Combined administration of FVIII and rFVIIa improves haemostasis in haemophilia A patients with high-responding inhibitors--a thrombin generation-guided pilot study.

Treatment of haemophilia A patients with inhibitors is challenging, and may require individually tailored regimens. Whereas low titre inhibitor patients may respond to high doses of factor VIII (FVIII), high-responding inhibitor patients render replacement therapy ineffective and often require application of bypassing agents. Thrombin generation (TG) assays may be used to monitor haemostasis and/or predict patients' response to bypass agents. In this study we defined by TG, the potential contribution of FVIII to recombinant activated factor VII (rFVIIa)-induced haemostasis in inhibitor plasma. Based upon results, prospectively designed individual regimens of coadministration of rFVIIa and FVIII were applied. Plasma samples from 14 haemophilia patients with inhibitors (including high titre inhibitors) were tested. The response to increasing concentrations of FVIII, rFVIIa or both was assayed by TG. Eight patients, chosen following consent and at physician's discretion, comprised the combined FVIII-rFVIIa therapy clinical study cohort. Combined spiking with FVIII/rFVIIa improved TG induced by rFVIIa alone in all inhibitor plasmas. Combined rFVIIa and FVIII therapy was applied during bleeding or immune tolerance to eight patients, for a total of 393 episodes. Following a single combined dose, 90% haemostasis was documented and neither thrombosis nor any complications evolved. During study period decline of inhibitor levels and bleeding frequency were noted. Pre-analytical studies enabled us to prospectively tailor individual therapy regimens. We confirmed for the first time that the in vitro advantage of combining FVIII and rFVIIa, indeed accounts for improved haemostasis and may safely be applied to inhibitor patients.

Type I mutation in the F11 gene is a third ancestral mutation which causes factor XI deficiency in Ashkenazi Jews.

BACKGROUND: Factor XI (FXI) deficiency is one of the most frequent inherited disorders in Ashkenazi Jews (AJ). Two predominant founder mutations termed type II (p.Glu117Stop) and type III (p.Phe283Leu) account for most cases. OBJECTIVES: To present clinical aspects of a third FXI mutation, type I (c.1716 + 1G>A), which is also prevalent in AJ and to discern a possible founder effect. METHODS: Bleeding manifestations, FXI levels and origin of members of 13 unrelated families harboring the type I mutation were determined. In addition, eight intragenic and five extragenic polymorphisms were analyzed in patients with a type I mutation, in 16 unrelated type II homozygotes, in 23 unrelated type III homozygotes and in Ashkenazi Jewish controls. Analysis of these polymorphisms enabled haplotype analysis and estimation of the age of the type I mutation. RESULTS: Four of 16 type I heterozygotes (25%) and 6 of 12 (50%) compound heterozygotes for type I mutation (I/II and I/III), or a type I homozygote had bleeding manifestations. Haplotype analysis disclosed that like type II and type III mutations, the type I is also an ancestral mutation. An age estimate revealed that the type I mutation occurred approximately 600 years ago. The geographic distribution of affected families suggested that there was a distinct origin of the type I mutation in Eastern Europe. CONCLUSIONS: The rather rare type I mutation in the FXI gene is a third founder mutation in AJ.

Point mutations regarded as missense mutations cause splicing defects in the factor XI gene.

BACKGROUND: Point mutations within exons are frequently defined as missense mutations. In the factor (F)XI gene, three point mutations, c.616C>T in exon 7, c.1060G>A in exon 10 and c.1693G>A in exon 14 were reported as missense mutations P188S, G336R and E547K, respectively, according to their exonic positions. Surprisingly, expression of the three mutations in cells yielded substantially higher FXI antigen levels than was expected from the plasma of patients bearing these mutations. OBJECTIVES: To test the possibility that the three mutations, albeit their positions within exons, cause splicing defects. METHODS AND RESULTS: Platelet mRNA analysis of a heterozygous patient revealed that the c.1693A mutation caused aberrant splicing. Platelet mRNA of a second compound heterozygote for c.616T and c.1060A mutations was undetectable suggesting its degradation. Cells transfected with a c.616T minigene favored production of an aberrantly spliced mRNA that skips exon 7. Cells transfected with a mutated minigene spanning exons 8-10 exhibited a significant decrease in the amount of normally spliced mRNA. In silico analysis revealed that the three mutations are located within sequences of exonic splicing enhancers (ESEs) that bind special proteins and are potentially important for correct splicing. Compensatory mutations created near the natural mutations corrected the putative function of ESEs thereby restoring normal splicing of exons 7 and 10. CONCLUSIONS: The present findings define a new mechanism of mutations in F11 and underscore the need to perform expression studies and mRNA analysis of point mutations before stating that they are missense mutations.

Three residues at the interface of factor XI (FXI) monomers augment covalent dimerization of FXI.

BACKGROUND: Human plasma factor XI is a homodimer, with each monomer comprising a catalytic domain and four homologous 'apple' domains. The monomers bind to each other through non-covalent bonds and through a disulfide bond between Cys321 residues in apple 4 domains. OBJECTIVE: To identify residues essential for dimerization in the FXI monomer interface. METHODS: Specificity-determining residues in apple 4 domains were sought by sequence alignment of FXI and prekallikrein apple domains in different species. Specific residues identified in apple 4 domains were mutagenized and expressed in baby hamster kidney (BHK) cells for evaluation of their effect on FXI dimerization, analyzed by non-reduced sodium dodecylsulfate polyacrylamide gel electrophoresis and size-exclusion chromatography. RESULTS: Among the 19 residues of the FXI monomer interface, Leu284, Ile290 and Tyr329 were defined as specificity-determining residues. Substitutions of these residues or pairs of residues did not affect FXI synthesis and secretion from transfected BHK cells, but did impair dimerization, despite the presence of cysteine at position 321. The double mutant 284A/290A yielded predominantly a monomer, whereas all other single or double mutants yielded monomers as well as disulfide-bonded dimers. CONCLUSIONS: The data suggest that Leu284, Ile290 and Tyr329 in the interface of FXI monomers are essential for forming non-covalently bonded dimers that facilitate formation of a disulfide-bonded stable FXI dimer.

Concomitant infusion of low doses of rFVIIa and FEIBA in haemophilia patients with inhibitors.

Patients with severe haemophilia A and an inhibitor may become refractory to FEIBA and/or recombinant factor VIIa (rFVIIa). Sequential therapy with both products has been reported in such patients. In this pilot study, we examined the safety and efficacy of combined rFVIIa and FEIBA therapy in patients with haemophilia A and inhibitors during bleeding episodes. We also tried to evaluate whether thrombin generation (TG), by various mixtures of these agents, can serve as a guide for tailoring therapy. TG was measured in plasma taken from eight haemophilia A patients. Increasing concentrations of rFVIIa, FEIBA or both were added ex vivo to the plasmas, and TG was induced by recalcification. Since low concentrations of rFVIIa and FEIBA had either an additive or a synergistic effect in all patients, the lowest combination, yielding TG comparable or lower than TG achieved with either FEIBA 100 U kg(-1) or rFVIIa 160 microg kg(-1) alone, was selected for the treatment of bleeding episodes. Five patients with a high titre of an inhibitor (8-1300 BU), including one previously refractory to infusions of rFVIIa at doses up to 400 microg kg(-1) X4 daily, were treated with combinations of 30-70 microg kg(-1) rFVIIa and 20-30 U kg(-1) FEIBA during a total number of 400 bleeding episodes with excellent haemostatic effect. No adverse events and no DIC were observed following these infusions. Concomitant infusion of low-dose rFVIIa and low-dose FEIBA, seems to be safe, efficacious and economical in patients refractory to rFVIIa and probably other haemophilia A patients with an inhibitor.

Familial factor VII deficiency with foetal and neonatal fatal cerebral haemorrhage associated with homozygosis to Gly180Arg mutation.

Inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder with a wide heterogeneous clinical pattern. Intracranial haemorrhage in infants has been previously reported in the severe form of the FVII deficiency and it has a high fatality rate. We report a family with high consanguineous relations, who experienced death of two baby girls, the first with prenatal manifestation of foetal hydrocephalus secondary to intracranial bleeding and the second with postnatal intracranial bleeding, both with less than 1% activity of FVII. Genetic analysis revealed that both parents are heterozygous and both daughters homozygous for a point mutation gG9639A in exon 7, predicting Gly180Arg substitution. This mutation was described previously in a compound heterozygous patient with mild bleeding manifestation. It seems that in this family, the mutation in its homozygous state is fatal and the lethal clinical expression can appear in utero at an early stage of gestation.

Effects of factor VIII inhibitor bypassing activity (FEIBA), recombinant factor VIIa or both on thrombin generation in normal and haemophilia A plasma.

Factor VIII inhibitor bypass activity (FEIBA) and recombinant factor VIIa (rFVIIa) are the common bypassing agents for treating haemophilia A or haemophilia B patients who developed an inhibitor to factor VIII or IX, respectively. As these preparations differ in their composition and mode of action, combined therapy, either sequential or simultaneous has recently been used for achieving haemostasis during bleeding episodes in patients who became refractory to FEIBA or rFVIIa when each was given alone. In this in vitro study, we show by a sensitive assay of thrombin generation that phospholipids present in FEIBA and other procoagulants contribute to FEIBA's activity and that exogenous phospholipids are essential for the activity of rFVIIa. We also demonstrate that the combination of FEIBA and rFVIIa has a marked synergistic effect on thrombin generation in plasma of a haemophilia A patient with a high titre of an inhibitor. It is conceivable that simultaneous administration of small doses of FEIBA and rFVIIa may be beneficial in treating haemophilia A patients, with an inhibitor to FVIII, who are resistant to conventional therapy.

Prerequisites for recombinant factor VIIa-induced thrombin generation in plasmas deficient in factors VIII, IX or XI.

BACKGROUND: Recombinant factor VIIa (rFVIIa) used for the treatment of hemophilia A or B patients with an inhibitor is hemostatically effective because it induces thrombin generation (TG), despite grossly impaired FVIII- and FIX-dependent amplification of FX activation. Tissue factor (TF) and or activated platelets were shown to be essential for the rFVIIa activity. OBJECTIVE: To evaluate the relative effects of TF and phospholipids on rFVIIa-induced TG in FVIII-, FIX- and FXI-deficient plasmas. METHODS: Phospholipids had an independent effect that was augmented by TF. The contribution of blood-borne TF in FVIII-, FIX- and FXI-deficient plasma to rFVIIa-induced TG was demonstrated by removing microparticles and use of anti-TF antibodies. RESULTS: At increasing concentrations of rFVIIa, the dependence of rFVIIa-induced TG on TF declined, but the presence of phospholipids was essential. rFVIIa was also shown to activate purified FIX and FX in the presence of phospholipids and absence of TF. rFVIIa-induced TG was dramatically augmented in FVIII- or FIX-deficient plasma in which the level of FVIII or FIX was increased to 1 or 2 U dL(-1). CONCLUSIONS: The data indicate that rFVIIa-induced TG is affected by TF, phospholipids, rFVIIa concentration, and the presence of FVIII and FIX.

Characterization of mutations causing factor VII deficiency in 61 unrelated Israeli patients.

Inherited factor (F)VII deficiency is rare in most populations but relatively common in Israel. The aim of this study was to characterize the molecular and functional defect in unrelated Israeli patients with FVII deficiency. Mutations were identified by direct sequencing of PCR-amplified genomic DNA fragments. Selected mutations were expressed in baby hamster kidney (BHK) cells and tested for binding to tissue factor (TF), activation by FXa and activation of FX. In 61 patients with FVII deficiency, the causative mutation in the FVII gene was discerned. The predominant mutation found in this and a previously reported cohort of 27 unrelated patients in Israel was Ala244Val substitution; of 121 independent mutant alleles defined in all 88 patients ascertained in Israel, 102 (84%) bore this alteration. Eleven additional mutations were identified of which one, Cys22Arg, is novel. Expression of the mutations in BHK cells revealed that four (Ala244Val, 11128delC, Leu300Pro and Cys22Arg) were cross-reacting material (CRM)- negative, and three (Ala294Val, Cys310Phe and Phe24del) were CRM-positive. As predicted by modeling, we observed no binding to TF of FVII Phe24del, diminished binding of FVII Cys310Phe and normal binding of FVII Ala294Val. The main defect of FVII Ala294Val was its inability to activate FX in the presence of TF. Coexpression of Ala294Val and Arg353Gln, a polymorphism known to affect FVII secretion, did not reveal an additive effect on FVII secretion, while coexpression of Ala244Val and Arg353Gln did yield an additive effect.

Severe factor XI deficiency caused by a Gly555 to Glu mutation (factor XI-Glu555): a cross-reactive material positive variant defective in factor IX activation.

During normal hemostasis, the coagulation protease factor (F)XIa activates FIX. Hereditary deficiency of the FXIa precursor, FXI, is usually associated with reduced FXI protein in plasma, and circulating dysfunctional FXI variants are rare. We identified a patient with < 1% normal plasma FXI activity and normal levels of FXI antigen, who is homozygous for a FXI Gly555 to Glu substitution. Gly555 is two amino acids N-terminal to the protease active site serine residue, and is highly conserved among serine proteases. Recombinant FXI-Glu555 is activated normally by FXIIa and thrombin, and FXIa-Glu555 binds activated factor IX similarly to wild type FXIa (FXIa(WT)). When compared with FXIa(WT), FXIa-Glu555 activates factor IX at a greatly reduced rate ( approximately 400-fold), and is resistant to inhibition by antithrombin. Interestingly, FXIa(WT) and FXIa-Glu555 cleave the small tripeptide substrate S-2366 with comparable k(cat)s. Modeling indicates that the side chain of Glu555 significantly alters the electrostatic charge around the active site, and would sterically interfere with the interaction between the FXIa S2' site and the P2' residues on factor IX and antithrombin. FXI-Glu555 is the first reported example of a naturally occurring FXI variant with a significant defect in FIX activation.

Seven novel mutations in the factor XIII A-subunit gene causing hereditary factor XIII deficiency in 10 unrelated families.

BACKGROUND: Hereditary factor (F)XIII deficiency is a rare bleeding disorder mostly due to mutations in FXIII A subunit. OBJECTIVES: We studied the molecular basis of FXIII deficiency in patients from 10 unrelated families originating from Israel, India and Tunisia. METHODS: Exons 2-15 of genomic DNA consisting of coding regions and intron/exon boundaries were amplified and sequenced. Structural analysis of the mutations was undertaken by computer modeling. RESULTS: Seven novel mutations were identified in the FXIIIA gene. The propositus from the Ethiopian-Jewish family was found to be a compound heterozygote for two novel mutations: a 10-bp deletion in exon 12 at nucleotides 1652-1661 (followed by 22 altered amino acids and termination codon) and Ala318Val mutation. The propositus of the Tunisian family was homozygous for C insertion after nucleotide 863 within a stretch of six cytosines of exon 7. This insertion results in generation of eight altered amino acids followed by a termination codon downstream. The propositus from Indian-Jewish origin was found to be homozygous for G to T substitution at IVS 11 [+1] resulting in skipping of exons 10 and 11. In addition to the Ala318Val mutation, three of the novel mutations identified are missense mutations: Arg260Leu, Thr398Asn and Gly210Arg each occurring in a homozygous state in an Israeli-Arab and two Indian families, respectively. CONCLUSIONS: Structure-function correlation analysis by computer modeling of the new missense mutations predicted that Gly210Arg will cause protein misfolding, Ala318Val and Thr398Asn will interfere with the catalytic process or protein stability, and Arg260Leu will impair dimerization.

A common ancestral mutation (C128X) occurring in 11 non-Jewish families from the UK with factor XI deficiency.

Factor XI (FXI) deficiency is a mild bleeding disorder that is particularly common in Ashkenazi Jews, but has been reported in all populations. In Jews, two FXI gene (F11) mutations (a stop codon in exon 5, E117X, type II, and a point mutation in exon 9, F283L, type III) are particularly common, but in other populations a variety of different mutations have been described. In the Basque region of France one mutation, C38R in exon 3, was found in eight of 12 families studied, haplotype analysis suggesting a founder effect. In the course of screening 78 unrelated individuals (including 15 Jewish and 12 Asian) we have found 10 Caucasian non-Jewish patients with the mutation C128X in exon 5. Individuals were investigated because of a personal or family history of bleeding, or finding a prolonged activated partial thromboplastin time. Individuals negative for the type II and type III mutations were screened by a combination of SSCP and heteroduplex analysis. The C128X mutation was found in 10 families (one previously described). Among three individuals with severe FXI deficiency, one was homozygous for the C128X mutation, and two were compound heterozygotes for the C128X and another mutation; other individuals were carriers of the C128X mutation. This is a nonsense mutation producing a truncated protein; individuals have FXI antigen levels concordant with FXI coagulant activity. Haplotype analysis of 11 families, including a further kindred previously reported from the USA, but which originally came from the UK (in which the index patient was homozygous for C128X), suggests a founder effect.

Inherited factor XI deficiency confers no protection against acute myocardial infarction.

BACKGROUND AND PURPOSE: Factor XI (FXI) contributes to thrombin generation thereby affecting fibrin formation and to down regulation of fibrinolysis by activation of thrombin-activatable fibrinolysis inhibitor (TAFI). The purpose of this study was to evaluate whether patients with severe FXI deficiency are protected against acute myocardial infarction (AMI). METHODS: The incidence of AMI in patients with severe FXI deficiency (FXI activity less than 15 U dL(-1)) whose age was 35 years or more was compared to the incidence of AMI in age and gender matched persons of the general population. Atherosclerotic risk factors were assessed in FXI deficient patients and blood was tested for prothrombotic parameters such as FV Leiden, prothrombin G20210A, lupus anticoagulant, and platelet membrane polymorphisms. The common mutations causing FXI deficiency in Jews were also examined. RESULTS: Of 96 patients with severe FXI deficiency (55 women and 41 men) 16 had a history of AMI (6 women and 10 men). The median age at the time of AMI was 64.5 for women and 58 for men. The calculated annual rate of AMI in men was similar to the expected in the general Israeli population, whereas in women it was almost 2-fold higher, but this difference did not reach statistical significance. One or more atherosclerotic risk factors were observed in 13 of 16 patients (81.3%) with AMI compared to 44 of 79 patients (55.7%) without AMI (P < 0.001). The frequency distributions of platelet polymorphisms and of prothrombotic polymorphisms were not different between patients with severe FXI deficiency who experienced or not an AMI. None of the patients had lupus anticoagulant. The common genotypes which cause FXI deficiency in Jews were similarly distributed in patients with and without AMI. CONCLUSIONS: Severe FXI deficiency does not confer protection against AMI.

Cerebrovascular events in patients with significant stenosis of the carotid artery are associated with hyperhomocysteinemia and platelet antigen-1 (Leu33Pro) polymorphism.

BACKGROUND AND PURPOSE: Although risk factors for carotid artery stenosis caused by atherosclerosis are known, it is unclear what triggers "activation" of the atherosclerotic plaques and the ensuing thromboembolic cerebral events. The aim of this study was to evaluate whether thrombophilic factors, platelet glycoprotein (GP) polymorphisms, and homocysteine are associated with a risk of ischemic events in patients with significant carotid stenosis. METHODS: Consecutive patients with >/=50% carotid stenosis, whether symptomatic (with ipsilateral ischemic events) or asymptomatic, who were evaluated and followed in a neurovascular clinic were tested for plasma levels of homocysteine, C677T mutation in methylenetetrahydrofolate reductase, G20210A mutation of factor II, factor V Leiden, antiphospholipid antibodies, and polymorphisms of platelet membrane GP: human platelet antigen (HPA)-1, GP Ia (C807T), and GP Ib (variable number of tandem repeats, Kozak, and HPA-2). RESULTS: Eighty-six asymptomatic and 67 symptomatic patients were evaluated. The former group was older (73.7+/-6.9 versus 69.5+/-9.1 years, P=0.02). Major risk factors for stroke were similar in both groups. In symptomatic patients versus asymptomatic patients, hyperhomocysteinemia was 3-fold more frequent (34.3% versus 12.8%, respectively; P=0.002) and HPA-1a/b was almost 2-fold more common (38.8% versus 20.9%, respectively; P=0.01). All other thrombophilic factors and platelet polymorphisms studied did not differ significantly between the 2 groups. Multivariate analysis revealed that hyperhomocysteinemia and the HPA-1a/b genotype conferred a significant risk of cerebral ischemic events, with odds ratios (95% CI) of 4.07 (1.7 to 9.7) and 3.4 (1.5 to 7.8), respectively. CONCLUSIONS: Hyperhomocysteinemia and HPA-1a/b are independent risk factors for ischemic events in patients with significant carotid stenosis.

Identification of a region in glycoprotein IIIa involved in subunit association with glycoprotein IIb: further lessons from Iraqi-Jewish Glanzmann thrombasthenia.

The most frequent mutation causing Glanzmann thrombasthenia in Iraqi-Jews (IJ-1) is an 11-bp deletion in exon 13 of the glycoprotein (GP) IIIa gene. This deletion predicts a frameshift that results in the elimination of the C406-C655 disulfide bond and a premature termination codon shortly before the transmembrane domain. To determine the contribution of each of these alterations to the thrombasthenic phenotype, Chinese hamster ovary or baby hamster kidney cells were cotransfected with normal GPIIb complementary DNA (cDNA) and the following GPIIIa cDNAs: normal, cDNA bearing IJ-1 mutation, 2011T>A mutated cDNA predicting C655S (single-letter amino acid codes) substitution, and 2019A>T mutated cDNA predicting Stop657. Elimination of the C406-C655 disulfide bond by C655S substitution did not affect GPIIb/IIIa surface expression or binding of the transfected cells to immobilized fibrinogen, whereas elimination of the transmembrane and cytoplasmic domains in IJ-1 and Stop657 mutants prevented both surface expression and binding of the transfected cells to immobilized fibrinogen. Immunohistochemical staining and immunoprecipitation demonstrated that the elimination of amino acids 657-762 in IJ-1 and Stop657 prevented intracellular GPIIb/IIIa complex formation, and differential immunofluorescence staining of GPIIIa and cellular organelles suggested that the truncated uncomplexed GPIIIa protein was retained in the endoplasmic reticulum. Because the use of GPIIIa Stop693 and normal GPIIb cDNAs yielded GPIIb/IIIa complex formation, though with lower efficiency, it is suggested that amino acids 657-692 of GPIIIa are essential for the intracellular association of GPIIb and GPIIIa. (Blood. 2001;98:1063-1069)

Identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in Israeli patients with Dubin-Johnson syndrome.

Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by conjugated hyperbilirubinemia and is caused by a deficiency of the multidrug resistance protein 2 (MRP2) located in the apical membrane of hepatocytes. The aim of this study was to identify the mutations in two previously characterized clusters of patients with Dubin-Johnson syndrome among Iranian and Moroccan Jews and determine the consequence of the mutations on MRP2 expression and function by expression studies. All 32 exons and adjacent regions of the MRP2 gene were screened by polymerase chain reaction and DNA sequencing. Two novel mutations were identified in exon 25. One mutation, 3517A-->T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G-->A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families. Use of four intragenic dimorphisms and haplotype analyses disclosed a specific founder effect for each mutation. The mutations were introduced into an MRP2 expression vector by site-directed mutagenesis, transfected into HEK-293 cells, and analyzed by a fluorescence transport assay, immunoblot, and immunocytochemistry. Continuous measurement of probenecid-sensitive carboxyfluorescein efflux revealed that both mutations impaired the transport activity of MRP2. Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. In contrast, expression of MRP2 (I1173F) was low and mislocated to the endoplasmic reticulum of the transfected cells. These findings provide an explanation for the DJS phenotype in these two patient groups. Furthermore, the close localization of the two mutations identify this region of MRP2 as important for both activity and processing of the protein.

The role of factor XI in thrombin generation induced by low concentrations of tissue factor.

Thrombin generation has been studied in the plasma of severely factor XI deficient patients under conditions in which contact activation did not play a role. In platelet-rich as well as platelet-poor plasma, thrombin generation was dependent upon the presence of factor XI at tissue factor concentrations of between 1 and 20 pg/ml i.e. approximately 0.01 to 0.20% of the concentration normally present in the thromboplastin time determination. The requirement for factor XI is low; significant thrombin generation was seen at 1% factor XI; at 10%, thrombin formation was nearly normalised. A suspension of normal platelets in severely factor XI deficient plasma did not increase thrombin generation. This implies that there is no significant factor XI activity carried by normal platelets, although the presence of factor XI and factor XI inhibitors in platelets cannot be ruled out.

Methionine synthase A2756G and methylenetetrahydrofolate reductase A1298C polymorphisms are not risk factors for idiopathic venous thromboembolism.

Hyperhomocysteinemia is a defined risk factor for venous thromboembolism (VTE). Several polymorphisms of genes encoding for enzymes acting in the remethylation pathway of homocysteine metabolism, ie, methionine synthase (MS) A2756G, methylenetetrahydrofolate reductase (MTHFR) C677T and MTHFR A1298C, can cause increased homocysteine levels particularly in patients with deficiencies of folic acid, vitamin B6, or B12 and hence be potential risk factors for VTE. Indeed, homozygous MTHFR C677T was shown to be a mild risk factor for VTE by some, but not by all, investigators. In this study, we assessed the risk exerted by MS A2756G and MTHFR A1298C in a cohort of patients with idiopathic venous thromboembolism. Homozygosities for MS A2756G and MTHFR A1298C were not found to be statistically significant risk factors for VTE. In addition, no interactions were observed among MS A2756G, MTHFR A1298C and MTHFR C677T in conferring a risk of VTE.