Factor V Protein Research Articles

Clinical Characterization and Molecular Analysis of Fourteen Chinese Patients with Factor V Deficiency.

Coagulation factor V (FV) functions as a vital cofactor that performs procoagulant roles in the coagulation system. We investigated 14 unrelated patients whose plasma FV levels were all below the reference range. FV activity (FV:C) and FV antigen were detected by one-stage clotting and ELISA, respectively. All 25 exons of the F5 gene in patients were amplified by the PCR, and they were sequenced directly. Haplotype analysis was performed with different polymorphisms on F5. Protein modeling was applied to analyze the potential molecular mechanisms. Of five patients with higher FV levels (FV:C > 10%), only one had minor bleeding symptoms. In contrast, of the remaining eight patients with lower FV levels (FV:C < 10%), six showed various bleeding manifestations. A total of 10 mutations were detected from 14 patients (6 were novel mutations). Interestingly, the homozygous p.Phe190Ser was found in five pedigrees, and haplotype analysis showed that they shared almost the same haplotype, indicating the common origin rather than a hotspot mutation. In silico analysis preliminarily investigated the potential pathogenic mechanism of the mutation. Modeling analysis showed that all six missense mutations would lead to conformational alterations in the FV protein. Among them, three (p.Gly1715Ser, p.Ser1753Arg, and p.Asp68His) would decrease hydrogen bonds. This is the largest genetic analysis of a single cohort of FV deficiency in Chinese. The study demonstrated that FV levels tended to be correlated with the probability of hemorrhage. The identification of a large number of unique FV-deficient pedigrees highlighted the screening for mutations in F5.

Full-scale network analysis reveals properties of the FV protein structure organization

Blood coagulation is a vital process for humans and other species. Following an injury to a blood vessel, a cascade of molecular signals is transmitted, inhibiting and activating more than a dozen coagulation factors and resulting in the formation of a fibrin clot that ceases the bleeding. In this process, the Coagulation factor V (FV) is a master regulator, coordinating critical steps of this process. Mutations to this factor result in spontaneous bleeding episodes and prolonged hemorrhage after trauma or surgery. Although the role of FV is well characterized, it is unclear how single-point mutations affect its structure. In this study, to understand the effect of mutations, we created a detailed network map of this protein, where each node is a residue, and two residues are connected if they are in close proximity in the three-dimensional structure. Overall, we analyzed 63 point-mutations from patients and identified common patterns underlying FV deficient phenotypes. We used structural and evolutionary patterns as input to machine learning algorithms to anticipate the effects of mutations and anticipated FV-deficiency with fair accuracy. Together, our results demonstrate how clinical features, genetic data and in silico analysis are converging to enhance treatment and diagnosis of coagulation disorders.

Analysis of 363 Genetic Variants in F5 via an Interactive Web Database Reveals New Insights into FV Deficiency and FV Leiden.

The inherited bleeding disorder Factor V (FV) deficiency and clotting risk factor FV Leiden are associated with genetic variants in the F5 gene. FV deficiency occurs with mild, moderate, severe, or asymptomatic phenotypes, and either dysfunctional or reduced amounts of plasma FV protein. Here we present an interactive web database containing 363 unique F5 variants derived from 801 patient records, with 199 FV deficiency-associated variants from 245 patient records. Their occurrence is rationalized based on the 2,224 residue sequence and new FV protein structures. The 199 FV deficiency variants correspond to 26 (13%) mild, 22 (11%) moderate, 49 (25%) severe, 35 (18%) asymptomatic, and 67 (34%) unreported phenotypes. Their variant distributions in the FV domains A1, A2, A3, B, C1 and C2 were 28 (14%), 32 (16%), 34 (17%), 42 (21%), 16 (8%), and 19 variants (10%), respectively, showing that these six regions contain similar proportions of variants. Variants associated with FV deficiency do not cluster near known protein-partner binding sites, thus the molecular mechanism leading to the phenotypes cannot be explained. However, the widespread distribution of FV variants in combination with a high proportion of buried variant residues indicated that FV is susceptible to disruption by small perturbations in its globular structure. Variants located in the disordered B domain also appear to disrupt the FV structure. We discuss how the interactive database provides an online resource that clarifies the clinical understanding of FV deficiency.

Factor V Atlanta: A Novel Mutation in the F5 Gene Reveals Potential New Cis-Acting Elements Involved in Regulating FV-Short and TFPI Levels

Coagulation factor V (FV) is procofactor essential to normal hemostasis. Recently, a point mutation in exon 13 of the F5 gene (FV-East Texas; FV-ET), which encodes the B-domain, has been shown to result in an inherited moderate bleeding diathesis termed East Texas Bleeding Disorder. The mutation (F5 c. A2350G, according to the latest annotated reference sequence for the F5 transcript, NM_000130.4, originally reported as A2440G) greatly enhances alternative splicing at an otherwise weak splice site, which leads to the production of a new FV isoform, FV-short. FV-short, via an exposed acidic region, binds with high affinity to tissue factor pathway inhibitor alpha (TFPIα) and is thought to stabilize the inhibitor in blood thereby increasing its concentration via changes in clearance mechanisms. High levels of TFPIα as well as other regulatory mechanisms contribute to inhibition of thrombin generation leading to a clinical bleeding phenotype. Another family with a different point mutation (FV-Amsterdam, F5 c.C2588G) downstream from FV-ET was recently identified and found to have similar clinical and laboratory characteristics. A 43 year-old male with a history of recurrent intramuscular hematomas underwent laboratory investigation to identify the etiology of his bleeding tendency. Laboratory testing revealed a prolonged PT and aPTT that failed to correct after 50:50 mixing with normal human plasma (NHP) and normal activity levels of factors II, V, VII, VIII, IX, X, XI, XII, and XIII. A lupus anticoagulant was not detected. A thrombin generation assay revealed a prolonged lag time versus control plasma (20 min vs. 6 min). These findings were similar to FV-ET, and further studies revealed that the concentration of total TFPI and TFPIα were elevated in the patient. Surprisingly however, the levels of TFPI were much higher compared to those measured in FV-ET. Relative to NHP, total TFPI and TFPIα were increased 11-and 22-fold, respectively for the patient compared to 4- and 5-fold for FV-ET (Table 1). Remarkably, a point mutation in exon 13 was not identified. Rather, analysis performed on genomic DNA identified a heterozygous 832 base pair (bp) deletion within exon 13 (c.2413-3244del). This deletion is predicted to result in the omission of 277 amino acids preceding a frameshift and translation of 12 non-native amino acids followed by a stop codon.While this alteration should result in a functionally null allele, causing the patient to have 50% FV levels, the clinical and biochemical findings do not align with this genomic data. Western blotting of the patient's plasma using anti-FV heavy or light chain antibodies revealed the presence of full length (FL)-FV (330 kDa) protein as well as a strong band at 250 kDa representing FV-short. Using recombinant FV-short as a control, quantitative western blotting revealed the patient has very high levels of FV-short (50 nM) compared to FV-ET plasma (5-7 nM; Table 1). To reconcile these data with the genetic information, we performed RT-PCR analysis of blood cell RNA. Several different FV transcripts were identified including a putative FL-FV transcript, a transcript containing the 832 bp frameshift deletion, and a FV-short transcript identical to that from FV-ET. The latter finding was unexpected as the 832 bp deletion is located 3' to the FV-ET donor splice site. We hypothesize that encoded within this novel 832 bp deletion is a sequence that functions as a cis-acting element which regulates FV-short production under normal physiologic conditions. FV-short is constituently expressed to a minor degree in unaffected individuals (~0.2-0.4 nM). Removal of this 832 bp sequence through genetic deletion is predicted to facilitate upregulation of FV-short biosynthesis via enhanced splicing at the FV-ET donor/acceptor splice sites. The identification of the FV-Atlanta deletion and the FV-ET and FV-Amsterdam point mutations highlight a complicated mechanism by which FV-short levels are modulated by alternative splicing. Since FV-short has a major influence on TFPIα levels, these collective findings are clinically relevant and may represent an additional level of regulation existing in the initiation and propagation of coagulation. [Display omitted] DisclosuresZimowski:National Hemophilia Foundation/Shire: Other: Clinical Fellowship Award Recipient. Denning:Expression Therapeutics: Employment. Jhita:Expression Therapeutics: Employment. Rivera:Expression Therapeutics: Employment. Escobar:Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kempton:Genentech: Membership on an entity's Board of Directors or advisory committees. Camire:Bayer: Consultancy, Research Funding; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Analysis of Phenotype and Genotype of A Family with Hereditary Coagulation Factor Ⅴ Deficiency Caused by A Compound Heterozygous Mutation

To analyze the molecular pathogenesis by analysis of phenotype and gene mutation in families with hereditary coagulation factor V (FⅤ) defect caused by complex heterozygous mutation. Plasma pro-thrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), FⅤ procoagulant activity (FⅤ∶C), FⅤ antigen (FⅤ∶Ag), and other related coagulation indexes were detected in the proband and his family members (3 generations 10 people). Using DNA direct sequencing to analyze all exons, flanks, 5' and 3' untranslated regions of F5 genes and the corresponding mutation site regions of family members, the mutation site was confirmed by reverse sequencing．The conservation of mutant amino acids was analyzed by ClustalX-2.1-win software. The PROVEAN and MutationTaster online bioinformatics software were used to predict the effect of mutation on protein function. Protein model and amino acid interaction at mutation sites was analyzed by Swiss-pdbviewer software. The PT and APTT of the proband were significantly prolonged compared with healthy controls (34.2 vs 13.2 s and 119.3 vs 36.0 s), while FⅤ∶C and FⅤ∶Ag extremely reduced (3% and 6%). The PT and APTT of the second-born, the third son, daughter, and grandson of the proband were slightly prolonged, and the FⅤ∶C and FⅤ∶Ag decreased to varying degrees. The related coagulant parameters of other family members were within normal range. Genetic analysis revealed that the proband had a c.911G>A heterozygous missense mutation on the exon 6 lead to p.Gly276Glu, and a c.5343C>G heterozygous missense mutation on the exon 16 lead to p.Ser1781Arg of the proband. The second-born, the third son, and grandson of the proband carry p.Gly276Glu heterozygotes, and the daughter carries p.Ser1781Arg heterozygotes, while the other family members were wild-type. The results of conservative analysis indicated that p.Gly276 and p.Ser1781 were highly conserved in homologous species. The two bioinformatics software predicted the same results, PROVEAN (score -6.214 and -12.79) indicated that the compound heterozygous mutation was a harmful mutation; MutationTaster (score 0.976 and 0.999) suggested that these mutations might cause corresponding disease. p.Gly276Glu protein model analysis showed that, the Glu side chain was prolonged and the molecular weight became larger, which would increase the steric hindrance between it and the surrounding amino acids, affect the normal local folding of the FⅤ protein, and eventually lead to the decrease of protein activity and content. This paper can not provide analysis of the spatial structure of p.Ser1781Arg mutant protein because of the lack of X ray 3 D structure file of FⅤ exon 16. The new compound heterozygous mutations (p.Gly276Glu and p.Ser1781Arg) identified in this study are the main reasons for the decrease in the FⅤ level of the family, among which p.Ser1781Arg is rarely reported at home and abroad.

Next generation sequencing in bleeding disorders: two novel variants in the F5 gene (Valencia-1 and Valencia-2) associated with mild factor V deficiency.

Inherited bleeding coagulation disorders (IBCDs) have a powerful diagnostic tool in next generation sequencing (NGS) that not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and helps to predict the clinical course and follow-up of a disease. In our group, targeted-NGS using a Custom SureSelect QXT Panel (Agilent Technologies, Inc., Santa Clara, CA, USA) was designed to screen for causal variants in 40 genes related with the coagulation cascade. In this work, we used NGS for screening all the coding and intronic boundary regions of F5 gene in two patients affected by factor V (FV) deficiency (parahemophilia). Two new mutations were found: c.4745A>G (p.Tyr1582Cys, NM_000130.4) and c.1999_2002dupAATT (p.Ser668ter; NM_000130.4), both located in exon 13 of the F5 gene. We designated them Valencia-1 and Valencia-2 respectively. Valencia-1 could provoke loss of the fifth cupredoxin domain of the FV, and would be responsible for its defective activity. Valencia-2 prematurely stops the translation of mRNA, resulting in a truncated FV protein which lacks completely the B domain and the light chain. NGS has permitted to describe an increasing number of FV deficiency-causing mutations and a better understanding of FV's structure and function. The description of deficiency-causing mutations will continue to increase our knowledge of the functional residues of FV, as well as those which are involved in the correct folding of the protein. In this sense, NGS is a useful tool for studying IBCDs, as permits studying the whole coagulation cascade at once and gives a global view of the patient's genetic background.

Subtype‐specific clinical and prognostic relevance of tumor‐expressed F5 and regulatory F5 variants in breast cancer: the CoCaV study

Background Tumor expression of certain coagulation factors has been linked to cancer progression. Single nucleotide polymorphisms (SNPs) in F5 (encoding the FV protein) have been found to be associated with breast cancer; however, the role of coagulation factor V (FV) in cancer pathogenesis remains undiscovered. Objectives We aimed to investigate the clinical significance of FV and the regulatory role of F5 gene variants in breast cancer. Patients/Methods A Scandinavian 503-sample breast cancer cohort and three public breast cancer datasets (GOBO, TCGA and KM plotter) were used to determine associations between F5 gene expression (tumor-specific), circulating FV, F5 SNPs, clinical characteristics and breast cancer survival. Immunohistochemistry (IHC) was used to detect FV antigen in tumors. Results F5 expression was 2-fold higher in breast tumors compared with normal tissue, and the presence of FV antigen in breast tumors was confirmed by IHC staining. F5 expression was increased in patients with hormone receptor negative tumors, triple negative tumors, HER2-enriched and basal-like tumors. In patients with basal tumors, high expression of F5 was associated with improved overall survival (hazard ratio, HR = 0.52, 95% confidence interval, 0.31-0.86). SNPs in F5 were associated with tumor size and luminal A tumors. The rs6427202-rs9332542 C-G haplotype, previously associated with breast cancer, displayed a cis-regulatory effect on F5 expression in tumors and plasma FV antigen levels. In silico mining supported this regulatory function. Conclusions FV was a possible marker of aggressive breast cancer, yet also a predictor of favorable outcome. Evaluation of FV expression may be clinically useful for prognosis and treatment decisions in aggressive breast cancer.

Novel insights into the regulation of coagulation by factor V isoforms, tissue factor pathway inhibitorα, and protein S

Factor V (FV) is a regulator of both pro- and anticoagulant pathways. It circulates as a single-chain procofactor, which is activated by thrombin or FXa to FVa that serves as cofactor for FXa in prothrombin activation. The cofactor function of FVa is regulated by activated protein C (APC) and protein S. FV can also function as an anticoagulant APC cofactor in the inhibition of FVIIIa in the membrane-bound tenase complex (FIXa/FVIIIa). In recent years, it has become clear that FV also functions in multiple ways in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. Of particular importance is a FV splice variant (FV-Short) that serves as a carrier and cofactor to TFPIα in the inhibition of FXa. FV-Short is generated through alternative splicing of exon 13 that encodes the large activation B domain. A highly negatively charged binding site for TFPIα is exposed in the C-terminus of the FV-Short B domain, which binds the positively charged C-terminus of TFPIα, thus keeping TFPIα in circulation. The binding of TFPIα to FV-Short is also instrumental in localizing the inhibitor to the surface of negatively charged phospholipids, where TFPIα inhibits FXa in process that is stimulated by protein S. Plasma FV activation intermediates and partially proteolyzed platelet FV similarly bind TFPIα with high affinity and regulate formation of prothrombinase. The novel insights gained into the interaction between FV isoforms, TFPIα, and protein S have opened a new avenue for research about the mechanisms of coagulation regulation and also for future development of therapeutics aimed at modulating coagulation.

Asp68His mutation in the A1 domain of human factor V causes impaired secretion and ineffective translocation

Congenital factor V (FV) deficiency is a rare inherited disorder. We determined the mechanism of a missense mutation, Asp68His, in the A1 domain of the FV protein, is associated with severe FV deficiency. We characterized the mutant FV-Asp68His protein using in vitro expression studies by using specific secretion and degradation pathway inhibitors and analysed the intracellular translocation of the mutant protein by immunofluorescence staining. The Asp68His mutation caused very low levels of FV protein in the conditioned media, with normal specific FV activity. Similar mRNA degradation rates between FV-wild-type (wt) and FV-Asp68His mRNA showed that the Asp68His mutation does not affect FV expression at the transcriptional level. A specific secretion pathway inhibitor, brefeldin A, was used to demonstrate that the lower efficiency of transport to the outside of the cell for FV-Asp68His mutant protein compared with that of the FV-wt protein. Furthermore, we showed that the Asp68His mutation resulted in increased intracellular degradation through a MG132-mediated proteasomal degradation pathway. In the transfected cell lysates, FV-wt protein had multiple posttranslational modified forms, but the FV-Asp68His protein was not completely glycosylated. We further observed that the FV-Asp68His protein was retrieved in the endoplasmic reticulum only and did not undergo transport to the Golgi apparatus, leading to impaired secretion. These results strongly suggest that the Asp68His mutation may result in intracellular defective trafficking and enhanced degradation, and impaired secretion of FV protein.

Functional characterization of a novel missense mutation, His147Arg, in A1 domain of FV protein causing type II deficiency

IntroductionCongenital factor V (FV) deficiency is a rare inherited disorder. Three compound heterozygous missense mutations, Asp68His, His147Arg, and Arg2074Cys, were observed in a Taiwanese patient with moderately severe FV deficiency. MethodThe novel His147Arg mutation in the A1 domain was investigated by protein modeling, followed by in vitro expression studies in COS-1 cells, to elucidate the molecular pathology associated with FV deficiency. ResultsThe His147Arg mutation was associated with normal antigen levels, both in cell lysates and conditioned media, whereas FV activity was significantly reduced to 63.5±17.0%. These observations correspond to a type II FV deficiency mutation. Protein modeling by short-duration molecular dynamics (MD) simulation showed that the His147Arg mutation was associated with a conformational change, which could disrupt the stability of FVa by interfering with His1817 coordination of the copper ion. In functional activation assays, the His147Arg mutation did not affect FV protein activation by thrombin; however, reduced cofactor activity of the FVa protein, due to an increased rate of dissociation of heavy and light chains, was observed. ConclusionOur results show that the His147Arg mutation in the A1 domain of FV does not impair synthesis or procoagulant activity. Instead, the His147Arg mutation appears to disrupt the stability of FVa, providing a potential explanation for the functional deficiency.

A novel factor V mutation causes a normal activated protein C ratio despite the presence of a heterozygous F5 R506Q (factor V Leiden) mutation

Activated protein C (APC) resistance (Dahlback et al, 1993) is a common risk factor for venous thromboembolism (Bucciarelli et al, 1999). This phenotype is highly associated with the F5 R506Q (F5 rs6025; factor V Leiden, FVL) mutation (Bertina et al, 1994). Heterozygous and homozygous discrepancies between F5 R506Q genotype and phenotype (APC resistance), known as pseudo-homozygosity, have been described (Castaman et al, 1997; Brugge et al, 2005). This incidental phenomenon suggests that, based on APC-resistance testing, there is a homozygous F5 R506Q mutation, yet, genetic testing only shows a heterozygous F5 R506Q mutation (Simioni et al, 1996). In such cases, polymorphisms located on the second factor V (FV) protein coding allele (i.e. wild-type for F5 R506Q) cause intracellular decreased FV protein production, leading to reduced FV activity (Castoldi et al, 1998). In contrast, the phenomenon “pseudo-wild-type” FVL, suggesting a wild-type F5 R506Q according to APC resistance testing but heterozygous F5 R506Q mutation by genetic testing, is infrequently described (Dargaud et al, 2003; Asselta et al, 2004). In these two studies, the polymorphisms responsible for the reduced FV protein levels were found to be located on the same FV protein coding allele, together with the F5 R506Q mutation.

Severe Factor V Deficiency Caused by a Novel Compound Heterozygous Mutation: Factor V G1617V and 1-Bp Insertion

Abstract 2268 Introduction:Congenital factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. There is poor correlation between FV activity in plasma and symptoms of bleeding tendency. In the present study, we identified a novel compound heterozygous mutation in the FV gene (F5) in a Japanese patient with severe bleeding symptoms.Patient and methods: The patient was a young boy in his teens. He suffered from intracranial hemorrhage after birth and he also experienced joints and muscle hemorrhage, afterwards. His plasma FV activity was less than 1% and FV antigen was 2%. Sequence analysis of F5, FV protein analysis, and recombinant mutant protein expression experiments were performed. And to evaluate the relation between bleeding symptoms and FV phenotypes, we analyzed the FV level of platelets in the patient and his family members. Our present and previous results were verified. Results:Sequence analysis of F5 in this patient revealed a novel compound heterozygous mutation, G1617V missense mutation in exon 14 and a 1-bp insertion in exon 16 (p.G1645V and c.5255–5259 ins A). The patient’s father had a G1617V mutation and his mother had a 1-bp insertion for the heterozygote state, respectively. The patient’s two sisters each had also a 1-bp insertion for the heterozygote state. We analyzed the expression of platelet F5 mRNA by semi-quantitative RT-PCR, which showed that platelet F5 mRNA from the patient and his family members were equal to the amount in healthy subjects. Platelet FV protein from family members examined by western blotting detected equal to those of healthy subjects, although the FV bands from the patient were detected only weakly. Platelet FV antigen and activity were measured with an ELISA and a functional assay based on prothrombin time. The patient’s platelet FV activity was 1.6% and antigen was 1%, compared with 100% for those of normal subjects. Platelet FV activity and antigen of the patient’s father, mother, elder sister and younger sister were 26% and 37.1%, 39.6% and 51.0%, 23.3% and 38.0%, and 35.4% and 56.6%, respectively. In the expression study of pMT2/FV-wild-type and FV-G1617V mutant recombinant proteins in HEK293 cells, the FV antigen levels produced by the FV-G1617V mutant in cell lysates was approximately 73% of wild-type. In conditioned media of the study, FV antigen and specific activity were reduced to approximately 4% and 6% of wild-type, respectively. Discussions:Although there was similar intracellular synthesis of the FV-G1617V mutant and wild-type FV, the results in the conditioned media suggested that secretion of the FV-G1617V mutant was impaired. Platelet FV protein was detected very low by Western blot and ELISA, though platelet F5 mRNA was confirmed to be normal by RT-PCR. Our previous results raise the possibility that in the case of severe FV deficiency, such as that with a 1 % or less plasma FV level, the amount of FV in platelets is important to cope with local bleeding. This patient's platelet FV activity and antigen levels were the lowest of those of a number of FV-deficient patients whom we have analyzed so far, and this patient had severe bleeding symptoms. Conclusion:Our previous studies support the concept that the severity of bleeding is related to FV protein in platelets. Taken together, we believe the severity of this patient's bleeding tendency is caused by a reduction in both platelet and plasma FV, especially by the highly significant decrease in platelet FV. Disclosures:No relevant conflicts of interest to declare.

Identification and Cloning of Novel Mutations In a Compound Heterozygous Factor V Deficient Patient

AbstractAbstract 2210 Introduction:Congenital factor V (FV) deficiency is a rare clotting disorder associated with mild to severe hemorrhagic symptoms and a prevalence of approximately 1 per million in the general population. Patients normally present with very low or unmeasurable levels of functional and/or immunoreactive FV and are usually homozygous or compound heterozygous for mutations located in the F5 gene. Heterozygotes typically have approximately half-normal levels of FV and are asymptomatic, whilst compound heterozygotes may show mild to severe bleeding diathesis. Patient Description:A proband now aged 73, was clinically diagnosed since childhood as having severe FV deficiency (<3% FV activity by clinical lab analysis), exhibiting severe bleeding tendency with surgery or dental extraction. Other symptoms included frequent nose bleeds, bruising easily and profound menorrhagia that led to eventual hysterectomy in mid-thirties. Additional routine clinical assays for clotting factors were normal. There was no history of parental bleeding tendency. Methods:DNA was isolated from peripheral blood leucocytes. Exonic and flanking intronic sequences of F5 were amplified by PCR and subjected to automated nucleotide sequencing. To investigate the role of identified mutations in FV function and protein secretion, wildtype (WT) FV and FV variants consistent with mutations identified for the patient using the QuikChange Site-Directed Mutagenesis Kit were cloned into the pED-FV-1033 expression vector. This produced recombinant single chain FV that requires proteolytic activation to express cofactor function. Plasmids encoding WT FV, and the mutation identified in the patient were transfected into baby hamster kidney cells (BHK) using Lipofectamine and stable clones were established. Secretion of FV protein was quantified by commercial enzyme immunoassay (EIA), and FV activity was evaluated using conventional prothrombin time (PT) and activated partial thromboplastin time (APTT) clotting assays. Western blot analysis using monoclonal antibodies to both FV heavy (FVaH) and light chains (FVaL) detected FV antigen and fragmentation profiles. Results:Based on the lack of parental bleeding, compound heterozygosity was probable for this FV deficient patient because the DNA sequence analysis revealed two novel mutations; the first changed the codon for Leu1821 to Ser (L1821S), the second changed Gly2192 to Cys (G2192C). Clotting assays showed low patient plasma FV activity of 0.5±0.015% for PT and 2.0±0.10% for APTT compared to normal pooled plasma. Western blot analysis demonstrated the patient FV banding pattern was comparable to normal plasma, whilst densitometric analysis of the specific bands showed that the patient had 9% of the normal FV antigen level. Recombinant FV secretion detected by EIA from various clones ranged from 0 to 16.6ng/mL. L1821S (28%) and G2192C (25%) mutants secreted lower FV protein compared to WT. Clotting assays (n=2) showed that Leu1821S activity levels were 15% that of WT in contrast to G2192C where no FV clotting activity was detectable. Compared to WT, L1821S had 55% and G2192C had only 1.4% of the specific activity.Western blot analysis demonstrated the L1821S banding pattern was comparable to WT whilst G2192C lacked several fragments. Prolonged thrombin (FIIa) pretreatment of FV clones confirmed WT and both mutants were cleaved to FVaL (∼74k Da) and FVaH (∼105 kDa). In addition, FIIa pre-treatment enhanced clotting activity for most of the clones evaluated. Interestingly, the inactive G2192C mutant acquired modest FV function after pre-incubation with FIIa. Conclusions:Two novel FV mutations that affect both function and secretion have been identified in a FV deficient patient and have been cloned. The L1821S mutation is located close to a FV metal ion binding site that may affect function. G2192C introduces a thiol into FV, reported in the literature to have deleterious effects on protein folding, which may explain attenuated secretion and lack of clotting activity. Only after prolonged pre-treatment with FIIa, G2192C function was acquired, which may suggest that this mutation stabilizes the pro-cofactor conformation of FV. Low in vitro secretion of mutant FV compared to WT corresponds to low patient plasma FV antigen, which is compounded by profound loss of function in G2192C. Further biochemical studies are underway to understand the effect of G2192C on FV activity. Disclosures:No relevant conflicts of interest to declare.

Novel Compound Heterozygous Factor V Deficiency with Severe Clinical Phenotype.

Abstract 3490Poster Board III-427 IntroductionCongenital factor V (FV) deficiency is a rare clotting disorder associated with mild to severe hemorrhagic symptoms and a prevalence of approximately 1 per million in the general population. Patients with FV deficiency normally show very low or unmeasurable plasma levels of functional and immunoreactive FV and are usually homozygous or compound heterozygous for mutations located in the FV gene. Heterozygous carriers have approximately half-normal levels of FV and are usually asymptomatic. Patient HistoryIn this study, a proposita now aged 71 is described, who has less than 3% FV activity and is clinically diagnosed as having severe FV deficiency. Since childhood she has exhibited severe bleeding tendency with surgery/dental extraction or trauma. Other symptoms include frequent nose bleeds, bruising easily and profound menorrhagia that led to eventual hysterectomy in her mid-thirties. Parents were clinically asymptomatic suggesting compound heterozygous FV deficiency for this individual. Her children, a son and a daughter, are also asymptomatic and have FV levels of approximately 50-60%. MethodsDNA was isolated from peripheral blood leucocytes and the FV exon and flanking intron sequences were amplified by PCR, and subjected to automatic DNA sequence analysis. FV levels were assayed in plasma using conventional clotting assays as well as immunoassays using monoclonal and polyclonal antibodies detecting several regions of FV. ResultsDNA sequence analysis revealed two mutations: the first in exon 17 (C > T) changed the codon for Leu-1821 to Ser (L1821S), the second in exon 25 (T > G) changed Gly-2192 to Cys (G2192C). Plasma clotting assays (n=2) showed FV activity levels of 0.5±0.015% for prothrombin time and 2.0±0.10% for activated partial thromboplastin time compared to normal pooled plasma. Western blot analysis using a polyclonal antibody demonstrated the patient FV banding pattern was comparable to normal plasma. Densitometric analysis of the specific bands showed that the patient had 9% of the FV antigen level compared to normal pooled plasma. ConclusionsTwo novel FV mutations have been identified. It has previously been reported in the literature that mutations involving thiols can have deleterious effects on protein folding and secretion. Consequently, the G2192C mutation could alter the FV protein folding and/or secretion which may explain the reduced level of FV antigen detected by immunoassay. In addition, the L1821S mutation is close to a putative activated FV metal ion binding site and may have an important effect on inter subunit reactions and subsequently FV function. Since antigen and activity are discordant, both FV function and secretion appear to be affected by these mutations. Future studies aim to introduce these unique mutations into recombinant FV protein to gain new insight into FV secretion, activity and function. Disclosures:No relevant conflicts of interest to declare.

Factor V Deficiency

Congenital factor V (FV) deficiency is a bleeding disorder associated with mild to severe hemorrhagic symptoms and a prevalence in the general population of 1 in 1,000,000 in the homozygous form. Patients with FV deficiency and clinically significant manifestations (mainly involving mucosal tracts) show very low or unmeasurable plasma FV levels and are usually homozygous or compound heterozygous for mutations located in the FV gene ( F5). Heterozygous carriers have approximately half-normal levels of FV and are usually asymptomatic. Replacement therapy for FV-deficient patients can only rely on administration of fresh-frozen plasma because specific FV concentrates are unavailable and FV is not present in cryoprecipitate or prothrombin complex concentrates. A total of 56 mutations have been published to date as being responsible for severe or moderately severe FV deficiency; more than two thirds of these are null mutations (mainly decreasing FV expression), with the remaining being missense mutations (usually impairing FV secretion). This article will provide a concise description of the FV protein and gene and will review the molecular, clinical, and therapeutic aspects of FV deficiency.

Modelling and expression studies of two novel mutations causing factor V deficiency

Human coagulation factor V (FV), a non-enzymatic cofactor of the prothrombinase complex, is required for the rapid generation of thrombin. FV deficiency is a rare autosomal recessive bleeding disorder. We describe two novel mutations, Tyr91Asn and Asp2098Tyr, found in two probands with a residual FV activity of 51% and 4%, respectively. Modelling and structural analysis of these mutations were performed following short-duration molecular dynamics (MD) simulation. Asp2098Tyr lead to abolishment of the highly conserved salt bridge Asp2098-Arg2171 presumably required for structural integrity of the C2 domain. MD studies suggest that additional conformational changes resulting from this mutation involve local rearrangements at Tyr2063 and Tyr2064 and so affect the phospholipid-membrane binding. MD modelling of the Try91Asn mutant revealed a conformational change nearby the Cu(2+) binding site that could affect overall stabilization of the heavy and light chains. These findings suggest that both mutations influence the structural integrity of FV protein. Transient expression data of wild-type and mutant FV variants in 293T human embryonic kidney cells showed FV-specific activity reduced to 26% for Asp2098Tyr and 56% for Tyr91Asn compared to that of wild-type. Thus, both the data from the short duration molecular dynamic simulation and from expression analysis indicate alterations of the FV protein variants that explain the clinical phenotype.

Molecular characterization of 3 factor V mutations, R2174L, V1813M, and a 5-bp deletion, that cause factor V deficiency

We identified 3 mutations in the factor V (FV) gene (F5) associated with FV deficiency in 3 unrelated Japanese patients. Patient 1 had severe bleeding symptoms (plasma FV activity, <1%; FV antigen, 9%) and was a compound heterozygote for a novel 5-bp deletion in exon 22 and the V1813M mutation. Patient 2 had moderate bleeding symptoms (plasma FV activity, <1%; FV antigen, 4%) and was homozygous for the V1813M mutation. Patient 3 had very mild symptoms (plasma FV activity, 1%; FV antigen, 5%) and was homozygous for the novel R2174L mutation. A study of recombinant protein expression revealed that the FV coagulant-specific activities in conditioned media for the FV-R2174L and FV-V1813M mutants were reduced to approximately 40% and 28% of wild-type FV, respectively. The amounts of FV-R2174L protein and messenger RNA in the platelets of patient 3 were similar to those of healthy subjects; however, the amount of FV-V1813M protein in patient 2 was decreased. Our data suggest that the severity of the bleeding tendency in patients with FV deficiency is correlated not only with plasma FV activity but also with the amount of FV protein in the platelets.

The Severity of Bleeding Tendency in 5 Patients with Factor V Deficiency Due to Distinct Kinds of Factor V Mutations.

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. It is poor correlation between FV levels in plasma and the severity of bleeding tendency. In the present study, we identified 5 mutations in the FV gene (F5) in 5 unrelated Japanese patients with reduced plasma FV activities associated with inherited FV deficiency. Their bleeding tendencies varied in severity from asymptomatic to severe. We hypothesized that the severity of bleeding symptoms with severe FV deficiency is correlated with FV levels in platelets, and performed recombinant mutant proteins expression experiments and analyzed of platelet FV. The data concerning 5 patient's FV levels in plasma and the bleeding symptoms are given in Table. The F5 mutations in 5 Japanese patients were identified by direct sequencing. One of the 5 patients was a compound heterozygote for FV mutations and carried a 5-base pair (bp) deletion in exon 22 (del.ACCCT) and V1813M. The other 4 patients were homozygous for a D68H, N468S, V1813M, and R2174L mutations, respectively. Four mutations except V1813M are newly identified mutations. These mutations were introduced independently by site-directed mutagenesis into a pMT2/FV mammalian expression plasmid containing the full-length FV cDNA, and the wild-type and mutant FV proteins were expressed in HEK293 cells. In the conditioned media, FV specific activities of the FV-D68H, FV-N468S, FV-V1813M, FV-R2174L, and 5bp del. mutants were an approximately 22%, 81%, 28%, 40%, and 19% of wild-type, respectively. On the other hand, analysis of platelet from patient by using RT-PCR showed that platelet F5 mRNA of FV-R2174L and FV-N468S was equal amount to that of normal subjects, although the amount of FV-V1813M platelet F5 mRNA was reduced. Platelet FV protein from patients was analyzed by western blotting and ELISA. Although the amount of platelet FV-R2174L protein was equal to that of normal platelets, platelet FV-V1813M protein was considerably reduced. In addition, the amount of FV-N468S protein in platelet was observed between that of normal subjects and FV-V1813M. These results indicate that the fact that sufficient amounts of FV are stored in platelet is required for local hemostasis. The results of FV-R2174L suggest that both functionality and amount of FV-R2174L in platelet is enough to cope with local bleeding, resulting very mild bleeding tendency. On the basis of present findings, we conclude that the severity of bleeding due to severe FV deficiency is correlated with not only plasma FV level, but also platelet FV.Five patient's FV levels in plasma and the bleeding symptomsPatient No.MutationFV activity (%)FV antigen (%)Bleeding symptoms1D68H44asymptomatic2N468S33asymptomatic3V1813M & 5-bp deletion<19severe4V1813M<14moderate5R2174L15very mild

Endocytosis and storage of plasma factor V by human megakaryocytes

Factor V is an essential coagulation cofactor that circulates in plasma and platelet alpha-granules where it is stored complexed to multimerin I (MMRN1). To gain insights into the origin and processing of human platelet factor V, and factor V-MMRN I complexes, we studied factorV in cultured megakaryocytes. Factor V mRNA was detected in all megakaryocyte cultures. However, like albumin, IgG and fibrinogen, factorV protein was detectable only in megakaryocytes cultured with exogenous protein. The amount of factor V associated with megakaryocytes was influenced by the exogenous factorV concentration. Similar to platelet factor V, megakaryocyte factor V was proteolyzed and complexed with megakaryocyte-synthesized MMRN1. With secretagogues, megakaryocytes released factor V, IgG, fibrinogen and MMRN1. Immunofluorescent and electron microscopy confirmed factorV uptake by endocytosis and its trafficking to megakaryocyte alpha-granules. These data provide direct evidence that human megakaryocytes process plasma-derived factor V into alpha-granules and generate factorV-MMRN I complexes from endogenously and exogenously synthesized proteins.

Factor‐V expression in platelets from human megakaryocytic culture

The origin of platelet-factor-V has long been discussed. To elucidate whether and when human platelet-factor-V is synthesized by megakaryocytes, we utilized in vitro-generated megakaryocytes capable of producing platelets. Factor-V gene was silent in purified progenitors and megakaryocytic precursors but was expressed in late culture phase and maintained also in platelets. Similarly, factor-V protein was expressed in mature proplatelet-bearing megakaryocytes (immunofluorescence analysis); it was also detectable in cultured megakaryocytes and platelets (Western blotting) and within permeabilized cultured platelets (flow cytometry). The absence of other cells in our culture system indicates conclusively that human megakaryocytes synthesize factor-V.