Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells.

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

A multi-center evaluation of TECHNOSCREEN® ADAMTS-13 activity assay as a screening tool for detecting deficiency of ADAMTS-13.

BACKGROUND: Quantifying A disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS-13) activity enhances thrombotic thrombocytopenic purpura (TTP) diagnosis but most assays are time consuming, technically demanding, and mainly available in reference centers. OBJECTIVE: Evaluate a simple, semiquantitative ADAMTS-13 activity screening test for early identification/exclusion of TTP. PATIENTS/METHODS: Plasma from 220 patients with suspected thrombotic microangiopathy at three reference centers were tested with TECHNOSCREEN® ADAMTS13 activity screening test in comparison with TECHNOZYM® ADAMTS-13 activity ELISA at two centers, and in-house fluorescence resonance energy transfer assay at the third center. The screening test indicates if ADAMTS-13 activity is at one of four level-indicator points: 0, 0.1, 0.4, or 0.8 IU/mL. RESULTS: Screen results were interpreted as binary data in that ADAMTS-13 activity was above or below the 0.1 IU/mL TTP clinical threshold. Combining all sites' data, the screen exhibited 88.7% sensitivity, 90.4% specificity, 74.6% positive predictive value, and 96.2% negative predictive value, comparable to published data for quantitative assays. Five samples with quantitative results below the threshold gave screen readings of 0.1 IU/mL and seven marginally above the threshold gave screen readings of zero. All would warrant plasma exchange while the level is quantified. Nine samples with normal/near normal results gave screens of zero and confirmatory quantifications would prompt early treatment withdrawal, as is current practice. One sample generated screen/quantitative results of 0.4/0.00 IU/mL respectively and was the only clear false-negative. CONCLUSIONS: The screening test provides more rapid ADAMTS-13 level evaluation than most currently available assays. Its simple operation renders it suitable for adoption in routine or specialist laboratory environments.

The significance of published polymorphisms in 14 cases of mild factor VII deficiency.

Factor VII (FVII) plays a critical role in the initiation of blood coagulation, and patients with dysfunctional or reduced levels of this protein are susceptible to mucosal bleeding. There is poor correlation between the clinical presentation and the phenotypic data; and in cases of a mild bleeding tendency, mild to moderate reductions in both FVII antigen and activity may be overlooked. The prevalence of FVII deficiency may therefore be underestimated. Polymorphic differences throughout the FVII gene are associated with variations in plasma FVII antigen and activity levels. This study highlights the significance of mild FVII deficiency, and examines the importance of seven previously published polymorphisms in such patients.

Development of a novel, rapid assay for detection of heparin-binding defect antithrombin deficiencies: the heparin-antithrombin binding (HAB) ratio.

INTRODUCTION: Qualitative and quantitative antithrombin deficiency predisposes to thrombosis, although patients with heparin-binding dysfunction have a lower incidence than other sub-types. Assays discriminating between qualitative sub-types are not widely available. PATIENTS/METHODS: Extended heparin incubation in antithrombin activity assays can overestimate levels in patients with HBDs. Plasmas from genetically proven HBD patients were assayed for antithrombin activity by factor Xa-inhibition and thrombin-inhibition at varying incubation times. Optimal pairings were assessed for generating a quantifiable discrepancy in HBDs by deriving a ratio between results from short and prolonged heparin-incubation assays respectively, the Heparin-antithrombin binding (HAB) ratio. Fourteen patients with hereditary antithrombin deficiency, including five with HBDs, were analysed. RESULTS: The FXa-inhibition assay with 30s and 300s incubations clearly identified a heterozygous p.Pro73Leu and homozygous p.Leu131Phe, giving HAB ratios of 0.24 and 0.67 respectively (reference range 0.90 - 1.01). However, three plasmas containing mutations with markedly reduced or absent heparin affinity (p.Lys146Glu, p.Gln150Pro, p.Arg79Cys) gave normal results. Nine antithrombin deficient plasmas were tested with the thrombin-inhibition assay and all generated reduced HAB ratios whilst two normal donors did not. The three available HBD plasmas generated lower values than non-HBD plasmas. The mildly reduced HAB ratios in non-HBD deficiencies may have been due to heparin cofactor II reacting with bovine thrombin during extended incubation. CONCLUSIONS: HAB ratio from FXa-inhibition assays distinguishes some but not all HBD from non-HBD antithrombins, and thrombin-inhibition assays may be diagnostically applicable with sub-type specific cut-offs.

Denaturing HPLC for mutation screening.

Denaturing High-Performance Liquid Chromatography (dHPLC) is probably the most versatile and one of the most widely used mutation screening technologies. It benefits from a combination of relative technical simplicity and a very high sensitivity (mutation detection rate), approaching 100%. DHPLC can reliably detect single-base mismatches in fragments between 150 and 500 bp, although detection in fragments up to 1,500 bp has been reported. The ability of dHPLC to detect both known and unknown mutations/SNPs, and its' high sensitivity and specificity (reproducibility) has put this technology at the forefront of genetic analysis for a wide variety of diseases.

Germline mosaicism resulting in the transmission of severe hemophilia B from a grandfather with a mild deficiency.

We report a family in which the normal pattern of X-linked inheritance of hemophilia B (Factor IX deficiency) is complicated by mosaicism in the proband's maternal grandfather. The proband, an infant with severe Factor IX deficiency, was initially thought to be a sporadic case. Testing of other family members identified his mother as a carrier of the disorder, and his asymptomatic maternal grandfather as having very mild FIX deficiency. The causative familial mutation was identified as a two base pair deletion (AG within codons 134-135) in the Factor IX gene. The grandfather was shown to be "heterozygous" for the deletion. Karyotype analysis confirmed him to be 46XY thereby ruling out Klinefelter syndrome. The proband's aunt, who as the daughter of a man with hemophilia is theoretically an obligate carrier, was found not to carry this familial mutation, and thus not to be a carrier of hemophilia B. The grandfather must therefore be an X chromosome somatic and germline mosaic, with consequent segregation of the affected and non-affected Factor IX genes. This observation underlines the importance of confirming carrier status even in those individuals assumed to be obligate carriers, and has implications for genetic counseling.

Eighteen unrelated patients with factor XI deficiency, four novel mutations and a 100% detection rate by denaturing high-performance liquid chromatography.

Factor XI (FXI) deficiency is an autosomal bleeding disorder of variable severity. Inheritance is not completely recessive as heterozygotes may display a distinct, if mild, bleeding tendency. Eighteen unrelated FXI-deficient patients were screened blind by fluorescent single-stranded conformation polymorphism (F-SSCP) analysis and denaturing high-performance liquid chromatography (dHPLC). Mutations were detected in 14 of the 18 patients ( approximately 78%) by F-SSCP and in all 18 patients by dHPLC. Dideoxy sequencing confirmed the mutations in all 18 patients: eight of the mutations being novel (four of which were in previously reported patients). This showed dHPLC to be a highly sensitive, reliable technique for mutation screening in heterogeneous disorders.