bluuebunny/crossref_metadata_2025 huggingface dataset:
Elderly Patients Treated with Tinzaparin (Innohep®) Administered once Daily (175 Anti-Xa IU/kg): Anti-Xa and Anti-IIa Activities over 10 Days
Authors: Pautas E. , Février M. , Wipff C. , Durand-Gasselin B. , Laurent M. , Andreux J.-P. , d’Urso M. , Gaussem P. , Siguret V.
Published: March 2018

Since low molecular weight heparins (LMWH) are partly eliminated by renal excretion, their pharmacodynamic profile may be modified in very elderly patients with age-related renal impairment. The aim of this prospective study was to determine whether tinzaparin (Innohep®) 175 anti-Xa IU/kg administered subcutaneously once daily over 10 days does accumulate in hospital patients greater than 70 years of age. Plasma anti-Xa and anti-IIa amidolytic levels and APTT were determined prior to the first injection (day 0), and then, at peak level i.e. 5 h after the second injection (day 2) and subsequently on days 5, 7 and 10.

Thirty consecutive inpatients (6 men, 24 women) requiring LMWHs at a curative dose for acute thromboembolic disease were included. Patients’ characteristics (mean ± SD) were: age 87.0 ± 5.9 years (range 71–96), body weight 62.7 ± 14.6 kg (range 38-90) and creatinine clearance 40.6 ± 15.3 mL/min (range 20–72). The mean actual dose of tinzaparin delivered was 174.8 anti-Xa IU/kg. Since no patient had an anti-Xa activity above 1.5 IU/mL, the dose of tinzaparin remained fixed over 10 days.

Anti-Xa and anti-IIa peak levels measured on day 2 were 0.66 ± 0.20 IU/mL (range 0.26–1.04) and 0.33 ± 0.10 IU/mL (range 0.18–0.55), respectively. Ex vivo anti-Xa/anti-IIa ratios were close to 2.1. APTT ratios (patient/control) were strongly correlated with anti-IIa activity (p <0.01). There was no progressive increase of the anti-Xa and anti-IIa activities after repeated administration of tinzaparin over the 10 day treatment period. No correlation was found between anti-Xa and anti-IIa activities and age, weight, or creatinine clearance. No major bleeding occurred during the study and only one minor haematoma at the injection site was reported. No thromboembolic complication or death occurred.

Tinzaparin may thus be administered safely at a treatment dose (175 anti-Xa IU/kg) in older patients with age-related renal impairment. Neither dose adjustment, nor serial anti-Xa activity monitoring seems to be required in patients with creatinine clearance above 20 mL/min during the first ten day treatment.
Some Factor VIII (FVIII) Inhibitors Recognise a FVIII Epitope(s) that Is Present only on FVIII-vWF Complexes
Authors: Lavend’homme Renaud , Peerlinck Kathelijne , Jacquemin Marc , Hoylaerts Marc , Jorieux Sylvie , Mazurier Claudine , Vermylen Jos , Saint-Remy Jean-Marie , Gilles Jean Guy
Published: March 2018

A mild haemophilia A patient (LE) with an Arg2150His mutation in the C1 domain of the factor VIII (FVIII) light chain was shown to have anti-FVIII antibodies inhibiting wild type but not self FVIII. Polyclonal anti-FVIII antibodies of this patient were purified by affinity adsorption using recombinant FVIII (rFVIII) and/or plasma-derived FVIII-von Willebrand factor (vWF) complexes. A distinct population of antibodies was obtained that bound to FVIII-vWF complexes but not to rFVIII, indicating that an epitope was created by the association of FVIII to vWF. Such antibodies belonged to the IgG2 isotype, but the FVIII epitopes to which they bind could not be mapped with precision due to vWF dependency. Depletion experiments showed that anti-FVIII antibodies recognising FVIII-vWF complex also distinguished wild-type from mutated self FVIII, indicating that the Arg2150His mutation alters the B cell epitope formed by the association of FVIII to vWF. To determine whether the Arg2150His substitution also alters the formation of the FVIII-vWF complex, the interaction between mutated or normal FVIII with vWF was evaluated in plasma. The dissociation rate of mutated FVIII from vWF was found to be significantly increased. The presence of an Arg2150His mutation therefore results in the disappearance of a FVIII B cell epitope generated by the association of FVIII with vWF. Patients carrying such an Arg2150His mutation and receiving infusion of wild-type FVIII may therefore be at risk of developing inhibitors to allogeneic FVIII only.
A Collaborative Study to Establish the 2nd International Standard for Antithrombin Concentrate
Authors: Walker A. Dawn , Heath Alan B. , Gray Elaine
Published: March 2018

Eighteen laboratories participated in a collaborative study to calibrate a replacement for the 1st International Standard for Antithrombin, Concentrate (88/548). Excellent agreement between laboratories, as indicated by mean % gcv of 3.3 and 5.9 for functional and antigenic assays, was observed when the candidate concentrate (96/520) was assayed against the 1st International Standard for Antithrombin, Concentrate (88/548). The functional potency was found to be 7.9% (p <0.05) lower than the antigenic potency. Based on the results and with the agreement of the participants of this study and the authorisation of the Expert Committee on Biological Standardisation of the World Health Organisation, the antithrombin concentrate, coded 96/520, has been established as the 2nd International Standard for Antithrombin, Concentrate, with labelled potencies for both functional (4.7 IU/ampoule) and antigenic (5.1 IU/ampoule) activities.
A Bispecific Antifibrin-antiplatelet Urokinase Conjugate (BAAUC) Induces Enhanced Clot Lysis and Inhibits Platelet Aggregation
Authors: Ruef Johannes , Nordt Thomas , Peter Karlheinz , Runge Marschall , Kübler Wolfgang , Bode Christoph
Published: March 2018

Thrombolysis is well established in the treatment of acute myocardial infarction. However, clinical application of thrombolytic agents has limitations with respect to efficacy and specificity. To achieve highly effective and at the same time clot-selective plasminogen activation urokinase was coupled to a bispecific antibody consisting of the mono-valent Fab’ from the antifibrin monoclonal antibody 59D8 and the monovalent Fab’ from the anti-glycoprotein GPIIb/IIIa monoclonal antibody 7E3. The bispecific antifibrin-antiplatelet urokinase conjugate (BAAUC) was synthesized and characterized. Assays with either immobilized platelets, GPIIb/IIIa or fibrin showed an increase in plasminogen activation compared to uncoupled urokinase by 10-fold, 58-fold and 13-fold, respectivley (p < 0.0001 each). In vitro clot lysis was performed on platelet-rich and fibrin-rich clots and revealed an up to 5-fold higher potency of BAAUC compared to uncoupled urokinase (p < 0.0001). In vitro platelet aggregation was effectively inhibited by the hybrid molecule, whereas urokinase had no effect. BAAUC and two monospecific urokinase-conjugates, UK-59D8-IgG and UK-7E3-(Fab’)2 were compared with each other with regard to similar tests. In vitro clot assays with platelet-rich and platelet-poor clots were performed. BAAUC achieved a significantly higher plasminogen activation compared to each of the monospecific conjugates (p < 0.05, respectively). We conclude that BAAUC, a bispecific plasminogen activator with antifibrin and antiplatelet properties has the potency to lyse both fibrin-rich and platelet-rich thrombi with high efficacy and to effectively inhibit platelet aggregation.
Persistent Impairment of Platelet Aggregation following Cessation of a Short-course Dietary Supplementation of Moderate Amounts of N-3 Fatty Acid Ethyl Esters
Authors: Maria Cerbone Anna , Cirillo Ferdinando , Coppola Antonio , Risè Patrizia , Stragliotto Eduardo , Galli Claudio , Giordano Mauro , Tremoli Elena , Di Minno Giovanni
Published: March 2018

The duration of the effect of a short-course (1-mo twice-daily) supplementation of moderate amounts (2.28 g) of n-3 fatty acid ethyl esters (FA) on platelet lipid composition and aggregation was compared with that of olive oil (3 g/d) supplementation in 14 healthy volunteers. The FA preparation employed contained eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) in a ratio of 1:1.4. A marked rise (p <0.05) in the plasma and platelet content of EPA and DHA, and minimal changes in the content of arachidonic acid (AA) were documented at withdrawal of the n-3 FA supplementation. EPA/AA and DHA/AA ratios in platelet phospholipids showed that the FA accumulation persisted 8-12 wks after stopping the supplementation (p <0.05). The aggregation of platelets in response to collagen or ADP, and thromboxane B2 (TXB2) formation were impaired at withdrawal. The impaired aggregation lasted 8-12 weeks (p always <0.05), whereas TXB2 formation returned to basal values 4 weeks after stopping the n-3 supplementation. No correlation was found between impaired aggregation and TXB2 formation. In contrast, the impaired sensitivity to ADP (p = 0.036) and, to a lesser extent, to collagen (p = 0.068) were related to changes in the intracellular pH (pHi) of the Na+/H+ reverse transport. No changes in platelet composition or function were observed either during or following olive oil supplementation. These results document a long-lasting impairment of platelet sensitivity to ADP and collagen; changes in the pHi values of the Na+/H+ reverse transport, and a simultaneous persistent accumulation of EPA and DHA in platelet phospholipids, after stopping a short-course dietary supplementation of moderate amounts of n-3 fatty acid ethyl esters.
Changes in the Levels of Soluble Adhesion Molecules and Coagulation Factors in Patients with Deep Vein Thrombosis
Authors: Quarmby J. W. , Collins M. , Lockhart S. M. , Burnand K. G. , Smith A.
Published: March 2018

Current biochemical markers of thrombosis, such as d-dimer, are of little value in demonstrating the presence of thrombus postoperatively, as their levels are elevated by surgery. Thrombosis involves adhesive interactions between the endothelium, platelets and leukocytes. The aim of the study was to determine which of a panel of haemostatic and adhesion factors are altered by the presence of thrombus, but not by surgery. These factors were measured in 20 patients with established spontaneous DVTs, 13 patients having hip replacement surgery and 28 control patients. Circulating levels of P-selectin, VCAM-1 and tissue factor were found to be increased when thrombus was present (p <0.018, p <0.0001, p <0.0028 respectively), but were not altered by surgery. The significance of these circulating factors in venous thrombosis remains to be established, but it is conceivable that they are the product of increased leukocyte trafficking and activity. Assay of VCAM-1, in particular, may be of use in the early detection of venous thrombi in postoperative patients.
Differences in Levels of Soluble E-selectin and VCAM-1 in Malignant versus non Malignant Mediterranean Spotted Fever
Authors: Tissot-Dupont Hervé , Grau Georges-Emile , Camoin-Jau Laurence , Raoult Didier , Sampol José , Dignat-George Françoise
Published: March 2018

In the present study, we investigated the plasma levels of soluble adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in 24 patients with Mediterranean spotted fever (MSF), 6 of whom with a malignant form. Measurements were performed on blood samples collected before treatment (T1), then twice during treatment (T2 and T3). Before treatment, MSF patients taken as a whole presented elevated levels of sICAM-1 and sVCAM-1 and normal levels of sE-selectin and sP-selectin compared to healthy controls. We found that sICAM-1 was elevated both in mild and malignant MSF. sE-selectin and sVCAM-1 were elevated only in patients with the malignant form and allowed to discriminate the two clinical subgroups. Their levels decreased after treatment with sE-selectin reaching control values at T2 whereas sVCAM-1 remained higher over the course of the malignant form. In patients with mild MSF, sP-selectin steadily increased after treatment, whereas it did not present any modification at any of the two sampling times in patients with the malignant form. Raised plasma levels of sE-selectin and sVCAM-1 reflect endothelial activation in malignant rickettsial disease and may be sufficiently early markers to influence the therapeutic decision.
Calibration of Local Systems with Lyophilized Calibrant Plasmas Improves the Interlaboratory Variability of the INR in the Italian External Quality Assessment Scheme
Authors: Chantarangkul Veena , Cesana Bruno , Mannucci Pier Mannuccio , Tripodi Armando
Published: March 2018

Calibration with lyophilized calibrant plasmas certified in terms of PT with International Reference Preparations for thromboplastin has been proposed to minimize the effect of coagulometers on the INR. Aim of this study was to test the ability of local calibration with lyophilized calibrant plasmas, combined with a modified statistical approach, to improve the interlaboratory variability of the INR measured on two test plasmas (one coumarin and one artificially-depleted) by participants in the External Quality Assessment Scheme (EQAS). Sets of lyophilized calibrant and test plasmas were sent to the participants in the EQAS, who were asked to determine PT with their own reagent/ instrument combination (local system). Results were returned as PT together with information on the type of local system, the stated International Sensitivity Index (ISI) and the geometric mean of PTs determined by testing with the local system fresh plasmas from 20 healthy subjects. Ninety-two participants using 9 and 11 brands of reagents and instruments returned results. The CV of the INR determined with the stated ISI for the coumarin (Mean INR = 4.39) and artificially-depleted (Mean INR = 4.23) test plasmas were 11.2% and 10.3% and were reduced on the average by 34% and 54%, respectively, when the INR was calculated with the local ISI.

In conclusions, results from this field study involving laboratories and testing systems representative of the real situation in oral anticoagulant monitoring in our country, indicate that local calibration by artificially-depleted plasmas, combined with the proposed statistical approach, is suitable to improve the interlaboratory agreement on the INR.
Monocyte Procoagulant Activity Induced by Adherence to an Artificial Surface Is Reduced by End-point Immobilized Heparin-coating of the Surface
Authors: Hamers Maria , Møller Anne-Sophie , Sakariassen Kjell , Barstad Marius
Published: March 2018

Heparin-coating improves the biocompatibility of blood contacting artificial surfaces. This led us to investigate the impact of heparin-coating (Carmeda AB, Stockholm) of polymetylmetacrylate on the expression of monocyte tissue factor procoagulant activity (TF-PCA) by surface adhesion. Also, the anticoagulant effect of heparin-coating in the presence or absence of adherent procoagulant monocytes was assessed. This is of particular interest, since activation of extrinsic coagulation by adherent monocyte TF-PCA may play a significant role in thrombin generation during extracorporeal circulation.

Monocytes exposed to heparin-coated or non-coated polymetylmetacrylate expressed TF-PCA. The heparin coat did not affect the rate of monocyte adhesion. However, heparin-coating reduced the induction of TF-PCA of non-adherent and adherent monocytes by 17 and 33% (p 0.001 and p 0.0003), respectively. Heparin-coating in the absence of monocytes, totally inhibited the clotting of recalcified plasma (p 0.003). In contrast, in the presence of adherent monocytes expressing TF-PCA, surface-bound heparin did not inhibit clotting. However, inclusion of heparin in a plasma concentration of 8.9 IU/ml totally inhibited the activation of coagulation.

It is apparent that heparin-coating of an artificial surface is an efficient means to inhibit coagulation of recalcified plasma, but much less so when procoagulant monocytes are adherent to the coated surface. The present findings are of clinical relevance, since monocytes will adhere to blood contacting surfaces of extracorporeal circuits or to implanted vascular prostheses and subsequently express TF-PCA, and this may promote thromboembolism.
Familial Clustering of Factor VIII and von Willebrand Factor Levels
Authors: Houwing-Duistermaat Jeanine , van Houwelingen Hans , Eikenboom Jeroen , Bertina Rogier , Rosendaal Frits , Kamphuisen Pieter
Published: March 2018

Recently, we found that high levels of clotting factor VIII (>150 IU/dl) are common and make an important contribution to thrombotic risk. The determinants of high factor VIII:C are unclear and might be partly genetic. Therefore, we tested the influence of age, blood group and von Willebrand factor (VWF) levels on factor VIII:C levels, and investigated whether factor VIII:C levels are genetically determined. We performed an analysis of 564 female relatives of hemophilia A patients, who had visited our center for genetic counseling. In univariate analysis, AB0 blood group, age and VWF antigen (VWF:Ag) levels all influenced factor VIII:C levels. After adjustment for the effect of VWF:Ag levels, both blood group and age still had an effect on factor VIII:C levels. In sister pairs, the Pearson correlation coefficient between factor VIII:C levels was 0.17 (p = 0.024) and this correlation remained positive (0.15, p = 0.046) after correction for the influence of VWF:Ag. In mother-daughter pairs, no correlation of factor VIII:C levels was found. The correlation of VWF:Ag levels in sisterpairs was 0.41 (p <0.001) and in mother-daughter pairs 0.44 (p <0.001), in line with the assumption that VWF:Ag levels are under control of autosomal genes. Familial influence on plasma factor VIII:C and VWF:Ag levels was investigated with a recently developed familial aggregation test. This test verifies whether familial aggregation of a particular parameter exists in a set of pedigrees. In 435 women from 168 families, factor VIII:C as well as VWF:Ag levels correlated significantly within families, which suggests a familial influence. The familial aggregation was more prominent for VWF:Ag levels than for factor VIII:C levels, possibly because the genetic effect on VWF:Ag levels is larger than on factor VIII:C levels. Our results support the presence of a familial influence on factor VIII:C as well as on VWF:Ag levels.

Our results support the presence of a familial influence on factor VIII:C as well as on VWF:Ag levels.
The Histidin-rich Glycoprotein Pro186/Ser Polymorphism Is not Related to Myocardial Infarction in the ECTIM Study
Authors: Poirier Odette , Nicaud Viviane , Ruidavets Jean-Bernard , Evans Alun , Arveiler Dominique , Luc Gerald , Bara Lucienne , Cambien François , Herrmann Stefan-Martin
Published: March 2018

The histidin-rich glycoprotein (HRG) may contribute to coronary heart disease as a consequence of its possible thrombophilic properties. To test this hypothesis we have investigated the Pro186/Ser polymorphism of the HRG gene, which is known to strongly affect plasma HRG levels, in a large multicenter case-control study of myocardial infarction (MI). The results failed to demonstrate any association between the polymorphism and MI or angiographically assessed coronary stenosis.
Nickel Enhances Collagen-induced Platelet Activation Acting by Increasing the Organization of the Cytoskeleton
Authors: Pulcinelli Fabio , Sebastiani Silvia , Pesciotti Marzia , Pignatelli Pasquale , Daniel James , Gazzaniga Pier Paolo
Published: March 2018

In keratinocytes, osteoclasts and enterocytes, Ni2+ acts as an agonist working through selective activation of the polyvalent cation-sensing receptor.

We report here that while Ni2+ alone had no direct ability to induce platelet aggregation or secretion, Ni2+ pretreatment produced these responses when platelets were stimulated with subthreshold concentrations of collagen. In addition, pretreatment with Ni2+ significantly enhanced collagen-induced phospholipase C activation and calcium mobilization. Platelet adhesion to collagen was increased and the inhibition of collagen-induced adhesion normally seen after cytochalasin D treatment was significantly diminished.

When Ni2+ was added to platelets alone, tyrosine phosphorylation of p60src was increased. Moreover, Ni2+ enhanced the amount of protein, especially actin, found in the low-speed Triton X-100 insoluble cyto-skeleton. Our results indicate that nickel, possibly acting via a platelet cation sensing receptor analogous to that which has been described in other cell types, may cause a rapid tyrosine kinase-dependent cyto-skeleton reorganization leading to enhanced adhesion of platelets to collagen and increasing collagen-dependent responses.
Fibrinogen, Plasminogen Activator Inhibitor-1, and Carotid Intima-Media Wall Thickness in the NHLBI Family Heart Study
Authors: Pankow James , Williams Roger , Evans Gregory , Province Michael , Eckfeldt John , Folsom Aaron
Published: March 2018

Several studies have linked higher plasma fibrinogen and plasminogen activator inhibitor (PAI-1) concentrations with increased risk of cardiovascular disease. We studied whether members of families with increased occurrence of coronary heart disease (CHD) have increased levels of fibrinogen and PAI-1 and whether subclinical carotid atherosclerosis is associated with these two hemostatic factors. Contrary to our hypothesis, fibrinogen and PAI-1 antigen levels were not different between high CHD risk families versus random families. Adjusted for age and family type, fibrinogen and PAI-1 were both associated positively with carotid intima-media thickness assessed by B-mode ultrasound. However, adjustment for lifestyle and medical covariates essentially eliminated these associations. These data suggest 1) elevated fibrinogen and PAI-1 do not explain clustering of CHD in families and 2) fibrinogen and PAI-1 may partly mediate the effects of other risk factors on carotid atherosclerosis, though the data are also consistent with them playing no causal role.
The Diagnostic Value of Thrombopoietin Level Measurements in Thrombocytopenia
Authors: Porcelijn Leendert , Folman Claudia , Bossers Bernadette , Huiskes Elly , Overbeeke Marijke , Schoot C. Ellen , de Haas Masja , Borne Albert E. G.
Published: March 2018

It has been reported that blood trombopoietin (TPO) levels can discriminate between thrombocytopenia due to increased platelet destruction and decreased platelet production. With our TPO ELISA and a glycocalicin ELISA we analysed a large group of patients in detail and could confirm and amplify the above notion in detail.

TPO levels were determined in plasma from 178 clinically and serologically well-defined thrombocytopenic patients: 72 patients with idiopathic autoimmune thrombocytopenia (AITP), 29 patients with secondary AITP, 5 patients with amegakaryocytic thrombocytopenia and 72 patients who suffered from various diseases (46 in whom megakaryocyte deficiency was not and 26 in whom it was expected). In addition, we measured the level of glycocalicin as a marker of total body mass of platelets.

In all patients with primary AITP and secondary AITP, TPO levels were within the normal range or in some (n = 7) cases only slightly increased. The level of glycocalicin was not significantly different from that of the controls (n = 95). The patients with amegakaryocytic thrombocytopenia had strongly elevated TPO levels and significantly decreased glycocalicin levels. Similarly, among the 72 thrombocytopenic patients with various disorders, elevated TPO levels were only found in patients in whom platelet production was depressed. The mean level of glycocalicin in these patients was decreased compared to that in controls and patients with AITP, but was not as low as in patients with amegakaryocytic thrombocytopenia.

In conclusion, all patients with depressed platelet production had elevated levels of circulating TPO, whereas the TPO levels in patients with an immune-mediated thrombocytopenia were mostly within the normal range. Therefore, measurement of plasma TPO levels provides valuable diagnostic information for the analysis of thrombocytopenia in general.

Moreover, treatment with TPO may be an option in AITP.
Inhibition of Thrombin-catalyzed Factor V Activation by Bothrojaracin
Authors: Arocas Véronique , Lemaire Charlotte , Bouton Marie-Christine , Bezeaud Annie , Bon Cassian , Guillin Marie-Claude , Jandrot-Perrus Martine
Published: March 2018

We have previously identified and characterized a potent and specific thrombin inhibitor, isolated from Bothrops jararaca, named bothrojaracin. Bothrojaracin interacts with the two positively charged recognition sites of thrombin referred to as exosite 1 and exosite 2, whereas it does not interact with the thrombin active site. Consequently, bothrojaracin inhibits thrombin-induced fibrinogen to fibrin conversion and platelet activation, without inhibition of thrombin-catalyzed cleavage of small synthetic substrates. In the present study, we show that bothrojaracin exerts an anticoagulant effect in plasma, illustrated by the prolongation of the aPTT. Using purified proteins, we observed that the anticoagulant effect of bothrojaracin was not only due to the inhibition of fibrinogen to fibrin conversion, but in addition to the inhibition of factor V activation by thrombin. Bothrojaracin decreased the rate of thrombin-catalyzed proteolysis of factor V and concurrently the generation of factor Va cofactor activity measured in a prothrombinase assay. We compared the effect of bothrojaracin with that of ligands binding specifically exosite 1 (hirudin C-terminal peptide SH54-65) or exosite 2 (heparin, prothrombin fragment 2). SH54-65 delayed thrombin catalyzed factor V activation whereas heparin or prothrombin fragment 2 did not. The thrombin derivatives β- and γ-thrombin, which are defective in their exosite 1, but present with a normally exposed exosite 2, had a reduced capacity to activate factor V, which was not further impaired by the exosite 2 ligands, bothrojaracin, heparin or prothrombin fragment 2. Altogether, our results provide further insight into the anticoagulant effect of bothrojaracin showing that it is a potent inhibitor of the feedback activation of factor V by thrombin, and thus of the up-regulation of its own production by thrombin. Inhibition of thrombin-catalyzed factor V activation by bothrojaracin is mainly mediated through the interaction of the inhibitor with thrombin exosite 1, whereas contribution of the interaction with exosite 2 does not appear to play a direct role in factor V recognition by thrombin.
Warfarin Resistance Is Associated with a Protein Component of the Vitamin K 2,3-Epoxide Reductase Enzyme Complex in Rat Liver
Authors: Cain Dean , Hutson Susan , Wallin Reidar
Published: March 2018

Warfarin, the most used drug in the world in long-term anticoagulation prophylaxis, targets the vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle in liver. Recently, the enzyme has been identified as a multicomponent lipid-protein enzyme system in the endoplasmic reticulum (ER) membrane (17). As the first step towards understanding genetic resistance to warfarin, we present in this paper data on VKOR from normal and a strain of warfarin resistant laboratory rats maintained in the United States. Metal induced in vitro assembly of the enzyme complex demonstrates that the glutathione-S-transferase (GST) enzyme component of the complex loses its GST activity upon formation of VKOR. Less VKOR activity is measured upon assembly of the complex from warfarin resistant rats. The GST activity measured in warfarin resistant rats, before assembly of the complex, is 10-fold less sensitive to warfarin inhibition than the GST activity measured in normal rats. Microsomal epoxide hydrolase (mEH) is the second component of VKOR. When incubated with the components of VKOR before assembly of the complex, antibodies raised against mEH prevented formation of the enzyme complex. Sequencing of mEH cDNAs from normal and warfarin resistant rats revealed identical sequences. The data suggest that the mutation responsible for genetic warfarin resistance is associated with the GST component of VKOR.
Contribution of Acute-Phase Proteins and Cardiovascular Risk Factors to Erythrocyte Aggregation in Normolipidemic and Hyperlipidemic Individuals
Authors: Weng Xiaoduan , Roederer Ghislaine , Beaulieu Raymond , Cloutier Guy
Published: March 2018

Background. Numerous studies have demonstrated that elevated concentrations of acute-phase proteins affect red blood cell (RBC) aggregation. Plasma lipids and lipoproteins were also shown to be correlated with RBC aggregation in hypercholesterolemia. However, whether acute-phase proteins promote RBC hyperaggregation in hyper-lipidemic patients is unknown. The main objective of the study was to identify the impact of acute-phase proteins such as fibrinogen (Fib), haptoglobin (Hp), ceruloplasmin (Cp), α1-acid glycoprotein (AGP), α1-antitrypsin (AT), immunoglobulin G (IgG), and albumin (Alb) on RBC aggregation in 35 hyperlipidemic patients. The influence of these proteins in 32 normolipidemic subjects was also determined.

Methods and Results. RBC aggregation parameters reflecting the kinetics of rouleau formation and the adhesive strength between RBCs were measured by laser reflectometry. Multivariate forward stepwise linear regression analyses were performed to study the relationship between RBC aggregation and these acute-phase proteins, total cholesterol (TC), triglycerides (TG), high (HDL-C) and low (LDL-C) density lipo-protein cholesterol, age, gender, body mass index (BMI), mean blood pressure (Mpressure), and smoking habit. The kinetics of rouleau formation was positively correlated with the linear combination of IgG and Hp (r = 0.76, p <0.0001) in hyperlipidemic patients, whereas IgG, smoking, AGP and gender were significant independent predictors in healthy subjects (r = 0.79, p <0.0001). The correlations obtained for the models predicting the adhesive strength between RBCs were 0.69 in patients (Alb, HDL-C, IgG, p <0.002) and 0.71 in healthy individuals (AGP, BMI, p <0.0001).

Conclusion. This study suggests that acute-phase proteins such as IgG, Hp, AGP and Alb influence significantly and in an independent way the level of RBC aggregation. The close association between RBC aggregation and cardiovascular risk factors further strengthens its clinical importance.
Home Treatment of Mild to Moderate Bleeding Episodes Using Recombinant Factor VIIa (Novoseven) in Haemophiliacs with Inhibitors
Authors: Aledort Louis , Beardsley Diana , Cooper Herbert , Davignon George , Ewenstein Bruce , Gilchrist Gerald , Gill Joan , Glader Bertil , Hoots W. Keith , Kisker C. Thomas , Lusher Jeanne , Rosenfield Cathy , Shapiro Amy , Smith Horace , Taft Edwin , Key Nigel
Published: March 2018

Objective. To assess the safety and efficacy of a fixed dose of recombinant activated factor VII (rFVIIa; NovoSeven™) in the home setting for mild to moderately severe joint, muscle, and mucocutaneous bleeding episodes in patients with haemophilia A or B with inhibitors. Design. Multicentre, open-label, single arm, phase III study of one year duration. Methods. Patients or their caregivers administered up to three doses of rFVIIa (90 μg/kg i.v.) at 3 h intervals within 8 h of the onset of a mild to moderate bleeding episode. Once the subject considered that rFVIIa had been “effective” with regard to haemostasis (after 1-3 injections), one further (maintenance) dose of rFVIIa was administered. Results. Of 60 patients enrolled, 56 experienced at least one bleed, and 46 completed the one year study. 614 of 877 bleeds (70%) were evaluable according to protocol definitions. Haemostasis was rated as “effective” in 92% (566/614) of evaluable bleeds after a mean of 2.2 injections. For successfully treated episodes, the time from onset of bleeding until administration of the first injection was 1.1 ± 2.0 h (mean ± SD). Twenty-four hours after initial successful response, haemostasis was reported as having been maintained in 95% of cases. Efficacy was comparable for muscle, joint and target joint, and muco-cutaneous bleeding episodes. In an intent-to-treat analysis of all 877 bleeding events, efficacy outcomes were equivalent to the evaluable bleeds, with an effective response in 88% of treated episodes. Treatment-related adverse events occurred in 32 (3% of all) bleeding episodes and consisted of re-bleeds/new bleeds in more than 50% (18/32) of these events. A single episode of superficial thrombophlebitis was the only thrombotic complication encountered, and there were no patient withdrawals due to adverse events. Development of FVII(a) antibodies could not be detected, and hypersensitivity reactions to rFVIIa were not reported. Conclusion. rFVIIa is effective and well tolerated when used in the home setting to treat mild to moderate bleeding episodes in patients with haemophilia A or B with inhibitors.
Rapid Hemophilia A Molecular Diagnosis by a Simple DNA Sequencing Procedure: Identification of 14 Novel Mutations
Authors: Vidal Francisco , Farssac Elisenda , Altisent Carme , Puig Lluís , Gallardo Dominique
Published: March 2018

We here describe a simple, efficient DNA sequencing procedure for hemophilia A molecular diagnosis. In severe patients we first test for the presence of factor VIII gene intron 22 inversion using a recently described single-tube PCR method. In moderate, mild, or inversion-negative severe patients we systematically sequence the promoter, all exons and splice junctions of factor VIII gene. Specially designed primers allow amplification of 23 PCR products under the same salt conditions and thermocycling parameters. The whole sequencing procedure, from blood extraction to mutation identification, can be readily done within 42 h when using regular instruments or in just 14 h when using a high-throughput sequencer. Thus, this is a versatile and cost-effective strategy with little hands-on time requirements. Since its implementation we have identified mutations in 45/46 hemophilia A patients, 14 of which are novel. Once the genetic defect has been identified, accurate genetic counseling is then easily performed.
Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Does not Inhibit In Vitro Thrombolysis by Pharmacological Concentrations of t-PA
Authors: D’Aprile Anna , Italia Alessandra , Gresele Paolo , Morser John , Semeraro Nicola , Colucci Mario
Published: March 2018

TAFI (thrombin activatable fibrinolysis inhibitor) is a plasma procarboxypeptidase that upon activation inhibits the fibrinolytic process by removing the C-terminal lysines from partially degraded fibrin. The generation of activated TAFI (TAFIa) has been suggested to represent a mechanism of thrombus resistance to thrombolytic therapy. However, the ability of TAFI to inhibit fibrinolysis by pharmacological concentrations of t-PA has not been properly investigated. We used an in vitro model consisting of 125I-fibrin blood clots submerged in auto-logous defibrinated plasma. Upon addition of t-PA (125-5000 ng/ml) and CaCl2 (25 mM), samples were incubated at 37° C, and clot lysis was measured at intervals from the radioactivity released into solution. The role of TAFI was assessed either by neutralizing the generated TAFIa with the specific inhibitor PTI (50 g/ml) or by enhancing TAFI activation through the addition of recombinant soluble thrombomodulin (solulin, 1 μg/ml). In our clot lysis model, activation of TAFI amounted to about 20% of inducible carboxypeptidase activity. Addition of PTI, however, produced a significant increase in the extent of lysis only at concentrations of t-PA equal to or lower than 250 ng/ml. When solulin was added to the plasma surrounding the clot, about 70% of TAFI was activated within 15 min. Under these conditions, inhibition of clot lysis was very marked in samples containing 125 or 250 ng/ml of t-PA, but negligible in those containing pharmacological concentrations of the activator (1000 and 5000 ng/ml). Additional experiments suggest that loss of fibrin-dependence by elevated concentrations of t-PA may be one of the mechanisms explaining the lack of effect of TAFIa. Our data indicate that, under our experimental conditions, clot lysis by pharmacological concentrations of t-PA is not influenced by TAFIa even after maximal activation of this procarboxypeptidase.
Vascular Endothelial Growth Factor Enhances the Expression of Urokinase Receptor in Human Endothelial Cells via Protein Kinase C Activation
Authors: Kroon Marielle , Koolwijk Pieter , Vermeer Mario , van der Vecht Bea , van Hinsbergh Victor
Published: March 2018

Among other proteolytic enzymes, the urokinase-type plasminogen activator (u-PA)/plasmin cascade contributes to cell migration and the formation of capillary-like structures in a fibrinous exudate. The u-PA receptor (u-PAR) focuses proteolytical activity on the cell surface of the endothelial cell and hereby accelerates the pericellular matrix degradation. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 enhance u-PA receptor expression in human endothelial cells. In this paper we show that the protein kinase C (PKC) inhibitors Ro31-8220 and GF109203X inhibit VEGF165-induced u-PAR antigen expression in human endothelial cells, whereas PKC inhibition had no effect on FGF-2-induced u-PAR antigen enhancement. In addition, inhibition of PKC activity had no effect on VEGF165-or FGF-2-induced proliferation in human endothelial cells. We conclude that VEGF165 induces u-PAR via a PKC-dependent pathway, whereas proliferation is induced via a different pathway probably involving tyrosine phosphorylation of proteins downstream of the VEGF receptors.
Platelet Shape Change Evoked by Selective Activation of P2X1 Purinoceptors with α,β-Methylene ATP
Authors: Rolf Michael , Brearley Charles , Mahaut-Smith Martyn
Published: March 2018

Simultaneous measurements of [Ca2+]i and light transmission were used to examine the relationship between P2X1 receptor activation and functional platelet responses. The P2X1 agonist α,β-MeATP evoked a transient [Ca2+]i increase and a reversible decrease in light transmission; both responses required external Ca2+ and the nucleotidase apyrase. The transmission response was due to shape change only, verified by scanning electron microscopy and insensitivity to Reopro, a GPIIbIIIa antagonist. α,β-MeATP stimulated smaller shape changes than ADP, however P2X1 responses had a lifespan of <2 h following resuspension in saline and may be considerably larger in vivo. A peak [Ca2+]i increase of >50 nM was required for detectable shape change. Overlap of concentration-response relationships for α,β-MeATP-evoked [Ca2+]i and shape change suggests that other second messengers are not involved. Therefore, the physiological P2X1 agonist ATP can contribute to platelet activation, in contrast to its previously described inhibitory action at metabotropic platelet purinoceptors.
Effect of Intradermal Tumor Necrosis Factor-α-induced Inflammation on Coagulation Factors in Dermal Vessel Endothelium
Authors: Kopp Christoph , Simonitsch Ingrid , Jilma Bernd , Jansen Burkhard , Exner Markus , Speiser Wolfgang , Kapiotis Stylianos , Chott Andreas
Published: March 2018

Inflammatory mediators were shown to exert procoagulant effects on cultured human endothelial cells (EC). In the present study the effect of intradermal application of tumor necrosis factor- (TNF-) on the expression of factors involved in regulation of coagulation at the EC surface, i. e. tissue factor (TF), thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI) was studied in humans in vivo. The endothelial expression of these factors was evaluated immunohistochemically in biopsies taken after intradermal application of 5000 U TNF- in 8 healthy volunteers. After 6 and 22 h biopsies were taken from the injection sites. At TNF- injected sites typical inflammatory changes, e. g. EC upregulation of adhesion molecules and accumulation of leukocytes were detected. In parallel we could document EC expression of TF, downregulation of TM and depletion of tissue factor pathway inhibitor (TFPI) in inflamed areas. Early depletion of endothelial I B at the site of inflammation after application of TNF- points to an activation of the NF- B pathway. Our data suggest that, as shown in in vitro experiments, TNF- activates the NF- B pathway and induces specific procoagulant changes of EC due to expression of TF, down-regulation of TM and depletion of TFPI in vivo in humans. This procoagulant shift in the haemostatic balance on the cell surface, caused by TNF- -induced inflammation, is likely to contribute to thrombosis associated with tissue inflammation in humans.
Activation Products of the Haemostatic System in Coronary, Cerebrovascular and Peripheral Arterial Disease
Authors: Bots M. L. , Haverkate F. , Meijer P. , Hofman A. , Kluft C. , Grobbee D. E. , van der Bom J. G.
Published: March 2018

To determine the presence of a ‘hypercoagulable state’ as assessed by indices of thrombin and plasmin generation and of the amount of fibrin that is lysed, in patients with stable coronary, cerebral and peripheral arterial disease a population-based cross-sectional study was performed. From a population-based cohort comprising 7983 men and women aged 55 years and over, we randomly selected 127 subjects with a history of myocardial infarction, 124 with a history of stroke and/or transient ischemic attack, 131 patients with peripheral arterial disease and 263 control subjects in the same age group without arterial disease. Subjects using anticoagulant drugs were not selected. F1+2, TAT, and PAP were not associated with a history of cardiovascular events, nor with peripheral arterial disease. In contrast, positive associations were found for D-Dimer. Mean D-Dimer level was 40 μg/l (95% CI 35,44) in control subjects; 53 μg/l (47, 61) in those with a history of myocar-dial infarction and 51 μg/l (45, 58) in those with a history of stroke and or transient ischemic attack. D-Dimer increased gradually with increasing severity of peripheral atherosclerosis; a decrease in ankle/arm systolic blood pressure ratio of 0.1 was associated with an increase in D-Dimer of 3.9 μg/l (p<0.01). This was more pronounced in subjects with higher F1+2, TAT and PAP concentration. In conclusion, the markers of onset of coagulation F1+2, TAT and PAP are not associated with the presence of arterial disease, but increased levels of these markers are necessary for the positive association between D-Dimer and arterial disease.
Treatment with Simvastatin and Low-dose Aspirin Depresses Thrombin Generation in Patients with Coronary Heart Disease and Borderline-high Cholesterol Levels
Authors: Undas Anetta , Undas Robert , Brożek Jan , Szczeklik Andrzej , Musiał Jacek
Published: March 2018

Aspirin and statins are beneficial in coronary heart disease across a broad range of cholesterol levels. We assessed the effects of low-dose aspirin (75 mg daily) on thrombin generation in patients with coronary heart disease and average blood cholesterol levels. We also investigated whether in patients with borderline-high cholesterol level who have been already taking aspirin, additional treatment with simvastatin would affect thrombin generation.

Seven-day treatment with low-dose aspirin decreased thrombin generation ex vivo only in patients with total cholesterol 5.2 mmol/L. In patients with higher cholesterol levels aspirin had no effect. In these patients, already taking low-dose aspirin, additional three-month simvastatin treatment resulted in a reduction of thrombin generation. This demonstrates that low-dose aspirin depresses thrombin generation only in subjects with desirable blood cholesterol levels, while in others, with borderline-high cholesterol, thrombin formation is being reduced following the addition of simvastatin.
Adhesive Interactions of Leukocytes, Platelets, and the Vessel Wall during Hemostasis and Inflammation
Authors: McEver Rodger
Published: March 2018

In response to hemorrhage, circulating platelets adhere to exposed subendothelial tissues and then recruit additional platelets into aggregates that function as procoagulant surfaces. In response to inflammatory stimuli, circulating leukocytes roll and then arrest on endothelial cells, and finally migrate into the surrounding tissues where they combat pathogens and repair tissue injury. These traditionally separate cellular adhesive contributions to coagulation and inflammation have become progressively linked. We now know that leukocytes roll and then arrest on activated platelets and that platelets roll on activated endothelial cells. Cell adhesion molecules that were first thought to contribute uniquely to either coagulation or inflammation are now known to contribute to both responses. This review focuses on multicellular adhesive interactions in the vasculature, with particular attention to the dual functions of the adhesion molecules P-selectin, P-selectin glycoprotein ligand-1 (PSGL-1), and glycoprotein Ib (GPIb) in coagulation and inflammation.
Increased Level of von Willebrand Factor Is Significantly and Independently Associated with Diabetes in Postinfarction Patients
Authors: Pancio George , Moss Arthur , Kalaria Vijay , Marder Victor , Weiss Harvey , Miller Watelet Luc , Sparks Charles , Zareba Wojciech ,   for THROMBO Investigators
Published: March 2018

Diabetes is an established risk factor for reinfarction and cardiac death in postinfarction patients. Since the underlying mechanism of diabetes-related risk is not fully understood we aimed to evaluate the association between lipids, thrombogenic factors and diabetes in postinfarction patients. The study population consisted of 1,045 post-infarction patients (846 non-diabetic, 125 non-insulin- and 74 insulin-requiring diabetics) with the following blood tests performed 2 months after an index myocardial infarction: lipoprotein (a), apolipoprotein-B, apolipoprotein-A, cholesterol, HDL cholesterol, triglycerides, insulin, von Willebrand factor (vWF), fibrinogen, factor VII, D-dimer, and plasminogen activator inhibitor (PAI-1). After adjustment for relevant clinical covariates, non-insulin-requiring diabetes was significantly (p <0.05) associated with elevated levels of (odd ratios per 1 log unit increase in parenthesis) vWF (1.74) and PAI-1 (1.42) whereas insulin requiring diabetes was associated with even more elevated levels of vWF (4.68), but not with increased levels of PAI-1. No significant differences in lipid levels were observed among three groups. In conclusion, increased level of von Willebrand factor is significantly and independently associated with diabetes in postinfarction patients, suggesting that endothelial damage is the primary mechanisms contributing to an increased occurrence of vascular and cardiac events in diabetic postinfarction patients.
Disseminated Intravascular Coagulation in Solid Tumors: Clinical and Pathologic Study
Authors: Wan Jim , Nguyen Nam , Hanrahan L. R. , Sigounas George , Sallah Sabah
Published: March 2018

Disseminated intravascular coagulation (DIC) is a well known hemostatic complication of solid tumors. We evaluated the occurrence of DIC in 1117 patients with solid tumors. Of these patients, 76 (6.8%) were diagnosed with DIC. There were a total of 145 bleeding and clotting episodes reported in the 76 patients. Thrombocytopenia, hypofibrinogemia, elevated D-dimer and fibrinogen degradation products were the most common coagulation abnormalities encountered in patients with DIC. In multivariate analysis, older age (p = .0001), male gender (p = .009), advanced malignancies (p = .027), breast cancer (p = .038) and the presence of necrosis in the tumor specimen (p = .004), emerged as independent factors significantly related to the occurrence of DIC in patients with solid tumors. Of the 76 patients, 25 (33%) achieved response to treatment of DIC as defined in the study. Patients with early stage and advanced malignancies who developed DIC had inferior survival when compared with their counterparts without DIC (p = .039 and p = .005, respectively). Taken together, this study indicates that certain clinical and laboratory features are more common in patients with solid tumors who developed DIC. The occurrence of DIC appears to have an independent effect on survival of patients with cancer. Cooperative studies are encouraged to better address the usefulness and optimal prophylactic heparin regimen in patients at risk for DIC.