bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Basis for Hemophilia A Caused by Mutations in the C Domains of Blood Coagulation Factor VIII
Authors: Gale Andrew , Pellequer Jean-Luc , Getzoff Elizabeth , Griffin John 
Published: March 2018

SummaryThree dimensional homology models for the C1 and C2 domains of factor VIII (FVIII) were generated. Each C domain formed a β-sandwich, and C1 was covalently connected to C2 in a head-to-head orientation. Of the >250 missense mutations that cause FVIII deficiency and hemophilia A, 34 are in the C domains. We used the FVIII C1-C2 model to infer the structural basis for the pathologic effects of these mutations. The mutated residues were divided into four categories: 15 conserved buried residues that affect normal packing of the hydrophobic side chains, 2 non-conserved buried residues that affect structure, 11 conserved exposed residues and 6 non-conserved exposed residues. The effects of all 34 missense mutations can be rationalized by predictable disruptions of FVIII structure while at most four mutations (S2069F, T2154I, R2209Q/G/L and E2181D) may affect residues directly involved in intermolecular interactions of FVIII/VIIIa with other coagulation factors or vWF.
Induction of Tissue Factor Procoagulant Activity in Myelomonocytic Cells Inoculated by the Agent of Human Granulocytic Ehrlichiosis
Authors: Behl Rajesh , Klein Marina , Dandelet Luke , Bach Ronald , Goodman Jesse , Key Nigel 
Published: March 2018

SummaryHuman granulocytic ehrlichiosis (HGE) is a recently recognized rickettsial tick-borne febrile illness that may occasionally be complicated by coagulopathy. The agent of HGE (aHGE) is an obligate intracellular pathogen, which replicates in endosomes within neutrophils and their precursors. We hypothesized that aHGE might cause DIC via induction of monocyte tissue factor procoagulant activity (TF PCA). Peripheral blood mononuclear cells (PBMNC) and HL-60 cells were used to model the effect of aHGE infection on monocytes/macrophages. Mononuclear cells inoculated with aHGE in vitro demonstrated approximately a 12-15-fold increase in TF PCA, with peak activity occurring at 8-12 h. HL-60 cells inoculated with aHGE also manifested a 4-6 fold induction of TF PCA, with maximal activity occurring at about 8 h. By comparison, E. Coli lipopolysaccharide (LPS) also induced an increase in TF PCA of an equivalent magnitude, and with a similar time course. Induction of TF did not require inoculation of HL-60 cells with live organism, since heat-inactivated aHGE still stimulated TF PCA expression in the target cells. Furthermore, filtered supernatants from heat-inactivated organisms induced TF PCA suggesting that the effect is due to a soluble mediator produced by the organism. Although aHGE is a gram negative organism, the soluble mediator did not appear to be classic endotoxin in that the supernatants tested negative for endotoxin by the Limulus Amoebocyte assay, and polymixin had no inhibitory effect on aHGE supernatants. We conclude that aHGE induces cells of the myelo-monocytic lineage to synthesize TF, which may contribute to the clinical coagulopathy that can be observed in this condition. An atypical soluble mediator or cellular component of the organism appears to be critically important in TF induction by aHGE.
Plasma Procarboxypeptidase U in Men with Symptomatic Coronary Artery Disease
Authors: Schatteman Katinka , Goossens Filip , Moor Elisabeth , Scharpé Simon , Strömqvist Mats , Hendriks Dirk , Hamsten Anders , Silveira Angela 
Published: March 2018

SummaryProcarboxypeptidase U (proCPU) is the plasma precursor of carboxypeptidase U (CPU, carboxypeptidase R, plasma carboxypeptidase B or activated thrombin-activatable fibrinolysis inhibitor, TAFIa). CPU removes C-terminal lysine residues that act as plasminogen binding sites from partially degraded fibrin, thereby down-regulating plasminogen activation and fibrinolysis. The present study was carried out as a pilot study to examine whether the plasma proCPU concentration is related to the presence of coronary artery disease (CAD) and/or to levels of established risk indicators for CAD, in a case-control study of 110 men requiring coronary artery bypass grafting (CABG) because of stable angina pectoris. The preoperative plasma proCPU level in the CABG patients was significantly higher than in population-based controls (1029 ± 154 vs. 974 ± 140 U/L, p <0.05). In addition, in a subset of the patients (n = 31) the proCPU concentration, which was significantly lower on the third postoperative day (−17 ± 10%), had increased significantly on the sixth day (+14 ± 12%) after surgery, compared with the preoperative level. In both patients and controls, proCPU concentration was strongly and positively associated with factor VII amidolytic activity and protein C activity, suggesting a common mechanism modulating the plasma levels of these proteins. Otherwise, statistically significant correlations with proCPU were group-specific. In the patients, proCPU correlated significantly with plasma fibrinogen and protein S. In the controls, proCPU correlated significantly with concentrations of cholesterol in plasma, VLDL and LDL. In addition, proCPU correlated significantly with C-reactive protein and haptoglobin levels in the controls only, indicating that also inflammatory mechanisms are involved in the regulation of plasma proCPU. These results suggest that a mechanism exists by which fibrinolytic function is impaired in a manner that is likely to result in more stable fibrin deposits and increase the risk of precocious CAD as well as early occlusion of venous bypass grafts.
Fibrin Down-regulates LPS- and PMA-induced Tissue Factor Expression by Blood Mononuclear Cells
Authors: Rossiello Maria , Italia Alessandra , Stramaglia Anna , Gesualdo Loreto , Grandaliano Giuseppe , Schena Francesco , Semeraro Nicola , Colucci Mario 
Published: March 2018

SummarySeveral studies indicate that fibrin may play a functional role in inflammation by modulating a variety of cellular functions. We investigated the effect of fibrin on tissue factor (TF) production by blood mononuclear cells (MNC). Citrated human blood was recalcified and incubated at 37° C for 1-4 h. The resulting clot was lysed by the addition of tissue plasminogen activator (t-PA) and MNC were isolated by density gradient centrifugation. A control blood sample was processed in the same way but omitting calcium addition and clot formation. Clot-and blood-derived MNC did not express detectable TF activity and antigen whatever the incubation time. Clot-derived MNC, however, generated on average 5 fold less TF (activity and antigen) than control cells, when stimulated with lipopolysaccharide (LPS, 1 µg/ml) for 3 h at 37° C. A reduced TF response of clot-derived cells was also observed at mRNA level as indicated by RT-PCR and in situ hybridization. The effect was dependent on the incubation time within the clot, could not be reversed by enhancing LPS concentration or by adding serum, and was maintained if LPS was replaced by the tumor promoter PMA. A reduced TF response was also found when washed MNC were incorporated for 1 h at 37° C within purified fibrin but not when the cells were incubated with fibrinogen, thrombin or fibrin split products alone, indicating that contact with fibrin was responsible for the inhibition of TF production. Fibrin-induced down-regulation of TF response to LPS and PMA by MNC may represent a negative feed-back aimed at limiting excessive blood clotting activation in immuneinflammatory diseases.
Issues with the Assay of Factor VIII Activity in Plasma and Factor VIII Concentrates
Authors: Kingdon Henry , Mann Kenneth , White Gilbert , Lundblad Roger 
Published: March 2018

SummaryA review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VIII concentrates such as Hemofil ® M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements, it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor Villa during the assay process.
Identification and Three-dimensional Structural Analysis of Nine Novel Mutations in Patients with Prothrombin Deficiency
Authors: Mannucci Pier , Lak Manijeh , Mancuso Giacomo , Mazzucconi Maria , Rocino Angiola , Jenkins P. , Perkins Stephen , Akhavan Sepideh 
Published: March 2018

SummaryProthrombin deficiency is an autosomal recessive disorder associated with a moderately severe bleeding tendency. In this study, 13 patients with prothrombin deficiency were screened for the presence of alterations in the prothrombin gene, and nine novel candidate mutations were identified. Of 11 patients with hypoprothrombinemia, ten are homozygous for five mutations and one patient is a compound heterozygote. The two patients with dysprothrombinemia are homozygous for two mutations. Eight of nine mutations are missense ones associated with single amino acid substitutions in the propeptide (Arg-1Gln, Arg-2Trp), the kringle-1 (Asp118Try) and kringle-2 (Arg220Cys) domains and the catalytic serine protease domain (Gly330Ser, Ser354Arg, Arg382His and Arg538Cys). The ninth mutation is an in-frame deletion of 3 bp that results in the omission of one amino acid (del Lys 301/302). The combination of these missense mutations with crystal structures for α-thrombin and the prothrombin fragments 1 and 2 resulted in new insight into the function of α-thrombin. The hypoprothrombinemia mutations were inferred to affect either the cleavage of the propeptide from the Gla domain, the stability of the kringle-1 and −2 domains, or the close association of the A and B chains of the serine protease domain. The dysprothrombinemia mutations were inferred to directly affect catalytic function through their location at the active site crevice or exosite 1 within the serine protease domain.
Identification of Type 2 von Willebrand Disease in Previously Diagnosed Type 1 Patients: a Reappraisal Using Phenotypes, Genotypes and Molecular Modelling
Authors: Nitu-Whalley Ioana , Riddell Anne , Pasi K. , Owens Dale , Enayat M. , Perkins Stephen , Jenkins P. , Lee Christine 
Published: March 2018

SummaryIn order to investigate the possibility that qualitative type 2 defects in von Willebrand factor (VWF) occurred in patients previously diagnosed with quantitative type 1 von Willebrand disease (VWD), the phenotypes and genotypes were reanalysed in 30 patients who exhibited discrepant VWF activity/VWF:Ag ratios of less than 0.7. The capacity of VWF to bind to glycoprotein Ib (GpIb) was reassessed using the ristocetin co-factor activity (VWF:RiCo) assay compared to an in-house and a commercial ELISA assay (based on a mAb directed against the GpIb binding site on VWF). This was supplemented by multimeric analysis and the amplification and sequencing of a 936 bp fragment of exon 28 of the VWF gene with the aim of identifying mutations in the A1 domain. On reappraisal, using the VWF:RiCo assay all patients demonstrated a disproportionately reduced VWF:RiCo/VWF:Ag ratio, indicative of a qualitative defect, while abnormal ratios were detected in only seven kindreds using the in-house ELISA assay and in only one kindred with the commercial ELISA assay. Eight single amino acid substitutions were found in nine kindreds, four of which were novel candidate VWF mutations and four previously described in association with type 2 VWD. In agreement with the phenotype, the novel VWF mutations were located in the VWF-A1 crystal structure at positions that corresponded to potential type 2M defects. This study underlines the difficulties of correct diagnosis of the subtype of VWD and emphasises the importance of using sensitive phenotypic assays, the relevance of the VWF:RiCo/ VWF:Ag ratio, multimeric analysis and molecular modelling analysis.
A Collaborative Study to Establish the 5th International Standard for Unfractionated Heparin
Authors: Walker A. , Mulloy Barbara , Barrowcliffe Trevor , Gray Elaine 
Published: March 2018

SummaryTwenty-four laboratories participated in a collaborative study to calibrate a replacement for the 4th International Standard for Unfractionated Heparin (82/502). Both candidate materials A and B, gave excellent intra- and inter-laboratory variations (majority of mean %gcv <10%) when assayed against the 4th International Standard. No major differences of potency estimates were found between methods, although the USP method generally gave lower potencies than the other methods and candidate B gave a greater variation between methods than A. Overall, this study showed that the differences between the candidates are marginal. Based on its narrower molecular weight profile, higher specific activity and slightly lower inter-method variation, candidate A, 97/578, was proposed and accepted in October, 1998, by the Expert Committee on Biological Standardisation of the World Health Organisation to be the 5th International Standard for Unfractionated Heparin with an assigned potency of 2031 IU/ampoule.
Identification of a Putative Binding Site for Negatively Charged Surfaces in the Fibronectin Type II Domain of Human Factor XII
Authors: te Velthuis Henk , Helmer-Citterich Manuela , Hack C. , Citarella Franca 
Published: March 2018

SummaryMonoclonal antibodies directed against functional sites of proteins provide useful tools for structure-function studies. Here we describe a mAb, KOK5, directed against the heavy chain region of human coagulation factor XII (FXII), which inhibits kaolin-induced clotting activity by preventing the binding of FXII to kaolin. Furthermore, mAb KOK5 enhances FXII susceptibility for cleavage by kallikrein and supports FXII autoactivation. Hence, mAb KOK5 likely is directed against the binding site of FXII for negatively charged surfaces. Screening of two phage-displayed random peptide libraries with mAb KOK5 selected phages that could be grouped on the basis of two amino acid consensus sequences: A) FXFQTPXW and B) HQ/LCTHR/KKC. Sequence A contains two motifs: one shares homology with FXII amino acid residues 30-33 (FPFQ), the second one with residues 57-60 (TPNF); both amino acid stretches belonging to the fibronectin type II domain of FXII. Sequence B also reveals homology with part of the fibronectin type II domain, i.e. the stretch 40-47 (HKCTHKGR). A three-dimensional model of FXII residues 28-65, obtained by homology modeling, indicated that the three amino acid stretches 30-33, 40-47 and 57-60 are close to each other and accessible for the solvent, i.e. in a form available for interaction with the monoclonal antibody, suggesting that mAb KOK5 recognizes a discontinuous epitope on the fibronectin type II domain of FXII. Peptides corresponding to FXII sequences 29-37 (FXII29-37) or 39-47 (FXII39-47), were synthesized and tested for the capability to inhibit FXII binding to negatively charged surfaces. Peptide FXII39-47 inhibited the binding of labeled FXII to kaolin and effectively prevented both dextran sulfate-and kaolin-induced activation of the contact system in plasma. Hence, we suggest that the fibronectin type II domain of FXII, in particular residues 39 to 47, contribute to the binding site of FXII for negatively charged surfaces.
Differential Expression of a Ligand Induced Binding Site (LIBS) by GPIIb-IIIa Ligand Recognition Peptides and Parenteral Antagonists
Authors: Haga Jason , Slack Steven , Jennings Lisa 
Published: March 2018

SummaryThe glycoprotein (GP) IIb-IIIa complex is an attractive anti-platelet target for the prevention of thrombotic events associated with coronary artery disease. Although GPIIb-IIIa antagonists inhibit GPIIb-IIIa binding to its ligands, the interactions have not been fully clarified, particularly with respect to their ability to induce structural changes in the complex that lead to exposure of neoantigenic epitopes or ligand-induced binding sites (LIBS). In this study we used the anti-LIBS monoclonal antibody (mAb) D3 to further define the activation states of purified active and inactive GPIIb-IIIa. We also compared the data obtained in the purified system to that observed with intact human platelets. Active GPIIb-IIIa expressed significantly greater high-affinity D3 LIBS sites compared to the inactive form. In addition, the ligand recognition peptides RGDS and H12 caused increased expression of the D3 epitope, with RGDS eliciting a much more potent response. The response of the purified GPIIb-IIIa to these peptides paralleled that observed with human platelets. To explore whether the platelet antagonists abciximab, eptifibatide and tirofiban induced expression of the D3 LIBS site, a modified competitive ELISA was developed. Our data indicate that the use of purified GPIIb-IIIa with this ELISA system provides a reproducible approach for exploring the interactions between GPIIb-IIIa and its antagonists. Whereas abciximab caused no detectable increase in the expression of the D3 epitope on purified GPIIb-IIIa, eptifibatide, tirofiban, RGDS, and H12 induced differential expression of the high-affinity LIBS. Studies with intact platelets suggested that abciximab blocked the binding of the D3 and LIBS6 mAbs, and that the pre bound anti-LIBS D3 sterically hindered abciximab binding.
Hormone Replacement Therapy with Estradiol and Risk of Venous Thromboembolism
Authors: Abdelnoor Michael , Sandset Per Morten , Høibraaten Else 
Published: March 2018

SummaryRecent epidemiological studies on hormone replacement therapy (HRT) containing mainly conjugated equine estrogens indicate increased risk for venous thromboembolism (VTE). The purpose of the present epidemiological study was to evaluate the effect of HRT containing natural estrogens, i.e., estradiol, on the risk of VTE. HRT formulations containing estradiol are commonly used in Scandinavia. The study was a population-based case-control study. Cases were consecutive females, aged 44-70 years, discharged from Ullevål University Hospital with the diagnosis of deep venous thrombosis or pulmonary embolism during 1990-1996. Fifty-one women with cancer-associated thrombosis were excluded from the study. Controls were randomly collected from the same source population and matched by age. The material comprised 176 cases and 352 controls, i.e., 2 controls for each case. Only formulations containing estradiol were used. The frequency of HRT use was 28% (50/176) in cases and 26% (93/352) in controls. The estimated matched crude odds ratio with 95% confidence interval was 1.13 (0.71-1.78), which indicates no significant association of overall use of HRT and VTE. The estimated adjusted odds ratio was 1.22 (0.76-1.94) performing multi-confounder adjustment using the conditional logistic model and adjusting for hypertension, coronary heart disease, diabetes mellitus, smoking habit, BMI, and the presence of previous VTE. Stratification for time of exposure indicated an increased risk of VTE during the first year of use with a crude odds ratio of 3.54 (1.54-8.2). This effect was reduced by extended use to a crude odds ratio of 0.66 (0.39-1.10) after the first year of use. We conclude that use of HRT containing estradiol was associated with a threefold increased risk of VTE, but this increased risk was restricted to the first year of use.
Incidence of Factor IX Inhibitor Development in Severe Haemophilia B Patients Treated with only one Brand of High Purity Plasma Derived Factor IX Concentrate
Authors: Laurian Yves , Rothschild Chantal , Navarro Robert , Guérois Claude , Gay Valérie , Durin Anne , Peynet Jocelyne , Sultan Yvette , Parquet Armelle 
Published: March 2018

SummaryFifteen previously untreated patients (Pups) with severe haemophilia B (factor IX activity ≤ 2 U/dl) only treated with one brand of plasma-derived high purity factor IX concentrate (FIX LFB) were studied. Age at first injection varied from 1 to 137 months and follow-up since this first injection from 21 to 86 months (median: 35). Cumulative exposure days (CED) were from 4 to over 100 (median: 26). Among these 15 Pups only one developed an inhibitor. Mutation analysis performed in all patients showed total gene deletion in the patient with inhibitor, partial gene deletion in another one, and missense mutations in 9 families. Mutation was not found in one patient. Actually, according to the data already published, only two patients were at high risk for inhibitor development in our population. Our study, although rather small, confirms the previously reported low incidence of inhibitors in haemophilia B. Large studies on incidence of FIX inhibitors are indeed difficult to perform, due to both the overall small number of severe haemophilia B patients and the low incidence of FIX inhibitors. Consequently, the impact of bias, such as prevalence of different types of gene defects in a given population, is major. Therefore, any study, dealing with incidence of FIX inhibitors in severe haemophilia B should report, for each patient, the type of gene defect.
The Three In-frame ATG, Clustered in the Translation Initiation Sequence of Human Factor IX Gene, Are Required for an Optimal Protein Production
Authors: Plantier Jean-Luc , Maurice Marielle , Attali Olivier , Enjolras Nathalie , Rodriguez Marie-Hélène , Négrier Claude 
Published: March 2018

SummaryThree in-frame potential methionine codons have been identified in human factor IX gene and are clustered at amino acids -46, -41 and -39. In view of initiating a gene therapy approach, human factor IX production has been evaluated after modifications of these first three in-frame translation start sites. To characterize the most efficient translation initiation context, five factor IX cDNA expression vectors directed by CMV promoter-enhancer were generated. These vectors contained different starting site combinations including one, two or three ATG. A quantitative analysis of factor IX production in stably transfected CHO cells and in a rabbit reticulocyte lysate cell free system revealed the ability of all single site to generate fully active factor IX. However, the factor IX production level increased with the ATG number and the wild type (WT) cDNA bearing the 3 ATG induced the highest protein production. A truncated intron I of factor IX, previously suggested of having an expression-augmenting activity, was also placed in the WT factor IX cDNA. In stably transfected CHO cells, a 8-fold increase in protein production was measured. These results show that at least in vitro, the presence of the three ATG seems to be crucial for a maximal factor IX production. The data also suggest that both the three ATG and the truncated intron I are required for an optimal factor IX production in a perspective of a human gene therapy of haemophilia.