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

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.

Real-time quantitative PCR analysis of factor XI mRNA variants in human platelets.

Coagulation factor XI (FXI) plays an essential role in blood coagulation. A deficiency of FXI is an unusual hemorrhagic diathesis in that the bleeding tendency can be highly variable, ranging from severe deficiencies with no symptoms to mild and moderate deficiencies requiring multiple blood transfusions for hemorrhages. This variability in bleeding has been attributed to a number of factors including the presence of a novel form of FXI associated with platelets, which ameliorates the bleeding in some cases of FXI deficiency. However, the nature of this platelet FXI molecule is controversial. Hsu et al. (J Biol Chem 1998; 273: 13787-93) suggest that it is a product of normal FXI - but lacking exon V whilst Martincic et al. (Blood 1999; 94: 3397-404) were unable to detect this alternatively spliced variant using RT-PCR. In order to resolve this controversy, we have employed the highly sensitive technique of real-time quantitative RT-PCR using RNA isolated from FXI-deficient patients. Our results indicate that the platelets of both normal and FXI deficient individuals contain FXI mRNA that is identical to the mRNA found in liver. An exon V deleted splice variant was not detected. Thus the FXI message is not alternatively spliced in platelets and therefore would not be able to produce an unusual FXI protein.

Validation of a quantitative SPR assay for recombinant FVIII.

Surface plasmon resonance was employed to establish a quantitative assay for recombinant FVIII (rFVIII) products using rFVIII as standard. The anti-FVIII monoclonal antibody ESH4 was immobilized onto a carboxymethyldextran surface. A range of rFVIII concentrations were injected over the surface and the binding response enhanced by the addition of a further monoclonal antibody ESH8. Validation using National Institute of Biological Standards and Controls (NIBSC) sixth International rFVIII concentrate standard gave inter- and intra-assay coefficient of variations (CVs) of 7.5 and 3.68% respectively for ESH4-rFVIII binding alone. Enhancement of the binding signal by secondary addition of ESH8 produced inter- and intra-assay CVs of 2.75 and 1.5%. The ESH4 immobilized chip was found to retain binding capacity following regeneration for at least 75 cycles. The assay was found to be unsuitable for quantitation of plasma derived FVIII product but may prove useful for monitoring of rFVIII production.

Evaluation of an automated screening assay for von Willebrand disease type 2N.

Evaluating the factor VIII (FVIII) binding activity of von Willebrand factor (VWF) is an important step in the diagnostic work-up of families affected by apparent mild haemophilia A. In von Willebrand's disease (VWD) type 2N (Normandy), mutations at the N-terminal end of the mature VWF subunit gene prevent the binding of FVIII. Individuals heterozygous for type 2N VWD are generally asymptomatic. Homozygotes and compound heterozygotes present with a clinical picture which mimics haemophilia A, with a markedly reduced FVIII : C activity and VWF within the normal range, but instead of exhibiting X-linked inheritance they show an autosomal recessive inheritance pattern. The distinction between haemophilia A and VWD type 2N has important implications for therapy and genetic counselling. We present a highly specific enzyme-linked immunosorbent assay screening method for the Normandy variant, which measures VWF : FVIII binding activity in parallel with VWF antigen, using monoclonal capture and detection antibodies. The assay is fully automated using a robotic microtitre plate processor, requiring minimal user intervention and providing the capacity to screen large numbers of patients.

Identification of an intronic regulatory element in the human protein C (PROC) gene.

Regulatory DNA elements responsible for human protein C (PROC) gene expression have previously been identified in the upstream promoter region and first (untranslated) exon of the gene. Here we show that an additional sequence element located more than 500 bp downstream of the core promoter within intron 1 further enhances PROC promoter-driven reporter gene expression in human hepatoma cells. In common with core promoter constructs used in previous studies, the activity of this 3'-extended regulatory region is diminished by a naturally occurring promoter mutation. However, in contrast to constructs lacking intronic sequence, the promoter/intron regulatory region is repressed rather than activated by the transcription factor HNF-1. Using both conventional alignment procedures and complexity analysis to study the human and canine PROC sequences, we identified two conserved intronic regions, which were tested for their involvement in gene regulation. High-level gene expression from the intron-coupled promoter was dependent upon the integrity of a 142 bp sequence element, a duplicate copy of which is located in an upstream region of the PROC gene that possesses enhancer activity. These findings emphasise the potential importance of intragenic sequences for gene regulation and serve to illustrate that the results of PROC promoter/reporter gene experiments are critically dependent upon the sequence context. The identification of such intragenic elements is relevant to the analysis of human genetic disease since it will facilitate the detection and functional evaluation of regulatory mutations and polymorphisms.

Heterozygous factor XI deficiency associated with three novel mutations.

To determine the utility of single-stranded conformation polymorphism (SSCP) analysis for screening mutations in the factor XI (fXI) gene, we investigated three patients with heterozygous factor XI deficiency. DNA sequence analysis confirmed three novel mutations; a CGC --> TGC (Arg308Cys) mutation in exon 9, a GCT-->GTT (Ala412Val) mutation in exon 11 and an AGC --> AGA (Ser576Arg) mutation in exon 15. We postulated on the structural implications of these missense mutations. Our results demonstrated that genotypic analysis is a useful tool for conclusive differentiation between heterozygous factor XI deficiency and normal subjects.

Immune tolerance therapy for haemophilia A patients with acquired factor VIII alloantibodies: comprehensive analysis of experience at a single institution.

Immune Tolerance Therapy for Haemophilia A Patients with Acquired Factor VIII Alloantibodies: Comprehensive Analysis of Experience at a Single Institution Eleven children with severe haemophilia A associated with the IVS 22 inversion and acquired high titre neutralising antibodies to factor VIII underwent immune tolerance induction. HLA class I and high resolution class II type is detailed for each patient. A three phase approach to immune tolerance induction was used. During phase 1, which lasted a median of six weeks, patients received factor VIII 100 IU/kg twice daily. Phase 2 comprised a factor VIII dose reduction to 100 IU/kg once daily, and continued for a median duration of 14 weeks. Subsequently 10 of the 11 patients satisfied the criteria of absent factor VIII neutralising activity by the Bethesda method, and a factor VIII elimination half life of greater than 5 h, allowing progression to phase 3, a further factor VIII dose reduction to 50 IU/kg three times weekly. A model for dose reduction as factor VIII tolerance evolves, based on pharmacokinetic analysis, is described.

Identification of a novel mutation in a non-Jewish factor XI deficient kindred.

The role of factor XI (FXI) in blood coagulation has been clarified in recent years by descriptions of FXI-deficient patients who are prone to excessive bleeding after haemostatic challenge. We have studied a large kindred of an Italian FXI-deficient patient with a previously undescribed mutation. The propositus, a 68-year-old woman, presented with a cerebral thromboembolic event but had no history of bleeding (FXI activity 1.6 U/dl). A sensitive ELISA failed to detect FXI antigen in the propositus. Sequence analysis of the entire FXI gene revealed a TGG to TGC transversion in codon 228 of exon 7 (FXI-W228C). This missense mutation results in a Trp to Cys substitution within the third apple domain of FXI. We conclude that this novel mutation occurred in a structurally conserved region and may therefore have interfered with either chain folding and secretion or stability of FXI and was responsible for the inherited abnormality seen in this kindred. It is unclear why this kindred does not exhibit a bleeding tendency but it may correlate with a FXI-like antigen and factor IX binding activity expressed on platelets.

Disruption of a binding site for hepatocyte nuclear factor 1 in the protein C gene promoter is associated with hereditary thrombophilia.

A heterozygous T-->C transition was detected in the putative promoter region of the protein C (PROC) gene in a patient with type I protein C deficiency and a history of recurrent venous thrombosis. This mutation occurred 14 bp upstream of the transcription initiation site and within a sequence strongly homologous to the consensus binding site for the liver-enriched transcription factor, hepatocyte nuclear factor 1 (HNF-1). Transfection experiments demonstrated that a CAT reporter gene construct containing 626 bp of the putative PROC gene promoter was capable of driving CAT expression in HepG2 hepatoma cells. Levels of CAT expression from constructs bearing the mutation were found to be drastically reduced by comparison with the wild-type, consistent with the reduced plasma protein C antigen levels observed in the patient. Gel retardation and cotransfection experiments demonstrated that the mutation abolished both the binding and the transactivating ability of HNF-1 observed with the wild-type PROC gene promoter. Further, the ability of the mutation to disrupt HNF-1 binding appears to be a function not only of the nature of the nucleotide substitution and its position within the recognition sequence, but also of the relative affinity of the wild-type binding site for HNF-1. This analysis is therefore indicative of a vital role for HNF-1 in the expression of the PROC gene in vivo. Taken together with the identification of a human hepatoma cell line which contains HNF-1 but which does not express protein C, these findings are consistent with the view that HNF-1 is necessary although not sufficient for PROC gene expression in the liver.

Clinical application of new technique that measures C4d for assessment of activation of classical complement pathway.

A new laser nephelometric technique that measures C4d for the assessment of the activation of the classical complement pathway was developed. C4d was isolated from other larger C4 related molecules at a final concentration of polyethylene glycol of 12% and then quantitated by laser nephelometry using a commercially available antiserum, which reacts with C4d determinants. C4d standard (100%) was produced by exhaustive activation of the classical pathway in pooled normal human serum using heat aggregated human immunoglobulin. Serial dilutions of the standard provided a reference curve against which clinical samples were read. Patients with rheumatoid arthritis showed significantly higher C4d values (mean 53.8%) than controls (21.7%; p less than 0.001). The technique proved accurate, rapid, and suitable for the routine laboratory evaluation of complement activation through the classical pathway, and it may be useful in the management of those conditions in which complement activation has a pathogenic role.

Activation of the alternative complement pathway: clinical application of a new technique to measure fragment Ba.

A new laser nephelometric technique for the measurement of the alternative complement pathway fragment Ba has been developed. Activation of the alternative complement pathway was assessed in 16 patients with Gram negative bacteraemia, six with Gram positive bacteraemia, 20 with rheumatoid arthritis, and 18 healthy subjects. Patients with Gram negative bacteraemia had significantly higher values of Ba (median 14.8%) than controls (9.3%) (p less than 0.01), while patients with Gram positive bacteraemia and rheumatoid arthritis had values similar to those of controls (10.1% and 9.5%). The technique proved sensitive and precise, and is suitable for the routine laboratory evaluation of complement activation through the alternative pathway.

Pathways to complement activation during cardiopulmonary bypass.

Complement activation was assessed in 34 patients undergoing cardiopulmonary bypass. Arterial concentrations of complement fragments Ba and C3d rose in all patients, the increase in Ba preceding that of C3d. At the same time as complement fragments were being generated the arterial neutrophil count fell. These findings suggest (a) that complement activation is initiated by the alternative pathway during cardiopulmonary bypass and (b) that complement activation mediates loss of neutrophils during bypass. Complement mediated loss of neutrophils during the analogous setting of haemodialysis is the result of leucosequestration in the pulmonary vasculature. During cardiopulmonary bypass the lungs are out of circuit, so that activated leucocytes may sequester in other target organs. This may be an aetiological factor in the multi-organ failure occasionally seen after uneventful cardiopulmonary bypass.