Letian Dai

The profibrinolytic effect of plasma thrombomodulin in factor XI deficiency and its implications in hemostasis

Summary.  Bleeding tendency in factor (F)XI deficiency may result from premature clot lysis due to insufficient thrombin activatable fibrinolysis inhibitor (TAFI) activation. Thrombomodulin (TM), upon binding to thrombin, is capable of modulating TAFI activation. In this study, we investigated the effects of plasma TM on fibrinolysis in FXI‐deficient patients. A clot lysis assay showed the defective down‐regulation of fibrinolysis in FXI‐deficient patients as compared with normal controls. To evaluate the effects of plasma TM on fibrinolysis, a monoclonal anti‐TM IgG was preincubated with plasma for 30 min. The presence of anti‐TM IgG significantly prolonged the clot lysis times both in the FXI‐deficient and normal plasma, indicating that plasma TM stimulated fibrinolysis. Furthermore, the presence of anti‐TM IgG not only reduced protein C activation, but also increased thrombin generation and TAFI activation. The profibrinolytic effect of plasma TM was inhibited in the assay by including either a monoclonal anti‐TAFI IgG or a specific TAFI inhibitor – carboxypeptidase inhibitor (CPI). Our results indicate that the impaired thrombin generation in FXI‐deficient patients leads to the defective down‐regulation of fibrinolysis, and that plasma TM stimulates fibrinolysis through APC pathway which inhibits TAFI activation. The profibrinolytic effect of plasma TM may contribute to the bleeding tendency observed in some FXI‐deficient patients.

Characterization of a causative mutation of hemophilia A identified in the promoter region of the factor VIII gene (F8)

Hemophilia A is an X-linked bleeding disorder of variable severity resulting from deficient or dysfunctional factor VIII protein. Genetic defects of the F8 gene are heterogeneous [1], and all the mutations reported in the Haemophilia A mutation Database [2] affect or disrupt theF8 gene coding sequence or its intron–exon boundaries. However, in 1 to 2% of cases, a causative mutation cannot be identified in the F8 gene [3], and prior to the recent publication by Bogdanova et al. [4], no causative mutations had been described in the F8 gene promoter. The F8 gene is regulated by a 1175-bp promoter that contains at least 19 binding sites for liver-specific and ubiquitous transcription factors, including, c/EBPa, c/EBPb, nuclear factor kappaB, HNF1, NF-Y andHLF [5–7].We have identified the same C>T substitution at position –219 (standard Human Genome Variation Society (HGVS) nomenclature; +1 refers to the first base of the ATG translational initiation codon, as per Figueiredo et al.) of the F8 gene promoter reported byBogdanova et al. [4] in a three-generation family with mild hemophilia A, and demonstrated its causality. A 61-year-old man admitted to hospital following an assault was found to have a low FVIII:C of 30.3 IU dL (normal range: 73–200 IU dL). A diagnosis of hemophilia A had not been previously assigned, but there was a strong personal history of bleeding. Molecular analysis was undertaken to determine the causative mutation of the apparent mild hemophilia A. Mutation screening, by denaturing high-performance liquid chromatography analysis, followed by dideoxy sequencing of all 26 exons and associated splice junctions of the F8 gene, failed to detect any variation from the normal sequence. Further phenotypic testing excluded an obvious alternative diagnosis, such as combined FV and FVIII deficiency or type 2N von Willebrand disease, with normal FV levels and vonWillebrand factor parameters, and a normal F8 binding assay. Subsequent analysis of the F8 gene promoter region, by polymerase chain reaction (PCR) amplification and dideoxy sequencing, detected a C>T substitution at position – 219. This base change was excluded as a common polymorphism by the failure to detect it in > 100 normal alleles. The possible influence of the –219C>T substitution on the F8 promoter was investigated. A 317-bp fragment from the human F8 gene promoter (positions –314 to +3; +1 refers to the first base of the ATG translational initiation codon) was amplified by PCR using patients or control DNA samples as a template, and ligated into a promoterless pGL3Basic luciferase reporter plasmid (Promega, Southampton, UK). PLC/PRF/5 cells (ATCC, CRL-8024), a hepatoma cell line proven to express FVIII [7,8], were cultured and transfected with both the firefly luciferase reporter plasmid (2 lg), containing either wild-type or mutant sequence, and an internal control plasmid (0.05 lg) containing the Renilla luciferase gene. After 48 h, cell lysates were assayed for luciferase activity using a dual-luciferase assay system (Promega). The firefly luciferase activity was calculated as fold induction compared to the transfected promoterless reporter plasmid, and normalized with respect to transfection efficiency using the Renilla luciferase activity. The relative value of luciferase activity for the mutant sequence was expressed as a percentage of the activity observed for the wild-type sequence. From three experiments, each with triplicate dishes, it was found that the plasmid containing the mutant sequence expressed 23% ± 3.2% (mean ± SEM) of the luciferase activity of the wild-type sequence. The results indicated that the presence of the –219T mutation significantly reduced the F8 promoter activity. Further analysis by electrophoretic mobility shift assay (EMSA) was performed to investigate the interaction of the F8 sequence with nuclear extracts from PLC/PRF/5 cells. Oligonucleotides corresponding to the wild-type and mutant sequences (positions –228 to –208 of the F8 promoter) were synthesized and then labeled with biotin-11-UTP, using a biotin 3¢-endDNA-labeling kit (Pierce, Northumberland,UK). The labeled oligonucleotides were then bound to PLC/PRF/5 cell nuclear extracts, prepared according to the method of Hoppe-Seyler et al. [9], and the protein–DNA complexes were electrophoresed on a 6% native polyacrylamide gel and Correspondence: Mike Mitchell, Centre for Haemostasis and Thrombosis, The Haemophilia Reference Centre, 1st Floor North Wing, St Thomas Hospital, London SE1 7EH, UK. Tel.: +44 0 207 188 2798; fax: +44 0 207 62

Characterisation of five factor XI mutations.

A large scale factor XI (FXI) mutation screening program identified a number of novel candidate mutations and previously reported mutations and polymorphisms. Five potential missense mutations were selected for further study; these included two novel missense mutations - Met-18Ile (p.Met1Ile) and Met102Thr (p.Met120Thr), two previously reported missense mutations - Tyr133Ser (Tyr151Ser) and Thr575Met (Thr593Met), and one amino acid substitution previously reported as a polymorphism - Arg378Cys (Arg396Cys). The substitutions were recreated by the site-directed mutagenesis of a FXI cDNA and stably expressed in a BHK-570 cell line. Subsequent analysis of both the conditioned media and cell lysates showed that three of the substitutions, Met-18Ile, Met102Thr andTyr133Ser, prevented secretion of the mutated protein from the transfected cell line, resulting in a cross-reactive material negative (CRM-) phenotype. The remaining two mutants, Thr575Met and Arg378Cys, secreted significant levels of FXI into the conditioned media; however, these mutant FXIs were shown to have negligible factor IX activation activity in an APTTbased assay. These results confirmed all five of the missense mutations as being causative of factor XI deficiency, despite one having been previously reported as a polymorphism (Arg378Cys) and one (Tyr133Ser) as a mild mutation - FXI:C 38 U/dl in a homozygous patient.

Homocysteine metabolism and the associations of global DNA methylation with selected gene polymorphisms and nutritional factors in patients with dementia.

Epigenetics (particularly DNA methylation) together with environmental and genetic factors, are key to understanding the pathogenesis of many diseases including dementia. Disturbances in DNA methylation have already been implicated in dementia. Homocysteine metabolism, with folate and vitamin B12 as essential cofactors, is integral to methylation processes. We evaluated in a case-control study the association of global DNA methylation, homocysteine, folate and vitamin B12 status with dementia. Selected polymorphisms of genes previously associated with dementia development and the influence of various factors on DNA methylation were also investigated. 102 patients with dementia (53 with Alzheimers disease, 17 with vascular dementia and 32 with mixed dementia) were recruited. The non-demented controls consisted of 45 age-matched subjects without dementia and 47 individuals with mild cognitive impairment. Global DNA methylation was determined by Imprint Methylated DNA Quantification Kit MDQ1 (Sigma-Aldrich, Gillingham, Dorset, UK). Plasma homocysteine, serum folate and vitamin B12 were determined by chemiluminescence. Plasma and erythrocyte 5-methyltetrahydrofolate and plasma methylmalonic acid (markers of folate and vitamin B12 status) were measured by HPLC. APOE, PON1 p.Q192R, MTHFR 677C>T (c.665C>T) and IL1B-511C>T polymorphisms were identified using PCR-RFLP methods. Patients with dementia had significantly higher concentrations of homocysteine (p=0.012) and methylmalonic acid (p=0.016) and lower folate (p=0.002) and plasma 5-methyltetrahydrofolate (p=0.005) than non-demented subjects. There was no difference in DNA methylation between patients and controls. A non-significant tendency to higher DNA methylation in patients with vascular dementia (p=0.061) was observed. Multivariate regression analysis of all recruited individuals demonstrated a significant positive association between DNA methylation and folate (p=0.013), creatinine (p=0.003) concentrations and IL1B-511T (p=0.002) and PON1 192R (p=0.049) alleles and negative association with fasting glucose (p=0.004). The biochemical results showed significantly lower folate and vitamin B12 status in demented patients than controls. Global DNA methylation was associated with markers of folate status, creatinine, glucose and PON1 and IL1B polymorphisms.

Molecular basis and bleeding manifestations of factor XI deficiency in 11 Turkish families.

Factor XI (FXI) deficiency is an autosomal bleeding disorder characterized by variable bleeding tendency. In the present study, the gene encoding FXI (F11) was analyzed by direct sequencing in 33 individuals belonging to 11 unrelated Turkish families, and the bleeding tendency was quantitatively assessed by means of a bleeding questionnaire in 27 individuals with low FXI clotting activity and/or mutated F11 gene. We identified 10 distinct mutations (five missense, three nonsense and two splice site), four of which were novel. No mutation was found in one family. Of the four novel mutations, homozygosity for a c.89T>C (p.Phe30Ser) mutation and compound heterozygosity for a c.646G>A (p.Asp216Asn) mutation with the known c.403G>T (p.Glu135*) type II Jewish mutation were associated with severe deficiency, whilst heterozygosity for the novel c.1655A>C (p.His552Arg) and c.1627G>A (p.Glu543Lys) mutations was associated with partial deficiency. p.Glu135* was found in 19% (5/27) of the mutated alleles. Bleeding score was positive in 57% (4/7) of individuals with severe and 39% (7/18) of those with partial deficiency. It was significantly correlated with clinical severity of bleeding (r = 0.43, P = 0.02), but not with FXI clotting activity (P > 0.05). There was no optimal cut-off level of the bleeding score that could predict FXI deficiency. We conclude that the spectrum of mutations found in this study reflects the genetic heterogeneity of FXI deficiency in the Turkish population. Quantitative assessment of the bleeding symptoms by a bleeding questionnaire seems to be useful for evaluating the severity of bleeding episodes, but it can not be recommended as a screening tool for FXI deficiency.