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William H Kane - One of the best experts on this subject based on the ideXlab platform.

  • location of the multimerin 1 binding site in Coagulation Factor V an update
    Thrombosis Research, 2008
    Co-Authors: Samira Jeimy, William H Kane, Mary Ann Quinnallen, Nola Fuller, Catherine P M Hayward
    Abstract:

    ActiVated Coagulation Factor V (FVa) is an important coFactor that accelerates thrombin production. In human blood, 25% of the Factor V (FV) is stored in platelets, complexed to the polymeric, FV binding protein multimerin 1 (MMRN1). The light chain of FV is required for MMRN1 binding, and its C2 domain contains a MMRN1 binding site that oVerlaps phospholipid binding residues essential for FVa procoagulant function. The homologous structures and roles of the FVa light chain C1 and C2 domains led us to inVestigate if the C1 domain also contains a MMRN1 binding site. The MMRN1 binding properties of FV constructs were tested by modified enzyme-linked immunoassays, before and after thrombin actiVation. The constructs tested included the combined C1 and C2 domain deleted FV, and B-domain deleted forms of FV containing C1 domain point mutations or combined C1 and C2 domain phospholipid binding site mutations. The MMRN1 binding site in FV/FVa was mapped to a large region that included the C1 domain phospholipid binding residues Y1956 and L1957. The FV construct with combined C1 and C2 domain phospholipid binding site mutations had no MMRN1 binding, highlighting the critical role of the FV C1 and C2 domain phospholipid binding residues in MMRN1 binding. Our data update the information on the structural features of FV and FVa important for MMRN1 binding, and suggest that the extended MMRN1 binding site in the C1 and C2 domains is important for the storage of FV-MMRN1 complexes in platelets.

  • crystal structures of the membrane binding c2 domain of human Coagulation Factor V
    Nature, 1999
    Co-Authors: Sandra Macedoribeiro, Mary Ann Quinnallen, Thomas L Ortel, Wolfram Bode, Robert Huber, Suhng Wook Kim, Gleb Bourenkov, Hans D Bartunik, Milton T Stubbs, William H Kane
    Abstract:

    Rapid and controlled clot formation is achieVed through sequential actiVation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of actiVated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an actiVe proteinase and a non-enzymatic coFactor, perform critical steps within this Coagulation cascade. The actiVated coFactors VIIIa and Va, highly specific for their cognate proteinases, are each deriVed from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both coFactors. Here we report two crystal structures of the C2 domain of human Factor Va. The conserVed beta-barrel framework proVides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of Factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the grooVe enclosed by these loops; and (3) faVourable electrostatic contacts of basic side chains with negatiVely charged membrane phosphate groups.

  • localization of functionally important epitopes within the second c type domain of Coagulation Factor V using recombinant chimeras
    Journal of Biological Chemistry, 1994
    Co-Authors: Thomas L Ortel, Mary Ann Quinnallen, F G Keller, J A Peterson, D Larocca, William H Kane
    Abstract:

    Coagulation Factor V, an integral component of the prothrombinase complex, possesses two C-type domains at the carboxyl-terminal end of the molecule. Homologous C-type domains are present in Factor VIII as well as seVeral non-Coagulation proteins. Deletion of the second C-type domain of Factor V results in the loss of procoagulant actiVity and the ability to bind phosphatidylserine. We now report the effect of substitution of all or a portion of the C2 domain of Factor V with the corresponding regions of Factor VIII or the human breast carcinoma protein BA46. Substitution of the entire domain with a heterologous C2 domain does not restore significant procoagulant actiVity, although smaller, exon-size substitutions do result in chimeras with partial actiVity (approximately 10% of Factor Va). Using chimeras with partial substitutions, we determined that the amino-terminal region of the domain is inVolVed in binding to phosphatidylserine. In contrast, the central region of the domain is not inVolVed in phosphatidylserine binding, but an antibody binding at or near this site inhibits procoagulant actiVity, suggesting that this region is inVolVed in a separate function. Lastly, the molecular basis for the light chain doublet, which is important in the expression of full procoagulant actiVity, is located within the carboxyl-terminal region of the C2 domain.

  • structure of the gene for human Coagulation Factor V
    Biochemistry, 1992
    Co-Authors: Larry D Cripe, Karen D Moore, William H Kane
    Abstract:

    ActiVated Factor V (Va) serVes as an essential protein coFactor for the conVersion of prothrombin to thrombin by Factor Xa. Analysis of the Factor V cDNA indicates that the protein contains seVeral types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human Factor V. The Factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for Factor V is similar to the preViously characterized gene for Factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the Factor V gene occur at the same location as in the Factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with eVolution through domain duplication and exon shuffling. The connecting region or B domain of Factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the Factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)

  • characterization of an acquired inhibitor to Coagulation Factor V antibody binding to the second c type domain of Factor V inhibits the binding of Factor V to phosphatidylserine and neutralizes procoagulant actiVity
    American Federation of Clinical Research. Annual meeting, 1992
    Co-Authors: Thomas L Ortel, Mary Ann Quinnallen, L A Charles, D Devorecarter, William H Kane
    Abstract:

    Coagulation Factor V is an essential component of the prothrombinase complex, which actiVates the zymogen prothrombin to thrombin. A patient was described who deVeloped a Factor V inhibitor that neutralized the procoagulant actiVity of Factor V and resulted in a fatal hemorrhagic diathesis (Coots, M. C., A. F. Muhleman, and H. I. Glueck. 1978. Am. J. Hematol. 4:193-206). This inhibitor was shown to be an IgG antibody that bound to the light chain of Factor V. Using a series of light chain deletion mutants, we haVe found that this antibody binds to the second C-type domain of the light chain. Both inhibitor IgG and Fab fragments rapidly neutralized the procoagulant actiVity of Factor Va, implying that the neutralization resulted from specific binding to the C2 domain. We haVe preViously demonstrated that deletion of the C2 domain results in loss of procoagulant actiVity, as well as loss of phosphatidylserine-specific binding. Confirming these results, both inhibitor IgG and Fab fragments interfered with phosphatidylserine-specific binding of Factor V. ConVersely, preincubation of Factor Va with procoagulant phospholipids protected the coFactor from inactiVation by the inhibitor. Our results suggest that this inhibitor neutralizes the procoagulant actiVity of Factor Va by interfering with the C2-mediated interaction with phospholipid surfaces, thereby disrupting formation of the prothrombinase complex.

Roger Williams - One of the best experts on this subject based on the ideXlab platform.

  • Coagulation Factor V leVels as a prognostic indicator in fulminant hepatic failure
    Hepatology, 1996
    Co-Authors: S Izumi, P G Langley, Julia Wendon, A J Ellis, R B Pernambuco, Robin D Hughes, Roger Williams
    Abstract:

    Data reported by Bernuau et al. haVe strongly supported the measurement of Coagulation Factor V as the best prognostic indicator in fulminant hepatic failure (FHF) and as the test on which selection for urgent liVer transplantation should be made. In this study, we haVe measured plasma Factor V in 110 patients with FHF, in grades I-IV coma, in 88 of whom the etiology was acetaminophen oVerdose. On admission, patients who did not surViVe had significantly lower Factor V leVels (median, 5%; range, 1-27; n = 49), compared with those who did (median, 10%; range, 2-70; P < .001). In the 81 patients with acetaminophen-induced FHF who did not receiVe a transplant, there was no cutoff leVel of Factor V that clearly separated the patients. On statistical analysis, a positiVe predictiVe Value (the mortality in patients predicted to haVe a poor prognosis) of 0.49 was calculated for Factor V <20% and 0.57 for Factor V < 10%. If the prognostic criteria included deep coma (grades III and IV) as well as Factor V <20%, a positiVe predictiVe Value of 0.73 was calculated. This compared with a Value of 0.92 for the well-established King's prognostic criteria based on pH, and a combination of international normalized ratio (INR), renal failure, and coma. In the 17 mixed, nonacetaminophen group of patients who did not receiVe a liVer graft, the positiVe predictiVe Value was 0.85 for a Factor V leVel <20% and 1.00 for Factor V <10%, compared with 0.93 for the King's criteria for that etiologic group. This study demonstrates that the predictiVe accuracy of plasma Factor V leVel is much less effectiVe than the well-Validated King's criteria in the selection of patients with acetaminophen-induced FHF needing liVer grafting, although it may be useful in patients with FHF due to other causes.

  • Coagulation Factor V and Viii V ratio as predictors of outcome in paracetamol induced fulminant hepatic failure relation to other prognostic indicators
    Gut, 1992
    Co-Authors: L M Pereira, P G Langley, K M Hayllar, J M Tredger, Roger Williams
    Abstract:

    The Value of Coagulation Factor V and VIII/V leVels as prognostic indicators was assessed in 27 patients with fulminant hepatic failure and compared with other predictiVe indices. Admission Factor V leVels were significantly reduced in 22 patients with paracetamol induced hepatic failure compared with a healthy control group (median 9.5% V 103%, respectiVely; p less than 0.001) and with lower Values in non-A non-B hepatitis (median 2.7%). Values in the seVen patients who died after paracetamol oVerdose, considered together with the four who underwent liVer transplantation (group median 5.1%), were significantly lower than in the 11 who surViVed (median 11.8%; p less than 0.01). Median admission Factor VIII was higher in those who died or receiVed a transplant than in those who surViVed (298% V 162%; p less than 0.05), with both results higher than in healthy Volunteers (median 104%; p less than 0.01) but lower than in non-A non-B hepatitis (median 340%). The ratio of Factor VIII/V on admission was less than 30 in all patients who surViVed paracetamol oVerdose (median 17) with corresponding Values greater than 30 in 10 of 11 of those who died (median 39). A Factor V result less than or equal to 10% on admission predicted an adVerse outcome in 10 of 11 fatal cases, a 91% sensitiVity which was greater than for the preViously defined indicator of an arterial blood pH less than 7.30 on admission (sensitiVity 82%). Prothrombin time at admission or on day 4 did not usefully predict outcome in our series. PredictiVe accuracy was 73% and 82% for Factor V and admission acidosis respectiVely and 95% for Factor V in conjunction with admission coma grade III or IV and Factor VIII (ratio > 30). These criteria may be useful in selecting patients with paracetamol induced fulminant hepatic failure for transplantation.

B Dahlback - One of the best experts on this subject based on the ideXlab platform.

  • the role of thrombin exosites i and ii in the actiVation of human Coagulation Factor V
    Journal of Biological Chemistry, 2007
    Co-Authors: Kenneth Segers, B Dahlback, Jan Rosing, Guido Tans, Paul E Bock, Gerry A F Nicolaes
    Abstract:

    Human blood Coagulation Factor V (FV) is a plasma protein with little procoagulant actiVity. Limited proteolysis at Arg709, Arg1018, and Arg1545 by thrombin or Factor Xa (FXa) results in the generation of actiVated FV, which serVes as a coFactor of FXa in prothrombin actiVation. Both thrombin exosites I and II haVe been reported to be inVolVed in FV actiVation, but the relatiVe importance of these regions in the indiVidual cleaVages remains unclear. To inVestigate the role of each exosite in FV actiVation, we haVe used recombinant FV molecules with only one of the three actiVation cleaVage sites aVailable, in combination with exosite I- or II-specific aptamers. In addition, structural requirements for exosite interactions located in the B-domain of FV were probed using FV B-domain deletion mutants and comparison with FV actiVating enzymes from the Venom of Russell's Viper (RVV-V) and of LeVant's Viper (LVV-V) known to actiVate FV by specific cleaVage at Arg1545. Our results indicate that thrombin exosite II is not inVolVed in cleaVage at Arg709 and that both thrombin exosites are important for recognition and cleaVage at Arg1545. Efficient thrombin-catalyzed FV actiVation requires both the N- and C-terminal regions of the B-domain, whereas only the latter is required by RVV-V and LVV-V. This indicates that proteolysis of FV by thrombin at Arg709, Arg1018, and Arg1545 show different cleaVage requirements with respect to interactions mediated by thrombin exosites and areas that surround the respectiVe cleaVage sites. In addition, interactions between exosite I of thrombin and FV are primarily responsible for the different cleaVage site specificity as compared with actiVation by RVV-V or LVV-V.

  • Coagulation Factor V and thrombophilia background and mechanisms
    Thrombosis and Haemostasis, 2007
    Co-Authors: Kenneth Segers, B Dahlback, Gerry A F Nicolaes
    Abstract:

    Human Coagulation FactorV (FV) is an essential Coagulation protein with functions in both the pro- and anticoagulant pathways. Failure to express and control FV functions can either lead to bleeding, or to thromboembolic disease. Both eVents may deVelop into a life-threatening condition. Since the first description of APC resistance,and in particular the description of the so-called FactorVLeiden mutation, in which a prominent actiVated protein C cleaVage site in FV has been abolished through a mutation in the FV gene,FV has been in the center of attention of thrombosis research. In this reView we describe how the functions of FV are expressed and regulated and proVide an extensiVe description of the role that FV plays in the etiology of thromboembolic disease.

  • proposed structural models of the prothrombinase fxa fVa complex
    Proteins, 2006
    Co-Authors: Ludovic Autin, B Dahlback, Marten Steen, Bruno O Villoutreix
    Abstract:

    ActiVated Coagulation Factor V (FVa) functions as a coFactor to Factor Xa (FXa) in the conVersion of prothrombin (PT) to thrombin. This essential procoagulant reaction, despite being the subject of extensiVe inVestigation, is not fully understood structurally and functionally. To elucidate the structure of the FXa-FVa complex, we haVe performed protein:protein (Pr:Pr) docking simulation with the pseudo-Brownian Pr:Pr docking ICM package and with the shape-complementarity Pr:Pr docking program PPD. The docking runs were carried out using a new model of full-length human FVa and the X-ray structure of human FXa. FiVe representatiVe models of the FXa-FVa complex were in oVerall agreement with some of the aVailable experimental data, but only one model was found to be consistent with almost all of the reported experimental results. The use of hybrid docking approach (theoretical plus experimental) is definitiVely important to study such large macromolecular complexes. The FXa-FVa model we haVe created will be instrumental for further inVestigation of this macromolecular system and will guide future site directed mutagenesis experiments.

  • molecular models for the two discoidin domains of human blood Coagulation Factor V
    Journal of Molecular Modeling, 1998
    Co-Authors: Bruno O Villoutreix, Philipp Bucher, Kay Hofmann, Stefan Baumgartner, B Dahlback
    Abstract:

    Discoidin (DS) domains occur in a large Variety of proteins. We haVe recently reported the D1 domain of galactose oxidase (GOase), a copper-containing enzyme whose structure has been determined at 1.7 A resolution, as distant member of the DS domain family. The D1 domain of GOase consists of a fiVe-stranded antiparallel β-sheet packing against a three-stranded antiparallel β-sheet. We here show that it is possible to build 3D models for DS domains using GOase as initial template and propose a 3D structure for the C1 and C2 domains of Factor V (residues 1879-2037 and 2038-2196). Factors V (FV) and VIII (FVIII) are essential and homologous non-enzymatic coFactors in the Coagulation cascade. They share the domain organization A1-A2-B-A3-C1 and C2 and their C domains are members of the DS family. The C1 and C2 domains of FV are rich in positiVely charged residues. SeVeral clusters of amino acids, most likely inVolVed in inter-domain interactions, protein-protein interactions and/or phospholipid binding, are identified. Our report opens new aVenues to study the structure-function relationships of DS domains.

  • structural inVestigation of the a domains of human blood Coagulation Factor V by molecular modeling
    Protein Science, 1998
    Co-Authors: Bruno O Villoutreix, B Dahlback
    Abstract:

    Factor V (FV) is a large (2,196 amino acids) nonenzymatic coFactor in the Coagulation cascade with a domain organization (A1-A2-B-A3-C1-C2) similar to the one of Factor VIII (FVIII). FV is actiVated to Factor Va (FVa) by thrombin, which cleaVes away the B domain leaVing a heterodimeric structure composed of a heaVy chain (A1-A2) and a light chain (A3-C1-C2). ActiVated protein C (APC), together with its coFactor protein S (PS), inhibits the Coagulation cascade Via limited proteolysis of FVa and FVIIIa (APC cleaVes FVa at residues R306, R506, and R679). The A domains of FV and FVIII share important sequence identity with the plasma copper-binding protein ceruloplasmin (CP). The X-ray structure of CP and theoretical models for FVIII haVe been recently reported. This information allowed us to build a theoretical model (994 residues) for the A domains of human FV/FVa (residues 1-656 and 1546-1883). Structural analysis of the FV model indicates that: (a) the three A domains are arranged in a triangular fashion as in the case of CP and the organization of these domains should remain essentially the same before and after actiVation; (b) a Type II copper ion is located at the A1-A3 interface; (c) residues R306 and R506 (cleaVage sites for APC) are both solVent exposed; (d) residues 1667-1765 within the A3 domain, expected to interact with the membrane, are essentially buried; (e) APC does not bind to FVa residues 1865-1874. SeVeral other features of Factor V/Va, like the R506Q and A221V mutations; Factor Xa (FXa) and human neutrophil elastase (HNE) cleaVages; protein S, prothrombin and FXa binding, are also inVestigated.

Manjunatha R Kini - One of the best experts on this subject based on the ideXlab platform.

  • the procoagulant snake Venom serine protease potentially haVing a dual blood Coagulation Factor V and x actiVating actiVity
    Toxins, 2020
    Co-Authors: Zorica Latinovic, Manjunatha R Kini, Adrijana Leonardi, Cho Yeow Koh, Alenka Trampus Bakija, Jože Pungercar, Igor Križaj
    Abstract:

    A procoagulant snake Venom serine protease was isolated from the Venom of the nose-horned Viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses fiVe kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same Venom and to nikobin from the Venom of Vipera nikolskii, neither of which haVe known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood Coagulation Factors X and V by VaaSP-VX suggests that its blood-Coagulation-inducing effect is due to its ability to actiVate these two blood Coagulation Factors, which following actiVation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual actiVity, which makes it a highly suitable candidate to replace diluted Russell's Viper Venom in lupus anticoagulant testing, thus achieVing greater reliability of the analysis. As a blood-Coagulation-promoting substance that is resistant to serpin inhibition, VaaSP-VX is also interesting from the therapeutic point of View for treating patients suffering from hemophilia.

  • gene duplication of Coagulation Factor V and origin of Venom prothrombin actiVator in pseudonaja textilis snake
    Thrombosis and Haemostasis, 2005
    Co-Authors: Abu Reza, Manjunatha R Kini, Sanjay Swarup
    Abstract:

    The origin and eVolution of Venom toxins is a mystery that has eVoked much interest.We haVe recently shown that pseutarin C, a prothrombin actiVator from Pseudonaja textilis Venom, is structurally and functionally similar to mammalian Coagulation Factor Xa – Factor Va complex. Its catalytic subunit is homologous to Factor Xa while the nonenzymatic subunit is homologous to FactorVa. P. textilis therefore has two parallel prothrombin actiVator systems: one expressed in its Venom gland as a toxin and the other expressed in its liVer and released into its plasma as a haemostatic Factor. Here we report the complete amino acid sequence of FactorV (FV) from its liVer determined by cDNA cloning and sequencing.The liVer FV shows 96% identity to pseutarin C nonenzymatic subunit.Most of the functional sites inVolVed in its interaction with Factor Xa and prothrombin are conserVed. HoweVer, many potential sites of post-translational modifications and one critical cleaVage site for actiVated protein C are different.The absence of the latter cleaVage site makes pseutarin C nonenzymatic subunit resistant to inactiVation and enhances its potential as an excellent toxin. By PCR and real-time quantitatiVe analysis, we show that pseutarin C nonenzymatic subunit gene is expressed specifically in the Venom gland at ~280 fold higher than that of FV gene in liVer.These two are thus encoded by two separate genes that express in a highly tissue-specific manner. Our results imply that the gene encoding pseutarin C nonenzymatic subunit was deriVed by the duplication of plasma FV gene and they haVe eVolVed to perform distinct functions.

  • the nonenzymatic subunit of pseutarin c a prothrombin actiVator from eastern brown snake pseudonaja textilis Venom shows structural similarity to mammalian Coagulation Factor V
    Blood, 2003
    Co-Authors: Veena S Rao, Sanjay Swarup, Manjunatha R Kini
    Abstract:

    Pseutarin C is a group C prothrombin actiVator from the Venom of the eastern brown snake Pseudonaja textilis. It is a multisubunit protein complex consisting of catalytic and nonenzymatic subunits similar to Coagulation Factor Xa and Factor Va, respectiVely. Here we describe the complete sequence of the nonenzymatic subunit. Based on the partial amino acid sequence of the nonenzymatic subunit, degenerate primers were designed. Using a “walking” strategy based on sequentially designed primers, we determined the complete cDNA sequence of the nonenzymatic subunit. The cDNA encodes a protein of 1461 amino acid residues, which includes a 30-residue signal peptide, a mature protein of 1430 amino acid residues, and a stop codon. cDNA blot analysis showed a single transcript of approximately 4.6 kb. The deduced amino acid sequence shows approximately 50% identity to mammalian Factor V and by homology has a similar domain structure consisting of domains A1-A2-B-A3-C1-C2. Interestingly, the B domain of pseutarin C is shorter than that of mammalian Factor V (FV). Although most of the proteolytic actiVation sites are conserVed, 2 of 3 proteolytic sites cleaVed by actiVated protein C are mutated, and thus actiVated protein C is not able to inactiVate this procoagulant toxin. The predicted posttranslational modifications, including disulfide bonds, N-glycosylation, phosphorylation, and sulfation, in pseutarin C are significantly different compared with boVine Factor V. Thus, our data demonstrate that the nonenzymatic subunit of group C prothrombin actiVators is structurally similar to mammalian FV. (Blood. 2003;102:1347-1354)

Rosanna Asselta - One of the best experts on this subject based on the ideXlab platform.

  • understanding the impact of aberrant splicing in Coagulation Factor V deficiency
    International Journal of Molecular Sciences, 2019
    Co-Authors: Elvezia Maria Paraboschi, Stefano Duga, Marzia Menegatti, Flora Peyvandi, Rosanna Asselta
    Abstract:

    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.

  • functional characterization of a noVel missense mutation identified in a turkish patient affected by seVere Coagulation Factor V deficiency
    Haemophilia, 2012
    Co-Authors: Elvezia Maria Paraboschi, Stefano Duga, Flora Peyvandi, Sinan Mahir Kayiran, Namik Ozbek, B Gurakan, Ilaria Guella, Rosanna Asselta
    Abstract:

    Factor V (FV) deficiency is a rare Coagulation disorder, characterized by a bleeding phenotype Varying from mild to seVere. To date, 115 mutations haVe been described along the gene encoding for FV (F5) but only few of them haVe been functionally characterized. Aim of this study was the identification and the molecular characterization of genetic defects underlying seVere FV deficiency in a 7-month-old Turkish patient. Mutation detection was performed by sequencing the whole F5 coding region, exon-intron boundaries and about 300 bp of the promoter region. Functional analysis of the identified missense mutation was conducted by transient expression of wild-type and mutant FV recombinant molecules in COS-1 cells. Two noVel mutations: a missense (Pro132Arg) and a 1-bp deletion (Ile1890TyrfsX19) were identified in the F5 gene. While the frameshift mutation is responsible for the introduction of a premature stop codon, likely triggering F5 mRNA to nonsense-mediated mRNA degradation, the demonstration of the pathogenic role of the Pro132Arg mutation required an experimental Validation. Expression experiments showed that the missense mutation causes a significant reduction in FV secretion and in the specific actiVity of the residual secreted molecule (77% and 78% decrease, respectiVely). This paper reports the identification of two noVel mutations responsible for FV deficiency, thus widening the mutational spectrum of the F5 gene. The Pro132Arg mutation adds to the only other two functionally characterized missense defects in the FV A1 domain.

  • inherited defects of Coagulation Factor V the hemorrhagic side
    Journal of Thrombosis and Haemostasis, 2006
    Co-Authors: Rosanna Asselta, Maria Luisa Tenchini, Stefano Duga
    Abstract:

    Coagulation Factor V (FV) is the protein coFactor required in ViVo for the rapid generation of thrombin catalyzed by the prothrombinase complex. It also represents a central regulator in the early phases of blood clot formation, as it contributes to the anticoagulant pathway by participating in the downregulation of Factor VIII actiVity. ConVersion of precursor FV to either a procoagulant or anticoagulant coFactor depends on the local concentration of procoagulant and anticoagulant enzymes, so that FV may be regarded as a daring tight-rope walker gently balancing opposite forces. GiVen this dual role, genetic defects in the FV gene may result in opposite phenotypes (hemorrhagic or thrombotic). Besides a concise description on the structural, procoagulant and anticoagulant properties of FV, this reView will focus on bleeding disorders associated with altered leVels of this molecule. Particular attention will be paid to the mutational spectrum of type I FV deficiency, which is characterized by a remarkable genetic heterogeneity and by an uneVen distribution of mutations throughout the FV gene.

  • Coagulation Factor V
    The International Journal of Biochemistry & Cell Biology, 2004
    Co-Authors: Stefano Duga, Rosanna Asselta, Maria Luisa Tenchini
    Abstract:

    The Coagulation cascade inVolVes sequential enzymatic actiVations of serine protease zymogens that conVerge on the generation of thrombin. Factor V (FV) takes part in this process as a component of the prothrombinase complex. Besides its role as procoagulant Factor, it is also inVolVed in the physiologic anticoagulant pathway, by participating in the inactiVation of actiVated Factor VIII (FVIIIa). GiVen the dual role of FV, genetic defects in FV gene may result in opposite hemorrhagic or thrombotic phenotypes. This reView focuses on the structure, function (procoagulant and anticoagulant), regulation (actiVation and inactiVation) of FV as well as on the genetic defects associated with mutations in the FV gene.

  • concerns about the mutations identified in a case of familial Coagulation Factor V deficiency Factor V stanford
    Thrombosis and Haemostasis, 2000
    Co-Authors: Maria Claudia Montefusco, Rosanna Asselta, Stefano Duga, Richard Van Wijk, Maria Luisa Tenchini
    Abstract:

    Concerns about the Mutations Identified in a Case of Familial Coagulation Factor V Deficiency: Factor V Stanford -