bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epitope Location on Tissue Factor Determines the Anticoagulant Potency of Monoclonal Anti-tissue Factor Antibodies
Authors: Moran Paul , Chiang Nancy , Kim Jin , Riederer Markus , Eigenbrot Charles , Kelley Robert , Kirchhofer Daniel 
Published: March 2018

SummaryTissue factor (TF), the cellular cofactor for the serine protease factor VIIa (F. VIIa), triggers blood coagulation and is involved in the pathogenesis of various thrombosis-related disorders. Therefore, agents which specifically target tissue factor, such as monoclonal antibodies, may provide promising new antithrombotic therapy. We mapped the epitopes of several anti-TF antibodies using a panel of soluble TF mutants. They bound to three distinct TF regions. The epitope of the 7G11 antibody included Phe50 and overlapped with a TF-F. VIIa light chain contact area. The common epitope of the antibodies 6B4 and HTF1 included residues Tyr94 and Phe76 both of which make critical contacts to the catalytic domain of F. VIIa. The antibodies D3 and 5G6 had a common epitope outside the TF-F. VIIa contact region. It included residues Lys165, Lys166, Asn199, Arg200 and Lys201 and thus overlapped with the substrate interaction region of tissue factor. The antibodies 5G6 and D3 were potent anticoagulants when infused to flowing human blood in an ex-vivo thrombosis model. Plasma fibrinopeptide A levels and fibrin deposition were completely inhibited. In contrast, 6B4 was a weak inhibitor in this ex-vivo thrombosis model, and HTF1 displayed no inhibition at all. These disparate activities were also reflected in TF-dependent F. X activation assays performed with human plasma. The potency differences could neither be explained by the determined binding affinities nor by the on-rates of antibodies. Therefore, the results suggest that antibody binding epitope and hence the particular mechanism of inhibition, is the main determinative factor of anticoagulant potency of anti-TF antibodies.
Occurrence of Amphoterin (HMG1) as an Endogenous Protein of Human Platelets that Is Exported to the Cell Surface upon Platelet Activation
Authors: Imai Shinji , Rauvala Heikki , Parkkinen Jaakko , Rouhiainen Ari 
Published: March 2018

SummaryAmphoterin (HMG1) is a 30-kD heparin-binding protein which is functionally associated with the outgrowth of cytoplasmic processes in developing neurones. Amphoterin has been shown to mediate adhesive and proteolytic interactions at the leading edge of motile cells. Recently it was shown that inhibition of amphoterin interactions with its cell surface receptor (RAGE) suppresses tumour growth and metastasis. In this work we have identified amphoterin polypeptide and its mRNA in human platelets. Amphoterin had a cytoplasmic localisation in resting platelets according to subcellular fractionation studies and immunogold electronmicroscopy. After platelet activation, part of amphoterin was associated with the external surface of plasma membrane. Externalisation of amphoterin during platelet activation was also detected in immunofluorescence studies. Amphoterin was detectable in human serum (0.2 ng/ml) but not in plasma. Resting platelets treated with PGI2 and forskolin bound to immobilised recombinant amphoterin independently of divalent cations. The binding induced a spicular morphology in platelets, and was effectively inhibited by heparin. Amphoterinbinding protein components on the platelet surface were not identified, but amphoterin bound to phosphatidylserine and sulfatide in lipid binding assays. Our results suggest that amphoterin is an endogenous protein in human platelets, which is exported to the cell surface during platelet activation. Interaction of amphoterin with the platelet surface may be mediated by sulfoglycolipids and phospholipids.
Effect of Sodium Arachidonate on Thrombin Generation through Platelet Activation – Inhibitory Effect of Aspirin
Authors: Scazziota Alejandra , Rouvier Jorge , Gonzalez Claudio , Altman Raul 
Published: March 2018

Summary
Background. Sodium arachidonate was used in this study to determine its capacity to generate thrombin through platelet activation. Whether aspirin prevent this effect was also investigated. Methods and Results. Seventeen healthy volunteers without and after 160 mg/day aspirin intake for 3-5 days were studied. Lag-time and TG at basal condition and after platelet stimulation by sodium arachidonate (AA) were measured in normal non-aspirinated as well as “in vivo” aspirinated platelet rich plasma. (PRP). The lag-time was statistically significant shorter in non-aspirinated PRP activated with AA compared with non-activated PRP. This effect was inhibited by aspirin. In non-aspirinated PRP, there was an increase of TG at 4 and 6 min. incubation when platelets were activated with AA but the difference disappeared after 8 min. incubation, (84 ± 71; 148 ± 58 and 142 ± 92 nmol/L respectively) compared with non-aspirinated, non-activated platelets (16 ± 23; 55 ± 56 and 111 ± 76 nmol/L at 4, 6 and 8 min, p < 0.0001, p < 0.0001 and p = 0.292, respectively). The AUCo→22 min were 520.6 ± 545.5 in nonaspirinated, non-stimulated PRP and 808.9 ± 617, in non-aspirinated PRP activated with sodium arachidonate (p = 0.014). Aspirin administered in vivo produced a decrease of TG in PRP activated with AA.
Conclusion. Platelet activated by AA trigged TG. This effect was inhibited by aspirin and could be an additional beneficial effect of aspirin in the prevention of thrombosis.
Genetic Risk Factors for Superficial Vein Thrombosis
Authors: Cattaneo Marco , Taioli Emanuela , De Stefano Valerio , Chiusolo Patrizia , Mannucci Pier Mannuccio , Martinelli Ida 
Published: March 2018

SummaryInherited thrombophilic states are associated with an increased risk for deep vein thrombosis (DVT), but whether they are also risk factors for superficial vein thrombosis (SVT) is uncertain. We assessed the risk conferred by inherited thrombophilic states in patients with a first episode of SVT in whom the coexistence of DVT had been ruled out by ultrasonography. Sixty-three patients with SVT, after exclusion of patients with varicose veins, malignant or autoimmune disease, and 537 healthy individuals were investigated. The G1691A mutation in the factor V gene, the G20210A mutation in the prothrombin gene, and deficiencies of the naturally occurring inhibitors of coagulation (antithrombin, protein C, protein S) were searched. The prevalence of each thrombophilic state was higher in patients than in controls. The odds ratios for SVT were 6.1 (95% confidence interval [CI], 2.6 to 14.2) in patients with the G1691A factor V mutation, 4.3 (95% CI, 1.5 to 12.6) in those with the G20210A prothrombin mutation, and 12.9 (95% CI, 3.6 to 46.2) in those with deficiencies of the naturally occurring inhibitors of coagulation taken together. Risks did not substantially change when the analysis was restricted to 43 patients who had SVT as their only thrombotic manifestation, being 4.3 (95% CI, 1.5 to 12.3) in patients with factor V mutation, and 3.6 (95% CI, 1.0 to 13.1) in those with the prothrombin mutation. Among the circumstantial risk factors investigated (surgery, trauma, prolonged immobilization, oral contraceptives and pregnancy or puerperium), pregnancy or puerperium was the most frequently associated with SVT, being present in 38% of women. Our findings indicate that inherited thrombophilic states are associated with an increased risk for SVT. Hence, a laboratory search of these alterations is recommended in patients with SVT, because it allows the identification of patients at high risk of DVT in whom antithrombotic prophylaxis is particularly warranted.
Prospective Evaluation of Hemostatic System Activation and Thrombin Potential in Healthy Pregnant Women with and without Factor V Leiden
Authors: Weltermann Ansgar , Philipp Karl , Hafner Erich , Kaider Alexandra , Kittl Eva-Maria , Brenner Brigitte , Mannhalter Christine , Lechner Klaus , Kyrle Paul , Eichinger Sabine 
Published: March 2018

SummaryNormal pregnancy is associated with alterations of the hemostatic system towards a hypercoagulable state and an increased risk of venous thromboembolism. The risk of venous thrombosis is higher in pregnant women with factor V Leiden (FVL) than in those with wildtype factor V. Routine laboratory assays are not useful to detect hypercoagulable conditions. A prospective and systematic evaluation of hemostatic system activation in women with and without FVL during an uncomplicated pregnancy employing more sensitive markers of hypercoagulability, such as prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), D-Dimer, or the endogenous thrombin potential (ETP), an indicator of the plasma’s potential to generate thrombin, has not been performed. We prospectively followed 113 pregnant women with (n = 11) and without (n = 102) FVL and measured F1+2, TAT, D-Dimer and the ETP at the 12th, 22nd and 34th gestational week as well as 3 months after delivery (baseline) in each subject. None of the women developed clinical signs of venous thromboembolism during pregnancy or postpartum. Pregnant women with and without FVL exhibited substantial activation of the coagulation and fibrinolytic system as indicated by a gradual increase of F1+2, TAT and D-Dimer throughout uncomplicated pregnancy up to levels similar to those found in acute thromboembolic events (p < 0.0001 by analysis of variance for each parameters). Levels of F1+2 and TAT were comparable between women with and without FVL, but levels of D-Dimer were significantly higher in women with FVL than in those without the mutation (p = 0.0005). The ETP remained unchanged in both women with and without FVL at all timepoints. Our data demonstrate a substantial coagulation and fibrinolytic system activation in healthy women with and without FVL during uncomplicated pregnancy. An elevated F1+2, TAT or D-Dimer level during pregnancy is not necessarily indicative for an acute thromboembolic event. The normal ETP in both women with and without FVL suggests that the capacity of the plasma to generate thrombin after in vitro activation of the clotting system is not affected by pregnancy. Higher levels of D-Dimer in women with FVL than in women with wildtype factor V at baseline as well as during pregnancy indicate increased fibrinolytic system activation in carriers of the mutation.
A Flow Cytometric Assay of Platelet Activation Marker P-Selectin (CD62P) Distinguishes Heparin-induced Thrombocytopenia (HIT) from HIT with Thrombosis (HITT)
Authors: Jy Wenche , Mao Wei Wei , Horstman Lawrence , Valant Peter , Ahn Yeon 
Published: March 2018

SummaryHeparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios ~2.5 vs. HIT ratios ~1.2; p <0.001). Eleven of 12 HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p < 0.0005). In a case of HIT with silent thrombosis, this assay gave a positive results prior to clinically evident thrombosis. In conclusion, this method distinguishes HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.
Platelet Aging In Vivo Is Associated with Loss of Membrane Phospholipid Asymmetry
Authors: Palomo Iván , Ocqueteau Mauricio , Soto Mónica , Aranda Eduardo , Mezzano Diego , Pereira Jaime 
Published: March 2018

SummaryThe mechanism(s) involved in the clearance of senescent platelets are largely unknown. The loss of membrane phospholipid (PL) asymmetry, with phosphatidylserine (PS) exposure appears to be an important signal for the ingestion by macrophages of apoptotic nucleated cells and it has also been suggested as a signal for the removal of aged erythrocytes. Accordingly, it seems possible that the clearance of normal aged platelets from circulation might be triggered by PS exposure. To investigate this, we determined PS exposure in human aging platelets taking advantage of the relationship between platelet density and platelet age and in dog platelets in a model of platelet aging in vivo. PS exposure was determined in two experimental conditions: 1) human platelet density subpopulations obtained by centrifugation in arabinogalactan gradients; 2) circulating canine platelets during decline in platelet count after suppression of thrombopoiesis following estradiol injection. PS exposure was determined by flow cytometry after labeling the cells with FITC-conjugated annexin V. The proportion of human platelets with exposed PS was significantly higher in high density (HD) platelets compared to low density (LD) platelets (11.3 ± 8.0% vs 5.2 ± 3.7%; p <0.05, respectively). In dogs, the proportion of cells with exposed PS rises dramatically with age, from 3.1 ± 0.4% before to 17.7 ± 12.3% ten days after estradiol injection. These findings suggest that platelet aging is associated with loss of phospholipid asymmetry and PS exposure on the outer leaflet of cell membrane, which may play an important role in the recognition and subsequent removal of senescent platelets.
Common Mutation of Plasminogen Detected in Three Asian Populations by an Amplification Refractory Mutation System and Rapid Automated Capillary Electrophoresis
Authors: Ooe Asako , Kida Masafumi , Yamazaki Tomio , Park Sang-Chul , Hamaguchi Hideo , Girolami Antonio , Ichinose Akitada 
Published: March 2018

SummaryCongenital deficiency and dysfunction of plasminogen (PLG) are associated with a mild thrombotic tendency. To facilitate the genetic diagnosis of dysPLGemia, we combined an amplification refractory mutation system and rapid automated capillary electrophoresis. Two different fluorescence-labeled PLG-specific primers for exon XV were designed so that each DNA amplified by PCR showed fluorescence of a different wavelength. Single peaks were detected for the normal and the mutant Ala601-Thr alleles, respectively. A study of 90 normal Caucasians revealed no individuals with the mutation, whereas its gene frequency was 0.021 in Japanese. This mutation was also detected in Korean and Chinese populations at gene frequencies of 0.016 and 0.015, respectively. All of the Korean and Chinese cases with the mutation had at least one haplotype I of the PLG gene, as did most Japanese cases. The high frequency of the Ala601-Thr mutation among these Asian populations may be due to the founder effect.
Prevention of Deep Vein Thrombosis after Hip Replacement
Authors: Samama Meyer , Lensing Anthonie , Büller Harry , Barre Jeanne , Vochelle Nicole , Beau Bertrand , Planès André 
Published: March 2018

SummaryConsecutive patients undergoing total hip replacement in 43 centres were randomly assigned to receive blindly either enoxaparin (40 mg) or tinzaparin (4,500 anti-Factor IU Xa), as once daily subcutaneous injections. The first injection was administered 12 h preoperatively. Efficacy was assessed by bilateral venography performed 12-14 days postoperatively. Efficacy and safety were blindly and centrally adjudicated. Among the 499 patients included, 440 had a venogram. The total incidence of DVTs was 44 (20.1%) of the 219 patients of the enoxaparin group and 48 (21.7%) of the 221 patients of the tinzaparin group. The upper limit of the 80% confidence interval of the difference between the two treatment groups was less than 5.0%. Therefore according to the protocol’s specifications equivalence was shown. Proximal DVTs occurred in 10.5% of the enoxaparin group (23 patients) and in 9.5% (21 patients) of the tinzaparin group. No overt major bleeding was observed. One patient in the enoxaparin group developed severe thrombocytopenia and died. The LMWH tinzaparin appears clinically to be as effective and safe as enoxaparin in the prophylaxis of deep vein thrombosis after total hip replacement, at the doses used and under the conditions of this study.
Inhibitors in German Hemophilia A Patients Treated with a Double Virus Inactivated Factor VIII Concentrate Bind to the C2 Domain of FVIII Light Chain
Authors: Laub R. , Giambattista Di , Fondu P. , Brackmann H.-H. , Lenk H. , Saenko E. L. , Felch M. , Scandella D. 
Published: March 2018

SummaryTo reduce the risk of transmission of hepatitis A virus, an Octaphar-ma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.
TFPIβ, a Second Product from the Mouse Tissue Factor Pathway Inhibitor (TFPI) Gene
Authors: Monroe Dougald , Oliver Julie , Roberts Harold , Chang Jen-Yea 
Published: March 2018

SummaryTissue factor pathway inhibitor (TFPI) contains three Kunitz domains separated by two connecting regions. We have cloned another naturally occurring TFPI gene product from a mouse lung cDNA library which we have called TFPI β. TFPIβ is derived from alternative splicing of the TFPI gene. Analysis of the cDNA shows that mouse TFPIβ protein is identical to TFPI from the N’-terminus through the second connecting region. However, mouse TFPIβ possesses neither a third Kunitz domain nor an Arg, Lys-rich C’-terminus but instead has a completely different C’-terminal (β-domain) sequence which is not homologous to any known protein. Northern blot analyses show that the tissues for mouse TFPIβ synthesis are heart and lung; in contrast, TFPI appears in Northern blots of heart and spleen. Both TFPIβ and TFPI messages first appear in 7-day-old mouse embryos, but only the TFPI mRNA persists until 17 days. Purified recombinant TFPIβ shows an apparent molecular weight of 38 kDa. Kinetic studies indicate that mouse TFPIβ is a slow-binding enzyme inhibitor for human factor Xa. In addition, heparin does not enhance the inhibition of factor Xa by mouse TFPIβ although it does accelerate factor Xa inhibition by TFPI.
Local Calibration of International Normalised Ratio Improves between Laboratory Agreement: Results from the UK National External Quality Assessment Scheme
Authors: Jennings I. , Woods T. A. L. , Preston F.E , Kitchen S. ,   behalf of UK NEQAS (Blood Coagulation) Steering Committee
Published: March 2018

SummaryIn the present study we have performed local calibration of International Normalised Ratio (INR) measurement systems in a large series of laboratories. We assigned INRs to five lyophilised plasma calibrants, one prepared from normal plasma and four using plasma from war-farinised patients, using different International Reference Preparations for Thromboplastin. These five calibrants, and two lyophilised test plasmas were analysed by 349 centres using 60 different thromboplastin instrument combinations.Plasma calibrants were assigned INRs using the WHO reference thromboplastin RBT-90 or the European reference thromboplastin CRM 149R. Each participating centre determined PTs of the calibrants with their local system. These PTs were then used to construct a local calibration graph relating PT to INR. The PTs of test plasmas were converted directly into INR using the local calibration model and into INR using the conventional method. The overall medians of conventionally derived INRs of two test plasmas analysed in 349 centres were 2.50 and 3.10, compared to 2.47 and 3.04 after local calibration where RBT-90 was employed to assign INRs to calibrants. Use of CRM 149R to assign INRs to calibrants led to a significant (p <0.0001) increase in INR to 2.7 and 3.36 respectively. When results were grouped according to the thromboplastin employed, agreement between results with different reagents was improved by local calibration. There was a significant reduction (p <0.01) in the spread of results in different centres as indicated by a reduction in coefficient of variation.
A Longitudinal Study of the Relationships between Haemostatic, Lipid, and Oestradiol Changes during Normal Human Pregnancy
Authors: Greer Ian , Rumley Ann , Stewart Grace , Shepherd James , Packard Chris , Lowe Gordon , Sattar Naveed 
Published: March 2018

SummaryIncreased activation of both blood coagulation and fibrinolysis occurs during normal pregnancy. The responsible mechanisms are unclear, but may include increases in both oestradiol and blood lipids. We, therefore, studied the associations between fasting serum oestradiol, plasma cholesterol and triglyceride, and Factor VII activity, PAI activity, t-PA antigen, fibrin D-dimer, and vWF antigen in 10 women, each sampled on 6 occasions between 10 weeks and 35 weeks during normal pregnancy. Strong and similar individual correlations were observed between increases in FVII, PAI, t-PA and D-dimer (but not vWF) and increases in both oestradiol and triglyceride. Associations between increments in plasma cholesterol and haemostatic factors (except for FVII), were somewhat weaker. We, therefore, suggest that oestradiol-induced hypertriglyceridaemia may be a cause of elevations in plasma Factor VII activity, PAI and t-PA, and fibrin turnover (D-dimer) during normal pregnancy, but is poorly related to the increase in vWF antigen.