bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of pathogens with the endothelium
Authors: Suttorp Norbert , Hippenstiel Stefan
Published: March 2018

The endothelium lines the inner surface of the vessel wall establishing a multifunctional, semi-permeable cellular barrier at blood-tissue interface. The large total surface of the endothelium is exposed to pathogens, pathogen-derived products as well as to agents of the activated host defense during an inflammatory reaction. The endothelium is not only specifically targeted by important infective agents like Rickettsiae (1) or Bartonella (2), it is involved in virtually most, if not all, acute inflammatory responses. Pathogens attack the endothelium by a wide variety of strategies, as different as activation of preformed receptor-mediated pathways in the endothelium, release of pore-forming exotoxins or intracellular replication and chronic parasitism. These pathophysiological forces affect the endothelial phenotype, resulting in endothelial barrier dysfunction, increased leukocyte-endothelial interaction, mediator release, and procoagulant activity. Moreover, endothelial responses retroact on the invading pathogen as well as on the host defense resulting in a complex and dynamic interaction (Fig. 1). Endothelial activation contributes considerably to inflammation and resulting clinical characteristics. In this context the endothelium is not just a passive victim, it rather aggravates the ongoing struggle with the pathogen. In this review we focus on some important mechanisms of the cellular microbiology of endothelial infection by bacteria and viruses.
Antithrombotic activity of LB30057, a newly synthesized direct thrombin inhibitor
Authors: Kang Kyu-Tae , Jung Byoung-In , Bae Ok-Nam , Lee Moo-Yeol , Chung Seung-Min , Lee Joo-Young , Lee Sang-Koo , Kim In-Chul , Chung Jin-Ho
Published: March 2018

An amidrazonophenylalanine derivative, LB30057, inhibits the catalytic activity of thrombin potently by interaction with the active site of thrombin, and has high water solubility. In the present study, we evaluated the effect of LB30057 on the biological activities of thrombin at various tissues, and determined whether thrombin inhibition by LB30057 could reduce the incidence of occlusive thrombosis in an in vivo animal model. Treatment with LB30057 to human plasma prolonged clotting times in a concentration-dependent manner. LB30057 suppressed significantly thrombin-induced phosphatidylserine (PS) exposure in platelets, suggesting that LB30057 could inhibit blood coagulation accelerated by PS exposure. In human platelets, soluble thrombin- and clot-induced platelet aggregation was inhibited by LB30057 potently. Consistent with this finding, LB30057 showed concentration-dependent inhibitory effects on serotonin secretion and P-selectin expression induced by thrombin in platelets. In the blood vessel isolated from the guinea pig, treatment with LB30057 resulted in a concentration-dependent inhibition of thrombin-induced vascular contraction. In vivo study revealed that LB30057 following oral administration significantly increased the time to occlusion and improved carotid arterial patency using rat carotid artery thrombosis model. All these results suggest that LB30057 is a potent inhibitor of biological activities of thrombin at various target tissues and, therefore, might be developed as an anti-thrombotic agent for treatment and prevention of thrombotic diseases.
Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood – different effects of collagen, TRAP and calcium ionophore A23187
Authors: Rånby Mats , Lindahl Tomas , Ramström Sofia
Published: March 2018

We have studied the effects of different platelet agonists on phosphatidylserine (PS) exposure and clotting times in blood without anticoagulants. Similar reductions in clotting time were obtained for collagen, TRAP-6 or calcium ionophore A23187 (50 μmol/L), in spite of huge differences in PS expression [6.7 ± 2.4%, 2.3 ± 0.5% and 99.9 ± 0.1%, respectively (mean ± SD, n = 5)]. Furthermore, the clotting times were much longer for samples with A23187 exposing the same amounts of PS as samples with collagen or TRAP-6. Annexin V reversed the clotting time reduction, but could not prevent coagulation. Addition of phospholipid vesicles containing 20% PS neither affected the clotting times nor induced clotting in recalcified, platelet-free plasma.

We conclude that platelet PS exposure is necessary, but not sufficient, for the coagulation amplification observed when platelets are stimulated via physiological receptors in a whole blood environment.
Prevention of Venous Thromboembolism in Internal Medicine with Unfractionated or Low-molecular-weight Heparins: A Meta-analysis of Randomised Clinical Trials
Authors: Laporte-Simitsidis Silvy , Tardy Bernard , Cucherat Michel , Buchmüller Andréa , Juillard-Delsart Daphné , Decousus Hervé , Mismetti Patrick
Published: March 2018

The prevention of venous thromboembolic disease is less studied in medical patients than in surgery.

We performed a meta-analysis of randomised trials studying prophylactic unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) in internal medicine, excluding acute myocardial infarction or ischaemic stroke. Deepvein thrombosis (DVT) systematically detected at the end of the treatment period, clinical pulmonary embolism (PE), death and major bleeding were recorded.

Seven trials comparing a prophylactic heparin treatment to a control (15,095 patients) were selected. A significant decrease in DVT and in clinical PE were observed with heparins as compared to control (risk reductions = 56% and 58% respectively, p <0.001 in both cases), without significant difference in the incidence of major bleedings or deaths. Nine trials comparing LMWH to UFH (4,669 patients) were also included. No significant effect was observed on either DVT, clinical PE or mortality. However LMWH reduced by 52% the risk of major haemorrhage (p = 0.049).

This meta-analysis, based on the pooling of data available for several heparins, shows that heparins are beneficial in the prevention of venous thromboembolism in internal medicine.
The Half-life of Infused Factor VIII Is Shorter in Hemophiliac Patients with Blood Group 0 than in those with Blood Group A
Authors: Mauser-Bunschoten Evelien , Zarkova Antoanette , Haan Els , Kruitwagen Cas , Sixma Jan , van den Berg H. , Vlot André
Published: March 2018

A considerable inter-individual variation in half-life of infused factor VIII is observed among patients with hemophilia A. The factors contributing to this wide range in factor VIII half-life are not known in detail. We analysed the pharmacokinetics of infused factor VIII in 32 patients with hemophilia A, comprising 20 brothers from 10 families, 3 and 4 brothers from 2 families, and 5 patients from 5 single families, respectively. Multiple linear regression analysis was used to asses the effect of several variables on factor VIII half-life. We found that the pre-infusion von Willebrand factor antigen levels (vWF:Ag) were positively correlated with factor VIII half-life (r = 0.52, p = 0.002), i. e., each variable was associated with about 27% of the variance of the other. In fraternal pairs, familial clustering was significant for AB0 blood group (p < 0.001), but could not be detected for factor VIII half-lives or pre-infusion vWF:Ag levels. vWF:Ag level (p = 0.001) and AB0 blood group (p = 0.003) significantly determined factor VIII half-life, whereas age, length, bodyweight, the presence or absence of a factor VIII gene inversion, and Rhesus phenotype did not. Patients with blood group 0 exhibited a statistically significant shorter factor VIII half-life than patients with blood group A (15.3 versus 19.7 h, respectively) (p = 0.003). Patients with blood group A and 0 differ in respect to the presence of anti-A antibodies in the latter. It is possible that these anti-A antibodies interact with endogenous vWF, thus affecting the half-life time of the factor VIII/vWF complex.
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

Three 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

Human 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

Procarboxypeptidase 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

Several 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

A 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

Prothrombin 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

In 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

Twenty-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

Monoclonal 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

The 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

Recent 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

Fifteen 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

Three 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.
Two Distinct Mutations Cause Severe Hemophilia B in Two Unrelated Canine Pedigrees
Authors: Gu Weikuan , Catalfamo James , Ray Jharna , Ray Kunal , Brooks Marjory
Published: March 2018

The molecular defects causing severe factor IX deficiency were identified in two distinct canine breed-variants. Both defects were associated with an absence of plasma factor IX coagulant activity and antigen. A large deletion mutation was found in 1 breed variant, spanning the entire 5’ region of the factor IX gene extending to exon 6. An approximately 5 kb insertion disrupted exon 8 of the second breed-variant. This insertion was associated with alternative splicing between a donor site 5’ and acceptor site 3’ to the normal exon 8 splice junction, with introduction of a new stop codon. The resultant transcript lacked most of the factor IX catalytic domain and 3’ untranslated region. Molecular analyses of canine hemophilia B define an experimental model for study of inhibitor formation and gene therapy strategies, and provide insight into spontaneous mutation mechanisms in the factor IX gene and on the X chromosome of mammalian species.
Laboratory Testing for von Willebrand’s Disease: An Assessment of Current Diagnostic Practice and Efficacy by Means of a Multi-laboratory Survey
Authors: Smith Julian , Petinos Penny , Hertzberg Mark , Koutts Jerry ,   the RCPA Quality Assurance Program (QAP) in Haematology Haemostasis Scientific Advisory Panel, Favaloro Emmanuel
Published: March 2018

We report an evaluation of current laboratory practice for the diagnosis of von Willebrand’s disease (VWD) by means of a multilaboratory survey. This assessment was undertaken with the RCPA Quality Assurance Program (QAP) in Haematology, which covers a wide geographic area encompassing Australia, New Zealand and Asia. A total of 25 laboratories actively involved in testing for VWD were selected to participate in a sample testing assessment exercise. Samples comprised 10 plasmas: (i) a normal plasma pool (in duplicate), (ii) this pool diluted to 50% (in duplicate), (iii) a normal individual (×1), (iv) severe Type 1 VWD (×1), (v) Type 2B VWD (×2 unrelated donors), (vi) Type 3 VWD (×1), (vii) Type 2A VWD (×1). Laboratories were asked to perform all tests available to them in order to establish a laboratory diagnosis of VWD, and then to comment on the possibility or otherwise of VWD. Overall findings indicated a wide variation in test practice, in the effectiveness of various test procedures in detecting VWD, and in the ability of various composite test panels to identify type 2 VWD subtypes. Firstly, while all laboratories (n = 25) performed tests for FVIII:C activity, von Willebrand factor ‘antigen’ (VWF:Ag) and a functional VWF assay [using the ristocetin cofactor assay (VWF:RCo; n = 23) and/or the collagen binding assay (VWF:CBA; n = 12)], only three laboratories carried out VWF:Multimer analysis. Secondly, for the three quantitative VWF assays, 10/25 (40%) laboratories performed all three, whereas 15/25 (60%) performed only two [VWF:Ag and VWF:RCo (n = 13); VWF:Ag and VWF:CBA (n = 2)]. Thirdly, a variety of assay methodologies were evident for VWF:Ag [ELISA, electro-immuno diffusion (EID), latex immuno-assay (LIA), and VIDAS assay] and VWF:RCo (platelet agglutination/‘aggregometry’ and a ‘functional VWF:RCo-alternative’ ELISA assay). Between method analysis for the quantitative VWF assays showed that the VWF:RCo yielded the greatest degree of inter-laboratory assay variation, and had the poorest overall performance with respect to sensitivity to low levels of VWF. The VWF:CBA also performed better than the VWF:RCo in terms of ability to detect functional VWF ‘discordance’ (i.e. Type 2 VWD). Within VWF:Ag method analysis showed that the EID assay procedure was associated with the greatest variation in assay
The Risk of Early Recurrent Venous Thromboembolism after Oral Anticoagulant Therapy in Patients with the G20210A Transition in the Prothrombin Gene
Authors: Minar Erich , Hirschl Mirko , Bialonczyk Christine , Stain Milena , Mannhalter Christine , Stümpflen Andreas , Schneider Barbara , Lechner Klaus , Kyrle Paul , Eichinger Sabine
Published: March 2018

A G20210A transition in the prothrombin gene is a common risk factor of venous thrombosis. The risk of recurrent venous thrombo-embolism in carriers of the 20210A allele is unknown and guidelines for secondary thromboprophylaxis in these patients are not available.

In a prospective multicenter trial, 492 patients with a history of objectively documented venous thromboembolism were followed for a mean observation time of 24 ± 16 months after discontinuation of oral anticoagulants. Forty-two patients (8.5%) were carriers of the 20210A allele. Three of the 42 patients with the G20210A mutation (7%) and 54 of 450 patients without the mutation (12%) experienced recurrent venous thrombosis. At 24 months, the probability of recurrence was 8% (95% CI 0-16.7) in patients with the mutation and was 12.2% (95% CI 8.8-15.6) in patients without the mutation.

In conclusion, the risk of early recurrent venous thromboembolism is not higher in patients with the G20210A mutation than in those without the mutation. Therefore, long-term secondary thromboprophylaxis with oral anticoagulants in heterozygous carriers of the 20210A allele is not justified.
Performance of a New Fibrin Monomer Assay to Exclude Deep Vein Thrombosis in Symptomatic Outpatients
Authors: Caliezi Christoph , Fünfsinn Nadja , Mauron Thomas , Baumgartner Iris , Sulzer Irmela , Ulrich Martin , Cottier Christoph , Biasiutti Franziska , Lämmle Bernhard , Wuillemin Walter
Published: March 2018

In this study we prospectively assessed the reliability of a new fibrin monomer assay in 106 outpatients with clinically suspected deep venous thrombosis of the lower limb. According to the results of the objective tests and using different cut-off points we calculated the sensitivity, specificity and negative predictive value of the fibrin monomer assay. The prevalence of deep vein thrombosis was 44.3% (31.1% proximal, 13.2% distal). Using a cut-off level of plasma fibrin monomer of 3.5 μg/ml, a sensitivity, specificity and negative predictive value of 100% (95% CI: 94-100%), 35.6% (95% CI: 23-48%) and 100% (95% CI: 86-100%), respectively, were obtained. The exclusion rate was 19.8% (95% CI: 12-27%) of all referred patients. These accuracy indices compared favourably with the respective results of a routine D-dimer ELISA used for comparison. Conclusion: This new fibrin monomer assay appears to be a reliable method for the exclusion of deep vein thrombosis in symptomatic outpatients.
A New Enzyme Immunoassay for Soluble Fibrin in Plasma, with a High Discriminating Power for Thrombotic Disorders
Authors: Laterveer-Vreeswijk G. H. , Lockwood D. , Szewczyk K. , Nieuwenhuizen W. , Bos R.
Published: March 2018

Fibrin formation is a multistep process initiated by thrombin. At first thrombin converts fibrinogen to fibrin molecules which in vivo form soluble complexes with fibrinogen. Soluble fibrin is considered to be an early biochemical marker for intravascular fibrin formation and impending thrombotic events, such as deep venous thrombosis (DVT), pulmonary embolism (PE) and disseminated intravascular coagulopathy (DIC).

A new enzyme immunoassay (EIA) was developed on the basis of a monoclonal antibody directed against a fibrin specific neo-epitope located on the gamma-chain of fibrinogen; γ-(312-324). In addition, it was possible to prepare a lyophilized reference material of thrombin-generated soluble fibrin, that allowed for full antigen recovery after reconstitution with buffer. Assay conditions, e.g. solid phase-Ig concentration and buffer composition, sample and conjugate dilution, and incubation times were optimised.

The present assay was found to be specific (no interference of homologous antigens) and reproducible (intra-assay CV 4-8%, inter-assay CV 4-9%), and therefore highly suited for measuring soluble fibrin levels in a plasma milieu. The median normal value for soluble fibrin was determined in plasma samples obtained from apparently healthy volunteers (n = 81) and found to be 0.040 μg/ml, with a range (10-90 percentiles) of 0.026-0.059 μg/ml.

A retrospective study showed that soluble fibrin levels were highly significantly increased in patients with a confirmed diagnosis of DIC (median 1.042 μg FEU/ml, range 0.160-2.319 μg/ml, n = 21, P <0.0001 vs normal), PE (median 0.527 μg FEU/ml, range 0.084-1.234 μg/ml, n = 29, P <0.0001 vs normal) and DVT (median 0.126 μg FEU/ml, range 0.059-0.878 μg/ml, n = 36, P <0.0001 vs normal), as determined by the Mann-Whitney U-Test.
Influence of Three Types of Automated Coagulometers on the International Sensitivity Index (ISI) of Rabbit, Human, and Recombinant Human Tissue Factor Preparations
Authors: Houbouyan L. L. , Aillaud M. F. , Denson K. W. E. , Johnston M. , Kitchen S. , Lindahl T. L. , Marren M. , Martinuzzo M. E. , Droullé C. , Tripodi A. , Vergnes C. , Besselaar A. M. H. P.
Published: March 2018

Five tissue factor reagents and three types of automated instruments for prothrombin time (PT) determination were studied in an international multicenter collaborative exercise. The purpose of this work was to determine the international sensitivity index (ISI) for each combination of reagent and instrument against the international reference preparation RBT/90. Each type of instrument was used by 3 or 4 centers to assess the interlaboratory variation of the ISI. The interlaboratory variation of the ISI for each combination of reagent and instrument ranged between 0.4% and 7.8% coefficient of variation. For three reagents, the mean ISI values for ACL (nephelometric) and STA (mechanical) were practically identical, but the mean ISI values for MLA (photo-optical) were at least 10% higher. For two other reagents prepared from rabbit tissue, the mean ISI values increased in the order ACL, STA, MLA. The widest range of mean ISI values was noted with one rabbit tissue factor reagent: 1.68 for ACL and 2.00 for MLA. Exclusion of patient specimens with INR <1.5 and INR >4.5 determined by the international reference preparation resulted in a slight decrease of the mean ISI.

The interlaboratory variation of the International Normalized Ratio (INR) was assessed from the results obtained with common lyophilized and deep-frozen plasmas. The use of instrument-specific ISI values resulted in reduced interlaboratory variation of the INR. It is recommended that thromboplastin manufacturers provide instrument-specific ISI values.
β2-Glycoprotein I Is Proteolytically Cleaved In Vivo upon Activation of Fibrinolysis
Authors: Oort Erica , Lisman Ton , Meijers Joost , Derksen Ronald , de Groot Philip , Horbach Daniëlle
Published: March 2018

β2-glycoprotein I (β2GPI) is a plasma glycoprotein with unknown physiological function(s). In in vitro experiments it has been demonstrated that β2GPI has both anticoagulant properties, such as the inhibition of factor X and prothrombin activation and procoagulant properties, such as the inhibition of the anticoagulant activity of activated protein C. Besides this, β2GPI bound to cardiolipin is recognized by anti-phospholipid antibodies (aPL).

In this study we demonstrate that β2GPI is very sensitive for cleavage between Lys317 and Thr318 by plasmin, resulting in two immunologically different cleaved forms. In vitro experiments show that these plasmin cleaved forms of β2GPI bind to negatively charged phospho-lipids with much lower affinity compared to intact β2GPI. Similar to plasmin, trypsin and elastase can also induce this proteolytical cleavage in β2GPI, whereas thrombin and factor Xa do not cleave β2GPI. The in vivo occurrence of the proteolytical cleavage was demonstrated by the finding that in plasmas of patients with disseminated intravascular coagulation(DIC) and in plasmas of patients treated with streptokinase, significant amounts of cleaved β2GPI (up to 12 μg/ml) are present.

During the development of DIC, the increase in levels of cleaved β2GPI is accompanied by a 70% decrease in the levels of intact β2GPI, whereas in streptokinase treated patients levels of intact β2GPI stay within the normal range.

This study demonstrates for the first time that during in vivo activation of fibrinolysis β2GPI is cleaved, which results in the formation of a form of β2GPI with much lower affinity for negatively charged phospholipids. Plasmin is most likely responsible for this modification.
Platelet Adhesion to Collagen Type I, Collagen Type IV, von Willebrand Factor, Fibronectin, Laminin and Fibrinogen: Rapid Kinetics under Shear
Authors: Simon Carl , Gear Adrian , Polanowska-Grabowska Renata
Published: March 2018

Extracellular matrix proteins in the blood vessel wall fulfill an essential role in haemostasis by promoting platelet adhesion at the site of vessel injury. We have combined a continuous-flow system with affinity chromatography to study platelet adhesion under conditions mimicking arterial flow and have examined the adhesion kinetics of unstimulated platelets to collagens type I and IV, von Willebrand factor (vWf), fibronectin, laminin and to fibrinogen. In the absence of red cells, in ACD-prepared plasma adhesion to collagens type I and IV or vWf was rapid, efficient (>50% in <1 s ) and independent of shear rates from 650 to 3400 s-1with kinetics following an inverse exponential decay curve. We introduced a simple mathematical model in which this type of kinetics arises, and which may be more generally applicable to various adhesion processes under flow conditions. The model is characterized by the rate of platelet deposition on the adhesive surface being proportional to the number of platelets in the flow. Adhesion to fibronectin was independent of shear rate, but revealed a lag phase of ~1.5 s before significant adhesion began. Laminin and fibrinogen supported efficient adhesion at low shear rates (650-1000 s-1), but a lag phase of ~1.5 s was seen at high shear rates (1700-3400 s-1). Control proteins (albumin and gelatin) supported minimal adhesion. Nonspecific adhesion to poly-l-lysine differed from that to other substrate proteins in that the kinetics were linear. In conclusion, human platelets adhered specifically, rapidly (within seconds) and efficiently to several proteins under flow conditions and the kinetics of adhesion depended on the protein serving as substrate as well as on shear rate.
Human Atheromatous Plaque Extracts Induce Tissue Factor Activity (TFa) in Monocytes and also Express Constitutive TFa
Authors: Teodorescu Victoria , Rand Jacob , Rosman Alan , Niemetz Julian , Muhlfelder Thomas
Published: March 2018

Tissue factor activity (TFa) is a major activator of the coagulation cascade and may play a role in atheroma-induced thrombosis. Mono-cyte-macrophages (MO-MF) generate considerable quantities of TFa when stimulated by a variety of inducers. To test the hypothesis that MO could be induced by atheromatous plaque to generate TFa, plaque extracts obtained from patients with obstructive atheromatous disease were used. These extracts were also assayed for constitutive TFa. The constitutive activity was variable from extract to extract but could be very high, up to 250 U TFa. The TFa induced in MO could be also very high, up to 200 U (i.e. 1/5 of the TFa of full strength rabbit brain thromboplastin). These findings point to a major role for MO-MF TFa in the induction or thrombosis by atheromatous plaque.