bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Cell Analysis of Factor VIII-specific T Cells in Hemophilic Mice after Treatment with Human Factor VIII
Authors: Sasgary Maria , Ahmad Rafi , Turecek Peter , Reipert Birgit , Schwarz Hans
Published: March 2018

A multi-parameter flow-cytometry assay was established suitable for analyzing T-cell-specific cell surface markers (CD3, CD4) together with intracellular cytokines on a single cell level. This assay was used to identify the frequency and the kinetic of different populations of factor VIII (FVIII)-specific CD4+ T cells in hemophilic E-17 mice after treatment with human FVIII. A clear temporal correlation was found between the appearance of FVIII-specific CD4+ T cells in the spleen and the detection of anti-FVIII antibodies in plasma. These cells and antibodies were detectable in all experiments after two doses of FVIII and in a few even after a single dose. The IFN-γ- producing T cells were the most prominent type of FVIII-specific T cells suggesting Th1-type T cells have an important role in regulating the anti-FVIII immune response in E-17 mice. IL-10-producing T cells were the second most dominant type. They were detectable after two doses of FVIII and increased in frequency after four. Cytokine co-expression studies analyzing IL-10 and IFN-γ- in the same cell indicated that there might be at least two types of IL-10 positive T cells, those cells that produce IL-10 only and in addition cells that produce IL-10 and IFN-γ-. Furthermore, FVIII-specific T cells producing IL-2 were found in all experiments after two doses of FVIII. In a few experiments IL-4-producing T cells were seen but in most experiments they were not detectable. In contrast, IL-4 could be found in supernatants of in vitro restimulated CD8– spleen cells.
Field Study of Lyophilized Calibrant Plasmas for Fresh Plasma INR Determination
Authors: Houbouyan-Réveillard L. L. , van den Besselaar A. M. H. P.
Published: March 2018

An alternative approach to INR estimation is for laboratories to calibrate their own local system using calibrant plasmas supplied by manufacturers or reference laboratories. The purpose of the present study was to investigate the within-laboratory variability of a calibrant plasma procedure using various sets of lyophilized plasmas. INR had been assigned to 13 calibrant plasmas in a previous multi-center study. Each of 10 other (“field”) laboratories measured PTs in the 13 calibrant plasmas and in 15 local fresh coumarin plasmas, using three different thromboplastin reagents. Each fresh coumarin PT was converted to INR using a calibration procedure with a set of 4 calibrants (1 normal + 3 abnormals). The abnormals of each set were either coumarin or artificial and were used with different assigned INR. When the INR had been assigned with a thromboplastin brand identical to the thromboplastin in the field laboratory, the procedure was named “reagent-specific” calibration. Otherwise the procedure was named “dissimilar” calibration. Using “reagent-specific” calibration procedures, relatively homogeneous INR were obtained for the fresh coumarin plasmas, whatever type of calibrant was used. In contrast, discrepant INR were obtained when “dissimilar” cross-species calibration procedures were used. The study was limited to 9 laboratories using the same type of coagulometer and one using a different type.
Coagulation and Inflammation in Acute Lung Injury
Authors: Carraway Martha , Ortel Thomas , Piantadosi Claude , Welty-Wolf Karen
Published: March 2018

The acute respiratory distress syndrome (ARDS) is a severe lung injury in patients with sepsis and other acute inflammatory insults, which is characterized by fibrin deposition in the pulmonary parenchyma, vasculature, and airspaces. Recent evidence suggests that progressive ARDS is closely linked to activation of inflammation and coagulation. Coagulation becomes activated by circulating endotoxin or bacteria, and a procoagulant state develops in the vascular and the alveolar compartments of the lung. This state is Tissue Factor (TF)-dependent and associated with increased elaboration of inflammatory cytokines. A similar procoagulant state is found in bronchoalveolar lavage of patients with ARDS, suggesting that extravascular coagulation contributes to lung inflammation. TF and other coagulation proteins, including Factor Xa, thrombin, and fibrin, also contribute to the pathogenesis of acute lung injury through multi-level interactions with inflammatory effectors, in which these proteins coordinately act as regulators of tissue injury responses. Each coagulation protein has direct and independent effects on inflammatory events that influences lung injury through changes in cytokine elaboration, inflammatory cell migration and activation, surfactant function, and repair mechanisms. New interventional strategies directed at procoagulant activity highlight the importance of the coagulation system to acute lung injury and suggest that blockade of initiation of coagulation may have therapeutic benefit in patients with ARDS.
Association of γA/γ’ Fibrinogen Levels and Coronary Artery Disease
Authors: Lovely Rehana , Falls Lisa , Al-Mondhiry Hamid , Chambers Charles , Sexton Gary , Ni Hanyu , Farrell David
Published: March 2018

γA/γ’ fibrinogen is a fibrinogen isoform that constitutes about 15% of total plasma fibrinogen. This isoform contains an additional binding site for zymogen factor XIII and for active thrombin, and forms fibrin clots that are resistant to fibrinolysis in vitro. Little is known about the variability of γA/γ’ fibrinogen levels in human populations, whereas total fibrinogen levels are known to increase with age and are higher in women than in men. In this report, evidence is presented that, in contrast to total fibrinogen levels, γA/γ’ fibrinogen levels showed no significant association with age or gender in a population of normal blood donors. A study of γA/γ’ fibrinogen levels in patients undergoing coronary angiography also showed that γA/γ’ fibrinogen levels were higher on average in coronary artery disease patients than in patients without coronary artery disease, and that this association was independent of total fibrinogen levels.
Characterization and Comparative Evaluation of a Novel PAI-1 Inhibitor
Authors: Gils Ann , Stassen Jean-Marie , Nar Herbert , Kley Joerg , Wienen Wolfgang , Ries Uwe , Declerck Paul
Published: March 2018

Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of both tissue-type plasminogen activator and urokinasetype plasminogen activator in plasma, is a well established risk factor in thrombotic diseases. Reduction of active PAI-1 levels may lead to a decreased tendency of thrombosis. Compounds that can suppress pharmacologically active PAI-1 levels are therefore considered as putative drugs.

In the present study, we describe the PAI-1 neutralizing properties and mechanism of a newly selected compound (i. e. fendosal, HP129) in comparison to four previously reported compounds (i. e. AR-H029953XX, XR1853, XR5118 and the peptide TVASS) using different assays. The inhibitory effect of these compounds on active PAI-1 was analyzed by a plasmin-coupled chromogenic assay (Coaset® t-PA), direct chromogenic assays (t-PA, u-PA) and quantification of complex formation by ELISA, SDS-PAGE and surface plasmon resonance. Comparative evaluation of the obtained IC50 values reveals large differences [i. e. IC50 of 15 µM (HP129) vs. >1000 µM (XR5118) determined at 37° C using SDS-PAGE] between the compounds studied.

Importantly, the relative potency of the various compounds is also dependent on the method used (10 to 170-fold differences in IC50 values). Characterization of the PAI-1 forms (i. e. active, non-reactive and substrate) generated upon inactivation reveals that the newly described compound HP129 induces a unique pathway (i. e. active to non-reactive conversion via a substrate-behaving intermediate) of inactivation compared to the other compounds.

Taken together, these data strongly suggest that the various compounds act through different mechanisms. In addition, the results stress the necessity for a careful selection of the method used for the evaluation of PAI-1 inhibitors, preferably requiring a panel of screening methods.
Importance of N-Terminal Residues in Plasminogen Activator Inhibitor 1 on its Antibody Induced Latency Transition
Authors: Ngo Thu-Hoa , Zhou Yan , Stassen Jean-Marie , Declerck Paul
Published: March 2018

The serpin plasminogen activator inhibitor-1 (PAI-1) is a wellknown risk factor for thromboembolic and cardiovascular diseases. Many efforts have been made to reveal structure-function relationship in PAI-1, including the understanding of its unique latency transition. In this study, we describe the molecular characterization of PAI-1 neutralization by MA-159M12, a monoclonal antibody against rat PAI-1.

Time-dependent inactivation of PAI-1, exposure of a trypsin cleavage site typically for the latent conformation and disappearance of elastase susceptibility revealed that MA-159M12 accelerated the active to latent, conformational transition (t1/2 120 ± 12 min and 18 ± 3.6 min in the absence and presence of MA-159M12, p <0.0001).

Cross-reactivity analysis of the antibody with various rat/human PAI-1 chimeras revealed that the epitope resides in αhA of rat PAI-1. Subsequent alanine-scanning mutagenesis and binding studies demonstrated that Pro2- Leu3- Pro4-Glu5 constitute the major residues of the epitope for MA-159M12. In conclusion, these findings demonstrate that, even though unexpected based on current knowledge on PAI-1 stability and function, interference with αhA results in a destabilisation of its active, inhibitory conformation. Therefore, αhA forms a putative target for the rational development of PAI-1 neutralizing components.
Expression and Characterization of Recombinant Murine Factor VIII
Authors: Doering Christopher , Parker Ernest , Healey John , Craddock Heather , Barrow Rachel , Lollar Pete
Published: March 2018

Hemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media. Subsequently, a two-step purification procedure resulted in 5,300-fold enrichment and a 70% yield. Highly purified recombinant murine fVIII had a specific coagulant activity of 660 units per nanomole. It underwent proteolytic processing by thrombin to yield an activated heterotrimer that demonstrated significantly greater stability than activated human fVIII. Recombinant murine fVIII was utilized to generate an anti-fVIII polyclonal antibody. Intravenous injection of recombinant murine fVIII into hemophilia A mice failed to induce a significant anti-fVIII immune response using a schedule that yielded high titer inhibitory antibodies to human fVIII. This may provide an important model for the study of immune tolerance to fVIII.
Two New β3 Integrin Mutations in Indian Patients with Glanzmann Thrombasthenia: Localization of Mutations Affecting Cysteine Residues in Integrin β3
Authors: Nair Sona , Li Jihong , Mitchell W. , Mohanty Dipika , Coller Barry , French Deborah
Published: March 2018

New mutations in the β3 integrin subunit have been identified in two unrelated Glanzmann thrombasthenia patients originating from India and Bangladesh. Both patients had histories of excessive bleeding and were found to have Glanzmann thrombasthenia based on absent ADPinduced platelet aggregation. Immunoblotting of platelet lysates of Patient 1 demonstrated reduced levels of αIIb and an unexpected high Mr β3 band of ∼260,000, with little or no normal-sized β3. Upon reduction, a weak β3 band of normal Mr was observed. Platelet lysates of Patient 2 demonstrated undetectable levels of β3. Sequence analyses identified homozygous mutations in the β3 genes of both patients. Patient 1 had a C506Y missense mutation resulting in the expression of an unpaired cysteine; we propose that the Mr ∼260,000 band is a disulfide-bonded β3 dimer. Patient 2 had an insertion mutation resulting in a frameshift and premature termination. Both mutations affect biogenesis of platelet αIIbβ3 receptors.

This work was supported in part by grants HL19278 (B.S.C.), AHA Heritage Affiliate, Ilma F. Kern Foundation in honor of Jonathan Halperin, MD (D.L.F.), The Charles Slaughter Foundation (D.L.F.), and a fellowship from an Institutional NRSA, NHLBI T32 HL07824-06 (to W.B.M.)
Clinical Use of a Rapid Collagen Binding Assay for von Willebrand Factor Cleaving Protease in Patients with Thrombotic Thrombocytopenic Purpura
Authors: Moll Stephan , Taylor Mark , Krizek Dennis , White Gilbert , Aronson David , Rick Margaret
Published: March 2018

A simple collagen binding assay (CBA) for measuring activity of the von Willebrand factor cleaving protease in clinical samples is described, and results of fifty masked plasmapheresis samples from patients with TTP/HUS and other diseases are presented.

There was 97.5% concordance between the CBA and a multimer gel assay. The CBA identified low protease activity in 78% of patients who had a clinical syndrome consistent with TTP/HUS and in 2 of 10 sick controls, giving it a positive predictive value of 0.94. The heterogeneity regarding the presence or absence of vWF protease activity in patients with TTP/HUS was confirmed by finding a low negative predictive value of 0.50 with the CBA. The CBA detected inhibitors of the protease in 26 of 29 patients (90%) with TTP/HUS and low protease activity levels. The CBA is a useful clinical assay for examining von Willebrand factor protease activity and detecting inhibitors against the protease.
The Endotoxin-induced Plasminogen Activator Inhibitor-1 Increase in Rabbits Is Not Tumor Necrosis Factor-α Dependent and Can Occur in the Absence of Interleukin-1β
Authors: Montes Ramón , Rodríguez-Whilhelmi Pablo , Hurtado Verónica , Matsukawa Akihiro , Montes Marta , Hermida José , Rocha Eduardo
Published: March 2018

The plasminogen activator inhibitor-1 (PAI-1)-dependent fibrinolytic inhibition occurring in endotoxemia contributes to disseminated intravascular coagulation (DIC). Previous findings suggest that tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are responsible for the increase in the level of PAI-1. These observations usually arose from mild endotoxemia models. We analyzed the effect of FR167653, an inhibitor of the TNF-α/IL-1β production, on the PAI-1 levels in rabbits given endotoxin at a dose sufficient to induce DIC: the steep plasma PAI-1 increase was not attenuated by FR167653, in spite of achieving efficient inhibition of the TNF-α production. No IL-1β was detected during endotoxemia. These results suggest that PAI-1 increase might be independent of TNF-α and IL-1β. If these findings applied to humans, therapeutic intervention directing these cytokines would not be useful for the treatment of fibrinolysis in patients with severe sepsis.
Rapid-SF: A Rapid Whole-Blood Screen for Soluble Fibrin Monomer
Authors: Hay Karen , Bull Brian
Published: March 2018

Thrombosis accounts for a high proportion of disability and death in the West. Although soluble fibrin (SF) assays have been shown to be good predictors of thrombosis, current quantitative assays are too complex or lengthy to provide timely results, while simpler methods are qualitative and lack sensitivity. We here describe a rapid, new, protamine-based whole-blood screening method for SF which is quantitative and suitable for point-of-care use. Citrated whole blood is mixed with reagent under controlled conditions and the time until development of an SF precipitate is measured. Negative samples remain precipitate-free for 300 seconds. Strongly positive samples develop precipitate in as little as 10 seconds. SF times are mathematically converted to arbitrary SF units (SFU). This Rapid-SF test provides a simple and reliable means of detecting the presence of SF, and is well-suited for whole-blood rapid screening in the emergency department, operating room or clinic.
Local Shear Conditions and Platelet Aggregates Regulate the Incorporation and Activity of Circulating Tissue Factor in
ex-vivo
Thrombi
Authors: Vele Oana , Nemerson Yale , Balasubramanian Viji
Published: March 2018

The presence of thrombogenic blood-borne or circulating tissue factor (cTF) has recently been demonstrated. These observations have implicated cTF to be a key determinant of thrombus propagation by depositing on platelets in nascent thrombi. Previously, we detected cTF by detergent solubilization and addition of phospholipids. We now report the direct demonstration of TF activity in ex-vivo thrombi. Collagen-coated substrates were exposed to native blood at shear rates of 0, 650, and 2000 s-1 for 10 min in a modified rotating Teflon cone and plate viscometer. Substrates were then gently rinsed to remove ‘loose’ (unadherent) components of blood. cTF activity was measured by adding a solution containing 10 nM FVIIa, 100 nM FX, and 5 mM CaCl2 to the substrates exposed to blood. Samples of this mixture were obtained at intervals for 30 min and the amount of Xa generated was quantified by adding a chromogenic substrate, Spectrozyme Xa, and measuring the increase in OD at 405 nm. Our studies show that a minimal amount of generated Xa (∼ 1nM) can be measured from ex-vivo thrombi. Static and shear samples generated the same amount of Xa, with the exception of blood subjected to 650 s-1 shear. At 650 s-1 shear rate, the amount of Xa generated reached a maximum of 4 nM at 5 min and then decreased to ∼1 nM. Immunohistological stains and fluorescent images demonstrate the presence of cTF antigen at 650 s-1 wall shear rate.
Intravenous Immunoglobulin Mediates an Increase in Anti-Platelet Antibody Clearance via the FcRn Receptor
Authors: Hansen Ryan , Balthasar Joseph
Published: March 2018

We have recently shown that intravenous immunoglobulin (IVIG) therapy leads to an increased rate of anti-platelet antibody clearance in an animal model of immune thrombocytopenia. The present study was performed to confirm the importance of the FcRn receptor in mediating this effect of IVIG. The pharmacokinetics of an anti-platelet antibody, 7E3, were studied in mice lacking expression of FcRn and in control mice, both in the presence and absence of IVIG. IVIG increased the clearance of 7E3 in mice with functioning FcRn receptors, with an average clearance value of 14.4 ± 1.4 ml/d/kg in IVIG treated mice vs. 5.2 ± 0.3 ml/d/kg in controls (P <0.001). In mice lacking expression of FcRn, IVIG treatment did not increase 7E3 clearance (61.0 ± 3.6 ml/d/kg vs. 71.5 ± 4.0 ml/d/kg in controls). Thus, these data support the hypothesis that IVIG increases antibody elimination via saturation of FcRn.
Thrombin binding to GPIbα induces platelet aggregation and fibrin clot retraction supported by resting αIIbβ3 interaction with polymerized fibrin
Authors: Dubois Christophe , Steiner Beat , Kieffer Nelly , Meyer Reigner Sylvie
Published: March 2018

We have investigated the mechanisms leading to platelet aggregation following thrombin interaction with the glycoprotein (GP) Ib-IX-V complex. We show that platelets desensitized for the two thrombin receptors, PAR-1 and PAR-4, are still able to aggregate in response to thrombin and that this aggregation can be inhibited by a monoclonal antibody (VM16d) that blocks thrombin binding to GPIbα, or by pretreatment of platelets with Mocarhagin, a protease that specifically cleaves GPIbα. The thrombin/GPIbα-initiated signaling cascade induces platelet shape change through activation of the Rho kinase p160ROCK, independent of calcium mobilization, transient MEK-1 phosphorylation as well as the cleavage of talin through a calcium-independent mechanism. This signaling cascade does not induce the exposure of high affinity αIIbβ3 integrin receptors, nor does it lead to µ-calpain cleavage of filamin or the integrin cytoplasmic tail. In contrast, we provide evidence that binding of thrombin to GPIbα induces fibrin binding to resting αIIbβ3 leading to fibrin-dependent platelet aggregation and clot retraction, that can be selectively inhibited by αIIbβ3 antagonists such as RGDS, the dodecapeptide or lamifiban, as well as by the fibrin polymerization inhibitor GPRP-amide.
Ruling out clinically suspected pulmonary embolism by assessment of clinical probability and D-dimer levels: a management study
Authors: Lutisan Johan , Marwijk Kooy Marinus , Kuipers Bart , Oostdijk Ad , van der Leur Jef , Büller Harry , Leclercq Monique
Published: March 2018

D-dimer test combined with clinical probability assessment has been proposed as the first step in the diagnostic work-up of patients with suspected pulmonary embolism (PE). In a prospective management study we investigated the safety and efficiency of excluding PE by a normal D-dimer combined with a low or moderate clinical probability. Of the 202 study patients this combination ruled out PE in 64 (32%) patients. The 3-month thromboembolic risk in these patients was 0% (95% CI, 0.0-5.6%).The prevalence of PE in the entire cohort was 29% (59 patients), whereas in the low, moderate and high clinical probability groups this was 25%, 26% and 50%, respectively. We conclude that ruling out suspected PE by a normal D-dimer combined with a low or moderate clinical probability appears to be a safe and efficient strategy. The accuracy of the clinical probability assessment is modest.
Angiogenesis: molecular mechanisms and functional interactions
Authors: Breier Georg , Augustin Hellmut
Published: March 2018

The German Priority Research Program “Angiogenesis” (www.angiogenese.de) hosts a biannual meeting in the Kloster Seeon in Southern Germany. The 2nd Kloster Seeon Meeting “Angiogenesis: Molecular Mechanisms and Functional Interactions” was held in September 2002. It included sessions on hypoxia, the biology of endothelial precursor cells, angiogenic growth factors including VEGFs, the angiopoietins, ephrins, and FGFs, mechanisms of vascular sprouting and cell-cell contacts during angiogenesis, angiogenic signaling, lymphangiogenesis, angiogenesis during tumor invasion and metastasis, and on novel angiomanipulatory therapies. This report summarizes the key findings reported during the platform presentations of the meeting.
Oxysterols suppress constitutive fibrinogen expression
Authors: Xia Hui , Redman Colvin
Published: March 2018

Elevated levels of both fibrinogen and cholesterol are risk factors in coronary artery disease. Previously we reported a metabolic link between fibrinogen and lipid metabolism in that HepG2 cells that were programmed by transfection of Bβ-fibrinogen cDNA to overexpress fibrinogen exhibited increased synthesis of cholesterol and increased secretion of apolipoprotein B. In this study we demonstrate that oxysterols, which participate in maintaining cholesterol homeostasis, also down regulate fibrinogen expression. Treatment of HepG2 cells with 25-hydroxycholesterol lowered fibrinogen Aα, Bβ and γ mRNA levels and inhibited fibrinogen synthesis and secretion but had no effect on α1-antitrypsin which, like fibrinogen, is an acute-phase protein. The inhibition of fibrinogen synthesis by oxysterols was maintained in interleukin-6 treated cells. Other oxysterols, that inhibit cholesterol synthesis by a feedback mechanism, also diminished fibrinogen expression in HepG2, rat H-4-II-E hepatoma cells and in primary human hepatocytes. Overexpression of SREBP-1 and SREBP-2 by transfection of HepG2 cells, or treatment with a synthetic LXRα agonist, which affect cholesterol metabolism, did not affect fibrinogen expression. We conclude that fibrinogen and cholesterol may share a novel common regulatory pathway.
Elucidation of the epitope of a latency-inducing antibody: identification of a new molecular target for PAI-1 inhibition
Authors: Naessens Dominik , Gils Ann , Compernolle Griet , Declerck Paul
Published: March 2018

The monoclonal antibody MA-33B8, directed against the serpin plasminogen activator inhibitor-1 (PAI-1), has unique functional properties as it induces acceleration of the active-to-latent transition (Verhamme I et al. J Biol Chem 274: 17511-7, 1999), resulting in PAI-1 activity neutralization. In this study, we have identified Lys88, Asp89, Lys176 and His229 as the major residues of the conformational epitope. Lysine88 and aspartic acid89, contributing the most, are located on the loop between α-helix D and β-strand 2A. Strikingly, these residues undergo dramatic conformational changes during latency conversion. The three-dimensional localization of this epitope and its differential exposure in active and latent forms of PAI-1 provide a molecular explanation for the underlying mechanism of MA-33B8. Rearrangements of hydrogen bonds in this region upon latency transition, strongly suggest that binding of MA-33B8 may result in the generation of energy required in the rate-limiting step of latency transition. Thus, this novel epitope reveals a new interaction site in PAI-1 as a putative molecular target to modulate PAI-1 activity.
Course of indicators of thrombin activity in the early phase of acute myocardial infarction: effect of fibrinolytic therapy and acute percutaneous coronary angioplasty
Authors: Kaden Jens , Haase Karl , Borggrefe Martin , Dempfle Carl-Erik
Published: March 2018

We evaluated assay systems for detection of in vivo thrombin activity in patients with acute myocardial infarction. The study included 31 consecutive patients with acute myocardial infarction treated either with fibrinolytic therapy (FLT), or acute PTCA. Blood samples were drawn at admission, after 30 min, 1 h, 3 h, 6 h, 12 h, 24, and 48 h. Assays related to the enzymatic action of thrombin included fibrinopeptide A ELISA, a novel latex-enhanced photometric immunoassay for soluble fibrin complexes, fibrin monomer antigen, D-dimer antigen, and soluble platelet glycoprotein V, which is released from the platelet surface by thrombin cleavage.

The soluble fibrin assay displayed a high degree of correlation with fibrinopeptide A, and no correlation with D-dimer antigen, indicating that this parameter allows monitoring of in vivo thrombin activity in patients with acute myocardial infarction. Fibrin monomer antigen, and D-dimer antigen, were influenced by FLT, indicating cross-reactivity with proteolytic fragments of fibrin. No significant changes in soluble glycoprotein V were observed.

The results show that the novel soluble fibrin assay appears to be a promising method for measurement of in vivo fibrin formation in patients with acute myocardial infarction, irrespective of the primary treatment decision for FLT or acute PTCA.
Homozygosity for the Protein S Heerlen Allele Is Associated with Type I PS Deficiency in a Thrombophilic Pedigree with Multiple Risk Factors
Authors: Espinosa-Parrilla Yolanda , Navarro Gemma , Morell Marta , Abella Eugènia , Estivill Xavier , Sala Núria
Published: March 2018

The multifactorial character of thrombotic disease is shown in a Spanish pedigree in which the propositus, with recurrent deep vein thrombosis, inherited the factor V R/Q506 mutation, the prothrombin 20210G/A variant and type III Protein S deficiency. Among 14 relatives carrying one or two of these three risk factors, thrombosis is present in a heterozygote for R/Q506 and in another for 20210G/A, who also had slightly positive antiphospholipid antibodies. Type I PS deficiency was also found in a young asymptomatic woman. PROS1 analysis showed coexistence of type III and type I PS deficiency to be associated with heterozygosity and homozygosity, respectively, for the P460 or PS Heerlen allele of the S/P460 variant. Analysis of PS values in this and other pedigrees segregating this variant revealed that not only free but also mean total PS levels are slightly but significantly lower in the SP460 heterozygotes than in the SS460 homozygotes. These findings strongly suggest a role of the P460 variant in the expression of the PS deficient phenotype.
A Reliable and Reproducible ELISA Method to Measure Ristocetin Cofactor Activity of von Willebrand Factor
Authors: Vauterin Stephan , Schlammadinger Agota , Mazurier Claudine , Deckmyn Hans , Vanhoorelbeke Karen , Cauwenberghs Nancy
Published: March 2018

The ristocetin induced binding of vWF to GPIb, which is routinely tested in a platelet agglutination assay, can be reproducibly studied in an ELISA where plasma vWF binds to a captured rGPIbα-fragment (His1-Val289) in the presence of ristocetin. This binding is specific since the vWF-GPIb interaction could (i) be blocked by inhibitory antiGPIb or anti-(vWF A1 domain) monoclonal antibodies (mAbs) and (ii) be enhanced by an anti-vWF mAb that also facilitates ristocetin induced platelet agglutination. Further studies were undertaken to determine whether the test could be used to differentiate vWF from patients with different types of von Willebrand’s disease. The median vWF:RiCof activity in controls (n = 24) was 0.75 U/ml, in type 1 vWD patients (n = 17) 0.28 U/ml, in type 2A (n = 18) 0.055 U/ml, in type 2B (n = 4) 0.094 U/ml and in type 3 (n = 3) <0.0005 U/ml. Moreover, the values correlated well with those obtained from the vWF:RiCof-agglutination assay (r = 0.873). The vWF:RiCof-ELISA has several advantages: the use of a recombinant fragment instead of donor platelets results in a more reproducible test with a low inter-and intra-assay variability (<14% CV), the test can further be readily automated and for a single determination, only minimal amounts of patient plasma are required (8 µl).
Glycoprotein Ib-binding Protein from the Venom of Deinagkistrodon acutus – cDNA Sequence, Functional Characterization, and Three-Dimensional Modeling
Authors: Chen Yuh-Ling , Tsai Kuo-Wei , Chang Tschining , Hong Tse-Ming , Tsai Inn-Ho
Published: March 2018

Agkicetin-C, a potent glycoprotein Ib antagonist from the venom of the Chinese pit viper, Deinagkistrodon acutus, has been purified and characterized (5). It is a disulfide-linked heterodimer containing subunits of 132 and of 123 amino acid residues. Herein, the complete amino acid sequences were resolved by cloning and nucleotide sequencing of the cDNAs. The sequences of its subunits are homologous to those of other snake venom proteins of the C-type (Ca2+-dependent) lectin superfamily. A three-dimensional model of agkicetin-C was constructed based on the crystal structure of habu coagulation factor IX/X-binding protein. By careful alignment of all the related sequences available and comparing the 3D-model of agkicetin-C with structures of other homologous proteins of different functions, some variable residues of agkicetin-C were identified, which possibly are responsible for the specificity of this distinct subtype of the C-type lectin-like venom proteins.
The Effect of Monoclonal Anti-human-platelet Antibodies on Platelet Kinetics in a Baboon Model: IgG Subclass Dependency
Authors: Pieters Henry , van Wyk Veronica , Cooper Susan , Roodt Jan , De Reys Stefan , Badenhorst Philip , Coetzee Lindi-Marie
Published: March 2018

We assessed the in vivo effect of six intact anti-human antiplatelet antibodies of two major IgG subclasses on platelet kinetics in baboons. Five of the six antibodies tested caused thrombocytopenia of varying degree when injected at a precalculated threshold value. An agglutinating IgG1 antibody (MA-8L4A12) caused a long-lasting, mild thrombocytopenia with a predominant uptake of radiolabelled platelets in the spleen, while the four IgG2 antibodies tested (MA-13G8E1, MA-2M5A6, MA-21K2E8 and MA-22M10) caused a severe, transient thrombocytopenia with uptake of platelets in the liver. Two of the IgG2 antibodies (MA-13G8E1 and MA-2M5A6) caused platelet activation and aggregation in vitro, whilst the other two did not elicit a platelet aggregation response. The platelet survival time was shortened with all five of the thrombocytopenia-inducing antibodies, while only one antibody (MA-2M5A6) had a significant effect on the bleeding time. This study indicates that the IgG subclasss may be a determining factor in the outcome of platelet sequestration in immune-induced thrombocytopenia.
Measurement of Tissue Factor Activity in Whole Blood
Authors: Santucci Richard , Erlich Jonathan , Labriola Joanne , Wilson Mark , Kao K. , Kickler Thomas , Spillert Charles , Mackman Nigel
Published: March 2018

High circulating levels of the procoagulant molecule tissue factor (TF) are associated with thrombosis in a variety of diseases including unstable angina, cancer, and sepsis. Currently, there are no clinical assays to measure the level of TF activity in whole blood. We present an assay called Tissue Factor Clotting Time (“TiFaCT™”) that detects fibrin formation in human blood. The mean baseline clotting time in a healthy population was 472 ± 94 s (mean ± SD, n = 150). Bacterial lipopolysaccharide (LPS or endotoxin) shortened the clotting time in a time-dependent manner. Inhibitory anti-TF antibodies prolonged the clotting time of LPS-stimulated blood, indicating that the shortened clotting time was due to induction of TF expression. Patients with unstable angina had shortened mean baseline clotting time (284 ± 86, n = 13) compared with healthy volunteers (474 ± 98, n = 30), suggesting that these patients had elevated levels of circulating TF. The TiFaCT assay should prove clinically useful in quantifying the levels of circulating TF in patients at risk of thrombosis.
Improvement of Walking Distance by Defibrotide in Patients with Intermittent Claudication
Authors: Marubini E. , Coccheri S. , Nenci G. G. , Violi F.
Published: March 2018

Defibrotide is an antithrombotic drug which enhances prostacyclin production and activates fibrinolytic system. The aim of this study was to investigate the improvement of walking distance in patients with intermittent claudication treated with defibrotide.

DICLIS was a double blind, placebo-controlled study which included patients with walking distance autonomy at a standardized treadmill test ≤350 ≥100 meters. A total of 310 patients were randomly allocated to placebo (n = 101), defibrotide 800 mg/day (n = 104) or defibrotide 1200 mg/day (n = 105).

During a one year follow-up, the Absolute Walking Distance (AWD) was measured six times (0, 30, 60, 90, 180, 360 days).

Similar improvement in walking distance was found in the three groups until the 90th day; thereafter placebo group showed no further increase, while AWD continued to increase in the defibrotide groups. Between the 180th and 360th day visits, AWD was significantly higher (P <0.01) in patients given defibrotide than in patients given placebo. No difference in efficacy was observed between the two dosages of defibrotide. No differences in side effects were observed among the three groups.

The results of the present trial suggest that long-term administration of defibrotide improves walking distance in patients with intermittent claudication.

Abbreviations: DICLIS = Defibrotide Intermittent CLaudication Italian Study, AWD = Absolute Walking Distance, IC = Intermittent Claudication, PVD = Peripheral Vascular Disease, LVOCF = Last Valid Observation Carried Forward, ITT = Intention-To-Treat

Appendix, please see p. 676.
Decreased Sialic Acid Content of Plasma von Willebrand Factor in Precapillary Pulmonary Hypertension
Authors: de Souza Bruno , Maeda Nair , Lopes Antonio
Published: March 2018

In pulmonary hypertension, defective von Willebrand factor protein (vWF) lacking large multimers is present in circulation. This is associated with evidence of chronic endogenous platelet activation. Since asialo vWF has been shown to promote platelet activation and aggregation, we decided to investigate possible changes in the sialic acid content of plasma vWF in patients with precapillary pulmonary hypertension. vWF-associated sialic acid was measured indirectly as a wheat germ agglutinin-reactive substance (WGA-RS, Western blotting), and directly, as a thiobarbituric acid-reactive substance (TBA-RS, spectrophotometric reading). In the sixteen patients we studied (ages 8–45 yr), circulating vWF concentration was 2.18 times normal (p <0.001). However, patient vWF subunit contained 19% (WGA-RS) to 24% (TBA-RS) less sialic acid than the normal protein (p <0.05 for both determinations). In five patients, vWF-associated sialic acid was below 50% normal. We conclude that circulating vWF is hyposialylated in precapillary pulmonary hypertension and speculate that this might influence its interaction with platelets in vivo in these patients.
Major Structural Defects in the Antithrombin Gene in Four Families with Type I Antithrombin Deficiency
Authors: Beauchamp N. J. , Makris M. , Preston F. E. , Peake I. R. , Daly M. E.
Published: March 2018

The molecular basis of quantitative antithrombin deficiency was investigated in four families predicted to have major antithrombin gene rearrangements. A 1,442 bp deletion and insertion of the sequence 5’T(n = 38-40)GAGACG was characterised in one case. Sequence surrounding the breakpoints contained two perfect, and one imperfect, inverted repeats which may have mediated formation of a stem loop structure on one strand during DNA replication potentiating the deletion. A 9,219 bp deletion spanning introns 2 to 5 was identified in a second family. The identical 6 bp sequence was upstream of each breakpoint and the 5’ breakpoint was located in a sequence of the Alu 3 repeat predicted to be susceptible to strand breakage during transcription. This may have promoted misalignment, and deletion, of one of the repeats and the intervening DNA. A novel 1.8 kb antithrombin gene fragment was present in DNA digests from affected members of the third family suggesting a partial antithrombin gene duplication event while in the remaining family, evidence supporting a complete gene deletion was obtained.
Macrophages Synthesize Factor X and Secrete Factor X/Xa-containing Prothrombinase Activity into the Surrounding Medium
Authors: Lunderius Carolina , Tomasini-Johansson Bianca , Pejler Gunnar
Published: March 2018

Activation of the coagulation cascade, mediated by various monocyte/macrophage procoagulants, is an important component in the pathology of inflammatory disease. The type of procoagulant expressed may vary between different monocyte/macrophage subtypes and may differ depending on how the cells are treated. In the present study we show that both murine peritoneal macrophages and human adherent synovial cells from rheumatoid arthritis lesions express prothrombinase activity that was inhibited by anti-Factor X antibodies. Northern blot analysis showed that Factor X was transcribed by the murine peritoneal cells and Western blot analysis showed the presence of Factor X antigen. Further experiments showed that the prothrombinase activity was secreted by the cells into the medium in a detergent-sensitive form, suggesting that the prothrombinase is released on small lipid-containing vesicles.
Increased Risk of Recurrent Venous Thromboembolism during Hormone Replacement Therapy
Authors: Qvigstad Erik , Arnesen Harald , Larsen Stig , Wickstrøm Egil , Sandset Per , Høibraaten Else
Published: March 2018

Recent observational studies suggest a 2-4 fold increased risk of venous thromboembolism (VTE) in women taking hormone replacement therapy (HRT). The present study was started before publication of these studies, and the aim was to determine if HRT alters the risk of VTE in high risk women. The study was a randomized, double-blind, and placebo-controlled clinical trial with a doubletriangular sequential design. Females with previously verified VTE were randomized to 2 mg estradiol plus 1 mg norethisterone acetate, 1 tablet daily (n = 71) or placebo (n = 69). The primary outcome was recurrent deep venous thrombosis (DVT) or pulmonary embolism (PE). Between 1996 and 1998 a total of 140 women were included. The study was terminated prematurely based on the results of circumstantial evidence emerging during the trial. Eight women in the HRT group and one woman in the placebo group developed VTE. The incidence of VTE was 10.7% in the HRT group and 2.3% in the placebo group. In the HRT group, all events happened within 261 days after inclusion. The sequential design did not stop the study, but strongly indicated a difference between the two groups. Our data strongly suggests that women who have previously suffered a VTE have an increased risk of recurrence on HRT. This treatment should therefore be avoided in this patient group if possible. The results also support those of recent epidemiological studies, which also indicate increased risk of VTE in non-selected female populations during HRT.
Lupus Anticoagulants and Thrombosis: Clinical Association of Different Coagulation and Immunologic Tests
Authors: Dlott Jeffrey , Norbis Francesca , Ruggeri Luisa , Cler Linda , Triplett Douglas , Barbui Tiziano , Galli Monica
Published: March 2018

The dilute Russell’s viper venom time (dRVVT) and the kaolin clotting time (KCT) are two among the most commonly used coagulation tests for the detection of lupus anticoagulants. The dRVVT seems superior to the KCT in identifying LA-positive patients at risk of thrombosis. However, this relationship is greatly influenced by both the source of reagents and the instrumentation employed to carry out the assays. Therefore, 4 dRVVTs (“home-made” dRVVT, DVV test, Bioclot LA, LA Screen), and one KCT (Kaoclot) were performed in two centers and compared for their retrospective correlation with the thrombotic complications of 72 patients with a previously established diagnosis of lupus anticoagulants. Two other assays (“home-made” KCT, and Colloidal Silica Clotting Time, CSCT) were performed in one of the two centers, and compared with Kaoclot for their clinical correlations in the same population of patients, 44 of whom (61%) had suffered from arterial and/or venous thrombosis. A rather good degree of inter-laboratory and inter-assay correlations of the different tests was found. However, a statistically significant association with thrombosis was found only with the coagulation profile generated using the “homemade” dRVVT. When the commercially available dRVVTs were used, none of the coagulation profiles remained associated with thrombosis. When the assays were analyzed separately, the association with thrombosis was statistically significant for LA screen (p = 0.0019), DVV test (p = 0.0043), and Bioclot (p = 0.0255), and of borderline significance for the “home-made” dRVVT (p = 0.0503) in one center. This last assay was also significantly associated with thrombosis in the other center (p = 0.0139). When venous and arterial thrombosis were considered separately, DVV test was statistically associated with venous thrombosis in both centers (p = 0.0076 and p = 0.0187, respectively), and LA screen in one center (p = 0.0303). No dRVVT was found to correlate with arterial thrombosis. Kaoclot, Colloidal Silica Clotting Time, and the “home-made” KCT did not correlate with thrombosis. The prevalence of IgG and/or IgM antibodies to cardiolipin, β2-glycoprotein I and prothrombin were 74%, 86% and 85%, respectively. Increased titers of IgG anticardiolipin antibodies were associated with arterial thrombosis (p = 0.0375), whereas IgM anti-β2-glycoprotein I antibodies were associated with venous thrombosis (p = 0.0433). In conclusion, these retrospective data support the notion that the dRVVT, rather than other coagulation or ELISA tests, are able to identify lupus anticoagulant-positive patients at risk of thrombosis. This property appears common to several commercially available dRVVT kits, making this type of assay the ideal target of future efforts of laboratory standardization.
Smooth Muscle Cell Migration Promoting Activity of Plasma Predicts Restenosis in Patients with Peripheral Arterial Occlusive Disease Undergoing Angioplasty
Authors: Janisch Sandra , Kvas Erich , Schnedl Wolfgang , Binder Bernd , Wojta Johann , Korninger Christian , Roller Regina
Published: March 2018

Efficacy of percutaneous transluminal angioplasty (PTA) is limited by restenosis occurring in a large proportion of patients. Smooth muscle cell (SMC) migration is a major pathomechanism of restenosis. We studied SMC migration inducing activity of plasma from patients with peripheral arterial occlusive disease (PAOD) undergoing PTA.

SMC migration was determined in a two-dimensional assay system after addition of 1/25 plasma dilutions. Mean increase in migration area was 65.5 ± 33.8% in normal controls and 67.7 ± 53.2% in patients with PAOD. 6 hours after PTA, plasmatic migration inducing activity was largely unchanged. In 19/30 patients with restenosis (6 months after PTA) migration promoting activity (82.7 ± 60.0) was significantly higher than in 11/30 patients with patent vessels (41.8 ± 21.0; p = 0.03). No correlation of clinical risk factors with outcome was observed. A weak correlation was found between plasmatic migration promoting activity and levels of epidermal growth factor and transforming growth factor-β.

The capacity of human plasma to stimulate SMC migration in tissue culture can be used to assess the risk for restenosis after PTA in patients with PAOD.
Rare Coagulation Disorders
Authors: Peyvandi Flora , Mannucci P. M.
Published: March 2018

The type of hemorrhagic manifestations that occur in patients with recessively transmitted coagulation disorders and their optimal treatment are not well established as for hemophilia A and B and von Willebrand disease, due to the rarity of these disorders. In a Muslim country like Iran where consanguineous marriages are frequent these disorders are less rare. We chose to evaluate the pattern of bleeding symptoms in 237 Iranian patients with the inherited deficiencies of fibrinogen, factor II, combined factor V and factor VIII, factor V, factor VII and factor X. Considering “severe” life-endangering hemorrhages such as those in the CNS, gastrointestinal tract and from the umbilical cord and those potentially handicapping such as hematomas and hemarthroses; and “mild” epistaxis, menorrhagia, hematuria, oral and postsurgical bleeding, it would appear the most severe diseases are factor X and factor II deficiencies. For the remaining defects only a minority of patients, even those with unmeasurable plasma levels, had life-endangering hemorrhages or muscoloskeletal disabilities as a consequence of hemarthroses and hematomas. The relatively mild severity of clinical manifestations in recessive coagulation disorders commands safety as the primary criterion in the choice of replacement material for treatment. Hence, virally inactivated plasma and factor concentrates should be the products of choice.
Hemostatic Activation under Anticoagulant Treatment: A Comparison of Unfractionated Heparin vs. Nadroparin in the Treatment of Proximal Deep Vein Thrombosis
Authors: Marchetti O. , Haeberli A. , Mombelli G. , Stricker H.
Published: March 2018

Background: Multiple clinical trials have been performed to compare standard heparin with low molecular weight heparin in the therapy of deep vein thrombosis, but little is known about the time course of the markers of hemostatic system during the treatment with these two heparin regimens.

Methods: Twenty patients with proximal deep vein thrombosis confirmed by duplex ultrasound and phlebography were randomly assigned to either unfractionated heparin (UH) given as an intravenous bolus of 80 U/kg followed by a constant infusion of 18 U/kg/h, or nadroparin 185 AXa IU/kg once daily subcutaneously. Oral anticoagulants were started at day 4. Markers of hemostatic activation (F1+2, FPA, TAT, D-dimer) were measured daily for 4 days. Primary end-points were the time course of these markers; secondary endpoints consisted in the evaluation of thromboembolic and hemorrhagic complications by clinical outcome and Marder score.

Results: Treatment with UH resulted in a rapid achievement of therapeutic heparin levels. UH reduced markers of fibrin formation and fibrinolysis more rapidly than nadroparin (p < 0.05). Within the nadroparin group activation of prothrombotic markers four hours after the subcutanous injection (peak level) was significantly lower when compared with the time prior to injection (trough level). Secondary endpoints showed no significant difference between the two groups.

Conclusion: Continuous intravenous perfusion of UH administered on a basis of a weight-adjusted nomogram controlled markers of the hemostatic system more rapidly than once-daily subcutaneously administered weight-adjusted nadroparin.