bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlates of thrombin generation in patients with advanced prostate cancer
Authors: Caprini Joseph , Goldshteyn Sofia , Dohnal James , Vesely Sara , Shevrin Daniel , Lind Stuart
Published: March 2018

Thrombin generation is increased in men with advanced prostate cancer. Thrombin has the ability to interact with, and affect the biology of, a variety of cell types including prostate cancer cell lines. We therefore looked for correlations between thrombin generation and other markers of disease activity in spot urine samples obtained from men with advanced prostate cancer. Excretion of part of the prothrombin activation peptide F1+2 (called here iF2), interleukin-6 (IL-6), the bone turnover marker deoxypyridinoline (DpD), and vascular endothelial growth factor (VEGF) were quantitated in spot urine samples collected from 37 men with hormone-refractory prostate cancer. Following log transformation of the data, significant correlations were found by univariate analysis between the excretion of a marker of thrombin generation (iF2) and IL-6, DpD and VEGF, as well as between IL-6 and DpD or VEGF excretion. No correlation was found between any marker and serum PSA level. After multivariate analysis, a significant correlation remained between thrombin generation and IL-6 excretion. Analysis of a second urine specimen obtained from 19 of the subjects 1 to 7 months after the first also revealed a significant correlation between thrombin generation and IL-6, DpD, and VEGF excretion. These data provide evidence of a correlation between thrombin generation/coagulation system activation and IL-6 generation in patients with cancer. They provide a rationale for studying the effects of inhibitors of thrombin generation upon the biology of prostate cancer.
High Plasma Concentration of Factor VIIIc Is a Major Risk Factor for Venous Thromboembolism
Authors: in 't Anker Pieternella , Koopman Marianne , Reitsma Pieter , Prins Martin , van den Ende Abraham , Büller Harry , Kraaijenhagen Roderik
Published: March 2018

Established risk factors, including deficiencies of protein C, protein S or antithrombin and the factor V Leiden and prothrombin mutation, are present in about one third of unselected patients with venous thromboembolism. In addition to these inherited thrombophilic defects, elevated plasma levels of factor VIIIc have been suggested to be important in the pathogenesis of (recurrent) venous thromboembolism. The objective of this study was to assess the relevance of factor VIIIc plasma concentration in consecutive patients with venous thromboembolism.

We studied the prevalence of elevated plasma levels of factor VIIIc in 65 patients with a proven single episode and in 60 matched patients with documented recurrent venous thromboembolism. The reference group consisted of 60 ageand sex-matched patients who were referred for suspected venous thromboembolism, which was refuted by objective testing and longterm clinical follow-up. To minimalize the influence of the acute phase, blood was obtained at least 6 months after the thromboembolic event and results were adjusted for fibrinogen and C-reactive protein. Factor VIIIc was re-determined several years after the first measurement in a subset of patients to evaluate the variability over time. To study a possible genetic cause, a family study was done.

In the control, single and recurrent episode group, the prevalences of plasma levels of factor VIIIc above 175 IU/dl (90th percentile of controls) were 10% (95% CI: 4 to 21%), 19% (95% CI: 10 to 30%) and 33% (95% CI: 22 to 47%), respectively. For each 10 IU/dl increment of factor VIIIc, the risk for a single and recurrent episode of venous thrombosis increased by 10% (95% CI: 0.9 to 21%) and 24% (95% CI: 11 to 38%), respectively. Both low and high plasma levels of factor VIIIc were consistent over time (R = 0.80, p = 0.01). A family study indicated a high concordance for elevated factor VIIIc plasma concentrations among first degree family members. Adjustment for fibrinogen, C-reactive protein and known thrombophilic risk factors did not change the observed association of elevated factor VIIIc with thrombosis.

Elevated plasma levels of factor VIIIc are a significant, prevalent, independent and dose-dependent risk factor for venous thromboembolism. It also predisposes to recurrent venous thromboembolism.
Hemostatic Parameters and Platelet Activation Marker Expression in Cyanotic and Acyanotic Pediatric Patients Undergoing Cardiac Surgery in the Presence of Tranexamic Acid
Authors: Levin Elena , Wu John , Alexander John , Reichart Clayton , Sett Suvro , Seear Michael , Devine Dana
Published: March 2018

We have investigated hemostatic parameters including platelet activation in 56 pediatric patients with or without cyanosis undergoing cardiopulmonary bypass (CPB) and cardiac surgery to repair congenital defects. Patients were participants in a study assessing the effects of tranexamic acid on surgery-related blood loss. Parameters monitored included blood loss, prothrombin F1.2, thrombin-antithrombin complexes, t-PA, PAI-1, plasminogen, fibrin D-dimer, and plasma factor XIII. Additionally, flow cytometry monitored platelet degranulation (P-selectin or CD63), as well as surface-bound fibrinogen, von Willebrand factor and factor XIIIa. Cyanotic patients had evidence of supranormal coagulation activation as both fibrin D-dimer and PAI-1 levels were elevated prior to surgery. While the extent of expression of Pselectin or CD63 was not informative, platelet-associated factor XIIIa was elevated in cyanotic patients at baseline. In both patient groups, CPB altered platelet activation state and coagulation status irrespective of the use of tranexamic acid.
A New Candidate Missense Mutation (Leu 1657 Ile) in an Apparently Asymptomatic Type 2A (Phenotype IIA) von Willebrand Disease Family
Authors: Guilliatt A. M. , Surdhar G. K. , Theophilus B. D. M. , Hill F. G. H. , Enayat M. S.
Published: March 2018

Type 2A von Willebrand disease (VWD) is mostly an autosomal dominantly inherited bleeding disorder characterised by a qualitative defect of von Willebrand factor (VWF). Mutation screening was used to screen the whole of VWF gene followed by direct sequencing to detect the mutation in a father and son diagnosed with type 2A (phenotype IIA) von Willebrand disease. A C5219 to A transversion was detected predicting Leucine to Isoleucine substitution in codon 1657. This novel missense mutation which was also identified by MboI restriction enzyme analysis, was found in both patient and his father but not in any other unaffected family member or 50 unrelated normal individuals. This substitution was reproduced by in vitro site directed mutagenesis of full-length VWF cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWF protein exhibited the full spectrum of VWF multimers, suggesting that the abnormal multimer seen in the patient results from increased proteolysis.
Prothrombin Activation Is Increased among Asymptomatic Carriers of the Prothrombin G20210A and Factor V Arg506Gln Mutations
Authors: Humphries Steve , Smillie Belinda , Li Lily , Cooper Jacqueline , Barzegar Samad , Rosenberg Robert , Miller George , Bauer Kenneth
Published: March 2018

The risk of venous thrombosis is increased in individuals who carry specific genetic abnormalities in blood coagulation proteins. Among Caucasians, the prothrombin G20210A and factor V Arg506Gln (FV R506Q) mutations are the most prevalent defects identified to date. We evaluated their influence on markers of coagulation activation among participants in the Second Northwick Park Heart Study, which recruited healthy men (aged 50–61 years) from nine general medical practices in England and Wales. They were free of clinical vascular disease and malignancy at the time of recruitment. Genotypes for the two mutations were analyzed using microplate array diagonal gel electrophoresis, and coagulation markers (factor XIIa; activation peptides of factor IX, factor X, and prothrombin; fibrinopeptide A) were measured by immunoassay. Factor VII coagulant activity and factor VIIa levels were determined by a functional clotting assay. Among 1548 men genotyped for both mutations, 28 (1.8%) and 52 (3.4%) were heterozygous for prothrombin G20210A and FV R506Q, respectively. The only coagulation marker that was significantly associated with the two mutations was prothrombin activation fragment F1+2 [mean ± SD, 0.88 ± 0.32 nmol/L in men with prothrombin G20210A (p = 0.002) and 0.89 ± 0.30 in men with FV R506Q (p = 0.0001) versus 0.72 ± 0.24 among non-carriers for either mutation]. This data provides conclusive evidence that heterozygosity for the prothrombin G20210A as well as the FV R506Q mutations in the general population leads to an increased rate of prothrombin activation in vivo.
Congenital Deficiency of Vitamin K Dependent Coagulation Factors in Two Families Presents as a Genetic Defect of the Vitamin K-Epoxide-Reductase-Complex
Authors: von Brederlow B. , Fregin A. , Rost S. , Wolz W. , Eberl W. , Eber S. , Lenz E. , Schwaab R. , Brackmann H. H. , Effenberger W. , Harbrecht U. , Schurgers L. J. , Vermeer C. , Müller C. R. , Oldenburg J.
Published: March 2018

Hereditary combined deficiency of the vitamin K dependent coagulation factors is a rare bleeding disorder. To date, only eleven families have been reported in the literature. The phenotype varies considerably with respect to bleeding tendency, response to vitamin K substitution and the presence of skeletal abnormalities, suggesting genetic heterogeneity. In only two of the reported families the cause of the disease has been elucidated as either a defect in the γ-carboxylase enzyme (1) or in a protein of the vitamin K 2,3-epoxide reductase (VKOR) complex (2).

Here we present a detailed phenotypic description of two new families with an autosomal recessive deficiency of all vitamin K dependent coagulation factors. In both families offspring had experienced severe or even fatal perinatal intracerebral haemorrhage. The affected children exhibit a mild deficiency of the vitamin K dependent coagulation factors that could be completely corrected by oral substitution of vitamin K.Sequencing and haplotype analysis excluded a defect within the γ-carboxylase gene. The finding of highly increased amounts of vitamin K epoxide in all affected members of both families indicated a defect in a protein of the VKOR-multienzyme-complex. Further genetic analysis of such families will provide the basis for a more detailed understanding of the structure-function relation of the enzymes involved in vitamin K metabolism.
Capillary Microscopic and Rheological Dimensions for the Diagnosis of von Willebrand Disease in Comparison to other Haemorrhagic Diatheses
Authors: Latza Reinhard , Mürsdorf Stefan , Mrowietz Christof , Kiesewetter Holger , Wenzel Ernst , Jung Friedrich , Koscielny Jürgen
Published: March 2018

It is known that angiodysplasia influence macrocirculation as well as microcirculation in patients with vWD. In the present study it was examined if intravital capillary microscopic dimensions (morphologic and dynamic) in skin (nailfold) in combination with rheologic parameters could give indications for the presence of vWD in patients with haemorrhagic diathesis.

Patients with vWD (n = 100; 92 type 1: definite type 1:78 and possible type 1:14; 8 type 2A) have in comparison to patients with other haemorrhagic diathesis [thrombocytopathy (n = 122), thrombocytopenia (n = 101), severe haemophilia A (n = 50) and severe haemophilia B (n = 20), congenital dysfibrinogenaemia (n = 22), oral anticoagulation with phenprocoumone (n = 112)] and to apparently healthy subjects (n = 100) a significantly increased capillary torquation (median index: 3.5), a venolar and an arteriolar capillary dilatation (median: 16.5 µm; median: 15.1 µm) and the highest part of microscopic bleedings (extravasates) with 40% in the video capillary microscopy as morphological changes. Only the congenital dysfibrinogenaemia appears with a larger dilatation in venolar capillaries (median: 14.5 µm). Microscopic bleedings are much less common in other haemorrhagic diatheses with a frequency between 4% and 13%.

In the vWD a significantly reduced duration of reactive hyperaemia (median: 150 sec). This is the only dynamic change that can be taken as a possible hint for a loss of flexibility within the precapillary vessels. A significantly reduced plasma viscosity (< 1.25 mPas) is typical for the vWD due to the increase of the shear stress in blood plasma because of the reduction of vWF-activities. Changes of the capillary morphology (dilatation, extravasates, capillary torquation) and the hypoplasmaviscosity are most sensitive for the vWD (75%, 65%, 40%, 80%) with a fairly high specifity (up to 93%) and a positive predictive value of 99%.

As a conclusion it seems reasonable to discuss the introduction of video capillary microscopy as a screening test for haemostasiological and angiological centers.
Characterization of Unfractionated Heparin: Comparison of Materials from the last 50 Years
Authors: Gray Elaine , Barrowcliffe Trevor , Mulloy Barbara
Published: March 2018

Physicochemical and anticoagulant characteristics of 27 samples from recent batches of commercially produced unfractionated heparin have been determined as part of the process of establishment of the 5th International Standard Unfractionated Heparin. They have been compared with current heparin standards (European Pharmacopoeia, United States Pharmacopoeia, Chinese), with the 4th International Standard Unfractionated Heparin, and with the three predecessor International Standards. The results indicate that the 4th International Standard Unfractionated Heparin, established in 1982, has significantly lower molecular weight and specific activity than recently produced heparin; this is also true of all preceding International Standard Heparins and of the United States Pharmacopoeial standard. The composition of commercial unfractionated heparin may therefore have changed over time; reasons for this are discussed.
An Assay to Quantify the Two Plasma Isoforms of Factor V
Authors: Hoekema Lico , Tans Guido , Rosing Jan
Published: March 2018

Blood coagulation factor V (FV) circulates in the blood in two forms, designated FV1 and FV2. In model systems containing purified proteins FV1 appears to be more thrombogenic than FV2. Recently, we reported that in plasma from carriers of the R2 haplotype, a polymorphism which encodes several amino acid changes in FV and which is associated with an increased risk of thrombosis, the FV1/FV2 ratio is shifted in favor of the more thrombogenic form FV1. Here we describe in detail the assay that enables quantification of the plasma levels of FV1 and FV2. FV present in highly diluted plasma samples was activated with thrombin and the FVa generated was subsequently quantified in two prothrombinase-based assay systems. In the first assay, which is performed at saturating amounts of FXa and phospholipid vesicles with a high mole fraction phosphatidylserine, FVa1 and FVa2 express the same cofactor activity in prothrombin activation. Hence, this assay quantifies the total FV level (FV1 + FV2) present in plasma. In the second assay, which is performed at suboptimal amounts of FXa and phospholipid vesicles with a low mole fraction phosphatidylserine, FVa2 has approximately an 8-fold higher cofactor activity than FVa1. Therefore, the response in this assay depends on the relative amounts of FV1 and FV2 in the plasma sample. Calibration curves made with samples containing known concentrations of purified FVa1 and FVa2 subsequently allowed calculation of the amounts of FV1 and FV2 present in plasma.
Tranexamic Acid Inhibits Fibrinolysis, Shortens the Bleeding Time and Improves Platelet Function in Patients with Chronic Renal Failure
Authors: Panes Olga , Muñoz Blanca , Pais Edgar , Tagle Rodrigo , González Fernando , Mezzano Sergio , Barriga Francisco , Pereira Jaime , Mezzano Diego
Published: March 2018

Background: A defect in platelet function is the main determinant of the prolonged bleeding time in chronic renal failure (CRF). We previously reported a significant correlation between platelet abnormalities and elevated plasma markers of plasmin and thrombin generation. Our aim was to explore the effect of inhibiting both plasmin action with tranexamic acid (TA) and thrombin production with low molecular weight heparin (LMWH), on the bleeding time (BT) and platelet function in patients with CRF. Methods: 37 patients with CRF (mean creatinine 8.6 ± 4.4 mg/dl) under conservative treatment, with prolonged BT, entered this study and received TA during 6 days, with (n = 24) and without LMWH (n = 13). BT, platelet aggregation/secretion, platelet granule contents, von Willebrand factor and parameters of coagulation and fibrinolysis were recorded before and at the end of treatment. Results: The BT was shortened in 26/37 (67%) patients. This effect was associated with significant improvement of platelet aggregation and secretion, with decrease to a normal range of fibrin/fibrinogen degradation products, mild increase in plasmin-antiplasmin complexes and pronounced reduction of circulating plasminogen. No differences were seen among patients with or without LMWH. No serious side effects or complications were observed. Interpretation: These findings indicate that the activation of fibrinolysis plays a significant role in the defect of primary hemostasis in patients with CRF. Inhibition of plasmin activity with TA shortens the BT and improves platelet function in the majority of patients with severe disease.
Anti Xa Monitoring during Treatment with Low Molecular Weight Heparin or Danaparoid: Inter-assay Variability
Authors: Iampietro R. , Woolley A. M. , Preston F. E. , Kitchen S.
Published: March 2018

If laboratory monitoring of low molecular weight heparin (LMWH) therapy is required the test of choice is the anti Xa activity assay. The relationship between anti Xa results obtained using different techniques is unknown. The aim of the present study was to compare anti Xa results obtained with eight different commercially available anti Xa activity assays (five chromogenic and three clotting based assays) in samples from patients receiving either therapeutic or prophylactic LMWH (enoxaparin or dalteparin) or danaparoid.

We have demonstrated that highly significant differences exist between results obtained using different techniques. The mean anti Xa activity in patients receiving treatment or prophylaxis with enoxaparin ranged from 0.28 to 0.64 iu/ml. A similar relationship was present in samples from patients treated with dalteparin, mean anti Xa results ranging from 0.43 to 0.69 iu/ml. The Heptest clotting assay as used here in combination with the Automated Coagulation Laboratory instrument, was associated with lower results than other clotting or chromogenic techniques. In patients receiving danaparoid for heparin induced thrombocytopaenia (HIT) mean results with three clotting based assays were 0.30 to 0.36 u/ml, compared to mean results of 0.47 to 0.65 u/ml for chromogenic assays.

Our data clearly indicate that the selection of anti Xa assay method could influence patient management since the dose required to achieve the therapeutic range would differ according to the assay employed. This is particularly important since the frequency of hamorrhagic side effects has been shown by others to be dose dependant, irrespective of the concomitant anti Xa activity results. In danaparoid therapy the clotting assays studied here should not be employed for monitoring without a modified target range, unless it can be demonstrated that the higher doses required to achieve the therapeutic range are safe.
Pretreatment with Soluble Thrombomodulin Prevents Intrasinusoidal Coagulation and Liver Dysfunction following Extensive Hepatectomy in Cirrhotic Rats
Authors: Kaido Toshimi , Yoshikawa Akira , Seto Shin-ichi , Yamaoka Shoji , Furuyama Hiroaki , Arii Shigeki , Imamura Masayuki , Takahashi Yasuo
Published: March 2018

The major cause of posthepatectomy liver dysfunction is supposed to be microcirculatory disturbance caused by imbalance of intrasinusoidal coagulation equilibrium. Thrombomodulin (TM) is a potent anticoagulant expressed on the endothelial cell surface that regulates the coagulation system by binding thrombin and accelerating the thrombin-catalyzed activation of protein C. Therefore, we examined the effect of soluble TM purified from human urine (UTM) on intrasinusoidal coagulation in cirrhotic rats. Dimethylnitrosamine-induced cirrhotic rats underwent 70% hepatectomy and received endotoxin 48 h after. UTM or vehicle alone was intravenously administered to each rat 30 min before endotoxin injection. UTM treatment attenuated the increases in cytosolic enzymes and serum hyaluronic acid level. The UTM supply improved the survival rate of the rats at 12 h after endotoxin challenge. Histologically, intrasinusoidal fibrin depositions and massive hepatocellular necrosis observed in control rats were scarcely found in UTM-treated rats. Immunohistochemical examination revealed that marked TM stains in sinusoidal endothelial cells were well preserved in UTM-treated rats. In conclusion, UTM administration prevented intrasinusoidal fibrin depositions and attenuated posthepatectomy liver dysfunction in cirrhotic rats.
Cloning and Expression of Canine Glycoprotein Ibα
Authors: Morateck Patricia , Fahs Scot , Warltier David , Montgomery Robert , Kenny Dermot
Published: March 2018

The interaction of the glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (vWF) is critical in initiation of haemostasis and thrombosis through platelet adhesion to damaged endothelium. The binding site for vWF resides within the GPIbα subunit of the complex. To further define the physiological function of platelet GPIbα we cloned and expressed the canine GPIbα cDNA. A canine platelet cDNA library was constructed and screened with a randomly primed 32P-labeled 1041-base-pair restriction fragment of the human GPIbα cDNA. Analysis of 23 clones demonstrated that the canine GPIbα cDNA is 2530 nucleotides in length and includes a short 5’ untranslated segment of 42 nucleotides followed by a signal peptide of 16 amino acids, a mature peptide of 645 amino acids and a 3’ noncoding region of 455 nucleotides. A single intron of 142 nucleotides, 6 nucleotides upstream from the ATG translation initiation codon was identified in the canine gene in a similar location to that present in the human gene.

Chinese hamster ovary cells that stably express human GPIbα and GPIX were transfected with the canine GPIbα cDNA. Canine GPIbα was expressed on the surface of these cells and bound vWF in the presence of botrocetin. The binding of vWF was inhibited by an anti-vWF human monoclonal antibody known to inhibit vWF binding to GPIbα. The results of this investigation will allow the development of reagents to study the physiological function of GPIbα in an animal model.
Thrombomodulin: A New Marker for Placental Abruption
Authors: Chan Daniel , Bruzek Debra , Copel Joshua , Hsu Chaur-Dong , Magriples Urania
Published: March 2018

Thrombomodulin (TM), a marker of endothelial cell damage, has been localized to the placental syncytiotrophoblast. A prospective cohort study of twenty-five pregnant women who were admitted with a clinical diagnosis of placental abruption was undertaken. Abruption was confirmed after delivery in eight cases (Group 1). Group 2 consisted of seventeen patients with no clinical or pathologic evidence of placental abruption after delivery. TM was significantly elevated in Group 1 (71.59 ± 5.35 vs. 48.29 ± 3.53 ng/ml, p = 0.001). The sensitivity and specificity of TM ≥60 ng/ml as a marker for abruption was 87.5 and 76.5%, respectively. In comparison, the sensitivity of an abnormal coagulation profile, maternal Kleihauer-Betke and ultrasound in patients with abruption was 0, 16.7 and 28.6%, respectively. TM is a highly sensitive and specific marker for acute placental abruption.
Monocyte Plasminogen Activator Inhibitor 2 (PAI-2) Inhibits u-PA-mediated Fibrin Clot Lysis and Is Cross-linked to Fibrin
Authors: Robbie Linda , Kinghorn Seonag , Exley Rachel , Booth Nuala , Ritchie Helen
Published: March 2018

Plasminogen activator inhibitor 2 (PAI-2) is a major product of activated human monocytes. Here we show that monocytes inhibited u-PA- but not t-PA-mediated fibrinolysis, by secreting PAI-2 into an overlying fibrin clot. Extracts of arterial and venous human thrombi were found to contain active PAI-2. PAI-2 was cross-linked to fibrin in a reaction catalyzed by two major transglutaminases (TG), tissue TG and factor XIII. The activity of PAI-2 was not affected by such cross-linking. Cross-linking of PAI-2 to fibrin was inhibited by Tridegin, a specific inhibitor of TG, and also by EDTA and iodoacetamide. The use of competitive peptides mimicking the loop between helices C and D of PAI-2 identified Gln 83 and 86 as residues important in cross-linking. This study defines a mechanism by which PAI-2 is localized to fibrin, where it acts as an effective inhibitor of u-PA-mediated fibrinolysis.
Epinephrine Promotes IL-8 Production in Human Leukocytes via an Effect on Platelets
Authors: Østerud Bjarne , Engstad Charlotte , Lund Trine
Published: March 2018

Interleukin-8 (IL-8) is generally accepted to be an important mediator of a number of acute and chronic inflammatory diseases and is produced by monocytes upon stimulation by lipopolysaccharide (LPS). Epinephrine has been reported by several groups to suppress activation of monocytes in response to LPS, and the aim of the present study was to examine the effect of epinephrine on LPS induced IL-8 production using whole blood as a model system. Epinephrine increased LPS induced IL-8 production in a dose-dependent manner in the whole concentration range (0.001–100 μM) and 1 μM epinephrine increased IL-8 levels with 125%. Epinephrine per se had no effect on IL-8 levels. The potentiating effect of epinephrine was mediated by blood platelets, since IL-8 levels in samples containing platelets and stimulated with LPS and epinephrine (1–100 μM) were significantly higher (p <0.05) than in control samples containing no platelets. This effect of platelets seemed to be due to platelet release products, since addition of 25 μl platelet lysate supernatant to whole blood increased LPS induced IL-8 production with 100% and a similar effect was observed in freshly isolated mononuclear cells resuspended in plasma. Upon addition of 50 μg/ml of the carboxyterminal peptide of platelet factor 4 (PF4(58-70)) to whole blood, LPS stimulated IL-8 levels were increased with 115%, whereas in mononuclear cells, 20 μg/ml PF4(58-70) enhanced IL-8 production with 40%. We demonstrate for the first time that epinephrine promotes LPS induced production of IL-8 in whole blood via an effect on blood platelets. This potentiating effect of platelets is shown to be due to platelet granule contents, and platelet factor 4 (PF4) is suggested to be one of several platelet granule proteins promoting LPS induced IL-8 production in whole blood.
Multicenter Evaluation of Lyophilized and Deep-frozen Plasmas for Assignment of the International Normalized Ratio
Authors: Houbouyan-Reveillard L. L. , Aillaud M. F. , Denson K. W. E. , Droullé C. , Johnston M. , Kitchen S. , Lindahl T. L. , Marren M. , Martinuzzo M. E. , Tripodi A. , Vergnes C. , van den Besselaar A. M. H. P.
Published: March 2018

The interlaboratory variation of the International Normalized Ratio (INR) in various external quality assessment schemes is still relatively high. This is partly caused by inaccuracy of manufacturers’ stated International Sensitivity Index (ISI) and/or local instrumentation effects. The interlaboratory variation and accuracy of INR determinations may be improved by a local calibration procedure based on lyophilized plasmas with assigned INRs. The purpose of the present study was to determine INR values for different types of lyophilized plasmas to be used for local calibration. A total of 13 lyophilized plasmas (one normal, six from coumarin-treated patients, six artificially depleted) were analyzed by 10 laboratories, each using five calibrated prothrombin time (PT) systems. INRs were calculated for each plasma using each laboratory’s specific ISI and mean normal prothrombin time values. In the same way, five deep-frozen pooled plasmas from coumarin-treated patients were analyzed. There were significant INR differences for the lyophilized plasmas between the prothrombin time systems. The differences were relatively small for the deep-frozen coumarin plasmas (CV 2.6-3.3%) and three lyophilized coumarin plasmas from one manufacturer (CV 3.7-4.8%). Important INR differences were observed for three lyophilized coumarin plasmas from another manufacturer (CV 9.5-14.1%) and several artificially depleted plasmas (CV 5.3-12.8%). The citrate concentrations in the artificially depleted plasmas were lower than those in the normal and coumarin plasmas. These differences should be considered in the selection and certification of plasmas as calibrants for local calibration of PT systems. The lyophilized plasmas’ INR values obtained in the present study will be used for a field study of local PT calibration to assess their efficacy.
A New Factor Xa Inhibitor (Lefaxin) from the Haementeria depressa Leech
Authors: Faria Fernanda , Kelen Eva Maria , Sampaio Claudio , Bon Cassian , Duval Nathalie , Chudzinski-Tavassi Ana Marisa
Published: March 2018

The salivary complex of the leech Haementeria depressa produces potent anticoagulant components. Among them, a protein named lefaxin inhibits factor Xa (FXa). Lefaxin was purified to homogeneity from dissected salivary complexes by gel filtration in Sephadex G-150 followed by two ion exchange chromatography steps in Mono-Q. Inhibition of FXa by lefaxin was demonstrated by the inhibition of its amidolytic activity, measured with chromogenic substrate S-2765 (apparent KI of 4 nM), and of its ability to inhibit thrombin generation in the prothrombinase complex (EC50 of 40 nM). Lefaxin has a molecular weight of 30 kDa and an isoelectric point of 5.7. It is made of a polypeptide chain whose N-terminal sequence shows no similarity with that of other FXa inhibitors (antistasin and ghilianten) isolated from leech saliva. On the other hand, the N-terminal sequence of lefaxin presents significant sequence similarity with nitric oxide carrier proteins myohemerythrin from the annelid Nereis diversicolor and prolixin S from the tick Rhodnius prolixus. Interestingly, prolixin S also proved to be an anticoagulant protein acting on FXa.
Relationship between Visceral Fat and PAI-1 in Overweight Men and Women before and after Weight Loss
Authors: Kockx M. , Leenen R. , Seidell J. , Princen H. M. G. , Kooistra T.
Published: March 2018

This study was aimed at evaluating the relationship between visceral fat accumulation and plasma plasminogen activator inhibitor-1 (PAI-1) levels in healthy, obese men and women undergoing weight loss therapy. The subjects, 25 men and 25 premenopausal women, aged between 26 and 49 years, with an initial body mass index between 28 and 38 kg/m2, received a controlled diet for 13 weeks providing a 4.2 MJ/day energy deficit. Magnetic resonance imaging was used to measure visceral and subcutaneous abdominal fat. Our results show that before weight loss visceral fat was significantly correlated with PAI-1 in men (r = 0.45; p <0.05), but not in women (r = -0.15; ns). The association between visceral fat and PAI-1 in men remained significant after adjustment for age and total fat mass, and multiple linear regression analysis showed a significant independent contribution of visceral fat to plasma PAI-1 levels. Both visceral fat areas and PAI-1 levels decreased significantly with weight loss in both men and women. Changes in visceral fat area were related to changes in PAI-1 in women (r = -0.43; p = 0.05) but not in men (r = -0.01; ns); however, this association in women disappeared after adjustment for total fat mass. We conclude that there is a relationship between visceral fat and PAI-1 in obese men but not in obese women, and that PAI-1 levels decrease substantially (52%) by weight loss, but this change is not related to changes in visceral fat mass per se.
Immunological Quantitation of Rabbit Plasminogen Activator Inhibitor-1 in Biological Samples
Authors: Ngo Thu-Hoa , Declerck Paul
Published: March 2018

Two immunoassays for the specific quantitation of rabbit plasminogen activator inhibitor-1 (PAI-1) antigen and activity in biological samples were developed and applied for the evaluation of PAI-1 in rabbits. Levels of PAI-1 antigen in rabbit plasma were 9.8 ± 4.6 ng/ml (mean ± SD, n = 6), with a corresponding value of 20.5 ± 13.5 ng/ml for PAI-1 activity. In rabbit serum PAI-1 antigen was 11.8 ± 4.9 ng/ml (n = 6) and PAI-1 activity was 2.9 ± 2.0 ng/ml (n = 6). Endotoxin injection (20 μg/kg, iv) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rabbit plasma, eventually resulting in a 40- to 90-fold increase (p <0.0001 vs baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.90, n = 6, p <0.05) and in plasma from endotoxin-treated rabbits (r = 0.98, n = 20, p <0.001). Analysis of PAI-1 antigen and activity in lysates of washed rabbit platelets revealed the absence of PAI-1 (i.e. <0.03 ng/108 platelets).

In conclusion, development of specific immunological assays allowed the quantitation of PAI-1 in rabbit samples. In striking contrast to other species (human, rat, mouse, pig) rabbit platelets lack detectable amounts of PAI-1 (i.e. >100-1000 fold lower vs other species studied). This observation may have important implications for the use of experimental rabbit models especially in studies on the role of platelets in various pathological conditions including thrombosis and atherosclerosis.
Lysophosphatidic Acid Activates Nuclear Factor Kappa B and Induces Proinflammatory Gene Expression in Endothelial Cells
Authors: Robson Simon , Nehls Volker , Palmetshofer Alois
Published: March 2018

The cellular phospholipid, lysophosphatidic acid (LPA), released by activated platelets and fibroblasts or, at high levels, from ovarian and cervical carcinomas is a powerful serum mitogen that may modulate several signaling pathways in endothelial cells (EC). Hence, LPA could function in a paracrine manner during EC-platelet interactions at sites of vascular injury.

Here, we demonstrate activation of the transcription factor nuclear factor kappa B (NF-κB) in EC following exposure to LPA. EC activation was further characterized by increased levels of mRNA transcripts encoding E-selectin, Intercellular Adhesion Molecule-1, Interleukin-8 and Monocyte Chemoattractant Protein-1. These effects were inhibited by preincubating EC either in the presence of mepacrine (to block phospholipase A2) or of pertussis toxin (to increase ADP-ribosylation of Gi proteins). No inhibition was observed in the presence of putative LPA receptor antagonists suramin or thrombospondin.

LPA induces a proinflammatory activation of endothelial cells that (i) involves Gi proteins; (ii) depends on phospholipase A2 activity; (iii) is associated with the activation of NF-κB and (iv) results in increased expression of proinflammatory genes. We propose that LPA release by activated platelets may directly modulate vascular inflammatory responses.

Abbreviations: EC, endothelial cells; LPA, lysophosphatidic acid; NF-κB, nuclear factor kappa B; PA, phosphatidic acid; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1, ICAM-1, intercellular adhesion molecule-1, vascular adhesion molecule-1; GAPDH, glycerol aldehyde phosphate dehydrogenase; MAPK, mitogen activated kinase, MEK, MAPK kinase, PAEC, porcine aortic EC.
Clinical Utility of Prothrombin Fragment 1+2, Thrombin Antithrombin III Complexes and D-dimer Measurements in the Diagnosis of Deep Vein Thrombosis following Total Hip Replacement
Authors: Cofrancesco E. , Corradi A. , Ravasi F. , Bertocchi F. , Cortellaro M.
Published: March 2018

Background: Measurements of prothrombin fragment 1+2 (F1+2), thrombin antithrombin III complexes (TAT) and D-dimer plasma levels have been proposed as non-invasive screening tests to exclude postoperative deep venous thrombosis (DVT). We investigated the diagnostic efficacy of these coagulation activation markers to rule out postoperative DVT in patients undergoing hip surgery under antithrombotic prophylaxis. Methods: In this substudy of a randomized double-blind thrombosis prophylaxis trial comparing three doses of desirudin (10, 15 or 20 mg b.i.d.) with unfractionated heparin (5000 IU t.i.d.) we used ELISA procedures to measure F1+2, TAT and D-dimer in 159 patients undergoing total hip replacement at baseline (day 0) and on postoperative days 1, 3 and 6. Bilateral venography was performed in all cases 8-11 days after surgery. Results: For the F1+2 assay sensitivity ranged from 73 to 83% in the three postoperative days investigated, and negative predictive value (NPV) from 68 to 74%. For TAT and D-dimer sensitivity ranged from 71 to 73% and from 71 to 83% and NPV from 61 to 65% and from 61 to 74% respectively. Interpretation: In terms of sensitivity and NPV F1+2 and D-dimer are equivalent and are superior to TAT. However, their accuracy is too low to rule out the presence of DVT after hip surgery under antithrombotic prophylaxis.
Factor V Leiden and Fatal Pulmonary Embolism
Authors: Bertina Rogier , Holmes Zandra , Spaargaren Caroline , van Krieken Joannes , Manten Bert , Vandenbroucke Jan , Reitsma Pieter
Published: March 2018

To investigate whether the factor V Leiden mutation increases the risk of fatal pulmonary emboli, we determined the presence of the factor V Leiden mutation in pathology material from two series of autopsies of patients from the Leiden University Hospital, The Netherlands. The first series consisted of consecutive autopsies in which pulmonary emboli were mentioned in the autopsy report; most patients of this series had major underlying disease. The second series consisted of autopsies in patients younger than age 70 in which pulmonary emboli were the sole cause of death and no major acquired risk factor for venous thrombosis was present. Extraction of DNA was done on newly prepared tissue from archival paraffin blocks. In the first series, the presence of factor V Leiden was determined in 44 patients, 1 of whom carried the mutation (2.3 percent; 95% confidence interval 0.06 to 12.0 percent). This prevalence is not different from the general population prevalence in The Netherlands. In the second series, factor V Leiden could be determined in 30 patients of whom 3 carried the mutation (10 percent; 95% confidence interval 2.1 to 26.5 percent), which would lead to a threefold relative risk. A large number of patients with diverse psychiatric diagnoses was present in the second series (eleven). We conclude that in the presence of severe illness, the factor V Leiden mutation plays no additional role in the development of pulmonary emboli. The relative risk of the very rare fatal pulmonary embolus that is the sole cause of death might also be less than the relative risk for deep-vein thrombosis in carriers of the factor V Leiden mutation.
Platelet cNOS Activity Is Reduced in Patients with IDDM and NIDDM
Authors: Bruno Guido , Trucco Fulvia , Zumpano Elena , Tagliabue Milena , Bisceglie Cataldo Di , Pescarmona Gianpiero , Martina Valentino
Published: March 2018

Several studies in vitro and in vivo suggest that the nitric oxide (NO) production is impaired in diabetes mellitus. Reduced levels of NO could contribute to vascular alteration facilitating platelet-vascular wall interaction, adhesion of monocytes to endothelium, vascular smooth muscle proliferation and by decreasing endothelium-dependent vasodilation. In this study we evaluated the activity of the constitutive nitric oxide synthase (cNOS) in platelets of patients with insulin-dependent diabetes mellitus (IDDM) and with non-insulin-dependent diabetes mellitus (NIDDM). When compared to that of normal subjects, cNOS activity is significantly lower in patients with IDDM and with NIDDM (1.57 ± 0.25 vs. 0.66 ± 0.10 fmol/min/10 9 PLTs and 1.57 ± 0.25 vs. 0.67 ± 0.08, respectively; p <0.005). These data demonstrate that the platelet cNOS activity is decreased in diabetes mellitus.
Lonomia obliqua Caterpillar Spicules Trigger Human Blood Coagulation via Activation of Factor X and Prothrombin
Authors: Donato José , Moreno Ronilson , Hyslop Stephen , Duarte Alaor , Antunes Edson , Le Bonniec Bernard , Rendu Francine , Nucci Gilberto de
Published: March 2018

In southern Brazil, envenomation by larvae of the moth Lonomia obliqua (Walker) may result in blood clotting factor depletion, leading to disseminated intravascular coagulation with subsequent haemorrhage and acute renal failure which may prove fatal. We have examined the effect of a crude extract of spicules from these caterpillars on in vitro hemostasis. The extract alone did not aggregate platelets and had no detectable effect on purified fibrinogen, suggesting that extract induces clot formation by triggering activation of the clotting cascade. In agreement with the presence of thrombin-mediated activity, hirudin prevented clot formation. The extract was found to activate both prothrombin and factor X, suggesting that the depletion of blood clotting factors results from the steady activation of factor X and prothrombin. Heating and diisopropylfluorophosphate abolished the procoagulant activity of the extract, indicating that the active component involved is a protein that may belong to the serine protease family of enzymes. The ability of hirudin to inhibit this coagulant activity suggests that this inhibitor could be beneficial in the treatment of patients envenomed by L. obliqua caterpillars.
Inhibition of Rat Platelet Aggregation by Mycalolide-B, a Novel Inhibitor of Actin Polymerization with a Different Mechanism of Action from Cytochalasin-D
Authors: Asai Fumitoshi , Saito Shinya , Ozaki Hiroshi , Fusetani Nobuhiro , Karaki Hideaki , Koike Hiroyuki , Sugidachi Atsuhiro
Published: March 2018

In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 μM) but was inhibited by higher concentrations (3 and 10 μM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 μM). Potentiation of ADP-induced aggregation by MB (0.3 μM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 μM). In contrast, cytochalasin-D (CD) at 3 μM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 μg/ml) or ADP (10 μM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 μM) and CD (3 μM) abolished both ADP (10 μM)- and collagen (3 μg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 μM) had no effects on intracellular Ca2+ concentrations in ADP (10 μM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 μM) was inhibited by MB (0.3-3 μM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 μM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.
Enhancement of Tissue-type Plasminogen Activator-induced Thrombolysis and Prevention of Reocclusion by Combination with a Humanized Anti-glycoprotein IIb/IIIa Monoclonal Antibody, YM337, in a Rhesus Monkey Model of Coronary Thrombosis
Authors: Sato Kazuo , Suzuki Ken-ichi , Sakai Yumiko , Taniuchi Yuta , Kaku Seiji , Yano Shinya , Inagaki Osamu , Tomioka Kenichi , Masuho Yasuhiko , Yanagisawa Isao , Takenaka Toichi , Kawasaki Tomihisa
Published: March 2018

We examined the effect of a humanized anti-glycoprotein IIb/IIIa monoclonal antibody, YM337, on thrombolysis with tissue-type plasminogen activator in a copper coil-induced coronary thrombosis model in rhesus monkeys. Fifty minutes after the formation of an occlusive thrombus, a test drug was administered by either i.v. bolus injection followed by continuous infusion (YM337, 0.25 mg/kg + 1.5 μg/kg/min) or i.v. bolus injection (aspirin, 17 mg/kg). Sixty minutes after induction of the occlusive thrombus, thrombolysis was initiated with tPA at a total dose of 0.5 mg/kg intravenously administered over 60 min, with 10% given as an initial bolus. The median time to reperfusion was significantly shortened by YM337 [saline, 60 min (n = 5); aspirin, 45 min (n = 5); YM337, 30 min (n = 5)]. The incidence of reocclusion was significantly decreased by YM337 (saline, 4/4; aspirin, 5/5; YM337, 1/5), and the median time to reocclusion was significantly prolonged by YM337 [saline, 30 min (n = 4); aspirin, 30 min (n = 5); YM337, 180 min (n = 5)]. YM337 significantly reduced the thrombus protein content at the end of experiment. ADP-induced platelet aggregation was completely inhibited by YM337. These results suggest that YM337 may be of clinical value as an adjunctive agent in thrombolytic therapy for patients with acute myocardial infarction.
A Glanzmann Thrombasthenia-like Phenotype Caused by a Defect in Inside-Out Signaling through the Integrin αIIbβ3
Authors: Shiraga Masamichi , Kinoshita Seiji , Ambo Hironobu , Kurata Yoshiyuki , Matsuzawa Yuji , Kunicki Thomas , Tomiyama Yoshiaki
Published: March 2018

Activation of the platelet integrin αIIbβ3, an essential step in platelet aggregation, is regulated by intracellular signal pathways (inside-out signaling). In this study, we characterize a 35-year-old Japanese female, HM, with a life-long history of mucocutaneous bleeding. HM showed a Glanzmann thrombasthenia-like phenotype with normal expression of αIIbβ3, and failure of platelet aggregation induced by various agonists. An activation-independent ligand mimic monoclonal antibody (mAb), OP-G2, and RGDS peptides bound normally to the patient‘s αIIbβ3„ while an activating anti-β3 mAb, AP5, induced normal aggregation of HM platelets. The nucleotide sequence of the entire coding region of the patient‘s αIIb and β3, including the cytoplasmic domains of each subunit, revealed no abnormalities. Agonist-induced phosphorylation of platelet pleckstrin and myosin light chain was not impaired. Recently, we proposed that a Na+/Ca2+ exchanger is involved in inside-out signaling, especially in the case of chymotrypsin-induced αIIbβ3 activation (Blood 88: 2594, 1996). However, chymotrypsin-induced platelet aggregation occurred normally in patient HM. Measurement of changes in cytosolic free calcium concentration ([Ca2+]i) revealed that the plateau level of [Ca2+]i after thrombin stimulation was significantly inhibited in patient HM. Our data suggest that patient HM exhibits a Glanzmann thrombasthenia-like phenotype associated with an abnormality in inside-out signaling which would otherwise activate αIIbβ3.