Mutations of factor VIII cleavage sites in hemophilia A

Hemophilia A is caused by a defect in coagulation factor VIII, a protein that undergoes extensive proteolysis during its activation and inactivation. To determine whether some cases of hemophilia are caused by mutations in important cleavage sites, we screened patient DNA samples for mutations in these sites by a two-step process. Regions of interest were amplified from genomic DNA by repeated rounds of primer- directed DNA synthesis. The amplified DNAs were then screened for mutations by discriminant hybridization using oligonucleotide probes. Two cleavage site mutations were found in a survey of 215 patients. A nonsense mutation in the activated protein C cleavage site at amino acid 336 was discovered in a patient with severe hemophilia. In another severely affected patient, a mis-sense mutation results in a substitution of cysteine for arginine in the thrombin activation site at amino acid 1689. This defect is associated with no detectable factor VIII activity, but with normal levels of factor VIII antigen. The severe hemophilia in this patient was sporadic; analysis of the mother suggested that the mutation originated in her gametes or during her embryogenesis. The results demonstrate that this approach can be used to identify factor VIII gene mutations in regions of the molecule known to be important for function.     

MR imaging of the acutely injured patient with cervical traction

Magnetic resonance (MR) imaging of the patient with acute cervical injury is important because of the potential prognostic significance of the appearance of the spinal cord at the time of injury. However, cervical traction may involve equipment incompatible with the magnetic environment, and transferring the patient to the imaging table may make it difficult to maintain traction. The authors describe a simple, inexpensive, and reliable method for providing cervical traction within the magnet room.     

Using Intervention Mapping to develop a programme to prevent sexually transmittable infections,including HIV,among heterosexual migrant men

Background  

There is little experience with carefully developed interventions in the HIV/STI prevention field aimed at adult heterosexual target groups in the Netherlands. The ability to apply intervention development protocols, like Intervention Mapping, in daily practice outside of academia, is a matter of concern. An urgent need also exists for interventions aimed at the prevention of STI in migrant populations in the Netherlands. This article describes the theory and evidence based development of HIV/STI prevention interventions by the Municipal Public Health Service Rotterdam Area (MPHS), the Netherlands, for heterosexual migrant men with Surinamese, Dutch-Caribbean, Cape Verdean, Turkish and Moroccan backgrounds.     

There are differences in cytogenetic abnormalities among histologic subtypes of the non-Hodgkin's lymphomas

Although many recurring chromosome abnormalities have been found in malignant lymphoma (ML) in recent years, their relationship to histology remains largely undefined. We have correlated, in the same tumor mass, chromosome findings with histology, defined by the International Working Formulation for Clinical Usage, in 120 patients. We find differences among histologies in the frequency of normal metaphases and the modal number of the predominant abnormal clone. In addition, most histologies have been significantly (P less than .01) associated with specific chromosome abnormalities. In particular, ML, follicular, predominantly small cleaved cell was associated with t(14;18)(q32;q21); ML, follicular, mixed small cleaved cell and large cell with t(14;18)(q32;q21) and trisomy 8; ML, follicular, predominantly large cell with trisomy 7 and breaks in 17q21-q25; ML, diffuse, mixed small cell and large cell with breaks in 11p; ML, diffuse, large cell with trisomy 21 and breaks in 2q and 9q; ML, large cell, immunoblastic with breaks at 6q21; and ML, small noncleaved cell with t(8;14)(q24;q32). Only the associations with t(14;18) and t(8;14) have been previously reported. The associated chromosome abnormality usually occurred in 30% to 70% of a given histology, raising the possibility that cytogenetics may add important prognostic information in lymphoma as it does in the acute leukemias.     

Increased release of plasminogen activator inhibitor type 2 accompanies the human mononuclear cell tissue factor response to lipopolysaccharide

Human peripheral blood mononuclear cells (PBM) respond to lipopolysaccharide (LPS) with increased release of a plasminogen activator (PA) inhibitor. This response is dose dependent and parallels the LPS-induced expression of PBM tissue factor activity. The PA inhibitors of control and LPS-stimulated PBMs appear identical as both are identified by antibodies to PA inhibitor type 2 of human placenta, but not by antibodies to type 1 inhibitor of bovine aortic endothelial cells. The PA inhibitor is specific for urokinase type PA as determined by the 125I-fibrin plate assay, and direct cleavage of 125I- plasminogen; it does not effectively inhibit tissue-type PA. The inhibitor forms an active site-dependent complex with 125I-urokinase, which then demonstrates an increase in mol wt from 33 kd to 68 kd on reduced sodium dodecyl sulfate (SDS) polyacrylamide gels. PBMs neither secrete nor express active PA. Hence, the exposure of PBMs to LPS results in conditions highly favorable to fibrin deposition and persistence: increased procoagulant and antifibrinolytic activities, accompanied by no measurable PA. Such modulation of these effectors may be important in the pathogenesis of fibrin characteristically found in tissue lesions of endotoxin-initiated processes.     

Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A

We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.