Factor X Activity Research Articles

Genotype Analysis of Patients with Hereditary Factor X Deficiency Enrolled in 2 Phase 3 Studies of Pdfx, a New High-Purity Factor X Concentrate

Introduction: Hereditary factor X (FX) deficiency is a rare, autosomal recessive bleeding disorder of variable severity, with an estimated prevalence of 1:500,000 to 1:1,000,000. Like hemophilia A and B, patients with severe FX deficiency commonly present with bleeding into joints, muscles, or mucous membranes. However, unlike the X-linked disorders of hemophilia A and B, hereditary FX deficiency occurs equally in both sexes due to the location of the FX gene (F10) on chromosome 13q34. A novel, high-purity, high-potency, plasma-derived FX concentrate (pdFX) has been developed for replacement therapy in patients with hereditary FX deficiency. This analysis examines the genotypic and phenotypic characteristics of subjects with hereditary FX deficiency enrolled in 2 prospective, open-label, multicenter phase 3 studies of pdFX. Methods: In study 1, subjects aged ≥12 years with moderate or severe FX deficiency (basal plasma FX activity [FX:C] of ≥1 and Results: Study 1 enrolled 16 subjects (aged 12-58 years [mean 27.1 years], 62.5% female) from the United Kingdom (n=3), Spain (n=4), the United States (n=2), Turkey (n=6), and Germany (n=1); two patients had moderate and 14 had severe FX deficiency. Among the 16 subjects, 13 separate mutations were identified, of which 6 were novel. Nine mutations were missense mutations, 2 were deletions, 1 was a nonsense mutation, and 1 was a splice-site mutation. Consistent with the low FX:C of Study 2 enrolled 2 male subjects (ages 55 years [United States] and 59 years [United Kingdom], respectively), both with mild FX deficiency (basal FX:C of 6 and 8 IU/dL, respectively) and compound heterozygous mutations. Of the 4 mutations identified in these 2 patients, 3 were novel. Of the subjects with moderate or severe FX deficiency (study 1), all 6 Turkish subjects had homozygous mutations resulting in an identical amino acid substitution (p.Gly262Asp). Two subjects in Spain and 1 each in the United States and Germany had mutations resulting in an identical amino acid substitution (p.Gly21Arg); one of these Spanish subjects was a compound heterozygote, with an additional missense mutation of p.Cys246Arg, while the other 3 subjects9 mutations were homozygous. Two UK subjects were each homozygous for 2 different missense mutations (p.Phe71Ser and p.Ile451Phe, respectively), and the remaining 4 subjects each had unique compound heterozygous mutations (p.Val298Met and p.Tyr384Leufs*57 [United Kingdom], p.Glu350Lys and p.Gly450Arg [Spain], p.Cys57Phe and c.70+4A>G splice site mutation [Spain], and p.Gln411* and exon 2 deletion [United States], respectively). The basal level of expressed FX protein (FX:Ag) in 1 patient (87 U/dL) was within the normal range (73-127 U/dL), whereas FX:Ag levels in all other patients (range, Each subject with mild FX deficiency (study 2) had unique compound heterozygous mutations (p.Tyr319His and c.71-1G>C splice site mutation [United Kingdom] and p.Cys90Arg and p.Gln416Leu [United States]). Basal FX:Ag levels in these patients (55 and 48 U/dL, respectively) were close to the normal range. Conclusions: In this analysis, 17 separate F10 mutations were identified in 18 subjects with mild to severe hereditary FX deficiency. Of the mutations identified, 9 are novel and have not previously been characterized. Support: Bio Products Laboratory Ltd. Disclosures Mitchell:Viapath: Employment, Other: employee of Viapath, which received funding from Bio Products Laboratory to perform genetic analysis. Kavakli:Baxter: Other: advisory board member and received educational and investigational support; Bayer: Other: advisory board member and received educational and investigational support; Novo Nordisk: Other: advisory board member and received educational and investigational support; Pfizer: Other: advisory board member and received educational and investigational support; Bio Products Laboratory: Other: received educational and investigational support; CSL Behring: Other: received educational and investigational support; Octapharma: Other: received educational and investigational support. Norton:Bio Products Laboratory: Employment. Austin:SOBI: Other: member of advisory board and received educational support; Pfizer: Other: member of advisory board and received educational support; Novo Nordisk: Other: member of advisory board and received educational support; CSL Behring: Other: member of advisory board and received educational support; Bio Products Laboratory: Other: member of advisory board and received educational support; Bayer: Other: member of advisory board and received educational support; Baxter: Other: member of advisory board and received educational support. Off Label Use: ALN-AT3 is an investigational drug for potential treatment of hemophilia. The data represent phase 1 data..

Frequency of the p.Gly262Asp mutation in congenital Factor X deficiency.

Congenital factor X (FX) deficiency is a rare bleeding disorder inherited as an autosomal recessive trait with an incidence of 1:500000-1000000. A total or partial deficiency of FX causes an impairment of clot formation, leading to a haemorrhagic disease, which manifests with bleeding symptoms of different severity, also unprovoked. We analysed the clinical manifestations, laboratory phenotype and genotype in 12 patients from Turkey affected with severe FX deficiency. Prothrombin time (PT), activated partial thromboplastin time (APTT), FX activity (FX:C) and FX antigen level (FX:Ag) were measured, and mutation analysis was performed for all patients. The most frequent bleeding episodes in patients were epistaxis and easy bruising (11/12, 91%), followed by haemarthroses (10/12, 83%). FX:C was <1% in 11 patients, and 4% in one. FX:Ag was reduced in all patients, consistent with type II deficiency. Direct sequencing of the factor X gene (F10) identified two different mutations: the novel 33bp in-frame deletion p.Thr176_Gln186, c.526_558del, which seems to be associated with milder bleeding symptoms and the c.785G>A, p.Gly262Asp missense mutation (previously reported as Gly222Asp), which is associated with severe bleeding symptoms. The p.Gly262Asp missense mutation was identified in 11 of the 12 patients in this study. Previously published cases on the same p.Gly262Asp mutation were Iranian patients originating from the border between Turkey and Iran suggesting that this mutation may be candidate as a good tool for mutational screening analysis in this area.

Efficacy of prophylaxis and genotype‐phenotype correlation in patients with severe Factor X deficiency in Iran

We aimed to evaluate the effect of regular prophylaxis with a Factor X (FX) concentrate for patients with severe FXD in Iran and to assess the correlation of the genotype and phenotype in these patients. Ten patients with severe FXD (FX activity <1%) were enrolled and characterized during 2010-2011. Prophylaxis with 20 IU FX P Behring per kg body weight was administered once a week. FX levels, were monitored at baseline, 15 and 30 min, 1, 3, 6, 12, 24, 48, 72 and 96 h after starting prophylaxis. All patients were followed for 1 year. The mean age of the patients was 15 ± 7.8 years (age range of: 6-27 years). One patient had anaphylactic reaction after the first infusion, and the treatment was stopped. During one-year follow-up after starting prophylaxis, no bleeding symptoms occurred in any patient who tolerated and remained on the prophylaxis programme and all of them had a FX level of 1% or above. The maximum level of FX activity has been observed at 15 min after starting prophylaxis. A level of 1.5-3.5% was detected after 96 h. Homozygous mutations p.Arg40Thr (Arg-1Thr), p.Gly51Arg and p.Glu69Lys were detected in patients with intracranial haemorrhage. In our patients, significant decrease in symptoms without any complication after administration of FX, was demonstrated in all except one patient who had an anaphylactic reaction. It seems that the dose of 20 IU kg(-1) could be probably the best choice for patients with severe FXD, who require regular prophylaxis.

Factor X deficiency and intracranial bleeding: who is at risk?

Very few mutations of the gene encoding for coagulation factor X (FX) have been found associated with intracranial haemorrhage (ICH) due to FX deficiency (FXD). No guidelines exist as to when prophylaxis in FXD should be started and how patients at risk for ICH can be identified. We report on a novel mutation causative for ICH in a family of Iranian origin and provide a summary of all published mutations in the FX gene related to ICH. The index patient is an infant with umbilical bleeding requiring blood transfusion in the postnatal period. The international normalized ratio (6.01) and activated partial thromboplastin time (117 s) were prolonged. Coagulation factor analysis was normal except for FX activity (<1%). At 4 months, the child suffered a spontaneous severe intracranial haemorrhage. The child was the product of a consanguineous union. Four of five available family members from three generations displayed minor bleeding symptoms and mildly reduced FX. Sequencing of FX gene demonstrated homozygosity for a novel duplication A (c.1402_1403dupA)* in exon 8 and heterozygosity in four family members. We compare this case to all 15 patients with FXD and ICH and their 11 known mutations described so far. This case illustrates a pattern of FXD (a male neonate with umbilical or gastrointestinal bleeding, very low FX:C (<1%) and an underlying homozygous genotype) who may be at high risk for ICH. In these cases, we recommend to start early prophylactic substitution of FX to prevent a possible life-threatening haemorrhage.

Effects of Aqueous Extract of Diospyros Kaki Calyx on Anti-thrombotic Activity in vitro and in vivo

ABSTRACT Objectives：The aim of this study is to research an anti-thrombus effect by Diospyros Kaki Calyx.Methods：The healthy human plasma were gained and used in vitro study such as factor X activity (FXa) inhibition, prothrombinase inhibition, prothrombin time (PT) an d activated partial thromboplastin time. Fifteen SD rats were divided into three groups；intact control group (or ally administrated with distilled water 5ml/kg) and two experimental group treated with extract of diospyros kaki calyx (EKC). Experimental rats were orally 600 mg/kg concentration of EKC and 200 mg/kg concentration of EKC. After an hour from administration, we anesthetized rats and made arteriovenous (AV) shunt rat models to study weight of thrombus, took whole blood to study content of thromboxane B2 and blood clotting time.Results：In vitro, EKC significantly increased inhibitory activity of FXa, prothrombinase compared with intact control group (*P<0.05). PT and aPTT were increased in EKC trea ted (600 mg/kg) group compared with intact control group (*P<0.05). In vivo, blood clotting time of experiment group treated with EKC 600 mg/kg were significantly increased compare with that of intact control group (p<0.05) and content of thromboxane B2 was significantly decreased in group treated with EKC 600 mg/kg in serum. The weight of thrombus were significantly reduced in group treated with EKC 600 mg/kg compa red with intact control group (p<0.05). But in vivo experiment study, those parameters of group treated with EKC 200 mg/kg were relatively decreased compared with those of intact control group without statistical significance.Conclusions：EKC has an antithrombic activity because of inhibition internal course such as FXa and prothrombin. And EKC inhibited a hole blood clotting in vivo experiment by low content of thromboxane B2.Key words：Diospyros Kaki Calyx, antithromboic activity, factor Xa assay, prothrombin time assay, thromboxane B2, arteriovenous shunt modle

Lactobacillus casei modulates the inflammation-coagulation interaction in a pneumococcal pneumonia experimental model

BackgroundWe have previously demonstrated that Lactobacillus casei CRL 431 administration improved the resistance to pneumococcal infection in a mouse model.MethodsThis study examined the effects of the oral administration of Lactobacillus casei CRL 431 (L. casei) on the activation of coagulation and fibrinolytic systems as well as their inhibitors during a Streptococcus pneumoniae infection in mice.ResultsThe alveolo-capillary membrane was damaged and the coagulation system was also activated by the infection. As a consequence, we could see fibrin(ogen) deposits in lung histological slices, increased levels of thrombin-antithrombin complex (TATc) in bronchoalveolar lavage (BAL) and plasma, decrease in prothrombin activity (PT) and prolonged activated partial thromboplastin time test (APTT) values. Factor VII (FVII) and factor X (FX) were decreased in plasma, whereas fibrinogen (F) and factor VIII (FVIII) were increased. The low levels of protein C (PC) in BAL and plasma proved damage on inhibitory activity. The infected animals showed reduced fibrinolytic activity, evidenced by an increase in plasminogen activation inhibitor-1 (PAI-1) in BAL and plasma. The pathogen induced an increase of TNF-α, IL-1β and IL-6 in BAL and serum a few hours after challenge followed by a significant decrease until the end of the assayed period. IL-4 and IL-10 in BAL and serum were also augmented, especially at the end of the experiment. The animals treated with L. casei showed an improvement of alveolo-capillary membrane, lower fibrin(ogen) deposits in lung and decrease in TATc. APTT test and PT, FVII and FX activity were normalized. L. casei group showed lower F levels than control during whole experiment. In the present study no effect of L. casei on the recovery of the inhibitory activity was detected. However, L. casei was effective in reducing PAI-1 levels in BAL and in increasing anti-inflammatory ILs concentration.ConclusionL. casei proved effective to regulate coagulation activation and fibrinolysis inhibition during infection, leading to a decrease in fibrin deposits in lung. This protective effect of L. casei would be mediated by the induction of higher levels of IL-4 and IL-10 which could regulate the anti-inflammatory, procoagulant and antifibrinolytic effects of TNF-α, IL-1β and IL-6.

Unexplained Discrepancies in the Activity—Antigen Ratio in Congenital FX Deficiencies With Defects in the Catalytic Domain

Studies on molecular biology have considerably enhanced our understanding of congenital coagulation disorders but have failed so far to supply tools for an adequate classification of defects. In fact, mutations in the same domain may give rise to different phenotypes. Conversely, mutations in different domains, controlled by different exons, may cause similar patterns. The 37 kindreds with congenital factor X (FX) deficiency, known to have a defect in the catalytic domain, have been evaluated in an attempt to investigate the genotype-phenotype relation. Discrepant results were obtained because about half kindreds showed a type I pattern, namely a concomitant decrease in FX activity and antigen. The other half showed a type II pattern, namely a decrease in FX activity with a normal or near normal FX antigen. In a few instances, the allocation of the kindred either to type I or to type II defect could not be reached, due to the lack of information about the antigen. The comparison of the kindreds in which the same mutation has been discovered by different investigations is not always possible also for lack of information. The study analyzes the need to have a multipronged approach to the study of congenital FX deficiency. The indication of a mutation in a given domain does not provide clear information about the phenotype.

The clinical and laboratory significance of cases of congenital FX deficiency due to defects in the Gla-domain

The Gla-domain of factor X (FX) is a 39 residue peptide, part of the light chain, and is characterized by the presence of 11 γ-carboxylglutaminic acid residues interspersed among other no-Gla residues. At least 15 cases of FX deficiency have been shown to be mutated in this area. In eight cases mutations involved only the Gla-domain, both at the homozygous or heterozygous level. In five of these eight cases mutation involved γ-carboxylglutaminic acid residues whereas another non-γ-carboxylglutaminic residue was involved in three cases. In the latter cases the mutation was always a Phe31Ser mutation. In the remaining cases the mutation in the Gla-domain was associated with mutations in other areas or domains. In the kindred with mutations only in the Gla-domain, FX activity via the extrinsic system varied from 1 to 10% of normal in homozygotes. In the heterozygous population it varied from 36 to 55%. Similar results were observed via the intrinsic system or in Russell viper venom dependent assays whenever these assays were available. Factor X antigen varied between 16 and 55% of normal in almost all cases. In one case it was reported to be normal. In another case it was reported to be low (2%). The bleeding tendency was usually mild to moderate. It was reported to be severe in the family with an associate homozygous factor VII deficiency, and in another instance, FX Algiers. Altogether the pattern seemed, with a few exceptions, fairly uniform so that a satisfactory genotype-phenotype relation could be inferred for the families with defects only in the Gla-domain. On the contrary, no firm conclusion could be drawn for kindred who showed mutations in the Gla-domain associated with defects in other domains.

A case of factor X (FX) deficiency due to novel mutation V196M, FX Hofu

The patient, a 20-year-old male, was found to have a slightly prolonged prothrombin time (PT). No episodes of bleeding were noted. The measurement of coagulation factors revealed that the level of factor X (FX) activity was solely deficient, 51% (normal range: 70-130% ), and that of FX antigen was 100%. Analysis of the entire FX gene revealed the novel missense mutation of GTG to ATG, resulting in the substitution of the 196th amino acid valine --> methionine. The mother and younger brother had a normal PT time and expressed no episode of bleeding. The mother exhibited a normal level of FX activity and antigen; however the younger brother showed a slight decrease in both the parameters. This mutation was not observed in the mother and younger brother. Polymorphism is not observed at this point in healthy persons. The present novel FX mutation was named FX Hofu.

Potent arterial antithrombotic effect of direct factor-Xa inhibition with ZK-807834 administered to coronary artery disease patients

It was the objective of this study to evaluate the anti-thrombotic potency of direct factor-Xa inhibition with ZK-807834 in stable coronary patients, using an ex-vivo model of arterial thrombus formation. Tissue factor pathway is important in atherothrombosis. Direct factor-Xa blockade may more potently reduce thrombosis and prevent coronary events. Badimon Perfusion Chamber 5-minute quantitative studies have shown 40-55% arterial thrombus reduction with abciximab, 23% with clopidogrel, but none with heparin. Coronary patients (n = 18, 59 +/- 9 years, 55% males) were blindly randomized to four groups receiving 24-hour infusion of a low, medium or high dose of direct factor- Xa inhibitor ZK-807834, or placebo. Arterial thrombus formation was measured in Badimon Chamber at baseline, end-of-infusion [EoI], and four hours and eight hours after EoI, and factor-X activity, prothrombin time [PT] ratio and plasma drug levels were measured simultaneously. For the low-, medium- and high-dose ZK-807834 groups, mean percent-reduction in thrombus size from baseline to EoI were 29%, 34% and 68%, respectively (p < 0.001), and at 8-h post EoI were 11%, 19% and 27%, respectively (p < 0.01). Mean PT-ratio prolongation showed a strong linear relationship (Pearson's r = 0.93) with ZK-807834 plasma concentration. Mean percent-reduction in factor-X activity from baseline was 13%, 42% and 58%, respectively. Placebo had no effect on thrombus size or factor-X activity. In conclusion, direct factor-Xa inhibition with ZK-807834 markedly reduces ex-vivo arterial thrombus formation and factor-X activity in a dose-dependent manner. Plasma levels of ZK-807834 show a strong linear correlation with PT ratio. This direct factor-Xa inhibitor may reduce the need for additional potent glycoprotein IIbIIIa inhibition.

Murine Model of Suppressed Intrinsic Pathway Using a FX Variant: FX Roma.

The complex interplay between the intrinsic and extrinsic pathways of coagulation is incompletely understood. The existence of Factor X variants that can be asymmetrically activated through one but not both of these pathways affords one strategy for analyzing the relationship of the two pathways. The Factor X Roma (FXRoma) variant, originally described in a patient with mild bleeding tendency (severe following trauma, De Stefano et al., Br J Haematol. 1988, 69(3):387–91), is due to a missense mutation (Thr318→Met) in exon 8. Coagulation testing revealed markedly decreased activity (1–3%) in the intrinsic pathway as measured by aPTT, but substantially higher activity (30–50%) in the extrinsic pathway as measured by PT. Using site-directed mutagenesis and transient transfection in HEK 293 cells, we confirmed this differential activity in extrinsic and intrinsic pathways for a variant human Factor X (FX) recombinant protein with a Thr318→Met change. FX antigen was assessed by ELISA as previously described (Larson PJ et al., Biochemistry. 1998, 37(14):5029–38) and FX activity by PT or aPTT; analysis of conditioned medium from FX wild-type and FX Thr318→Met showed respectively: antigen 100%, 82%; intrinsic activity 120 U/mg (100%), 1.8 U/mg (1.5%); extrinsic activity 190 U/mg (100%), 81 U/mg (41%). To further study this variant, we constructed a mouse homozygous for the analogous mutation (Thr318→Met) in the murine FX gene. This residue is conserved in human, murine and canine FX. We utilized mouse ES cells in which exon 8 of the murine FX gene had previously been deleted and replaced with a neomycin resistance gene and a partial HPRT gene through a targeted recombination event. These cells were used for a second electroporation event with a targeting construct carrying an exon 8 cassette containing the Thr318→Met substitution, and the missing portion of the HPRT gene. After selection on HAT medium, correctly targeted ES cells were micro-injected into blastocysts and implanted into pseudopregnant mice to generate chimeric mice. Heterozygous offspring of chimeric males were intercrossed to obtain animals homozygous for the FXRoma mutation. Coagulation testing revealed that FXRoma heterozygotes showed the expected 52.1±5.9% intrinsic activity and 142.8±25.1% extrinsic activity. More importantly, FXRoma homozygotes showed 8.5±2.7% intrinsic activity and 107.7±41.1% extrinsic activity. Thus the murine FXRoma mutation demonstrates a coagulation profile similar to that observed with the human variant. Thus the FXRoma mouse can model the effect of impeded intrinsic pathway more accurately than e.g. a FIX knockout mouse, because both FX and FIX can act as substrates for the Factor VIIa-Tissue Factor complex and all the components of the Xase complex will be essentially at normal or near-normal levels. The ability to carry out studies of long-term effects of an impeded pathway, and to analyze the response of these animals to prothrombotic stimuli, will allow us to determine the safety and efficacy of therapeutic approaches based on impeding the intrinsic pathway.

Molecular characterization of two novel mutations causing factor X deficiency in a Chinese pedigree

Factor X (FX) deficiency is a rare bleeding disorder inherited as an autosomal recessive trait. In this study, we investigated the molecular basis of FX deficiency in a Chinese pedigree. The proposita showed a markedly prolonged activated partial thromboplastin time and a mild prolongation of prothrombin time. The levels of FX antigen and FX activity were 58.6% and 2.5%, respectively. Molecular analysis revealed that the proposita was compound heterozygous for two novel mutations: IVS1 + 1G > A and G1185A (Arg347His). The aberrant transcripts from the IVS1 + 1G > A mutant allele were not detected by analyzing the splicing pattern of ectopic transcripts in leukocytes of the patient with nested polymerase chain reaction after reverse transcription. We thus hypothesize that the mRNA molecules originating from the IVS1 + 1G > A mutation were rapidly destroyed in vivo. Site-directed mutagenesis of FX cDNA was used to introduce FXG1185A mutation, and wild-type as well as mutant FX proteins were expressed by transient transfection in HEK 293 cells. Normal FX antigen levels both in the conditioned media of cells expressing the mutant and in cell lysates were detected by an enzyme-linked immunoadsorbent assay. Evaluation of wild-type and mutant coagulant activity demonstrated that the FX molecules carrying the Arg347His mutation have dramatically decreased activity.

Impaired prothrombinase activity of factor X Gly381Asp results in severe familial CRM+ FX deficiency

We investigated three members of a large Omani family affected by severe factor X (FX) deficiency (coagulant activity <1%) and showing marked differences in the onset of severe hemorrhagic symptoms. All patients were homozygous for a novel FX mutation (Gly381Asp) in the structurally conserved region of the serine protease active site. Expression levels of recombinant 381D-FX were similar to those of wt-FX, indicating the presence of a severe CRM+ FX deficiency, a poorly investigated condition. The 381D-FX was normally activated and did not show a detectable amidolytic activity. Instead, we observed a residual activity in a prothrombin-time based assay (1%) and in prothrombinase assays both in plasma (1%) and in purified systems (3%). Comparison with FX variants characterized by reduced activation suggests that mutations affecting FX activity might result in a more pronounced impairment of coagulation and thus in severe hemorrhagic phenotype. In addition, this study indicates that the hemorrhagic heterogeneity observed in FX deficiencies is only partially explained by molecular analysis of FX gene.

Reduced Activation of the Gla19Ala FX Variant via the Extrinsic Coagulation Pathway Results in Symptomatic CRMred FX Deficiency

We characterized a symptomatic CRMred factor X (FX) deficiency produced by the Glu19Ala mutation in the gamma-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19Ala-FX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation. The study of this FX deficiency provides an "in vivo" and "in vitro" model for the investigation of Gla domain interactions.

A New Factor X Defect (Factor X Padua 3): A Compound Heterozygous Between True Deficiency (Gly380→Arg) and an Abnormality (Ser334→Pro)

We report a novel mutation in Factor X (FX) gene which results in a phenotype without any bleeding tendency. The proband has been found to be a compound heterozygote between a novel FX true deficiency (Gly380→Arg) and a previously reported dysfunctional mutation Ser334→Pro (FX Marsiglia). Prothrombin time (PT) and partial thromboplastin time (PTT) were moderately prolonged and were fully corrected by the addition of normal serum. Her FX activity level varied between 8% and 19% of normal according to the method used whereas the FX antigen level was 40% of the normal control value. All the exons and intron/exon junctions of the FX gene were studied using a combined approach of polymerase chain reaction and conformation sensitive gel electrophoresis. A transversion G to A in exon 8 resulting in the replacement of Gly380 by Arg was found in the proband, in the father and in a proband's brother, whereas heterozygous FX Marsiglia was present in the proband's mother and her sister. Gly380 is strictly linked to Ser379, a component of the catalytic triad. The substitution of Gly for Arg causes the introduction of a large charged amino acid which could affect the catalytic function of FX leading to secretion problem, accounting for the cross-reactive material (CRM) negative phenotype.

Factor XSanto Domingo. Evidence that the severe clinical phenotype arises from a mutation blocking secretion.

Factor X (FX) is a vitamin K-dependent plasma protein required for the intrinsic and extrinsic pathways of blood coagulation. FXSanto Domingo is a hereditary FX deficiency which is characterized clinically by a severe bleeding diathesis. The proposita has a FX activity of less than 1% and a FX antigen of less than 5%. We have determined the molecular basis of the defect in the FXSanto Domingo gene by amplification of all eight exons with polymerase chain reaction and subsequent sequence analysis. The patient is homozygous for a G----A transition in exon I at codon -20 (numbering the alanine at the NH2 terminus of the mature protein as +1), resulting in the substitution of arginine for glycine in the carboxy-terminal part of the signal peptide. This amino acid change occurs near the presumed cleavage site of the signal peptidase. We hypothesized that the mutation might prevent cleavage by the signal peptidase which in turn would impair proper secretion of the FX protein. To test this hypothesis, we compared the expression of wild type and mutant FX cDNA in a human kidney cell line. Wild type and mutant constructs in the expression vector pCMV4 were introduced into the human embryonic kidney cell line 293 by calcium phosphate transfection. FX antigen levels in the supernatant of the cells harboring the wild type construct were 2.4 micrograms/10(7) cells per 24 h, whereas antigen levels in media from cells containing the FXSanto Domingo construct were undetectable. No FX antigen was detected in the cell lysates of cells transfected with the mutant construct. To insure that the difference in protein levels was not due to a difference in steady state levels of mRNA, Northern analysis was performed on RNA from the cell lysates of both constructs. The results showed a transcript of the same size, present in roughly equal amounts, in both cases. Thus, the defect in the signal sequence of FXSanto Domingo exerts its effect posttranscriptionally. FXSanto Domingo is the first described example of a bleeding diathesis due to a mutation in the signal sequence.

Effects of warfarin on vitamin K-dependent coagulation factors in Atlantic salmon and rainbow trout with special reference to factor X

Plasma factor X (FX) was severely depleted in Atlantic salmon and rainbow trout after 18 days of oral warfarin administration; this response was further reflected in prolonged prothrombin time. Thus, both species require vitamin K for the maintenance of normal haemostasis. FX activity in controls of rainbow trout was 3-to 5-fold that of salmon. The FX of both species show several of the features of human FX: it is activated by RVV, inhibited by SBTI, inactivated in the presence of heparin, and adsorbs to insoluble barium precipitates. Rainbow trout sustained warfarin poisoning better than did salmon, with mortalities occurring 8 days later in trout. After 18 days all remaining fish had developed a severe haemorrhagic anaemia with anisocytosis, poikilocytosis, leucopenia, and thrombocytopenia.

Molecular defect (Gla+14----Lys) and its functional consequences in a hereditary factor X deficiency (factor X "Vorarlberg").

Factor X (FX) "Vorarlberg" is a congenital FX deficiency characterized clinically by a mild bleeding tendency. Homozygous individuals have a FX activity of less than 10% in the extrinsic system and 25% in the intrinsic system. FX antigen is 20%. Using molecular techniques, two point mutations were detected in the coding sequence of the FX Vorarlberg gene: a G----A at base pair 160 in exon II resulting in a change of Gla14 (GAA) to Lys (AAA); a G----A at base pair 424 in exon V resulting in a change from Glu102 (GAG) to Lys (AAG). The mutations abolished a TaqI restriction site in exon II and an MnlI site in exon V. To determine whether these mutations are present on one or on both alleles, restriction analyses of amplified exon II and exon V fragments were performed. Analysis of the pedigree showed that the genotype for the mutation on exon II (homozygous versus heterozygous) correlates with the severity of the phenotypic coagulation defect. We therefore conclude that the mutation in exon II is responsible for the functional defect in FX Vorarlberg. We have also purified the mutant FX protein from patient plasma. Purified FX Vorarlberg is indistinguishable from normal FX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its activity is 15% of normal FX upon activation with factor VIIa/tissue factor, 75% upon activation with factor IXa/factor VIIIa, and 100% upon activation with RVV. Activation at varying Ca2+ concentrations shows that the affinity of FX Vorarlberg for Ca2+ is decreased. Factor Xa Vorarlberg is able to convert prothrombin at a normal rate but also shows decreased affinity for Ca2+ in this interaction. Upon addition of Ca2+, FX Vorarlberg does not undergo the same conformational change as normal FX. Our data show that FX Vorarlberg has a decreased affinity for Ca2+ which impedes a normal conformational change. This leads to a decreased rate of activation by factor VIIa/tissue factor and by factor IXa. The decrease is much more marked for the extrinsic than for the intrinsic pathway.

An unusual factor‐X inhibitor in leprosy

Two cases of Hansen disease demonstrating a lupus-like inhibitor directed against the activation of coagulation factor X are described. Each case showed factor-X activity markedly depressed by an inhibitor when measured in one assay system; yet normal levels were measured by three alternate factor-X assay systems.