bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Arterial Thrombus Formation in Two Canine Models: Comparison of Ancrod, a Fibrinogen-Depleting Agent, the Thrombin-Inhibitor r-Hirudin, and the Glycoprotein IIb/IIIa-Receptor Antagonist Ro 43-8857
Authors: Rübsamen K , Hornberger W , Kirchengast M
Published: July 2018

Inhibition of arterial thrombus formation by ancrod, a fibrinogen depleting agent isolated from a snake venom, r-hirudin, an inhibitor of thrombin-mediated fibrinogen cleavage, or the glycoprotein (GP)IIb/IIIa-receptor antagonist Ro 43-8857 interfering with fibrinogen binding to platelets, was evaluated in two canine models. As a marker of platelet-dependent thrombus formation, cyclic blood flow reductions (CFR) were induced in the left coronary artery (LAD) of mongrel dogs by mechanical injury of the endothelium combined with critical stenosis. In the second model CFRs were induced by thrombolysis of a copper coil-induced thrombus in the carotid artery. Blood flow rate during the reocclusion phase was used as an additional parameter of efficacy.

The frequency of CFRs used as indicator of platelet aggregation and adhesion was significantly diminished by all treatments in both the carotid artery- and the LAD-model. In the LAD-model, following ancrod treatment, CFRs were correlated with plasma fibrinogen concentrations. Carotid artery blood flow after reperfusion, used as indicator of occlusive thrombus formation, rapidly declined to zero in the control group but remained at a high level after treatment with ancrod or r-hirudin. Ro 43-8857 at the selected dose improved blood flow rate only to a minor degree but prolonged the bleeding time from a mean value of 87.2 ± 10.9 s (n = 24) to values >300 s in 50% of the animals.

Our results indicate that CFRs as indicator of platelet aggregation and adhesion are inhibited by either treatments. Blood flow as indicator of occlusive thrombus formation, however, is effectively improved by ancrod and r-hirudin only. Inhibition of fibrinogen binding to platelet GPIIb/IIIa receptors alone was found to be a less potent antithrombotic principle in this model.
Haemophilia A: Mutation Type Determines Risk of Inhibitor Formation
Authors: Schwaab R , Brackmann H-H , Meyer C , Seehafer J , Kirchgesser M , Haack A , Olek K , Tuddenham E G D , Oldenburg J
Published: July 2018

The formation of factor VIII antibodies is a major problem for replacement therapy of haemophilia A patients. Antibodies occur in 5-30% of patients with severe haemophilia A. The reason for antibody formation is still unknown. In this study we correlate for the first time different factor VIII gene mutations, stop- and missense mutations, large and small deletions and intrachromosomal intron 22 recombinations to antibody formation. A total of 364 patients with known inhibitor status of our institute, of the database, and of 3 studies representing intron-22-inversion data are included. The results show that the risk for developing factor VIII antibodies is strongly related to stop mutations, large deletions and intrachromosomal recombinations. A probable explanation could be the complete lack of endogenous circulating factor VIII protein in these cases. Other factors that might be important for the pathogenesis of inhibitor formation, e. g. the antenatal period, as well as possible therapeutic effects, are discussed.
Changes in Haemostatic Factors and Activation Products after Exercise in Healthy Subjects with Different Ages
Authors: van den Burg P J M , Hospers J E H , van Vliet M , Mosterd W L , Bouma B N , Huisveld I A
Published: July 2018

We studied exercise-induced changes in coagulation and fibrinolytic factors and activation products in different age categories. Thirty-eight sedentary males, divided in three age categories (cats I-III; 20-30, 35-45 and 50-60 y) were subjected to a standardized exercise test.

Pre-exercise levels (cats I-III resp) of FVII:c (105 ± 5, 121 ±6 and 123 ± 7% NP), fibrinogen (2.35 ± 0.12, 2.55 ± 0.10 and 2.66 ± 0.09 mg/ml), prothrombin activation fragment F1+2 (0.80 ± 0.10,0.80 ± 0.11 and 1.22 ±0.16 nM), t-PA (5,2 ± 0.6, 9.2 ± 1.0, 8.6 ± 1.2 ng/ml) and PAI-1 (42.8 ± 7.5, 67.6 ± 7.6, 62.2 ± 10.9 ng/ml) showed differences that seemed related to age. Regression analysis revealed associations with anthropometry (FVII:c, fibrinogen, F1+2, t-PA, PAI-1) rather than with age.

Exercise-induced changes in coagulation (increase in von Wille-brand factor and FVIII:c and a shortening of APTT) and fibrinolytic potential (increase in t-PA and u-PA) were of comparable magnitude for the three age categories. Hardly any change in F1+2 (6%) was observed, while thrombin-antithrombin complexes (93%), plasmin-antiplasmin complexes (79%) and D-dimer (77%) almost doubled during maximal exercise.

We conclude that anthropometric differences play a more significant role than age on constitutive levels of haemostatic factors in participants up to 60 years of age. The magnitude of exercise-induced changes is comparable in the age categories under study, and simply superimposed on constitutive (pre-exercise) levels. Clear evidence for prothrombin activation is lacking, but plasmin formation is enhanced during exercise.
Interspecies Comparison of Platelet Aggregation, LIBS Expression and Clot Retraction: Observed Differences in GPIIb-IIIa Functional Activity
Authors: Jennings Lisa K , White Melanie M , Mandrell Timothy D
Published: July 2018

We examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species’ platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.
Fast and Reproducible Vascular Neointima Formation in the Hamster Carotid Artery: Effects of Trapidil and Captopril
Authors: Matsuno H , Stassen J M , Hoylaerts M F , Vermylen J , Deckmyn H
Published: July 2018

Neointima formation was induced in the hamster carotid artery by mechanical intraluminal injury with a catheter covered with roughened dental cement. Neointimal thickening occurred as early as 7 days after denudation and further increased during the next 1 to 2 weeks. Proliferation indices of smooth muscle cells (SMCs) showed the highest proportion of proliferating cells in the media and neointima respectively 1 and 5 days after the vascular injury. Transmission and scanning electron microscopy of damaged carotid artery sections as well as immuno-histochemical stainings of von Willebrand factor (vWF) confirmed that reendothelialization was progressive and already complete on day 14, at which time the neointima formation was almost complete.

In order to pharmacologically characterize this model further, the effects on neointima formation of trapidil (triazolopyrimidine), a platelet-derived growth factor (PDGF) antagonist, and captopril, an angiotensin converting enzyme inhibitor, were investigated. Trapidil administered orally twice daily at total doses of 25, 50 and 100 mg/kg/day, started 3 days prior to infliction of injury and up to 7 or 14 days after the catheterization, significantly reduced neointima formation. Captopril administered orally three times daily at a total dose of 100 mg/kg/day, equally reduced neointima formation, with 100 mg/kg/day trapidil being more effective than 100 mg/kg/day captopril 7 days after injury. When the treatment by either one of these drugs was arrested on day 7, neointima formation resumed quickly.

The hamster appears to be a small, reproducible and fast model for the study of SMC proliferation, requiring only relatively small amounts of experimental drugs. The model furthermore is sensitive to substances known to reduce neointima formation in other animal models.
Quantitative Comparison of Fibrin Degradation with Plasmin, Miniplasmin, Neurophil Leukocyte Elastase and Cathepsin G
Authors: Kolev Krasimir , Komorowicz Erzsébet , Owen Whyte G , Machovich Raymund
Published: July 2018

The relative contribution of plasmin, miniplasmin, PMN-elastase and cathepsin G to the fibrin-gel dissolution is studied. The global kcat/KM ratios are determined as a measure of the fibrinolytic catalytic efficiency using spectrophotometric kinetic analysis of the competition between fibrin and synthetic peptide substrates for the proteases, turbi-dimetric assay for fibrin dissolution and gel-filtration of the partially degraded fibrin. When the substrate is fibrin polymerized in the presence of 3 mM Ca2+, the value of this ratio is 4.3 × 105 M-1·s-1 for plasmin, 1.9 × 105 M-1·s-1 for miniplasmin, 5.0 × 104 M-1·s-1 for PMN-elastase and 2.2 × 103 M-1·s-1 for cathepsin G. When fibrin is polymerized without addition of Ca2+, the kcat/KM values are increased by a factor of 2.3 for plasmin, 2.0 for miniplasmin and 1.6 for cathepsin G, whereas that of PMN-elastase is unchanged. Progressive crosslinking of fibrin decreases the catalytic action of all studied proteases, but no change in their relative contribution to fibrinolysis is observed. When plasmin inhibitor (at physiological concentration) is also crosslinked to fibrin, the most efficient fibrinolytic enzymes are miniplasmin and PMN-elastase. The effect of 6-aminohexanoate on the formation of fibrin degradation products by plasmin and miniplasmin suggests that the high-affinity lysine binding site in the N-terminal kringle domain of plasmin is involved in the interactions with the native polymerized fibrin, whereas the fifth kringle found in both enzymes participates in binding to newly exposed lysine residues. These results provide a quantitative basis for the evaluation of fibrinolytic efficiency and support the concept of synergistic fibrinolysis.
Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa
Authors: Tokita Shigeru , Arai Morio , Yamamoto Naomasa , Katagiri Yasuhiro , Tanoue Kenjiro , Igarashi Koji , Umeda Masato , Inoue Keizo
Published: July 2018

To study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.
Primary Binding Domain of Bovine von Willebrand Factor Fragment Expressed in E. coli
Authors: Bakhshi Meenakshi R , Sinha Dipali , Vora Renu K , Budzynski Andrei Z , Kirby Edward P
Published: July 2018

Bovine vWF cDNA has been cloned from a bovine endothelial cell library. A fragment of this cDNA, corresponding to amino acid sequence Leu 469-Ser 723, called primary adhesion domain (PAD-1), and containing the binding sites for platelet glycoprotein Ib (GPIb), heparin and collagen, has been expressed in E. coli. The reduced and alkylated form of fragment PAD-1 inhibited native vWF binding to GPIb. Fragment PAD-1 bound to heparin and botrocetin in a specific and dose dependent manner as did the native vWF. In a solid-phase assay, fragment PAD-1 bound to calf skin collagen in contrast to a human vWF recombinant fragment (Ser 445-Val 733) which was inactive in the same assay. The studies presented in this paper demonstrated that the A1 domain of bovine vWF contained the GPIb, heparin, botrocetin as well as collagen binding sites and that integrity of the disulfide bond (Cys 509-Cys 695), did not seem to be essential for binding of bovine vWF fragment to GPIb.
Elevated Fibrinopeptide A and B Levels during Thrombolytic Therapy: Real or Artefactual?
Authors: Weitz Jeffrey I
Published: July 2018

There is good evidence that thrombolytic therapy induces a procoagulant state that retards the lytic process and triggers reocclusion. Thus, both in experimental animal systems and in humans, potent inhibitors of platelets and antithrombin Ill-independent thrombin inhibitors have been shown to be better than heparin at accelerating thrombolysis and preventing reocclusion. These in vivo observations have lent credence to the concept that elevated FPA levels that are suppressed by heparin reflect systemic activation of coagulation during the thrombolytic process. However, this concept may not be correct. Thus, when heparin is given in conjunction with plasminogen activators only modest increases in FPA levels are found, and our data suggest that this is due, at least in part, to the activity of enzymes other than thrombin. In the absence of concomitant heparin, thrombolytic therapy causes a much greater increase in the FPA values. These high FPA values are rapidly reduced by heparin, even though heparin has limited antithrombotic activity in the setting of pharmacologic thrombolysis. Based on these considerations, we believe that plasma FPA and desarginine FPB levels should not be used as specific markers of thrombin activity during the course of thrombolytic therapy and suppression of elevated FPA values by heparin should not be accepted as evidence that heparin is effective in this clinical setting.
Infusion of Phospholipid Vesicles Amplifies the Local Thrombotic Response to TNF and Anti-Protein C into a Consumptive Response
Authors: Taylor F B , He S E , Chang A C  K , Box J , Ferrell G , Lee D , Lockhart M , Peer G , Esmon C T
Published: July 2018

Inflammation often is considered a contributing factor to both thrombosis and disseminated intravascular coagulation. The molecular mechanisms that dictate which of these clinical manifestations will result from the inflammatory stimulus remain obscure. Bacterial infection and certain tumors are common initiators of the disseminated intravascular coagulant response. Complement activation resulting from bacterial infection shares with selected tumors the capacity to generate or release membrane particles that lack functional adhesion receptors and hence could circulate to amplify a disseminated intravascular coagulant response. We developed a model of venous thrombosis that resulted in localized thrombus formation without disseminated intravascular coagulation. The model involves infusion of tumor necrosis factor, blockade of protein C and a partial decrease in venous flow caused by ligation of the superficial femoral vein without obstruction of the deep femoral vein. Infusion of phospholipid vesicles into this model resulted in amplification of a localized thrombotic response into a consumptive response.

Seven different groups of animals were studied. The first three groups established the conditions necessary to produce deep vein thrombosis. The second four groups established the conditions necessary to produce disseminated intravascular coagulation. The infusion of phospholipid vesicles plus tumor necrosis factor and anti-protein C antibody resulted in consumption of fibrinogen, the production of thrombin/antithrombin complexes, a fall in platelet count, and venous thrombosis. Without ligation and catheterization phospholipid vesicles failed to produce the consumptive response. We conclude, therefore, that phospholipid vesicles can amplify a local thrombotic response into a consumptive response, and that vesiculation accompanying inflammation is one means by which localized coagulant activity may be amplified to produce disseminated intravascular coagulation.
Characterization of Fcγ Receptors on Human Megakaryocytes
Authors: Wu Zhanhe , Markovic Boban , Chesterman Colin N , Chong Beng H
Published: July 2018

Megakaryocyte and platelet Fcγ receptors (FcR) are of importance in the pathophysiology of immune complex-mediated thrombocytopenias such as heparin-induced thrombocytopenia. In this study, FcγR proteins and mRNAs in normal human megakaryocytes were examined. FcγR proteins were studied with immunocytochemical staining, dual colour flow cytometry and immunoprecipitation using monoclonal antibodies against FcγR I, FcγR II and FcγR III. FcγR mRNAs were measured with biotinylated cDNA or oligonucleotide probes using a novel quantitative in situ hybridization technique. Using these techniques, FcγR II protein and mRNA, but not FcγR I and FcγR III proteins and transcripts were detected in megakaryocytes. Further, transcript analysis showed that megakaryocytes contain only the transcript of FcγR IIA gene but no transcripts of FcγR IIB nor FcγR IIC genes; FcγR IIA transcripts with and without the transmembrane (TM) exon are present in approximately equal proportions. In contrast, neutrophils and macrophages also contain FcγR IIA transcript but FcγR IIA transcript with the TM exon predominates suggesting cell lineage-specific FcγR IIA expression. FcγR IIA transcript lacking the TM exon predicts the presence of a potential soluble form of FcγR in platelets and megakaryocytes which may have a physiological role as it can compete with the membrane-bound FcγR IIA for binding of IgG-containing immune complexes and thus protect these cells from excessive binding and injurious effects of immune complexes.
Fibrinogen Promotes Adhesion of Monocytic to Human Mesothelioma Cells
Authors: Shetty Sreerama , Kumar Anuradha , Pueblitz Siegfried , Emri Saleh , Gungen Yucel , Johnson Alice R , Idell Steven
Published: July 2018

Adhesion between monocytic and mesothelioma or pleural meso-thelial cells influences stromal remodeling in pleural neoplasia. We found that cultured monocytic cells (U937) adhere to either human pleural mesothelioma (MS-1) or mesothelial (MeT5A) cells in vitro. 125I-fibrinogen bound specifically and saturably to either cell line, and specific fibrinogen binding increased upon stimulation of these cells with proinflammatory agents such as phorbol myristate (PMA), lipo-polysaccharide (LPS) or tumor necrosis factor (TNF-±). We purified the fibrinogen receptor protein from a membrane fraction of MS-1 cells and identified it by immunoprecipitation as intercellular adhesion molecule (ICAM-1). Anti-ICAM-1 antibody or antisense oligonucleotides inhibited fibrinogen-mediated cell adhesion and binding of 125I-fibrinogen to mesothelioma or mesothelial cells. Cultured monocytic cells adhere to either mesothelioma or mesothelial cells, and the interaction is promoted by fibrinogen binding ICAM-1 at the cell surface. ICAM-1 is expressed by mesothelioma cells and CD 1 lb by macrophages in the fibrinous mesothelioma tumor stroma. The data suggest a common mechanism by which monocytic cells could adhere to either malignant mesothelioma cells or the mesothelial surface in pleural neoplasia.
Reduced Effect of Aspirin on Thrombus Formation at High Shear and Disturbed Laminar Blood Flow
Authors: Marius Barstad R , Ørvim Una , Hamers Maria J A.G , Tjønnfjord Geir E , Brosstad Frank R , Sakariassen Kjell S
Published: July 2018

Aspirin is the most commonly used antithrombotic drug in primary and secondary prophylaxis against cardio- and cerebrovascular disease. In previous studies from our laboratory it was demonstrated that the effect of aspirin on collagen-induced thrombus formation in a parallelplate perfusion device with laminar blood flow is shear rate dependent. Although aspirin did not affect collagen-induced thrombus formation at 650 s-1 (medium sized arteries), a significant inhibition of thrombus formation by approximately 38% at 2,600 s-1 (moderately stenoses in medium sized arteries) was observed. At present we have extended these studies to thrombus formation at the apex of eccentric stenoses in a parallel-plate perfusion chamber device. The stenoses reduced the cross-sectional area of the blood flow channel of the perfusion chambers by 60 or 80%, introducing disturbed laminar flow and apex wall shear rates of 2,600 and 10,500 s-1, respectively. The corresponding wall shear stresses were 80 and 315 dynes/cm2, respectively.

Aspirin reduced the platelet thrombus volume at the 60% stenosis by 45% (p <0.03), and the fibrin deposition by 70% (p <0.004). However, none of these parameters were affected by aspirin at the 80% stenosis. These observations may at least partly explain why aspirin has a limited clinical effect in preventing arterial thrombus formation in atherosclerotic vessels at high shear and disturbed blood flow. In contrast, thrombus formation in blood from one patient with Glanzmann’s thrombasthenia and two patients with von Willebrand disease subtype 2M was almost abolished at this blood flow condition. Thus, blocking the function of either von Willebrand factor or glycoprotein IIb/IIIa may represent better antithrombotic approaches for such critical events than blocking the prostaglandin metabolism by aspirin. The lack of effect of aspirin on thrombus formation at the 80% stenosis may reflect shear-induced platelet activation at the stenosis inlet region, since shear-induced platelet aggregation in rotational viscometers is not affected by aspirin at shear stresses exceeding 100 dynes/cm2.
Evidence for Cultured Human Vascular Smooth Muscle Cell Heterogeneity: Isolation of Clonal Cells and Study of their Growth Characteristics
Authors: Benzakour Omar , Kanthou Chryso , Kanse Sandip M , Scully Michael F , Kakkar Vijay V , Cooper David N
Published: July 2018

The monoclonal theory of atherosclerosis postulates that the initial vascular smooth muscle cell (VSMC) proliferative event involves the expansion of a single cell or a sub-population of cells thus implying differences in the replicative potential of VSMC. Using the technique of limited dilution, VSMC clones derived from animal tissues have been previously isolated and shown to be morphologically heterogeneous. However, the same technique applied to human VSMC (HVSMC) has been unsuccessful, possibly because HVSMC do not grow when plated at very low densities. In this report, the anchorage-independent growth of HVSMC in semi-solid medium was studied. Platelet-derived growth factor-BB (PDGF-BB) and to a lesser extent PDGF-AB and basic fibroblast growth factor (bFGF) induced colony formation. This assay provided a tool for the isolation of HVSMC clones. In terms of their growth characteristics and responsiveness to several growth factors, isolated HVSMC clones and the original parental cell population exhibited marked heterogeneity.
Reliable Rapid Blood Test for the Exclusion of Venous Thromboembolism in Symptomatic Outpatients
Authors: Turkstra Franktien , van Beek Edwin J R , ten Cate Jan W , Buller Harry R
Published: July 2018

In this study we assessed the reliability of a rapid bed-side whole blood D-dimer assay prospectively in patients with clinically suspected venous thromboembolism, referred to the Academic Medical Centre, Amsterdam. In consecutive outpatients with clinically suspected deep vein thrombosis or pulmonary embolism we measured the sensitivity, specificity and negative predictive value of the assay compared to the outcome of standard diagnostic tests and 3-month follow-up. A total of 234 patients were included; the prevalence of venous thromboembolism was 29%. A sensitivity, specificity and negative predictive value of 100% (95% Cl: 95% -100%), 58% (95% Cl: 50% - 65%) and 100% (95% Cl: 96% -100%), respectively, were obtained. The exclusion rate was 41% of all referred patients. Conclusion: The SimpliRED whole blood D-dimer assay appears to be a simple and reliable method for the exclusion of venous thromboembolism in symptomatic outpatients.
A Pilot Study on Bolus Administration of Recombinant Staphylokinase for Coronary Artery Thrombolysis
Authors: Vanderschueren S , Collen D , Van de Werf F
Published: July 2018

Recombinant staphylokinase (Sak) is a highly fibrin-specific thrombolytic agent but the optimal dose and mode of administration remain to be defined. Intravenous (i.v.) infusion over 5 min of 20 mg Sak in 12 patients with acute myocardial infarction induced complete coronary patency (TIMI perfusion grade 3) in 7 patients (58%) within 60 min. In 3 of the 5 patients with no or suboptimal flow (TIMI grade 0,1 or 2) at 60 min, an additional 10 mg i.v. bolus of Sak resulted in TIMI grade 3 flow at 90 min. No major treatment-related complication occurred. Residual fibrinogen and α2-antiplasmin levels at 90 min were 110 ± 6.0% and 98 ±4.1% (mean ± SEM) of baseline, respectively. Median antibody-related Sak-neutralizing activity was low at baseline (0.0 μg/ml) and after 1 week (0.5 μg/ml) but increased from day 10 on (to 4.0 μg/ml).

Thus, bolus thrombolysis with Sak may induce efficient coronary artery recanalization while preserving circulating fibrinogen and α2-antiplasmin. Comparative trials of coronary thrombolysis with double-bolus Sak appear to be warranted.
Effect of Thrombomodulin on the Molecular Recognition and Early Catalytic Events in Thrombin-Protein C Interaction
Authors: De Cristofaro Raimondo
Published: July 2018

A viscosity perturbation method allowed to compute the second order rate constant, k±15 for the formation of thrombin-Protein C complex, both in the absence and presence of thrombomodulin (TM) at pH 8.00 and 37° C. In the absence of TM the second order rate constant was found equal to 7.9 ± 0.6 × 103 M-1 sec-1, whereas it was enhanced to 9.9 ± 0.4 × 104 M-1 s-1 by a saturating (100 nM) TM concentration. Addition of 5 mM Ca++ to solution containing 100 nM TM induced a further increase of k+1 value up to 7.3 ± 0.5 × 105 M-1 s-1. Moreover, it was demonstrated that the thrombin-PC complex undergoes the acy-lation reaction more rapidly than it dissociates to form free thrombin and substrate (stickiness ratio = 2.4 ± 0.9). This tendency is even favored when thrombin is bound to TM both in the absence and presence of Ca++ (stickiness ratio = 9 ± 6 in the absence of Ca++ and 16 ± 10 in the presence of Ca++). Altogether these results demonstrate that TM is able to positively affect both the molecular encounter and the kinetics of the early catalytic events of the thrombin-Protein C interaction.
HLA Genotype of Patients with Severe Haemophilia A due to Intron 22 Inversion with and without Inhibitors of Factor VIII
Authors: Oldenburg J , Picard J K , Schwaab R , Brackmann H H , Tuddenham E G D , Simpson E
Published: July 2018

Molecular genetic studies have shown that development of antibodies to factor VIII (inhibitors) occurs most frequently in patients with severe haemophilia due to major gene lesions including inversions, stop codons and large deletions. Previous studies of HLA type were performed on inhibitor and non-inhibitor patients with diverse uncharacterised mutations which may have confounded detection of significant associations. We therefore selected a group of patients with a single mutation type, the prevalent intron 22 inversion, with or without inhibitors, to determine HLA genotype. Seventy-one such patients, 42 without and 29 with inhibitors (13 high, 9 low and 7 transient responders) were genotyped for MHC Class I HLA-A, -B, -C and Class II HLA-DQA, -DQB and -DRB loci. No strong correlation of any HLA-allele to inhibitor or non-inhibitor status was found. However, alleles of the haplotype HLA-A3, HLA-B7, HLA-C7, HLA-DQA0102, HLA-DQB0602, HLA-DR15 occurred more often in inhibitor patients. Since the alleles of this extended haplotype are common in the North European population only a very strong association would achieve statistical significance. Further studies of groups of patients similar to those studied here will be needed to confirm or exclude this association.
Coagulation Activation Markers in the Prediction of Venous Thrombosis after Elective Hip Surgery
Authors: Cofrancesco E , Cortellaro M , Corradi A , Ravasi F , Bertocchi F
Published: July 2018

Background. Despite prophylaxis, deep vein thrombosis (DVT) after hip surgery continues to occur frequently. Thus it would be helpful if before surgery patients at higher risk of DVT could be identified and more adequate prophylaxis given. As part of an international study on the prevention of DVT after total hip replacement, we investigated whether preoperative levels of three coagulation activation markers, prothrombin fragment F1+2 (F1+2), thrombin-antithrombin III complexes (TAT) and D-dimer, correlate with results of postoperative venography.

Methods. 159 patients undergoing total hip replacement were randomized to receive 10, 15 or 20 mg desirudin bid or 5000 IU unfractionated heparin tid immediately before surgery and then for 11 days, until bilateral venography was performed. Preoperative F1+2, TAT and D-dimer plasma levels were measured using ELISA procedures. As no difference among anticoagulant treatments or in the interaction between treatments and DVT was detected for any of the three variables, results are reported as pooled data.

Findings. The frequency of DVT was 18.8% in the low (0.75-1.33 nM) vs 65.7% in the high third of distribution (1.77-3.47 nM) of F1+2 (p ˂.001), 27.3% in the low (2.00-2.50 μg/1) vs 57% in the high third (5.10-61.00 μg/l) of TAT (p = .042), and 29.4% in the low (39-59 μg/1) vs 57.1% in the high third (129-651 μg/1) of D-dimer (p = .051).

Interpretation. Preoperative F1+2, TAT and D-dimer levels are associated with the risk of development of DVT after total hip replacement.
Expression and Characterization of Recombinant Porcine Plasminogen Activator Inhibitor-1
Authors: Bijnens A P , Knockaert I , Cousin E , Kruithof E K O , Declerck P J
Published: July 2018

Porcine models are, among other animal models, very suitable for in vivo investigations in the vascular field especially with respect to the possible relationship between atherosclerosis and thrombosis. In order to use this model to define the in vivo role of PAI-1, the characterization of porcine PAI-1 and its availability for the generation of immunological tools are a prerequisite. Porcine plasminogen activator inhibitor-1 (poPAI-1) cDNA was isolated from a cDNA library prepared from cultured porcine aortic cells and characterized in comparison with PAI-1 cDNA’s from other species including human, bovine, rabbit, rat and murine. Subsequently the DNA sequence coding the mature protein was cloned into an appropriate vector for expression in Escherichia coli and recombinant porcine PAI-1 was purified and characterized. On SDS-PAGE the apparent molecular weight was estimated to be 45 kDa, identical to the molecular weight of human PAI-1. The purified recombinant porcine PAI-1 (rpoPAI-1) had a specific activity of 508,800 ± 800 U/mg (mean ± SD, n = 3) towards human tissue-type plasminogen activator (ht-PA) and a functional half-life in vitro of 2.1 ± 0.8 h (n = 3). Incubation with a two fold molar excess of ht-PA (n = 3) or human urokinase-type plasminogen activator (hu-PA, n = 2) followed by analysis by SDS-PAGE revealed reaction products corresponding to active (71 ± 7% resp. 96 ± 3.6%), latent (12 ± 0.4% resp. 2.6 ± 2.4%) and substrate (16.6 ± 6.8% resp. 1.5 ± 1.3) forms. Inactivated samples of porcine PAI-1 could be reactivated with guanidinium chloride up to 52% of its original specific activity towards t-PA and u-PA. The second order rate constant of inhibition of ht-PA was 1.64 ± 0.37 1 07M-1 s-1 (n = 9). In gel filtration rpoPAI-1 in buffer eluted at a volume corresponding to 24 kDa, whereas in the presence of porcine plasma, the molecular form containing PAI-1 activity eluted at a volume corresponding to 330 kDa, presumably as a consequence of binding of active PAI-1 to vitronectin.

Taken together, these data demonstrate that no obvious functional differences exist between human and porcine PAI-1.
Impaired Prothrombin Consumption in Bernard-Soulier Syndrome Is Corrected In Vitro by Human Factor VIII
Authors: Bellucci S , Girma J P , Lozano M , Meyer D , Caen J P
Published: July 2018

The Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein lb (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII.'C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.
Outcome of Neonates with Vein of Galen Malformation Presenting with Severe Heart Failure: A Case Series
Authors: De Rosa Gabriella , De Carolis Maria , Tempera Alessia , Pedicelli Alessandro , Rollo Massimo , Luca Ersilia , De Luca Daniele , Conti Giorgio , Piastra Marco , Morena Tony
Published: July 2018

Background Neonatal presentation of vein of Galen aneurysmal malformations (VGAMs) complicated by cardiac failure and pulmonary hypertension is frequently associated with a poor prognosis. Interventional neuroradiology with embolization can offer a chance for survival, although neurological damage can represent a limitation.

Objective This article determines if aggressive intensive care and drug management of cardiac failure before urgent embolization can influence morbidity and mortality.

Patients and Methods Twelve infants (7 boys, 5 girls) were diagnosed with symptomatic vein of Galen malformations in the neonatal period during the period 2000 to 2014. Due to high output cardiac failure, endovascular treatment was attempted as soon as stabilization was achieved.

Results Endovascular procedures successfully reverted cardiac failure in 5 patients who survived without significant neurological damage, while in 7 patients the causes of death were refractory cardiac failure, multiorgan failure, and severe brain damage. Bidimensional echocardiography assessment was performed at presentation and after early embolization procedures.

Conclusion Aggressive intensive care approach to heart failure and pulmonary hypertension leading to early neurointervention results in good survival rates with low morbidity even in cases of high-risk neonatal VGAM. Combined hemodynamic treatment can improve outcome in neonates with cardiac failure secondary to VGAM, although there is the risk of precipitating systemic hypoperfusion and renal failure. A moderate prematurity may not prevent both interventional approach and good outcome.
Predicting the Outcome of Total Knee Arthroplasty Using the WOMAC Score: A Review of the Literature
Authors: Walker Lucy Camilla , Clement Nick D. , Deehan David J.
Published: July 2018

It is estimated that up to a third of recipients of total knee arthroplasty (TKA) experience chronic pain postoperatively. However, there are no clear indications within the literature that predict which patients are at higher risk of being dissatisfied with their TKA. The Western Ontario and McMaster University Osteoarthritis Index (WOMAC) is one of the most commonly used, patient-reported outcome measures in patients with lower limb osteoarthritis. This review discusses the available evidence surrounding the predictability of the outcome of TKA using the WOMAC score as well as considering further patient factors that have been implicated in the level of improvement post TKA. It may be concluded from the available literature that a combination of knee scores and patient factors would be the most accurate way of predicting those patients most likely to have a good outcome from their TKA. There is some disparity within the literature about which patient factors and reported outcome measure scores lead to a positive postoperative outcome. Patient expectations following the procedure also need to be evaluated, as objective measures on a scoring system do not necessarily equate with the subjective patient experience.
Value of Systemic Staging in Asymptomatic Early Breast Cancer
Authors: Soares Gregório Pinheiro , Pereira Allan Andresson Lima , Vilas Boas Mariana Silva , Vaisberg Victor Van , Magalhães Maria Cristina Figueroa , Linck Rudinei Diogo Marques , Mano Max Senna
Published: July 2018

Objective Metastases are rare in early breast cancer (EBC), and international guidelines recommend against routine systemic staging for asymptomatic patients. However, imaging exams remain widely employed in the clinical practice. The aim of the present study is to evaluate the value of imaging for systemic staging in EBC.

Methods A retrospective analysis of newly-diagnosed breast cancer (BC) patients was performed. Clinical data including BC subtype, stage, presence of symptoms at diagnosis and instrumental procedures performed for staging were recorded.

Results A total of 753 patients were included, with a median age of 57 years. The majority of the patients underwent at least 1 imaging procedure (91%); had invasive ductal carcinoma (83.5%); histological grade 2 (51.4%); stage II (61.8%); and luminal subtype (67.9%). Among the 685 (91%) patients who underwent any radiologic staging, distant metastases (DMs) were detected in 32 (4.7%). In the univariate analyses, stage IIb and pathological lymph node involvement (pN1) showed a statistically significant association with the presence of DMs, versus only a trend for triple negative and human epidermal growth factor receptor 2 (Her2) positive subtype. In an exploratory analysis performed in this same subgroup, when unfavorable biology (triple negative or Her2 positive) was present, patients had a DM rate of 14.4%, one of the highest reported at this stage of the disease.

Conclusion Early breast cancer has a low prevalence of DM at the initial evaluation, and systemic staging of asymptomatic, unselected patients is not warranted as a routine practice. However, we have identified subgroups of patients to whom a full staging could be indicated.
Some multidimensional integrals in number theory and connections with the Painlevé V equation
Authors: Basor Estelle , Ge Fan , Rubinstein Michael O.
Published: July 2018

We study piecewise polynomial functions γk(c) that appear in the asymptotics of averages of the divisor sum in short intervals. Specifically, we express these polynomials as the inverse Fourier transform of a Hankel determinant that satisfies a Painlevé V equation. We prove that γk(c) is very smooth at its transition points and also determine the asymptotics of γk(c) in a large neighbourhood of k = c/2. Finally, we consider the coefficients that appear in the asymptotics of elliptic aliquot cycles.
Ticks and dust mites: invasive and health-affecting borderline organisms.
Authors: Simoni S. , Grandi G.
Published: July 2018

According to the situations and cases presented here, organisms belonging to tick and house dust mite groups can be included within alien invasive pests representing a risk for human health. Rhipicephalus sanguineus, the brown dog tick, a vector for many tick-borne pathogens, is able to efficiently colonize households at quite wide ranges in temperature and relative humidity. Cases of the spread of this tick by movements of pets and people are reported. House dust mites and main storage mites - although not strictly alien species - can act not only as allergens' disseminators but even as potential indicators of environmental quality in different settings (newly colonized areas, both geographic and within human dwellings). The impact of alien mites affecting agricultural production has been generally measured by the ecological consequences on biodiversity, but the evaluation of the effects of these species on human health has been mostly neglected. Trading, travelling and modified home conditions can increase the chance for diffusion of ticks and mites affecting human health, i.e. causing higher exposures to vector-borne pathogens and allergens, respectively. A more interdisciplinary approach that takes into account both the dynamics in ecological evaluation and the consequences on human health of these organisms is needed.
Is a Reported Penicillin Allergy Sufficient Grounds to Forgo the Multidimensional Antimicrobial Benefits of β-Lactam Antibiotics?
Authors: Sakoulas George , Geriak Matthew , Nizet Victor
Published: July 2018

The majority of patients with reported penicillin allergy are not allergic when tested or challenged. Penicillin allergy testing has been shown to significantly reduce annual healthcare expenditures. Data have emerged showing β-lactams have multidimensional antibacterial effects in vivo, far beyond what is appreciated in standard bacteriological susceptibility testing media. These include enhancing bacterial killing by the innate immune system. Supporting the clinical relevance of these secondary underappreciated effects are recent clinical and pharmacoeconomic analyses that show worse outcomes in patients with reported penicillin allergies who receive non-β-lactam antibiotics when compared to their non-penicillin-allergic counterparts. This is particularly relevant in the treatment of Staphylococcus aureus bacteremia. This article reviews the tremendous advantages offered by β-lactam therapy and makes a strong case that the debunking of false penicillin allergies through a detailed allergy history and penicillin allergy testing should be a vital component of antimicrobial stewardship practices.
Assessment of Students’ Perception of the Nutrition Curriculum in a Canadian Undergraduate Medical Education Program
Authors: Hanninen Stacy , Rashid Mohsin
Published: July 2018

Background

Nutrition plays an important role in diseases, and physicians need to be proficient in providing nutrition counselling to patients. There is limited information regarding nutrition education in Canadian medical schools.

Objective

The objective of this study was to investigate students’ perspectives about nutrition training provided in the undergraduate medical education program at Dalhousie University.

Methods

All medical students in their second, third, and fourth years of training at Dalhousie University were surveyed online with a 23-item questionnaire that included 10 nutrition competencies.

Results

Of 342 students, 89 (26%) completed the survey. Using a five-point Likert scale, ranging from one, indicating ‘very dissatisfied/strongly disagree’ to five, indicating ‘very satisfied/strongly agree,’ the mean overall satisfaction with nutrition curriculum was 2.9 ± 0.81. Perceived competency in nutrition assessment had the highest mean satisfaction rating (3.98 ± 0.89). There was more variance on perceived competency, with other aspects of training including basic nutrition principles (3.51 ± 0.92), disease prevention (3.14 ± 1.12), disease management (3.48 ± 1.00), role of dietitians (2.97 ± 1.05), credible nutrition sources (3.14 ± 1.09), dietary assessment (2.82 ± 1.11), lifecycle nutrition (2.67 ± 1.09), food security (2.4 ± 0.95) and malnutrition (2.74 ± 0.93). Med-4 students agreed significantly more than Med-2 students regarding confidence about their understanding of the role of dietitians. Students recommended a longitudinal nutrition program, inclusion of dietitians as educators, and provision of evidence-based resources in the curriculum. The majority (79%) agreed that more nutrition instruction is needed. Satisfaction with nutrition education has not improved since 2010, despite curricular changes.

Conclusions

Medical students’ satisfaction with nutrition education remains problematic. They want more nutrition training. Ongoing assessment and student feedback is important to make changes and improvements in the nutrition curriculum.
Stochastic Variational Inference for Bayesian Phylogenetics: A Case of CAT Model
Authors: Dang Tung , Kishino Hirohisa
Published: July 2018

The pattern of molecular evolution varies among gene sites and genes in a genome. By taking into account the complex heterogeneity of evolutionary processes among sites in a genome, Bayesian infinite mixture models of genomic evolution enable robust phylogenetic inference. With large modern data sets, however, the computational burden of Markov chain Monte Carlo sampling techniques becomes prohibitive. Here, we have developed a variational Bayesian procedure to speed up the widely used PhyloBayes MPI program, which deals with the heterogeneity of amino acid profiles. Rather than sampling from the posterior distribution, the procedure approximates the (unknown) posterior distribution using a manageable distribution called the variational distribution. The parameters in the variational distribution are estimated by minimizing Kullback-Leibler divergence. To examine performance, we analyzed three empirical data sets consisting of mitochondrial, plastid-encoded, and nuclear proteins. Our variational method accurately approximated the Bayesian phylogenetic tree, mixture proportions, and the amino acid propensity of each component of the mixture while using orders of magnitude less computational time.