Arg2074Cys missense mutation in the C2 domain of factor V causing moderately severe factor V deficiency: molecular characterization by expression of the recombinant protein

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 173-177 ◽  
Author(s):  
Stefano Duga ◽  
Maria Claudia Montefusco ◽  
Rosanna Asselta ◽  
Massimo Malcovati ◽  
Flora Peyvandi ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Factor V ◽  
Metabolic Labeling ◽  
C2 Domain ◽  
Missense Mutations ◽  
Wild Type ◽  
Intracellular Degradation ◽  
Factor V Deficiency

Abstract Factor V (FV) deficiency is a rare bleeding disorder whose genetic basis has been described in a relatively small number of cases. Among a total of 12 genetic defects reported in severely or moderately severe deficient patients, 3 were missense mutations and in no case was the mechanism underlying the deficiency explored at the molecular level. In this study, a homozygous missense mutation at cDNA position 6394 in exon 23 of the FV gene was identified in a 22-year-old Italian patient. This mutation causes the replacement of arginine 2074 with a cysteine residue (Arg2074Cys) in the C2 domain of the protein. The effect of the Arg2074Cys mutation on FV secretion, stability, and activity was investigated. Site-directed mutagenesis of FV cDNA was used to introduce the identified mutation, and wild-type as well as mutant FV proteins were expressed by transient transfection in COS-1 cells. An enzyme immunoassay detected low FV antigen levels both in the conditioned media of cells expressing the mutant protein and in cell lysates. Metabolic labeling and pulse-chase experiments confirmed that the mutation caused an impaired secretion of FV associated with rapid intracellular degradation. In addition, evaluation of wild-type and mutant coagulant activity demonstrated that the FV molecules carrying the Arg2074Cys mutation have reduced activity. These findings, beside confirming the structural and functional importance of the arginine 2074 residue, demonstrate that its substitution with a cysteine impairs both FV secretion and activity.

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 ◽  
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.

A Novel In-Frame Deletion in Domain C1 of Factor V Gene Results in Profound Decrease in Factor V Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2151-2151
Author(s):  
Jaewoo Song ◽  
Hyun-Sook Kim ◽  
Singyoung Kim ◽  
Jong-Rak Choi ◽  
Yoo-Hong Min ◽  
...  
Keyword(s):  
Coagulation Factor ◽  
Factor V ◽  
C2 Domain ◽  
Functional Importance ◽  
Intracellular Degradation ◽  
Truncating Mutation ◽  
Factor V Deficiency ◽  
C1 Domain ◽  
Factor V Gene ◽  
Partial Deficiency

Abstract Coagulation factor V acts as the cofactor of activated factor X of prothrombinase complex is composed of six domains which are A1, A2, B, A3, C1, C2 arranged from N to C-terminal. Crystalography of C2 domain has been reported along with its three spike-like structures at the base which are important for interaction with phospholipids. But the functional importance of C1 domain which closely resembles C2 domain largely remains unidentified. We have experienced a family with hereditary factor V deficiency whose proband was a compound heterozygote of in-frame deletion located to domain C1 and truncating mutation of domain B. The proband was 25 year old male who suffered from bleeding after tooth extraction. Prothrombin time and activated partial thromboplastin time were both prolonged (35.7 sec, 111.7 sec respectively), and coagulation factor activities were all normal except for factor V which was 4%. The factor V antigen level measured by ELISA method was 3%. We sought for mutations of factor V gene by PCR direct sequencing targeting whole coding region. A truncating mutation (3481C>T, R1133X) was found in exon 13, where most of the other mutations have been reported. It has already been reported by Van Wijk et al. in 2001. The same mutation was found in his twin brother (factor V activity 5%) but in only one of two sisters exhibiting partial deficiency (factor V activity, 45% and 50% and antigen level, 25% and 37% each). In addition In-frame deletion (nt 6026 del 6 bp, corresponding to deletion of N1982, S1983) in C1 domain was also found in the proband and also in his twin brother and one sister who has not R1133X explaining the partial deficiency in two sisters each possessing different mutations. The putative structural and functional importance of N1982, S1983 was sought by examining protein model based on the crystal structure of bovine factor Va that is inactivated by protein C. N1982, S1983 are located on a loop region that is exposed on surface of domain C1 and have close contact with another loop in A3 domain. This model suggests the possibility that N1982 and S 1983 contribute to maintaining the stable conformation attributable to hydrogen bond formation between K1980 and N1986 of domain C1 with D1604 of domain A3. Mutations implicated in hereditary factor V deficiency involving domains other than A or B are mostly located in or affect the integrity of C2 domain. To the best of our knowledge only five mutations involving C1 domain have been reported till now. Four were truncating mutations and splicing error resulting in gross abnormality in protein structure. One missense mutation in this domain was reported to be subject to increased intracellular degradation. R1985A near to N1982 and A1983 also caused decreased factor V level in scanning mutagenesis study. The novel in-frame deletion can also be susceptible to accelerated degradation. And the in-frame deletion in our patient may also result in unstable factor 5 structure which enhances intracellular degradation. But the possibility of functional defect including decreased phospholipid binding or attenuated cofactor function due to incorrect positioning of domain A3 relative to domain C1, cannot be ruled out and should be further investigated.

The R2-haplotype associated Asp2194Gly mutation in the light chain of human factor V results in lower expression levels of FV, but has no influence on the glycosylation of Asn2181

2003 ◽  
Vol 89 (03) ◽  
pp. 429-437 ◽  
Author(s):  
Richard Dirven ◽  
Hans Vos ◽  
Rogier Bertina ◽  
Marijn Kolfschoten
Keyword(s):  
Human Factor ◽  
Light Chains ◽  
Factor V ◽  
Missense Mutations ◽  
Wild Type ◽  
Molecular Weights ◽  
Apc Resistance ◽  
Increased Risk ◽  
The Individual ◽  
Lower Expression

SummaryThe R2 haplotype of the FV gene spans from exon 8 through 25 and comprises several strongly linked polymorphisms in the FV gene, including some missense mutations. Carriership of the R2-FV allele has been associated with reduced plasma FV levels, increased FV1/FV2 ratios and mild APC resistance. Some studies have reported that carriership of the R2-FV allele is associated with an increased risk of venous thombosis. At this moment, the individual contribution to the R2-associated phenotypes of the different mutations linked to the R2 haplotype of FV is unclear. The main objective of our study was to obtain insight in the influence of the R2-related Asp2194Gly mutation on FV expression, FV structure and FV function using B-domainless rFV mutants. Replacing Asp at position 2194 by Gly resulted in a more than threefold reduction of rFV expression compared to rFV wild-type. Therefore, we propose that the R2-linked Asp2194Gly mutation is an important determinant of the association of the R2-FV allele with lower FV levels. Furthermore, the light chains from Asp2194Gly containing rFV mutants showed similar molecular weights as the light chains of the non-glycosylated rFVwt or the plasma FV2 isoform, indicating that glycosylation at Asn2181 is not stimulated by the presence of a glycine in position 2194. Finally, the apparent K d for dissociation of the FXaVa complex (K 1/2Xa) was not higher in rFV mutants with the Asp2194Gly mutation than for rFVwt, suggesting that also the affinity for negatively charged phospho-lipids is not affected by substitution of Asp into Gly at position at 2194.

Molecular characterisation of Tyr530Ser and IVS16–1G>T mutations causing severe factor V deficiency

2010 ◽  
Vol 104 (09) ◽  
pp. 536-543 ◽  
Author(s):  
Weidong Zheng ◽  
Yanhui Liu ◽  
Ying Luo ◽  
Zhihong Chen ◽  
Yan Wang ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Coagulation Factor ◽  
Underlying Mechanism ◽  
Procoagulant Activity ◽  
Site Directed Mutagenesis ◽  
Factor V ◽  
Wild Type ◽  
Site Mutation ◽  
Bioinformatics Analyses ◽  
Mutant Transcript

SummaryOur previous study reported a missense mutation (Tyr530Ser) and a splicing site mutation (IVS16–1G>T) in blood coagulation factor V (FV) gene in a two-year-old Chinese boy. However, the linkage between the mutations and severe FV deficiency and the underlying mechanism has not been elucidated. The present study was designed to investigate the effect of the two mutations and the possible pathogenetic mechanism. FV procoagulant activity showed tremendous decrease in the patient with two mutations. The bioinformatics analyses predicted that IVS16–1G>T mutation may cause the entire exon 17 of FV to be skipped in transcription and thereby result in a deletion mutant. To confirm the predicted results, the fragment of exon 16 to exon 18 containing IVS16–1G>T mutation was obtained by PCR and site-directed mutagenesis. IVS16–1G>T mutant and wild-type constructs were transfected into COS-7 cells. Sequence analysis showed that mutant transcript lacked the entire 180-bp length of exon 17. Moreover, compared to wild-type, the expression of the two mutant proteins was decreased and the procoagulant activity was also reduced when the deletion mutant cDNA and Tyr530Ser site mutant cDNA were transfected into COS-7 cells, respectively. Our results indicate that Tyr530Ser and IVS16–1G>T could be separately responsible for severe FV deficiency, while the phenotype in the proband could be caused by the combination effect of the two defects.

The Mutations SER2117→LEU, and TRP/TRP2063/64→MET/PHE in the Hydrophobic Spikes of the Factor V C2 Domain Lead to Tenacious Phospholipid Binding and Suggest a Mechanism through Which Phosphatidylserine Activates the Prothrombinase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2703-2703
Author(s):  
Gary E. Gilbert ◽  
Valerie A. Novakovic ◽  
Randal J. Kaufman ◽  
Hongzhi Miao ◽  
Steven W. Pipe
Keyword(s):  
Specific Activity ◽  
Phospholipid Membranes ◽  
Unexpected Result ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Hydrophobic Residues ◽  
Phospholipid Binding ◽  
Prothrombinase Complex ◽  
Type Factor

Abstract Factor V (fV) binds to phospholipid (PL) membranes via a motif localized to the C2 domain. We and others have shown that PL binding is mediated by two pairs of hydrophobic residues, each displayed at the tips of β-hairpin turns. The homologous hydrophobic residues in the C2 domain of factor VIII also contribute to PL binding. We hypothesized that the solvent-exposed hydrophobic residues of the fV C2 domain make specific contacts that influence membrane affinity and activity of fV. To test this hypothesis we have prepared fVIII/fV hybrid mutants in which amino acid(s) of the fV C2 domain were changed to the homologous residues of fVIII (Mutants #1 W/W 2063/2064 M/F, #2 L/S 2116/2117 L/L, and #3 W/W/S 2063/2064/2117 M/F/L). Mutants were expressed in COS-1 cells and purified by FPLC. The specific activity of the fV/FVIII hybrids #2 and #3 exceeded those of wild type factor V by approx. 10-fold and approx. 4-fold, respectively, in a prothrombin time assay with factor V deficient plasma. Apparent PL affinites were evaluated in a prothrombinase complex assay with limiting phospholipid. The apparent affinities are 9.8 and 21-fold higher than wild type factor V for mutants #2 and #3 which contain the S→L change in the second hydrophobic spike. An unexpected result was that mutants 1–3 supported prothrombinase activity in the absence of added phospholipid, in contrast to wild type fV. We hypothesized that this activity resulted from phospholipid that was not dissociated from fV during purification. This hypothesis was supported because activity was eliminated by incubation of the factor V mutants with phospholipase A2 or by incubation with lactadherin. The tenacity of the phospholipid binding was further investigated by washing immobilized wild type fV and fV mutants with CHAPS prior to elution from an FPLC column. fV mutants #2 and #3 retained activity after the CHAPS wash, free of added PL and activity remained inhibitable by lactadherin, further illustrating the tenacious phospholipid affinity. We utilized Mutants #1 & 2 in the absence of added PL to evaluate the mechanism through which soluble phosphatidylserine with 6-carbon acyl chains (C6PS) enhances activity of the prothrombinase complex. In the absence of phospholipid vesicles C6PS enhanced activity of Mutant 1 equivalent to wild type fV (> 20 fold). However, enhancement of mutant 2 activity, which presumably retained phospholipid via residues 2116/2117, was < 3-fold. This suggests that C6PS functions to activate wild type fV, in part, by engaging the free LS 2116/2117 hydrophobic spike. Together, these data indicate that the hydrophobic spikes of factor V influence the specific activity of factor V, that the high affinity reversible binding of fV to phospholipid membranes is readily perturbed by mutations, and that activation of the prothrombinase complex by C6PS and phospholipid membranes likely involves engagement of amino acids 2116/2117.

Molecular Mechanism of Type I Congenital Heparin Cofactor (HC) II Deficiency Caused by a Missense Mutation at Reactive P2 Site: HC II Tokushima

2001 ◽  
Vol 85 (01) ◽  
pp. 101-107 ◽  
Author(s):  
Yasuhiko Kanagawa ◽  
Toshio Shigekiyo ◽  
Ken-ichi Aihara ◽  
Masashi Akaike ◽  
Toshio Matsumoto ◽  
...  
Keyword(s):  
Molecular Pathogenesis ◽  
Nucleotide Position ◽  
Type I ◽  
Metabolic Labeling ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Intracellular Degradation ◽  
Atherosclerotic Lesions ◽  
Old Japanese ◽  
Digestion Pattern

SummaryWe found a 66-year-old Japanese patient with type I congenital heparin cofactor (HC) II deficiency manifesting multiple atherosclerotic lesions. To investigate the molecular pathogenesis of our patient, we performed sequencing analysis and expressed recombinant human wild-type and mutant HC II molecules in COS-1 and CHO-K1 cells. Sequencing analysis following amplification of each of all 5 exons and its flanking region showed a single C to T transition at nucleotide position 12,854 in exon 5, which changed a Pro443 codon (CCG) to Leu codon (CTG). Because this mutation generates a new Bbv I site, the Bbv I digestion pattern of the PCR-amplified exon 5 fragments from each family member was analyzed. In all cases, the patterns were consistent with the activities and antigen levels of plasma HC II in those members. Transient transfection, metabolic labeling and pulse-chase experiments followed by immunoprecipitation analysis showed that the recombinant mutant HC II molecules were secreted from COS-1 cells in reduced amounts compared with the wild-type, and that an enhanced intracellular association of the mutant molecules with a chaperone, GRP78/BiP, was observed in CHO-K1 cells. Northern blot analysis indicated that the mutant HC II mRNA was transcribed at a similar level as that of wild-type.Immunohistochemical staining of the transfected cells revealed that COS-1 cells expressing the mutant HC II molecules were stained mainly in the perinuclear area. We conclude that the impaired secretion of the mutant HC II molecules, due to intracellular degradation, is the molecular pathogenesis of type I congenital HC II deficiency caused by a Pro443 to Leu mutation at reactive P2 site.

Factor V Arg2074Cys: A Novel Missense Mutation in the C2 Domain of Factor V

2002 ◽  
Vol 87 (05) ◽  
pp. 923-924 ◽  
Author(s):  
Anna Bossone ◽  
Francesca D’Angelo ◽  
Rosa Santacroce ◽  
Domenico De Lucia ◽  
Maurizio Margaglione
Keyword(s):  
Missense Mutation ◽  
Factor V ◽  
C2 Domain

Conservative Mutations in the Membrane-Binding Motif of Factor V C2 Domain to Residues of Pseudonaja Textilis Venom Factor V Confer Phosphatidylserine-Independent Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2243-2243
Author(s):  
Valerie A Novakovic ◽  
Hongzhi Miao ◽  
Steven Pipe ◽  
Gary E. Gilbert
Keyword(s):  
Amino Acids ◽  
Human Factor ◽  
Membrane Binding ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Hydrophobic Membrane ◽  
Prothrombinase Complex ◽  
Membrane Affinity ◽  
Prothrombinase Activity

Abstract Abstract 2243 Toxicity of venom from the eastern brown snake (Pseudonaja textilis) is related to a prothrombin activator protein complex (pseutarin C) that is homologous to the factor Va/factor Xa complex. A previous study has found that the factor V-homologous subunit of this protein (pt-fV) is constitutively active and does not require anionic membranes to function (Bos et al. 2010, Blood). We have previously found that conservative mutation of the amino acids on the hydrophobic membrane binding regions (called spikes) of factor V (W2063M/W2064F/S2117L) can produce increased prothrombinase activity, increased membrane binding affinity and apparent phospholipid-independent prothrombinase activity. However, the membrane-independent activity is caused by retention of phospholipid by factor V through the purification process. We hypothesized that the P.textilis venom-derived factor V has an increase in lipid affinity due to differences in the membrane-interactive spikes. Sequence alignment of the P.textilis venom-derived factor V with bovine and human factor V revealed 5 amino acids located in the putative membrane-binding region (four on spike 3 and one in a region targeted by a small-molecule inhibitor of membrane binding for both factor VIII and factor V) that differed in the venom-derived factor V versus the consensus sequence of mammalian factor V. A mutant factor V that incorporated these five mutations (L2116M, S2117T, S2118T, E2119S, and S2183Y) (factor VMTTS/Y) was expressed in COS cells. After purification utilizing ion exchange chromatography, factor VMTTS/Y showed phospholipid-independent activity that could be inhibited with phospholipase A2. Subsequently, factor VMTTS/Y was washed extensively with CHAPS during purification to prevent phospholipid from co-purifying. Activity was measured with a prothrombin time assay with plasma lacking factor V. Specific activity was 1183 units/mg vs. 676 units/mg for wild type human factor V. Steady state kinetics of the prothrombinase complex with factor VMTTS/Y were assessed with varying concentrations of phospholipid vesicles. In the presence of membranes containing excess phosphatidylserine (15:20:65 PS:PE:PC), factor VMTTS/Y (5 pM) showed 39% greater Vmax than wild type human factor V and 3-fold higher apparent membrane affinity. With limiting phosphatidylserine (2:20:78 PS:PE:PC), factor VMTTS/Y (10 pM) showed 64% greater Vmax and 2-fold higher apparent membrane affinity. Factor VMTTS/Y, purified with a CHAPS wash, did not show lipid-independent activity but did support prothrombinase activity on membranes lacking PS or other negatively charged lipid (20:80 PE:PC). On these vesicles factor VMTTS/Y (50 pM) had a Vmax that was 8-fold higher than wild type factor V (see figure). These data indicate that the apparent phospholipid-independent activity results from higher membrane affinity or from greater activity on minimal phospholipid retained by factor V during purification. They imply that toxicity of pseutarin C may result, in part, from procoagulant activity on cell membranes that do not support the mammalian prothrombinase complex. Furthermore, they indicate that the precise manner in which the C2 domain of factor V binds to a phospholipid membrane influences the Vmax of the prothrombinase complex. Disclosures: No relevant conflicts of interest to declare.

Inherited factor V deficiency associated with a novel heterozygous missense mutation (p.G493R) in a patient with excessive surgical bleeding

2009 ◽  
Vol 102 (10) ◽  
pp. 787-789 ◽  
Author(s):  
Kitti B. Kovács ◽  
Bernadett Tisza ◽  
Komáromi István ◽  
László Muszbek ◽  
Zsuzsanna Bereczky
Keyword(s):  
Missense Mutation ◽  
Factor V ◽  
Heterozygous Missense Mutation ◽  
Factor V Deficiency ◽  
Surgical Bleeding
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