Severe coagulation factor V deficiency caused by 2 novel frameshift mutations: 2952delT in exon 13 and 5493insG in exon 16 of factor 5 gene

Abstract A male infant with severe bleeding tendency had undetectable factor V activity. Sequence analysis of the proband's DNA revealed one base deletion in exon 13 (2952delT) and one base insertion in exon 16 (5493insG) in heterozygous form. Both mutations introduced a frameshift and a premature stop at codons 930 and 1776, respectively. The proband's father and mother were heterozygous for 2952delT and for 5493insG, respectively. Both mutations would result in the synthesis of truncated proteins lacking complete light chain or its C-terminal part. In the patient's plasma, no factor V light chain was detected by enzyme-linked immunosorbent assay. The N-terminal portion of factor V containing the heavy chain, and the connecting B domain was severely reduced but detectable (1.7%). A small amount of truncated factor V–specific protein with a molecular weight ratio of 236 kd could be immunoprecipitated from the plasma and detected by Western blotting. This protein, factor VDebrecen, corresponds to the translated product of exon 16 mutant allele.

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Coagulation Functions with Clinical Phenotype and Inhibitory Mechanisms in Acquired Factor V Inhibitors.

Blood ◽

10.1182/blood.v120.21.2232.2232 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2232-2232

Author(s):

Tomoko Matsumoto ◽

Keiji Nogami ◽

Kenichi Ogiwara ◽

Midori Shima

Keyword(s):

Light Chain ◽

Thrombin Generation ◽

Clinical Phenotype ◽

Waveform Analysis ◽

Severe Bleeding ◽

Functional Evaluation ◽

Factor V ◽

Bleeding Tendency ◽

Coagulation Function ◽

Apc Resistance

Abstract Abstract 2232 Development of acquired factor V (aFV) inhibitors rarely occurs, but its clinical phenotype varies from asymptomatic to life-threatening bleeding. A recent systematic report describes that little bleeding symptom is present in 20% at the diagnosis for patients with acquired FV (aFV) inhibitors. However, the coagulation function and its mechanism(s) on the different clinical phenotype are poorly understood. In this study, we examined the coagulation function on aFV inhibitors by using comprehensive coagulation assays, thrombin generation test (TGT) and clot waveform analysis (CWA). TGT was performed using tissue factor (0.5 pM), phospholipid (PL; 4 μM) and ellagic acid (0.3 μM). CWA, that evaluates the parameters of min1 as maximum coagulation velocity and min2 as maximum coagulation acceleration, was performed on MDA-II® system. We tested 7 cases with aFV inhibitors. Four cases were asymptomatic (FV:C; 3.6±3.4 IU/dl, inhibitor 5.8±3.3 BU/ml: non-B group), and 3 cases had severe bleeding tendency (2.9 ± 4.5 IU/dl, 66 ±51 BU/ml; B group). In TGT, all cases in both groups little showed the thrombin generation within 60 min, independently of FV:C level and clinical phenotype, showing little informative in functional evaluation for aFV inhibitors. However, in a PT-based CWA, the clotting time observed in non-B group was markedly shorted compared to that in B group (62.2±17.0/112±15 sec; p=0.006). In addition, both parameters in non-B group were significantly greater than those in B group ( min1 ; 2.89±1.10/0.98±0.29 dT/dt; p=0.014) and ( min2 ; 0.75±0.40/0.15±0.07 d2T/dt2; p=0.028), suggesting that CWA was useful for the prediction and monitoring of hemorrhagic symptoms in patients with aFV inhibitors. To confirm the distinct mechanism(s) on both groups, the IgGs from aFV plasmas were immune-purified using protein G-Sepharose. In the reactant mixtures with normal plasma and aFV IgGs, all parameters obtained in CWA were similar to those obtained in patients' plasmas. SDS-PAGE and western blotting revealed that 2 cases in B group reacted the light chain of FV(a). However, 2 cases in non-B group reacted the heavy chain, and other 2 cases were reacted with both chains (heavy>light), indicative of the distinct epitopes of IgGs in both groups. Since the light chain contains the PL-binding site(s), the effects of aFV IgGs were examined on the FV-PL binding in an ELISA. All IgGs in B group inhibited this binding (by 40–90%) dose-dependently, whilst little affected in non-B group. Since FV acts as a cofactor of activated protein C (APC) on inactivation of FVIIIa, the effects of aFV IgGs on the ability of APC on FVIIIa inactivation were examined using intrinsic FXa generation assay. The APC sensitivity ratio (APCsr) was expressed as ratio of amounts of generated FXa in the absence of APC relative to its presence. A low level of APCsr indicates the reduction in FVIIIa inactivation, consequently APC resistance. APCsr values in B group were >2.0 within normal range, whilst those in non-B group were decreased to 1.5, supportive of APCR in non-B group. Based on these findings, we propose that severe bleeding tendency in B group would be appeared through negligible prothrombinase activity, since aFV IgGs blocked the FV(a)-PL binding. While, clinical phenotype in non-B group would be asymptomatic, since aFV IgGs unaffect the FV(a)-PL binding and further cause the APC resistance. In addition, various clinical phenotypes in aFV inhibitors appear to be dependent on the recognizing epitope of these IgGs. Disclosures: No relevant conflicts of interest to declare.

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Understanding the Impact of Aberrant Splicing in Coagulation Factor V Deficiency

International Journal of Molecular Sciences ◽

10.3390/ijms20040910 ◽

2019 ◽

Vol 20 (4) ◽

pp. 910

Author(s):

Elvezia Paraboschi ◽

Marzia Menegatti ◽

Flora Peyvandi ◽

Stefano Duga ◽

Rosanna Asselta

Keyword(s):

Ex Vivo ◽

Coagulation Factor ◽

Missense Variant ◽

Premature Termination Codon ◽

Severe Bleeding ◽

Factor V ◽

Bleeding Tendency ◽

Biallelic Mutations ◽

Cryptic Splice Sites ◽

The Impact

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.

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Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽

10.1182/blood.v83.8.2180.bloodjournal8382180 ◽

1994 ◽

Vol 83 (8) ◽

pp. 2180-2190

Author(s):

MD Rand ◽

M Kalafatis ◽

KG Mann

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Activated Protein C ◽

Coagulation Factor ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Factor V ◽

Platelet Factor ◽

Factor Va ◽

Plasma Factor

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

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Development of antibodies to thrombin and factor V with recurrent bleeding in a patient exposed to topical bovine thrombin

Blood ◽

10.1182/blood.v76.10.2011.2011 ◽

1990 ◽

Vol 76 (10) ◽

pp. 2011-2016 ◽

Cited By ~ 160

Author(s):

JL Zehnder ◽

LL Leung

Keyword(s):

Enzyme Linked Immunosorbent Assay ◽

Recurrent Bleeding ◽

Severe Bleeding ◽

Factor V ◽

Bleeding Events ◽

Bovine Thrombin ◽

Normal Plasma ◽

Human Thrombin ◽

Immunomodulatory Therapies ◽

Bovine Factor

Abstract A 65 year old patient who was exposed to topical bovine thrombin during cardiac surgery developed markedly prolonged clotting times and a severe bleeding diathesis. Mixing studies with normal plasma failed to correct the clotting times. Platelet transfusions, immunosuppressive and immunomodulatory therapies were ineffective, but plasmapheresis was effective in decreasing clotting times and in the resolution of clinical bleeding events. The patient's purified IgG reacted with bovine thrombin by immunoblotting and enzyme-linked immunosorbent assay (ELISA). However, the IgG reacted minimally with human thrombin. In view of the severe bleeding, a coexisting inhibitor was sought. The patient's factor V activity was 1% of normal and was not corrected by mixing with normal plasma, demonstrating the presence of an inhibitor against factor V. The patient's IgG reacted with both bovine and human factor V. Immunoblotting localized the site of antibody binding to the light chain of activated bovine factor V. Detectable amounts of bovine factor V were found in commercial bovine thrombin preparations by ELISA. The data suggest that patients exposed to topical bovine thrombin may develop antibodies to thrombin and factor V. Anti-thrombin antibodies may mask coexisting factor V inhibitors responsible for clinical bleeding.

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The Thrombogram in Rare Inherited Coagulation Disorders: Its Relation to Clinical Bleeding

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613258 ◽

2002 ◽

Vol 88 (10) ◽

pp. 576-582 ◽

Cited By ~ 181

Author(s):

Raed Al Dieri ◽

Flora Peyvandi ◽

Elena Santagostino ◽

Muriel Giansily ◽

Pier Mannuccio Mannucci ◽

...

Keyword(s):

Lag Time ◽

Peak Height ◽

Factor Vii ◽

Clotting Factor ◽

Severe Bleeding ◽

Factor V ◽

Bleeding Tendency ◽

Factor X ◽

Factor Xi ◽

Factor Concentration

SummaryWe investigated the relation between clotting factor concentration, the parameters of the thrombin generation curve (the thrombogram) and the severity of clinically observed bleeding in patients with congenital deficiency of prothrombin (n = 21), factor V (n = 22), factor VII (n = 22), factor X (n = 10), factor XI (n = 7) and factor XII (n = 6). The parameters used were: area under the curve (endogenous thrombin potential, ETP), peak concentration of thrombin attained and lag time before manifest formation.Peak height and ETP varied linearly with the concentration of prothrombin. For the other factors these parameters hyperbolically approached to the 100% limit with increasing clotting factor concentration. Half normal ETP was seen at about the following concentrations: prothrombin (50%), factor V (1%), factor VII (2%), factor X (5%) and factor XI (1%). As a rule, the peak height was somewhat more sensitive to clotting factor decrease than the ETP was.In all the patients with severe bleeding symptoms the ETP was less than 20% of normal. Bleeding tendency was absent or mild in patients with an ETP of 30% or higher. This value (except for prothrombin) is already obtained at concentrations of clotting factor of 1%-2%, which corroborates the clinical observation that a severe bleeding tendency is only seen in severe clotting factor deficiencies (less than 1%). The one exception was a patient with factor VII deficiency and severe bleeding, who showed a normal ETP value, albeit with a decreased peak height and a prolonged lag-time.

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BIOSYNTHESIS AND EXPRESSION OF FACTOR V IN MAGAKARYOCYTES

10.1055/s-0038-1642955 ◽

1987 ◽

Author(s):

R W Colman ◽

A Gewirtz ◽

D L Wang ◽

M M Huh ◽

B P Schick ◽

...

Keyword(s):

Endothelial Cells ◽

Membrane Proteins ◽

Guinea Pig ◽

Light Chain ◽

Critical Role ◽

Small Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Factor V ◽

Single Chain ◽

Activation Peptide

Coagulation factor V (FV), is a single chain, multifunctional glycoprotein of Mr 350,000 which interacts with a variety of hemostatic proteins such as factor Xa, prothrombin, thrombin and protein C, on the surface of platelets and vascular endothelial cells. FV serves as both a cofactor and substrate in the generation of thrombin and plays a critical regulatory role in both physiologic hemostasis and pathologic thrombosis. The biosynthesis of FV and its subsequent expression are therefore expected to be precisely controlled and may differ in the three sites of synthesis - hepatocytes, endothelial cells, and megakaryocytes (MK). We have previously demonstrated that each guinea pig MK contains 500 times as much FV as in a platelet, as quantified by a competitive enzyme-linked-immunosorbent assay and expresses FV by cytoimmunofluorescence. De novo biosynthesis was demonstrated by incorporation of S-methionine into FV purified on a immunoaffinity column. The purified MK protein exhibited both FV coagulant activity and antigenicity. However, MK FV was more slowly activated by thrombin, more stable in the absence of Ca and exhibited a slightly higher M of 380,000 compared to plasma FV. Similar studies have documented biosynthesis in human MK. In addition, all morphologically recognizable MK enriched by elutriation from human bone marrow contained FV as documented by both monospecific polyclonal and monoclonal antibodies (MAb) to FV. All these cells bound FV since a murine MAb reacting with the light chain of FV (B38) labeled all cells. In contrast, 68% of cells synthesized FV since B10, a MAb to the activation peptide recognizing FV but not FVa, labeled this fraction. To determine whether immature nonnorphologically recognizable MK expressed FV, we identified these cells with an antiserum to human platelet glycoproteins and then probed them with B38. Seventy percent (70%) of such small cells expressed FV. In contrast, no small cells in MK colonies cloned in FV deficient medium expressed FV while only 40% of such colonies contained cells which expressed FV.To further probe the regulation of FV in MK we attempted to correlate the synthesis of FV as probed by MAb B10 with geometric mean cell diameter, stage and ploidy. No significant correlation of FV with any of these indicators of MK maturation. In contrast, preliminary studies suggest that low doses of tetradecanoyl phorbol acetate augment both the number of MK containing FV and the level of FV expressed by individual cells. Thus, FV synthesis may be regulated independent of size, stage, or ploidy and protein kinase C may play a role.To further define the molecular nature of FV in MK we found that purified FV was converted from a monomer to high Mr multimers by an enzyme derived from MK. These multimers resulting from covalent crosslinking since they were stable to SDS, 100° C and reducing agents. The responsible enzyme appeared to be MK FXIIIa since it required C, was inhibited by agents which react with the active site thiol group and was blocked by pseudoamine donor substrates such as putrescine. In addition, FXIIIa was directly demonstrated in guinea pig MK by a specific activity stain. Other investigators have established that FV became irreversibly associated with platelet cytoskeletons after exposure to thrombin. tested whether FXIIIa might mediate this association by performing ligand blotting of platelet membrane proteins using 125I-FV(FV*). Only actin of all the membrane proteins was detected by radioautography. The binding of FV* to the cytoskeleton was dependent in the presence of Ca and FXIIIa. In purified systems crosslinked complexes containing FV* or radiolabeled actin were detected in separate experiments. In whole platelets, the formation of the heteropolymer, after thrombin stimulation, was inhibited by antibodies to FXIII a chain, FV activation peptide (B10) or actin. Endogenous platelet FV was also dependent on FXIII for incorporation into the platelet cytoskeleton after thrombin stimulation. When thrombin-treated FV was crosslinked to actin only the activation peptide (150 kDa) was crosslinked. The light chain or heavy chain of FVa were not involved. Thus FXIIIa play an important role in the binding of FV in platelets to the cytoskeleton during activation and secretion.Further studies of FV in megakaryocytes are necessary to define the regulation of biosynthesis and the control of expression which dictate its critical role in hemostasis and thrombosis.

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A Review of Coagulation Abnormalities of Autoimmune Acquired Factor V Deficiency with a Focus on Japan

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0041-1740149 ◽

2021 ◽

Author(s):

Akitada Ichinose ◽

Tsukasa Osaki ◽

Masayoshi Souri

Keyword(s):

Coagulation Factor ◽

Nationwide Survey ◽

Autoimmune Disorder ◽

Antibody Detection ◽

Extensive Literature ◽

Factor V ◽

Bleeding Tendency ◽

Factor X ◽

Timely Initiation ◽

Fv Antibody

AbstractCoagulation factor V (or FV for the purpose of medical safety) is an essential cofactor of coagulation factor X in the common pathway of coagulation; severe FV deficiency leads to a bleeding tendency. Although both congenital and acquired FV deficiencies are widely recognized, FV deficiency also presents as an autoimmune disorder. A nationwide survey on autoimmune coagulation factor deficiencies (AiCFDs) conducted in Japan by our Japanese Collaborative Research Group identified 24 new patients with autoimmune FV deficiency (AiFVD) in the past 5 years. Furthermore, our extensive literature search confirmed that 177 AiFVD cases have been reported in previous articles published from Japan. Patients with AiFVD in Japan were predominantly men, with age similar to those with other AiCFDs. AiFVD was confirmed as a relatively mild type of bleeding diathesis, associated with lower mortality rate than that for AiFVD and other AiCFDs reported in previous studies. Patients with AiFVD had variable FV inhibitor titers and both neutralizing anti-FV autoantibodies and nonneutralizing counterparts. Although spontaneous resolution occurs in some patients, timely initiation of hemostatic and immunosuppressive therapies helps arrest the bleeding and eliminate anti-FV antibodies, resulting in a high cumulative recovery rate. Immunological anti-FV antibody detection is recommended to avoid missing AiFVD cases for the presence of nonneutralizing anti-FV autoantibodies. Further investigation is necessary to clarify the long-term prognosis and optimal management of AiFVD.

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Identification of the Multimerin Binding Region within the C2 Domain of Human Factor V Using Constructs Generated by Alanine-Scanning and Site-Directed Mutagenesis.

Blood ◽

10.1182/blood.v104.11.124.124 ◽

2004 ◽

Vol 104 (11) ◽

pp. 124-124

Author(s):

Samira B. Jeimy ◽

Rachael A. Woram ◽

Nola Fuller ◽

Mary Anne Quinn-Allen ◽

Gerard Nicolaes ◽

...

Keyword(s):

Conformational Changes ◽

Light Chain ◽

Coagulation Factor ◽

Procoagulant Activity ◽

Site Directed Mutagenesis ◽

Factor V ◽

C2 Domain ◽

Wild Type ◽

Factor Va ◽

Covalently Linked

Abstract Activated coagulation factor V is a key non-enzymatic cofactor that is an essential component of the prothrombinase complex. In blood, much of the procoagulant factor V is stored in platelets, as a complex with the α-granule protein multimerin, for activation-induced release during clot formation. Presently, the molecular nature of multimerin - factor V binding has not been determined, although multimerin is known to interact with the light chain of factor V and Va. Using modified enzyme-linked immunoassays and recombinant factor V constructs, we previously found that discontinuous regions in the C2 domain of factor V were important for binding multimerin, and that these regions overlapped with areas in factor V important for its procoagulant function. Specifically, four (S2183T, W2063A/W2064A, K2060Q/K2061Q, K2060Q/K2061Q/W2063A/ W2064A) full-length, site-directed C2 mutants, and 12 (W2063A, W2064A (W2063, W2064)A, R2074A (R2072, R2074)A (K2101, K2103, K2104)A, L2116A (K2157, H2159, K2161)A, R2171A, R2174A, E2189A (R2187, E2189)A) B domain deleted, charge to alanine constructs had significantly reduced multimerin binding (p< 0.01), relative to the corresponding wild-type. In the present study, we evaluated multimerin-factor V binding with a new assay that used affinity purified, recombinant multimerin immobilized onto microtitre wells to test the binding of recombinant factor V constructs. Because results from the new binding assays were in agreement on the regions of the C2 domain important for multimerin binding, the new assay was used to examine the effect of thrombin on factor V-multimerin binding. Thrombin exposure led to significant dissociation of preformed multimerin-factor V complexes (p<0.01). In addition, thrombin cleaved factor Va had significantly reduced multimerin-binding in assays using antibodies against the factor Va heavy chain and light chain (p<0.01). Recently, our lab identified that platelets contain forms of factor V covalently linked to multimerin via cysteine 1085 in the factor V B-domain. After recombinant factor V was activated by thrombin, there was no detectable binding of the liberated B-domain to multimerin (p<0.001). Nonetheless, the B domain of factor V appeared to enhance factor V binding to multimerin, as factor V constructs synthesized without the B-domain had reduced multimerin binding even after conversion to factor Va, compared to wild-type factor V. Based on the overlap between multimerin-binding and procoagulant, PS binding regions in the C2 domain of factor V, we assessed the effect of multimerin on factor V procoagulant activity in one stage and two stage prothrombinase assays. However, multimerin did not neutralize factor V procoagulant activity when tested in molar excess. Our study indicates that multimerin binding of factor V is modulated by conformational changes in factor V upon activation, and that the factor V B-domain may function to enhance binding to multimerin. The dissociation of multimerin-factor V complexes by thrombin suggests multimerin might be important for delivering and localizing factor V onto platelets, prior to prothrombinase assembly.

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Identification of a Novel Factor V A2 Domain Tyrosine Deletion Mutation in a Patient with Factor V Deficiency and Bleeding.

Blood ◽

10.1182/blood.v104.11.4033.4033 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4033-4033

Author(s):

Carol D. Jones ◽

Fernando Negro ◽

Katherine Darnell ◽

James L. Zehnder

Keyword(s):

Amino Acids ◽

Frameshift Mutation ◽

Coagulation Factor ◽

Severe Bleeding ◽

In Vitro Mutagenesis ◽

Factor V ◽

Coding Region ◽

Base Pair Deletion ◽

A2 Domain ◽

Factor Function

Abstract The gene for coagulation Factor V (FV) is located on chromosome 1q23. FV deficiency shows an autosomal recessive mode of inheritance; heterozygotes are generally not clinically affected. The homozygous clinical phenotype occurs in approximately 1 per million individuals with variable severity of bleeding. Thus, genotype-phenotype correlations are likely to shed light on functionally important residues of FV. Here we describe a case of FV deficiency with a severe bleeding phenotype. The proband is a male infant from Argentina. His parents are unrelated. He was born healthy with no bleeding from the umbilical stump or other symptoms. He presented at eight months with a CNS hemorrhage, then suffered a second massive subdural bleed at nine months of age. Both episodes required surgical drainage and treatment with fresh frozen plasma He continues to receive prophylactic FFP infusions and has some residual neurologic impairment. The proband’s FV activity ranges from 2–14%. Two siblings are unaffected. His father’s FV activity is 50% and his mother’s is 70%. We performed DNA sequencing spanning the entire coding region of the proband’s FV gene and found two heterozygous mutations: a heterozygous single base pair deletion, del 2952T in exon 13, located in the B-domain of the FV protein, causing a frameshift mutation followed by a premature termination codon 3 amino acids downstream; and a novel 3-bp deletion in exon 10. This deletion is in-frame and results in the deletion of Y478. The del 2952T frameshift mutation was present in the father, while the del Y478 mutation was present in the mother. Y478 is in the A2 domain of FV and adjacent to another tyrosine, Y477. Evidence suggests that these tyrosine residues are important for co-factor function. Tyrosine residue sulfation has been shown to be required for full activity of the homologous co-factor, FVIII, as well as for hirudin. These sulfated tyrosines and surrounding acidic amino acids have been proposed to be important in interactions with the thrombin anion binding exosite; in the case of hirudin, sulfation of a carboxy-terminal tyrosine increases the affinity for thrombin 10-fold. The homologous tyrosines, Y718 and Y719 appear to be sulfated in FVIII. FV has been shown to be sulfated, but the precise location of the FV sulfation sites has not yet been determined. One of this patient’s FV alleles is nonfunctional due to a frameshift and a premature trancation of translation. With respect to the other allele, we hypothesize that, like FVIII, one or both of FV tyrosines 477 and 478 is sulfated, and that deletion of Y478 may result in disruption of FV co-factor function. In vitro mutagenesis and expression studies to characterize the functional consequences of the del Y478 and/or del Y477 are in progress.

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

Blood ◽

10.1182/blood.v110.11.1764.1764 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1764-1764

Author(s):

Keiko Shinozawa ◽

Kagehiro Amano ◽

Takashi Suzuki ◽

Hiroshi Inaba ◽

Katsuyuki Fukutake

Keyword(s):

Coagulation Factor ◽

Normal Subjects ◽

Japanese Patients ◽

The Other ◽

Hek293 Cells ◽

Poor Correlation ◽

Factor V ◽

Bleeding Tendency ◽

Wild Type ◽

Mammalian Expression

Abstract 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 symptoms Patient No. Mutation FV activity (%) FV antigen (%) Bleeding symptoms 1 D68H 4 4 asymptomatic 2 N468S 3 3 asymptomatic 3 V1813M & 5-bp deletion <1 9 severe 4 V1813M <1 4 moderate 5 R2174L 1 5 very mild

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