. The FXa-inhibitory activity of TFPIα is synergistically stimulated by FV-Short and protein S. The circulating FV-Short/protein S/TFPIα complex concentration is normally low (≈0.2 nM) but provides an anticoagulant threshold. In the East Texas bleeding, the concentration of the complex, and thus the threshold, is increased 10-fold, which results in bleeding manifestations. The anticoagulant properties of FV were discovered during investigations of individual patients and follow the great tradition of bed-to-bench and bench-to-bed research in the coagulation field.

DNA methylation changes and increased mRNA expression of coagulation proteins, factor V and thrombomodulin in Fuchs endothelial corneal dystrophy.

Late-onset Fuchs endothelial corneal dystrophy (FECD) is a disease affecting the corneal endothelium (CE), associated with a cytosine-thymine-guanine repeat expansion at the CTG18.1 locus in the transcription factor 4 (TCF4) gene. It is unknown whether CTG18.1 expansions affect global methylation including TCF4 gene in CE or whether global CE methylation changes at advanced age. Using genome-wide DNA methylation array, we investigated methylation in CE from FECD patients with CTG18.1 expansions and studied the methylation in healthy CE at different ages. The most revealing DNA methylation findings were analyzed by gene expression and protein analysis. 3488 CpGs had significantly altered methylation pattern in FECD though no substantial changes were found in TCF4. The most hypermethylated site was in a predicted promoter of aquaporin 1 (AQP1) gene, and the most hypomethylated site was in a predicted promoter of coagulation factor V (F5 for gene, FV for protein). In FECD, AQP1 mRNA expression was variable, while F5 gene expression showed a ~ 23-fold increase. FV protein was present in both healthy and affected CE. Further gene expression analysis of coagulation factors interacting with FV revealed a ~ 34-fold increase of thrombomodulin (THBD). THBD protein was detected only in CE from FECD patients. Additionally, we observed an age-dependent hypomethylation in elderly healthy CE.Thus, tissue-specific genome-wide and gene-specific methylation changes associated with altered gene expression were discovered in FECD. TCF4 pathological methylation in FECD because of CTG18.1 expansion was ruled out.

Impaired factor V-related anticoagulant mechanisms and deep vein thrombosis associated with A2086D and W1920R mutations.

Factor V (FV) plays pivotal roles in both procoagulant and anticoagulant mechanisms. Genetic mutations, FV-W1920R (FVNara) and FV-A2086D (FVBesançon), in the C1 and C2 domains of FV light chain, respectively, seem to be associated with deep vein thrombosis. However, the detailed mechanism(s) through which these mutations are linked to thrombophilia remains to be fully explored. The aim of this study was to clarify thrombotic mechanism(s) in the presence of these FV abnormalities. Full-length wild-type (WT) and mutated FV were prepared using stable, human cell lines (HEK293T) and the piggyBac transposon system. Susceptibility of FVa-A2086D to activated protein C (APC) was reduced, resulting in significant inhibition of APC-catalyzed inactivation with limited cleavage at Arg306 and delayed cleavage at Arg506. Furthermore, APC cofactor activity of FV-A2086D in APC-catalyzed inactivation of FVIIIa through cleavage at Arg336 was impaired. Surface plasmon resonance-based assays demonstrated that FV-A2086D bound to Glu-Gly-Arg-chloromethylketone active site-blocked APC and protein S (P) with similar affinities to that of FV-WT. However, weakened interaction between FVa-A2086D and phospholipid membranes was evident through the prothrombinase assay. Moreover, addition of FVa-A2086D to plasma failed to inhibit tissue factor (TF)-induced thrombin generation and reduce prothrombin times. This inhibitory effect was independent of PC, PS, and antithrombin. The coagulant and anticoagulant characteristics of FV(a)-W1920R were similar to those of FV(a)-A2086D. FV-A2086D presented defects in the APC mechanisms associated with FVa inactivation and FV cofactor activity, similar to FV-W1920R. Moreover, both FV proteins that were mutated in the light chain impaired inhibition of TF-induced coagulation reactions. These defects were consistent with congenital thrombophilia.

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.