bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Antibodies Induced by Human Factor VIII in a Murine Knockout Model of Hemophilia A
Authors: Reipert Birgit , Ahmad Rafi , Turecek Peter , Schwarz Hans
Published: March 2018

To investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy, knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.
In situ Analysis of Tissue Factor-Dependent Thrombin Generation in Human Atherosclerotic Vessels
Authors: Westmuckett Andrew , Lupu Cristina , Goulding David , Das Saroj , Kakkar Vijay , Lupu Florea
Published: March 2018

Tissue factor (TF) is expressed in human atherosclerotic plaques where it may contribute to the thrombogenicity of the lesions and their progression toward unstable syndromes and acute myocardial infarction. In this study we tested the hypothesis that thrombin generation takes place in the lesion. Localisation of TF, factor VII (FVII), factor X/Xa (FX/Xa), thrombin, thrombin receptor PAR-1 and FXa receptor EPR-1 was done by immunostaining, ligand binding, or immunogold electron microscopy. Quantitation of TF antigen was done using a modified ELISA on fixed tissue sections. The amount of antigen was correlated with the pattern and intensity of immunostaining as detected on consecutive sections using confocal microscopy. TFdependent generation of FXa on cryosections was used to assess the functional activity of TF. Active thrombin was detected using hirudin as a specific probe, followed by anti-hirudin IgG. Our light microscopy and immunogold electron microscopy results showed that the factors involved in TF-dependent coagulation are localised in atherosclerotic plaques in close proximity and colocalise with active thrombin and fibrin deposits. We have detected 3 to 7-fold increase of TF antigen and TF-dependent FXa generation in atherosclerotic vessels as compared with controls. Hirudin binding proved that active thrombin is present within the lesions. In conclusion, our data show that active coagulation factors are generated within atherosclerotic lesions and co-localise with their cellular receptors. These findings may suggest possible roles of the TF-dependent coagulation pathway in the intramural fibrin deposition and the progression of the atherosclerotic lesions.
Thrombophilic Polymorphisms Are Common in Women with Fetal Loss without Apparent Cause
Authors: Sarig G. , Weiner Z. , Younis J. , Blumenfeld Z. , Lanir N. , Brenner B.
Published: March 2018

An association between fetal loss and thrombophilia has recently been described but has not been yet fully elucidated. We have evaluated prospectively the prevalence of the three common thrombophilic polymorphisms (TP) factor V G1691A (Leiden), thermolabile-methyl-enetetrahydrofolate reductase (TL-MTHFR) C677T and factor II G20210A mutations, in 76 women with fetal loss (≥3 in first, ≥2 in second, ≥1 in third trimester) without apparent cause and 106 controls without fetal loss. Thirty seven out of 76 (49%) of the women in the fetal loss group had at least one TP compared to only 23/106 (22%) in the control group (p = 0.0001). Factor V-Leiden was more common in the fetal loss group 24/76 (32%) compared to the control group 11/106 (10%) (OR = 4.0, 95% CI: 1.8-8.8, p <0.001). Five of the 76 patients (7%) were homozygous for factor V-Leiden compared to none of the controls (p = 0.012). A trend, albeit no statistically significant difference was found between women with fetal loss and control groups regarding factor II G20210A (8% vs. 4% respectively, OR = 2.2, 95% CI: 0.6-8.0, p = 0.23) and MTHFR C677T (18% vs. 10% respectively, OR = 1.95, 95% CI: 0.83-4.6, p = 0.12). Combined TP were documented in 6/76 (8%) patients compared to 1/106 (1%) in controls (OR = 9.0, 95% CI: 1.1-76, p = 0.02). Second or third trimester fetal loss were more common cause of pregnancy termination in 37 patients with TP compared to 39 patients without TP (57/158 (36%) vs. 23/135 (17%) respectively, (p = 0.0004). Thrombophilic polymorphisms are common in women with fetal loss without apparent cause and are associated with late pregnancy wastage. Combinations of TP increase the risk for fetal loss.
Helicobacter Pylori Infection and the Risk of Myocardial Infarction: Role of Fibrinogen and Its Genetic Control
Authors: Zito Francesco , Di Castelnuovo Augusto , D’Orazio Andria , Negrini Riccardo , De Lucia Domenico , Benedetta Donati Maria , Iacoviello Licia
Published: March 2018

The contribution of Helicobacter pylori (HP) infection to the risk of myocardial infarction was evaluated. The role of fibrinogen and its genetic control as a possibile mechanism by which HP may influence myocardial infarction risk was explored in this context. A case-control study was performed in 101 patients with myocardial infarction and in 101 controls.

HP infection was associated with an increased risk of myocardial infarction independently for confounding variables (OR 4.1, CI95: 1.8-9.4). HP infection was significantly associated with higher levels of fibrinogen, both in cases and in controls. Furthermore, there was an additive effect of HP infection and B2 allele of BclI fibrinogen poly-morphism in increasing fibrinogen levels. HP infection showed a stronger effect on the risk of myocardial infarction in B2 allele carriers (OR 7.6, CI95: 1.8-31.6) as compared to subjects carrying the B1B1 genotype (OR 3.3, CI95: 1.2-9.2).

We showed that a previous HP infection is a risk factor for myocardial infarction. An increase in fibrinogen levels is a possible mechanism by which HP may act. Concomitant conditions, like a genetic predisposition in increasing fibrinogen levels, seem to further increase the effect of HP on myocardial infarction risk.
A Novel Splice Acceptor Site Mutation which Produces Multiple Splicing Abnormalities Resulting in Protein S Deficiency Type I
Authors: Tatewaki Hideki , Iida Hiroko , Nakahara Mutsuko , Tsuda Hiroko , Kinoshita Sachiko , Kanaji Taisuke , Yoshida Nobuyuki , Miyazaki Sumio , Hamasaki Naotaka
Published: March 2018

In an attempt to explore the molecular mechanisms for protein S deficiency, a patient with such a deficiency was examined at the DNA, RNA and protein levels. Nucleotide analyses revealed that the proband, the mother and the grandmother had a G → C substitution in the invariant AG dinucleotide at the splicing acceptor site of intron A/exon 2. This patient was heterozygous for this substitution and the mutant allele was inherited from the proband’s mother and grandmother. Reverse transcription-polymerase chain reaction analysis demonstrated several kinds of splicing abnormalities such as exon skipping and cryptic splicing, in addition to correct splicing. Semiquantitation of mRNA for the protein S gene revealed that the amount of the proband’s mRNA was reduced to 60% of normal. Thus, this mutation impaired the normal processing of mRNA for the protein S gene, resulting in the subject’s severe protein S deficiency.
Regulation of Prothrombinase Activity by Protein S
Authors: Butenas Saulius , Golden Neal , Mann Kenneth , van't Veer Cornelis
Published: March 2018

The independent effect of protein S as prothrombinase inhibitor has been proposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrations of protein S (300 nM) equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low phospholipid concentrations (~0.2 μM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd ~ 10-8 M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin generation rates at low phospholipid in the presence of protein S based on the diminished available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the phospholipid prior to the addition of factor Xa, factor Va and pro-thrombin. The results indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course of the physiological thrombin generation. The effect of protein S was therefore studied on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation when surface is provided at physiologic concentrations using isolated platelets (2 × 108/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effects of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generation, indicate that protein S and protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.
The Effects of Unfractionated Heparin on Survival in Patients with Malignancy – A Systematic Review
Authors: Hettiarachchi Rohan , Vink Roel , Büller Harry , Smorenburg Susanne
Published: March 2018

Clinical and experimental studies have suggested that unfractionated heparin (UFH) effects malignancy progression. We reviewed all published clinical reports concerning the effects of UFH, as compared to no treatment on survival of cancer patients. Studies were classified on methodological strength and subdivided as to whether therapeutic or prophylactic dosages of UFH were used. Mortality rates after 3 years were extracted or calculated. One randomized study that evaluated the use of UFH in therapeutic dosages in patients with small cell lung carcinoma reported on an improved survival (odds ratio (OR) 0.64; 95% confidence interval (CI): 0.25 to 1.62). A detrimental effect was observed in 2 randomized studies which investigated the effects of intraportal UFH treatment in a prophylactic dose after surgery for gastrointestinal cancer (OR 1.66; 95% CI: 1.02 to 2.71). In contrast, level 2 studies in which either therapeutic or prophylactic dosages of UFH on mortality of patients with gastrointestinal cancer were evaluated, showed OR of 0.58 (95% CI; 0.11-3.13) and 0.65 (95% CI 0.51 to 0.84), respectively. We conclude that there is no convincing evidence of either positively or negatively effects of UFH on survival of patients with malignancy.

Harry R. Büller is an Established Investigator of the Dutch Heart Foundation
Expression of Tissue Factor and Tissue Factor Pathway Inhibitor in situ in Laryngeal Carcinoma
Authors: Zacharski Leo , Ruciñska Monika , Zimnoch Lech , Jaromin Jacek , Rózañska-Kudelska Malgorzata , Kisiel Walter , Kudryk Bohdan , Wojtukiewicz Marek
Published: March 2018

The blood coagulation mechanism may support tumor progression by several mechanisms including promotion of cell proliferation and angiogenesis. Immunohistochemical procedures were applied to AMeX-fixed sections of twelve cases of squamous cell carcinoma of the larynx obtained at surgical resection to determine the presence and distribution of tissue factor (TF), tissue factor pathway inhibitor (TFPI), other coagulation factors, fibrinogen, and fibrin in situ. TF antigen was present in normal squamous epithelial cells and tumor cells, predominantly in immature tumor cells in the vicinity of the host-tumor interface. Tumor cells stained also for factors VII and X. Staining for TFPI antigen was demonstrated in the connective tissue stroma adjacent to the tumor, in microvascular endothelial cells, and in normal squamous epithelial cells. Fibrinogen and factor XIIIa were distributed throughout the tumor connective tissue stroma. Fibrin (thrombin-cleaved fibrinogen) was detected at the host-tumor interface and along the margins of tumor nodules. Tumor cells in carcinoma of the larynx express a functional, TF-initiated pathway of blood coagulation. Interpretation of these findings together with the results of clinical trials of inhibitors of TF-induced coagulation activation versus effects of inhibitors of TF expression suggest novel approaches to the experimental therapy of laryngeal carcinoma.
Matrix Gla Protein Synthesis and Gamma-carboxylation in the Aortic Vessel Wall and Proliferating Vascular Smooth Muscle Cells
Authors: Cain Dean , Sane David , Wallin Reidar
Published: March 2018

Matrix GLA protein (MGP) is an inhibitor of calcification in the arterial wall and its activity is dependent upon vitamin K-dependent γ-carboxylation. This modification is carried out by a warfarin sensitive enzyme system that converts specific Glu residues to γ-carboxyglutamic acid (GLA) residues. Recent studies have demonstrated that the γ-carboxylation system in the arterial wall, in contrast to that in the liver, is unable to use vitamin K as an antidote to warfarin.

By use of immunohistochemistry we demonstrate that MGP is expressed in the arterial wall and immunocytochemistry localized the MGP precursors to the endoplasmic reticulum in vascular smooth muscle cells. Resting smooth vascular muscle cells in the aortic wall and proliferating cells from explants of the aorta have all the enzymes needed for γ-carboxylation of MGP. However, when compared to the liver system, expression of the enzymes of the γ-carboxylation system in vascular smooth muscle cells is different. Of particular interest is the finding that the specific activity of the warfarin sensitive enzyme vitamin K epoxide reductase is 3-fold higher in vascular smooth muscle cells than in liver. DT-diaphorase, which catalyses the antidotal pathway for vitamin K reduction in liver, is 100-fold less active in resting vascular smooth muscle cells than in liver. Data obtained from an in vitro γ-carboxylation system suggest that the antidotal pathway catalyzed by DT-diaphorase in the vessel wall is unable to provide the carboxylase with enough reduced vitamin K to trigger γ-carboxylation of MGP. This finding provides an explanation to the inability of vitamin K to work as an antidote to warfarin intoxication of the arterial wall. Therefore the vitamin K dependent γ-carboxylation system in the arterial wall share a common feature with the system in bone cells by being unable to utilize vitamin K as an antidote.
Antithrombin Cambridge II (Ala384Ser): Clinical, Functional and Haplotype Analysis of 18 Families
Authors: Daly M. E. , Tait R. C. , Walker I. D. , Brown K. , Beauchamp N. J. , Preston F. E. , Gyde H. , Harper P. L. , Carrell R. W. , Perry D. J.
Published: March 2018

Thirty-one individuals from 18 unrelated families with antithrombin deficiency have been identified as having a single point mutation within codon 384 (13268 GCA→TCA) resulting in an alanine to serine substitution. Six families (11 individuals) were identified by the screening of individuals with thromboembolic disease or with a family history of thromboembolic disease, whilst the remaining 12 families (20 individuals) were identified by screening of asymptomatic blood donors. Four individuals had a history of venous thrombotic disease, a further 2 gave a history of superficial thrombophlebitis but the remaining 25 individuals were asymptomatic. Affected individuals demonstrated normal immunological levels of antithrombin but a decrease in anti-IIa activity in the presence of heparin. Haplotype analysis was used to examine the possibility of a founder effect to explain the high frequency of this non-CpG mutation. 29/31 individuals showed a single common “core” haplotype, the only variation existing in the number of copies of an (ATT)n repeat polymorphism – 13, 14, 15 or 17. The results suggest that at most there are four independent origins for this mutation.
The Canine Factor VIII cDNA and 5’ Flanking Sequence
Authors: Cameron Cherie , Notley Colleen , Hoyle Susan , McGlynn Leslie , Hough Christine , Kamisue Seiki , Giles Alan , Lillicrap David
Published: March 2018

Factor VIII is a trace plasma glycoprotein involved as a cofactor in the activation of factor X by factor IXa. Inherited deficiency of factor VIII results in the X-linked bleeding disorder hemophilia A which has been documented in humans, horses, sheep and dogs. In this report, the putative proximal promoter, 5’ untranslated region, complete coding sequence and 3’ untranslated region of the canine factor VIII gene have been characterized. When compared to the human gene, the 5’ flanking region shows conservation of transcription factor binding sites in the 5’ untranslated region. Alignment of the amino acid sequence with that of the previously reported human, mouse and pig proteins demonstrates sequence identity of between 77 and 92% for the A1, A2, A3, C1 and C2 domains but an identity of only between 44 and 62% for the central B domain. The three thrombin cleavage sites are conserved in the canine sequence as are the protein C cleavage sites and the von Wille-brand factor binding region. In addition, all six tyrosine residues that are known to undergo sulfation in the human protein are conserved in the dog. The 3’ untranslated region of the canine gene extends 1.5 kilo-bases. The initial 700 basepairs of this sequence are highly GC rich and the sequence terminates with 2 alternative potential polyadenylation sites. The knowledge of this sequence, in combination with a well described canine model of hemophilia A, provides the necessary starting point for studies addressing the long-term evaluation of factor VIII gene therapy using a homologous transgene.
Characterization of Soluble Thrombomodulin Fragments in Human Urine
Authors: Furutani Masayuki , Hiraishi Sayuri , Ishii Hidemi , Nakano Masahiko
Published: March 2018

The soluble thrombomodulin (TM) subspecies in human urine detected by polyclonal anti-human TM IgG were isolated and characterized. 105, 85, 80, 56, 33, 31 and 28 kDa subspecies under reducing conditions was comparable to 78, 66, 56, 200, 52, 30 and 25 kDa under non-reducing conditions, respectively, in the two-dimensional electrophoresis. Each subspecies under non-reducing conditions, except the 200 and 52 kDa molecules, was constituted of single subspecies, whereas the 200 and 52 kDa molecules were constituted of the tetramer of the 56 kDa subspecies of reducing conditions and a dimer of the 33 kDa subspecies, respectively. NH2-terminal amino acid sequences of the 105, 85 and 80 kDa subspecies maintained Ala1-Pro2-Ala3- of intact human TM, however, 56, 33, 31 and 28 kDa subspecies started from Glu137-Gln138-, Gln214-Gly215-, Ser228-Val229- and Ala240-Ile241-, respectively. All subspecies obtained under non-reducing conditions exhibited cofactor activity for thrombin-dependent protein C activation ranging from 58 to 162 pmol APC/min/nmol TM at 0.4 mM Ca2+ indicating that all of the subspecies maintained the fourth to sixth repeat of epidermal growth factor-like structure of intact TM. 85, 80, 56, 33, 31 and 28 kDa subspecies were suggested to lack both chondroitin sulfate glycosaminoglycan (CSGAG), transmembrane and cytoplasmic domains of intact TM, while 105 kDa subspecies lack only CSGAG from the results of kinetic properties and the interaction with phospholipid vesicles composed from phosphatidylcholine and phosphatidylethanolamine.
Effect of SR121566A, a Potent GP IIb-IIIa Antagonist on Platelet-mediated Thrombin Generation In Vitro and In Vivo
Authors: Herault J. P. , Peyrou V. , Savi P. , Bernat A. , Herbert J. M.
Published: March 2018

The effect of SR121566A, a new non-peptide GP IIb-IIIa antagonist was studied in vitro with regard to thrombin generation in platelet rich plasma and in vivo on stasis-induced venous thrombosis in the rabbit. SR121566A inhibited ADP-, arachidonic acid- and collagen-induced human platelet aggregation with IC50 values of 46 ± 7.5, 56 ± 6 and 42 ± 3 nM, respectively. In the same experimental conditions, SR121566A strongly inhibited thrombin generation triggered by low concentrations of tissue factor. SR121566A reduced in a dose-dependent manner both the area under the curve and the thrombin peak concentration but did not affect the lag phase (defined as the time until 10 nM thrombin was generated). Aspirin (100 µg/ml) did not affect thrombin generation.

One hour after intravenous administration to rabbits, SR121566A exhibited a potent ex vivo inhibitory effect against ADP-, arachidonic acid- and collagen-induced platelet aggregation. The ID50 were 0.6 ± 0.25, 0.7 ± 0.08 and 0.13 ± 0.08 mg/kg, respectively. The ability of aspirin and SR121566A to affect venous stasis was determined in a stasis-induced venous thrombosis model in rabbits under high and low thrombogenic challenges. While aspirin was ineffective in both conditions, SR121566A significantly inhibited thrombus formation under low thrombogenic challenge demonstrating for the first time that a potent non-peptide platelet GP IIb-IIIa antagonist inhibits thrombin generation in vivo and exhibits a strong antithrombotic effect with regard to stasis-induced venous thrombosis. These results therefore confirm the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation but also emphasise the key role of platelets in the development of venous thrombosis, most likely through activation of the GP IIb-IIIa complex.
Microvesicles Bind Soluble Fibrinogen, Adhere to Immobilized Fibrinogen and Coaggregate with Platelets
Authors: Solum Nils Olav , Brosstad Frank , Pedersen Turid , Kveine Marit , Holme Pål André
Published: March 2018

In the present study we have investigated whether platelet derived microvesicles can bind soluble fibrinogen, bind to immobilized fibrinogen, and coaggregate with platelets. Flow cytometry was used for studies on binding of soluble fibrinogen and coaggregation, whereas ELISA wells were used to study binding of microvesicles to immobilized fibrinogen. Biotinylated microvesicles produced by stimulation with A23187, thrombin or SFLLRN of platelets which had been surface-labelled with biotin, were used both for the coaggregation experiments and for the binding studies with immobilized fibrinogen. Unlabelled microvesicles and biotinylated fibrinogen were employed when studying binding of soluble fibrinogen to the microvesicles. For the flow cytometry, the biotinylated proteins were reacted with avidin or streptavidin which was PE-conjugated, whereas the same substances were conjugated with alkaline phosphatase for the ELISA studies. The microvesicles formed after stimulation of platelets by SFLLRN or A23187 clearly bound the soluble, biotinylated fibrinogen. Moreover, isolated biotinylated microvesicles added to washed platelets prior to activation, were associated to the microaggregates that formed after stimulation. A significant binding of biotinylated microvesicles to immobilized fibrinogen could also be detected. The binding of micro-vesicles to soluble and immobilized fibrinogen and association to platelets was clearly specific and at least partly dependent on the GPIIb-IIIa complex, as all of these phenomena could be prevented or reduced by addition of the c7E3 Fab which blocks the activated form of this receptor complex. From these in vitro results it is clear that microvesicles can bind to immobilized fibrinogen, bind soluble fibrinogen and are able to coaggregate with platelets. It may be speculated that these results also reflect a haemostatic role of microvesicles in vivo.
International Collaborative Study for the Calibration of a Proposed Reference Preparation for Thromboplastin, Human Recombinant, Plain
Authors: Chantarangkul Veena , Negri Barbara , Clerici Marigrazia , Mannucci Pier Mannuccio , Tripodi Armando
Published: March 2018

Stocks of the International Reference Preparation (IRP) for thromboplastin, human, plain, coded BCT/253 and held by the World Health Organization (WHO) are nearly exhausted and must be replaced. For practical reasons the choice of the replacement candidate was restricted to two available human recombinant preparations which were coded as X/95 and Y/95 and calibrated in an international collaborative study involving 19 laboratories from Europe, Australia, Canada and Argentina. To minimize the differences between routes of calibration, the two candidates were calibrated against the existing WHO-IRP from human, rabbit and bovine origin and the final ISI was the resultant average value. On the basis of predefined criteria (i.e., within- and between-laboratory precision of the calibration and the conformity to the calibration model), X/95 was the preferred candidate. The assigned ISI (SE of the mean) value is 0.940 (0.0060) and the interlaboratory coefficient of variation 4.7%.
Circulating ICAM-1 and VCAM-1 in Peripheral Artery Disease and Hypercholesterolaemia: Relationship to the Location of Atherosclerotic Disease, Smoking, and in the Prediction of Adverse Events
Authors: Seigneur Martine , Steiner Michael , Miller J. , McCollum Charles , Blann Andrew
Published: March 2018

We examined the relationship of soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) with smoking and hypercholesterolaemia in peripheral artery disease (PAD). Serum samples were obtained from 119 patients with objectively-proven PAD, 39 patients with hypercholesterolaemia but asymptomatic for PAD, and 132 age and sex matched asymptomatic controls. Using ELISAs, we found increased sICAM-1 and sVCAM-1 (both p <0.01) in the patients with PAD relative to the controls, but no significant change in patients with hypercholesterolaemia. However, the effect for sVCAM-1 was lost when smoking was entered as a covariate. Only sICAM-1 was higher in patients with PAD in the femoral/iliac arteries compared to the carotid arteries (p <0.05). In a 39-month follow-up of 112 patients with PAD, increased ICAM-1 weakly (univariate p <0.05) predicted those 57 whose disease progressed (i.e. to end points such as myocardial infarction and arterial surgery). However, high fibrinogen was a much better (univariate p = 0.001, multivariate p <0.05) predictor of disease progression. We suggest (i) that increased levels of sVCAM-1 in atherosclerosis are due to smoking, (ii) that increased sICAM-1 is independent of this risk factor, (iii) that both these changes are independent of hypercholesterolaemia, and (iv) that increased sICAM-1 is a weak predictor of disease progression in peripheral atherosclerosis.
Evaluation of Thrombopoiesis in Thrombocytopenic Disorders by Simultaneous Measurement of Reticulated Platelets of Whole Blood and Serum Thrombopoietin Concentrations
Authors: Yoneyama Akiko , Shirai Jun , Ishida Tateru , Shoda Eriko , Miyazaki Kouji , Sunaga Sinji , Horie Ryoichi , Aoki Katunori , Koike Kazuhiko , Ogata Iturou , Tahara Tomoyuki , Kato Takashi , Nakahara Kazuhiko , Kariya Toshitugu , Higashihara Masaaki , Koike Yukako
Published: March 2018

To evaluate thrombopoiesis in thrombocytopenic disorders, we simultaneously determined reticulated platelet counts in whole blood by FACScan flow cytometry and serum thrombopoietin (TPO) concentrations by a sensitive sandwich ELISA. The subjects were 40 healthy volunteers and 45 thrombocytopenic patients. In idiopathic thrombocytopenic purpura (ITP), the percentage of reticulated platelets was significantly elevated (5.61 ± 2.02%: mean ± SD) relative to normal controls (2.17 ± 0.90%), but serum TPO concentrations (1.91 ± 1.27 fmol/l) did not differ significantly from the normal range (1.43 ± 0.62 fmol/l). The patients with aplastic anemia (AA) had decreased reticulated platelet counts and markedly increased serum TPO concentrations (13.65 ± 10.64 fmol/l). In thrombocytopenic patients with liver cirrhosis (LC), the absolute number of reticulated platelets (1.65 ± 1.11 × 109/l) decreased similarly that in AA. However, serum TPO concentrations (1.38 ± 0.50 fmol/l) did not increase in contrast to AA. Our findings suggested a possible dual mechanism of thrombocytopenia in LC; that is, thrombocytopenia in LC results from the decreased TPO production primarily in the liver adding to an increase in platelet sequestration in the spleen.
Increased Potency and Decreased Elimination of Lamifiban, a GPIIb-IIIa Antagonist, in Patients with Severe Renal Dysfunction
Authors: Nordal Knut , Midtvedt Karsten , Goggin Timothy , Brosstad Frank , Lehne Gustav
Published: March 2018

Activation of the platelet membrane receptor glycoprotein (GP) IIb-IIIa is essential for thrombus formation. The novel nonpeptide GPIIb-IIIa antagonist, lamifiban, represents a promising approach for antiplatelet therapy in patients with cardiovascular disease. Since renal impairment frequently occurs in these patients, we designed a phase I study to assess the tolerability, pharmacodynamics and pharmacokinetics of lamifiban in patients with renal impairment. Four healthy volunteers (Group 1) with creatinine clearance (CLCR) >75 ml/min, eight patients (Group 2) with mild to moderately impaired renal function (CLCR 30-74 ml/min) and eight patients (Group 3) with severe renal impairment (CLCR 10-29 ml/min) were studied. They received stepwise increased doses of lamifiban intravenously (IV). There was a linear relationship between the systemic clearance of the drug and renal function (R2 = 0.86). The mean plasma concentration required for half-maximal inhibition of thrombin-receptor agonist peptide (TRAP) induced platelet aggregation (EC50) ex vivo was 21, 28 and 11 ng/ml in Groups 1, 2 and 3. The patients in Group 3 were sensitized to the anti-platelet effect allowing an 18-fold dosage reduction without compromising the pharmacodynamics. In conclusion, the decreased clearance of lamifiban may act in concert with increased potency of the drug in patients with severe renal impairment, and the drug dosage should be reduced accordingly.
Regulation of Gelatinase Activity in Mice with Targeted Inactivation of Components of the Plasminogen/Plasmin System
Authors: Silence J. , Lemmens G. , Frederix L. , Collen D. , Lijnen H. R.
Published: March 2018

To investigate a potential physiological role of the plasminogen/ plasmin system in activation of the matrix metalloproteinase (MMP) system, the distribution of latent and active MMP-2 (gelatinase A) or MMP-9 (gelatinase B) was monitored in aorta extracts and in serum-free conditioned cell culture medium obtained from wild-type (WT) mice and from mice with deficiency of tissue-type plasminogen activator (t-PA–/–), urokinase-type plasminogen activator (u-PA–/–), plasminogen activator inhibitor-1 (PAI-1–/–) or plasminogen (Plg–/–).

In aorta extracts, the contribution of active MMP-2 to the total MMP-2 level ranged between 7 and 16% for the different genotypes, whereas active MMP-9 was not detected.

The contribution of active 58 kDa MMP-2 to the total MMP-2 level (active plus latent) ranged between 14 and 29% (mean of 3 experiments) for fibroblasts of the different genotypes, and between 18 and 32% for smooth muscle cells, and was relatively constant in time (7-72 h). The contribution of active 83 kDa MMP-9 to the total MMP-9 level ranged between 15 and 29% for fibroblasts of the different genotypes and was relatively constant in time (24-72 h); corresponding values were 17 to 57% for smooth muscle cells, with the exception of Plg–/– smooth muscle cells which had undetectable levels of active MMP-9. Addition of plasmin(ogen) to the cell culture medium of fibroblasts did not significantly affect the distribution of active and latent MMP-2, but resulted in an approximately two-fold enhancement of the contribution of active MMP-9. In macrophages of Plg–/– mice, active MMP-9 was detected only when the cells were cultured in the presence of plasminogen.

These data indicate that activation of proMMP-2 occurs independently of the physiological plasminogen activators and of plasmin(ogen) in all the cell types evaluated. Activation of proMMP-9 was enhanced in the presence of plasmin(ogen), but active MMP-9 was also detected in fibroblasts of Plg–/– mice, indicating that in vivo activation may occur via plasmin(ogen)-independent mechanisms.
Neutrophils Rolling on Immobilised Platelets Migrate into Homotypic Aggregates after Activation
Authors: Buckley Christopher , Simmons David , Nash Gerard , Rainger G. E.
Published: March 2018

Interactions between platelets and leucocytes are implicated in the pathology of thrombotic vascular disease. Using a flow-based adhesion assay we have investigated a novel route for the formation of neutrophil aggregates on the surface of immobilised activated platelets. Neutrophils perfused over a platelet monolayer formed numerous rolling attachments but rapidly stopped and spread after the superfusion of N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor (both at 10–7 M). Subsequent integrin-mediated migration across the platelet monolayer enabled formation of homotypic neutrophil aggregates, which was significant within 2.5 min of receipt of either stimulus. Aggregates increased in size with time and had an average projected area of ~500 μm2 after 10 min. Increasing size was correlated with an increasing tendency for movement downstream and large aggregates sometimes tumbled in that direction. The formation and stability of homotypic aggregates was dependent on several adhesive mechanisms. Antibody blockade demonstrated that interactions involving CD11a/ CD18 and ICAM-3, between αvβ3-integrin and CD31 and between L-selectin and an unidentified counter-ligand were all required for the complete aggregatory response. Furthermore, blockade of L-selectin allowed initial aggregation which then reversed, suggesting that this receptor might regulate the interactions between other adhesion molecules that directly supported cell-cell adhesion. We propose that this novel route for leucocyte aggregation could promote vascular occlusion in thrombotic vessels or at distal sites in the event of embolisation.
A Three-Dimensional Consideration of Variant Human Fibrinogens
Authors: Everse Stephen , Spraggon Glen , Doolittle Russell
Published: March 2018

Recently reported X-ray structures for large core fragments derived from human fibrinogen and fibrin make it possible to correlate structural and functional anomalies of known genetic variants. Here we examine a variety of amino acid replacements previously reported for hereditary dysfibrinogenemias, most of which are associated with impaired fibrin polymerization. For many of these we have modeled in the mutant amino acid and considered the structural consequences. We have also examined the cases of a small deletion and a large insertion, as well as the impact of substitutions in the GPRPam ligand that was co-crystallized with fragment double-D.
Heparin and Cancer
Authors: Ornstein Deborah , Zacharski Leo
Published: March 2018

Heparin has been the subject of intensive investigation for decades by both basic and clinical scientists because of its usefulness as a therapeutic anticoagulant (1). In addition to its effects on coagulation, however, heparin exhibits many other activities which seem to have little to do with anticoagulation (2). Goerner first observed an effect on the natural history of malignancy in 1930 when he demonstrated that heparin inhibited tumor growth in experimental animals (3). A substantial body of literature on heparin and cancer has developed during the ensuing decades. A recent increase in interest has resulted from observations made during prospective, randomized clinical trials which compared unfractionated heparin (UH) with low molecular weight heparin (LMWH) for the prevention and treatment of venous thromboembolism (VTE). Several studies have shown improvement in short to intermediate term survival in the subset of patients with cancer who received LMWH. This improved outcome has been emphasized in two published meta-analyses (4, 5), and the trend is supported by the results of a more recent prospective study (6). The improved cancer outcome could not be attributed to prevention of VTE in any of the studies; therefore, it is reasonable to hypothesize that a beneficial effect of heparin may be mediated by mechanisms independent of anticoagulation.

Heparin has several properties that may plausibly explain its effect on experimental and human malignancy. The purpose of this paper is to summarize the literature on heparin and cancer and review mechanisms by which heparin may exert antineoplastic activity. The goal is to encourage both definitive clinical trials of heparin on cancer outcome and further studies of drug mechanisms in human malignancy. LMWH, with its favorable pharmacokinetic attributes, is suitable for chronic outpatient administration and is an excellent candidate for further investigation. Such a departure from conventional experimental cytotoxic chemotherapy holds promise for development of novel forms of growth regulatory therapy that interrupt pathways of cancer progression. In this review, “heparin” will refer to unfractionated heparin (UH), and “UH” and “LMWH” will be used to distinguish between the two molecular forms.
Comparison between von Willebrand Factor (VWF) and VWF Antigen II in Normal Individuals and Patients with von Willebrand Disease
Authors: Mazurier Claudine , Romeuf Christophe de
Published: March 2018

Von Willebrand disease is characterised by a quantitative (type 1) or qualitative (type 2) decrease in von Willebrand factor (vWF) a multimeric glycoprotein involved in primary haemostasis. The propep-tide of von Willebrand, also named vWF antigen II (vWF:AgII), is released from platelets and endothelial cells and circulates in plasma as a glycoprotein of 100 kD. In the present study, we attempted to determine whether vWF:AgII level may provide information on the synthesis of vWF, specially in patients with von Willebrand disease (vWD). To elucidate that point, we developed an ELISA and quantify the vWF:AgII in normal individuals and in various vWD patients. The propeptide molar concentration was found to be 5nM as compared to 31 nM for mature vWF. In normal individuals, the level of vWF:AgII was significantly decreased in females from O and A blood groups. In type 2 vWD patients the level of plasma vWF:AgII appears normal in the patients with normal level of platelet vWF. In type 2 B vWD characterised by increased affinity of mature vWF for platelet glyco-protein Ib, the vWF:Ag II in contrast to the vWF antigen (vWF:Ag) was not decreased. In type 2A vWD patients the level of vWF:AgII was decreased in patients with absence of high molecular weight vWF in platelets and plasma but normal in patients with increased sensitivity to proteolysis. Finally, in type 1 vWD, some studied patients have a parallel decrease in vWF:AgII and vWF:Ag whereas in others, the vWF:Ag levels were much more affected than corresponding vWF:AgII levels, as observed in some type 2 vWD patients. Thus, in contrast to that already described, the plasma vWF:AgII level cannot discriminate type 1 from type 2 vWD patients. We conclude that the vWF:AgII measurement provides additional information on the mechanisms responsible for vWD and might also contribute to the classification of vWD patients.
Monitoring Heparin Therapy Using Activated Partial Thromboplastin Time – Results of a Multicenter Trial Establishing the Therapeutic Range for SILIMAT, a Reagent with High Sensitivity to Heparin
Authors: Boutière Brigitte , Horellou Marie-Hélène , Trzeciak Marie Christine , Samama Meyer , Toulon Pierre
Published: March 2018

APTT is widely used for laboratory monitoring of treatment with unfractionated heparin (UFH). However, since its sensitivity to heparin varies significantly from one reagent to another, the therapeutic range had to be defined for each brand of APTT reagent. As an example, SILIMAT (bio-Mérieux) is a new APTT reagent containing rabbit brain phospholipids and micronized silica as an activator. Since its high sensitivity to heparin has been previously reported, a multicenter trial was carried out in an attempt to define the therapeutic range of APTT performed using this new reagent. For that purpose, 170 blood samples drawn for routine coagulation testing from 170 different patients treated with UFH were analyzed. A single batch of two different APTT reagents were used on KC10 coagulometers: SILIMAT and Automated APTT (Organon-Teknika) whereas the anti-Xa activity was evaluated by a chromogenic substrate-based assay. The same methodology was used in all the centers. In order to obtain a plasma anti-Xa activity within the therapeutic range i.e. between 0.30 and 0.70 IU/ml, the APTT ratios were found between 1.90 and 5.40 for SILIMAT, which corresponded to clotting times of the patients plasma between 63 and 178 s. The APTT ratios were significantly lower when evaluated using Automated APTT (between 1.70 and 4.10), with clotting times between 53 and 127 s. In addition, a good correlation was found between the Anti-Xa activity and APTT for both reagents (r >0.65). However, it is not possible to make recommendations regarding the therapeutic ranges without restrictions. Although about 70% of the patients with an anti-Xa activity between 0.30 and 0.70 IU/ml had an APTT in the above defined ranges, the degree of concordance between the two assays is not absolute. Actually more than 30% of the patients had discordant anti-Xa activity and APTT and more than a quarter of the patients included in the above defined therapeutic range for APTT had an anti-Xa activity outside the 0.30-0.70 IU/ml range, whatever the reagent used. In conclusion, to define the therapeutic ranges of APTT using the recommended method is practicable but some critical points could be raised, suggesting that a better method is awaited in order to improve the standardization.
Population Pharmacokinetics of Recombinant Factor VIIa in Volunteers Anticoagulated with Acenocoumarol
Authors: Nony Patrice , Erhardtsen Elisabeth , Delair Sylvie , Ffrench Patrick , Dechavanne Marc , Boissel Jean-Pierre , Girard Pascal
Published: March 2018

This study establishes a population PK model for FVII clotting activity (FVII:C) after injection of recombinant activated factor VII (rFVIIa) to healthy volunteers. Twenty eight volunteers, anticoagulated with acenocoumarol, received one or two rFVIIa injections, with dose ranging from 5 to 320 μg/kg. The FVII:C kinetic was fitted to a 2 compartment model, with continuous “endogenous perfusion” mimicking endogenous activity. Estimated clearance was 2.4 l/h (20% inter-individual variability and 9% inter-period variability). The volume of distribution at steady-state appeared to be significantly dose dependent: 78 ml/kg for doses ≤20 μg/kg and 88 ml/kg for doses >20 μg/kg respectively, with 16% inter-individual variability. The dose producing 50% of the maximum drop of INR was estimated to be 2.2 μg/kg. The model will be used to better define the dosage regimen for future clinical developments.
Warfarin or Acenocoumarol: Which Is better in the Management of Oral Anticoagulants?
Authors: Barcellona Doris , Vannini Maria Luigia , Fenu Lara , Balestrieri Cinzia , Marongiu Francesco
Published: March 2018

Warfarin is employed more frequently than acenocoumarol because of its longer half-life (36 h), theoretically providing more stable anticoagulation, and avoiding factor VII fluctuations that potentially occur during acenocoumarol treatment (half-life 10 h).

The aim of our study was to compare acenocoumarol with warfarin in the same group of 103 patients who started oral anticoagulation with acenocoumarol and then changed to warfarin. In these patients we compared the previous period of six months on acenocoumarol treatment (July-December 1996) with a new six-month period on warfarin (July-December 1997). We wished to know whether warfarin could improve the quality and the stability of oral anticoagulation of our patients and whether there was a difference between the two drugs in the weekly mean dose per patient. Moreover in order to detect the possible daily fluctuation of factor VII, we evaluated a further group of 54 patients. A subgroup of these patients was treated with warfarin while another received acenocoumarol.

In the first group of patients, 1,158 and 1,064 PTs were carried out with acenocoumarol and warfarin, respectively. The percentage of PTs in the therapeutic range was 59% with acenocoumarol and 62% with warfarin (p = 0.4). The mean number of visits per patient was 12 and 11, and the mean number of visits in the therapeutic range was 7 and 7, respectively. The last check in file method did not show any difference between the two drugs. Overdose states were 51 (4.4%) with acenocoumarol and 30 (2.8%) with warfarin (p = 0.4). A good correlation (r = 0.92) was found between the acenocoumarol and the warfarin weekly mean dose. The mean warfarin/acenocoumarol weekly dose ratio was 2.08 (range: 1.25-3.30; CI 95%: 1.99-2.16).

In the second group of patients, factor VII levels with both drugs were higher 24 h after administration than 16 h after, showing that their daily fluctuation was independent of the drug’s half-life, since factor VII levels in patients with a low vitamin K intake were not increased.

Our results showed that warfarin did not appear to be better than acenocoumarol in the performance of an Anticoagulation Clinic in terms of PTs within the therapeutic range per patient. It seems that the behaviour of factor VII was affected by the intake of vitamin K rather than by the short half-life of acenocoumarol.
Retrospective Neuropathological Review of Prion Disease in UK Haemophilic Patients
Authors: Ironside James , Bell Jeanne , Giangrande Paul , Ludlam Christopher , Esiri Margaret M. , McLaughlin James , Lee Christine
Published: March 2018

In 1996, the CJD surveillance unit in Edinburgh, UK described nvCJD which was thought to be the human equivalent of bovine spongiform encephalopathy (BSE). The identification of prion protein in the tonsil of an affected individual has raised the question of transmission of nvCJD via blood products. This study examines the post mortem brains of 33 patients who were treated with clotting factor concentrate of predominately UK donor source during the years 1962-1995. The brains were examined by conventional histological methods and also for the prion protein using monoclonal antibodies KG9 and 3F4. No evidence of spongiform encephalopathy was found and the immunocytochemistry was negative for PrP in all cases. It is concluded that, at present, there is no evidence for the transmission of nvCJD via clotting factor concentrate to patients with haemophilia.