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PubMed2002

Factor V Leiden and factor II G20210A in preeclampsia and HELLP syndrome.

BACKGROUND: The association of factor V and factor II mutations with preeclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome, and a possible role of the two thrombophilic mutations in the pathogenesis of the diseases have been previously investigated. The results, however, are still inconclusive and contradictory. METHODS: A case-control study was performed over an interval of 24 months, on 111 subjects with preeclampsia and 111 normal pregnant women matched for age and parity, without previous thromboembolic disorders. The subjects were tested for the mutation A1691G in the factor V gene (R506Q or Leiden mutation) and the mutation A20210G in the factor II (prothrombin) gene. The Student's t-test and the chi2-test were applied when appropriate, and odds ratios and 95% confidence intervals were calculated. RESULTS: Fourteen patients with preeclampsia (12.6%) had at least one of the two mutations, as compared with six controls (5.4%). Factor V Leiden was found in eight patients with preeclampsia (7.2%) and in five controls (4.5%). Factor II G20210A was detected in eight preeclamptic women (7.2%) and in one normal pregnant woman (0.9%)(p = 0.041). In the subgroup of 32 preeclamptic subjects with HELLP syndrome, factor V Leiden was found in three patients (9.3%) and factor II G20210A in two (6.2%). CONCLUSIONS: The prevalence of factor V and factor II mutations is increased in patients with preeclampsia; the thrombophilic mutations may interact with other pathogenic factors to determine the clinical features of the disease and of its complications.

Adult↗
PubMed2002

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

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.

Adult↗
PubMed2002

Molecular basis of quantitative factor V deficiency associated with factor V R2 haplotype.

To investigate the molecular mechanisms of the quantitative factor V (FV) deficiency associated with the FV R2 haplotype, 4 missense mutations, Met385Thr, His1299Arg, Met1736Val, and Asp2194Gly, identified in the R2 haplotype allele, were analyzed by in vitro expression studies. The FV variant carrying all 4 mutations showed a markedly lower steady-state expression level than wild-type FV because of low synthesis rate and impaired secretion of the mutant protein. The Asp2194Gly mutation was found to play a key role in the impaired secretion of the mutant FV by interfering with its transport from the endoplasmic reticulum to the Golgi complex. The deleterious effect of the Asp2194Gly mutation was shown to be dominant among the 4 mutations. The Met385Thr mutation and His1299Arg mutation had no effect on steady-state expression levels, but the secretion rates of the mutant proteins were moderately decreased by these mutations. The His1299Arg mutation partially impaired glycosylation in the C-terminal part of the B-domain of the mutant FV, which was supposed to affect the secretion rate, but not the steady-state expression level. It was also suggested that the Met385Thr mutation partially impairs posttranslational modification of the mutant FV without affecting the steady-state expression level. No deleterious effect of the Met1736Val mutation was observed in terms of expression and intracellular processing. Our in vitro data strongly suggest that the naturally existing R2 haplotype mutant FV, which carries all 4 mutations, has the potential to result in quantitative FV deficiency in vivo owing to impaired expression of the mutant protein when the Asp2194Gly mutation is present.

Amino Acid Substitution↗
PubMed1992

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.

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.

Antigen-Antibody Reactions↗
PubMed2002

Lower birth-weight in neonates of mothers carrying factor V G1691A and factor II A(20210) mutations.

BACKGROUND AND OBJECTIVES: Inherited thrombophilia has been associated with fetal and maternal complications of pregnancy. It is reasonable to suppose that an imbalance of maternal hemostasis could lead to decreased fetal growth. DESIGN AND METHODS: We retrospectively investigated the birth-weight of neonates in relation to the presence of factor V G1691A and factor II A(20210) mutations in the mothers. Overall, 755 women (194 with a history of unexplained recurrent pregnancy loss, 202 with gestational hypertension with or without proteinuria, 359 with at least one uneventful pregnancy) and 1100 alive neonates were considered. RESULTS: Among 980 neonates from mothers without mutations, 136 (13.9%) weighed <2500 grams, whereas 34 out 123 (27.6%) neonates from mothers carrying the factor V G1691A or factor II A(20210) mutation were under this birth-weight (OR: 2.4, 95%CI: 1.5- 3.7). Adjusting for diagnosis, parity, and age, the risk of having a baby <2500 grams was 2.0 (95%CI: 1.1-3.6) in women carrying factor V G1691A or factor II A(20210) mutation. When we analyzed all the neonates according to growth centiles and the presence of a thrombophilic mutation in the mothers, we found 142 (14.5%) and 28 (22.8%) neonates under the 10th centile from mothers without and with thrombophilic mutations, respectively (OR: 1.7, 95%-CI: 1.1-2.7). Adjusting for confounding variables (diagnosis, parity and age), the association between thrombophilic mutations and <10th growth centile did not change (OR: 1.7, 95% CI: 1.0-3.0). INTERPRETATION AND CONCLUSIONS: Mothers carrying the factor V G1691A or factor II A(20210) mutation have a significantly higher risk of delivering neonates with a lower birth-weight.

5' Untranslated Regions↗
PubMed1998

Inhibitory anti-factor V antibodies bind to the factor V C2 domain and are associated with hemorrhagic manifestations.

Factor V inhibitors may develop as spontaneous autoantibodies, as alloantibodies after exposure to bovine thrombin preparations, or in factor V-deficient patients after plasma therapy. Clinical manifestations range from asymptomatic laboratory abnormalities to life-threatening hemorrhage. We have characterized the anti-factor V antibodies from 12 patients diagnosed with factor V inhibitors. In 8 patients, hemorrhagic complications (5 autoantibodies and 3 bovine thrombin-induced alloantibodies) developed, and 4 were asymptomatic (2 autoantibodies and 2 alloantibodies). The IgG fractions from all 12 patients immunoprecipitated the factor Va light chain, but only the 8 IgG fractions associated with hemorrhage inhibited factor V activity in a prothrombinase assay. Nine IgG fractions, including the 8 patients with hemorrhage, immunoprecipitated the isolated second C-type domain (C2). The 8 IgG fractions from the symptomatic patients also immunoprecipitated recombinant chimeras containing only the N-terminal third of the factor V C2 domain, and isolated recombinant C2 domain abrogated the inhibitory effect of the antibodies. Five of the inhibitory IgG fractions blocked binding of factor V to phosphatidylserine. These results suggest that inhibitory anti-factor V antibodies are associated with hemorrhagic manifestations and frequently bind to a common region within the C2 domain, whether originating spontaneously or after exposure to bovine thrombin.

Adolescent↗
PubMed1994

The mechanism of inactivation of human factor V and human factor Va by activated protein C.

The cleavage of human factor V and human factor Va by human activated protein C (APC) was analyzed in the absence and presence of phospholipid vesicles containing 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS). Membrane-bound human factor V (250 nM) is cleaved by APC (2.5 nM) to give M(r) = 200,000, 70,000, 45,000, and 30,000 fragments and an M(r) = 22/20,000 doublet. These fragments are released after four sequential cleavages of the membrane-bound procofactor at Arg306, Arg506, Arg679, and Lys994. No cofactor activity is observed following thrombin treatment of the membrane-bound APC-cleaved procofactor. In the absence of a membrane surface, no cleavage of factor V by APC is observed, and following thrombin activation factor Va retains full cofactor activity. Membrane-bound human factor Va (600 nM) loses more than 90% of its initial cofactor activity after 10 min of incubation with APC (10.9 nM), and virtually no cofactor activity is observed after 1 h of incubation. Under similar conditions but in the absence of PCPS vesicles, factor Va is cleaved but retains approximately 80% of its initial cofactor activity after 2 h of incubation with APC. In the presence of PCPS vesicles, the APC related loss of activity is correlated with cleavage of the heavy chain and appearance of fragments of M(r) = 45,000, 30,000, and of 28/26,000, and 22/20,000 doublets. These products correspond to three cleavages of the heavy chain (at Arg306, Arg506, and Arg679). Cleavage at Arg506 of factor Va precedes and appears to be required for cleavage at Arg306 and Arg679. In the absence of membrane, proteolysis at Arg506 produces an M(r) = 75,000 fragment which corresponds to the NH2-terminal portion of the human factor Va heavy chain (residues 1-506), and a carboxyl-terminal doublet of M(r) = 28/26,000 (residues 507-709) which is cleaved by APC at Arg679 to generate an M(r) = 22/20,000 doublet and an M(r) = 6,000 peptide. No cleavage of the light chain of the human cofactor is observed in the presence or absence of PCPS vesicles following 2 h of incubation with APC. Our data demonstrate that inactivation of human factor V and human factor Va only occurs in the presence of a membrane surface after cleavage at Arg306. However, while this cleavage site is exposed on membrane-bound human factor V, cleavage at Arg506 on the heavy chain of factor Va appears necessary for complete exposure of the cleavage site at Arg306.

Amino Acid Sequence↗
PubMed2001

An extensive interaction interface between thrombin and factor V is required for factor V activation.

The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.

Amino Acid Substitution↗
PubMed2001

Five novel mutations in the gene for human blood coagulation factor V associated with type I factor V deficiency.

Coagulation factor V (FV) plays an important role in maintaining the hemostatic balance in both the formation of thrombin in the procoagulant pathway as well as in the protein C anticoagulant pathway. FV deficiency is a rare bleeding disorder with variable phenotypic expression. Little is known about the molecular basis underlying this disease. This study identified 5 novel mutations associated with FV deficiency in 3 patients with severe FV deficiency but different clinical expression and 2 unaffected carriers. Four mutations led to a premature termination codon either by a nonsense mutation (single-letter amino acid codes): A1102T, K310Term. (FV Amersfoort) and C2491T, Q773Term. (FV Casablanca) or a frameshift: an 8-base pair deletion between nucleotides 1130 and 1139 (FV Seoul(1)) and a 1-base pair deletion between nucleotides 4291 and 4294 (FV Utrecht). One mutation was a novel missense mutation: T1927C, C585R (FV Nijkerk), resulting in the absence of mutant protein despite normal transcription to RNA. Most likely, an arginine at this position disrupts the hydrophobic interior of the FV A2 domain. The sixth detected mutation was a previously reported missense mutation: A5279G, Y1702C (FV Seoul(2)). In all cases, the presence of the mutation was associated with type I FV deficiency. Identifying the molecular basis of mutations underlying this rare coagulation disorder will help to obtain more insight into the mechanisms involved in the variable clinical phenotype of patients with FV deficiency.

Adolescent↗