bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a Novel 33-kDa Ser/Thr Kinase that Phosphorylates the Cytoplasmic Tail of Protease-activated Receptor 1 (Thrombin Receptor) in Human Platelets
Authors: Ido Masaru , Hayashi Tatsuya , Gabazza Esteban , Suzuki Koji 
Published: March 2018

SummaryStimulation of human platelets with thrombin or thrombin receptor agonist peptide (TRAP/ Ser-Phe-Leu-Leu-Arg-Asn) resulted in phosphorylation of the protease-activated receptor 1 (PAR1). However, protein kinase(s), capable of phosphorylating PAR1 upon activation of this receptor, has not been as yet identified in human platelets. The present study was undertaken to assess the presence of protein kinase(s) that may interact with PAR1 using a procedure based on the ability of protein kinase to undergo renaturation and phosphorylate a protein substrate fixed in a gel. We employed a fusion protein that was prepared using a glutathione S-transferase (GST) and the cytoplasmic tail of PAR1 (Pro368-Thr425)(GST-PAR1) or a reverse sequenced peptide of this domain (GST-rPAR1). The results showed that treatment of platelets with thrombin induced about 10-fold increase in the activity of the 33-kDa Ser/Thr protein kinase, which was also activated by TRAP, but not by hirudin-treated thrombin or diisopropylfluorophosphate-inactivated thrombin, suggesting that it is activated through PAR1. Furthermore, treatment of platelets with thromboxane A2 analog, STA2, led to an activation of this protein kinase and phosphorylation of PAR1. In conclusion, the present study provides evidence of homologous and heterologous activation of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of PAR1.
Aspirin Potentiates LPS-induced Fibrin Formation (FPA) and TNF-α-synthesis in Whole Blood
Authors: Haug K.B. , Joø G.B. , Westvik Å.-B. , Øvstebø R. , Kierulf P. , Osnes L.T.N. 
Published: March 2018

SummaryThe effect of aspirin on LPS-incubation of whole blood was investigated. Aspirin induced a concentration dependent increase (2.5–5-fold at 5 mM aspirin) in LPS-induced appearance of TNF-α and fibrinopeptide A (FPA) in plasma, despite the concomitant increase in the inhibitory cytokine IL-10. Aspirin substantially raised the levels of LPS-induced TF-mRNA and TNFα-mRNA in monocytes isolated from whole blood. The median ratio for TF-/β-actin mRNA increased from 1.5 ± 0.44 in the presence of LPS-alone, to 2.5 ± 0.51 when 5 mM aspirin was added. The TNFα/β-actin mRNA ratios were 1.8 ± 0.4 and 5.5 ± 2.7 respectively. Addition of exogenous PGE2 before incubation nearly abrogated the effect of aspirin on TNF-α, substantiating the role of PGE2 as a regulator of TNF-α synthesis, whereas the effect on FPA was small. Thus, in the presence of LPS in this whole blood model, aspirin apparently had a pro-inflammatory rather than an anti-inflammatory effect.
Platelets Release Thrombopoietin (Tpo) upon Activation: another Regulatory Loop in Thrombocytopoiesis?
Authors: Folman Claudia , Linthorst Gabor , van Mourik Jan , van Willigen Gijsbert , de Jonge Evert , Levi Marcel , de Haas Masja , von dem Borne Albert 
Published: March 2018

SummaryThrombopoietin is produced at a constant rate by the liver and kidney and is removed from the circulation upon binding and subsequent uptake via the Tpo receptor, c-Mpl, expressed by platelets and megakaryocytes. Apart from uptake, this study shows that platelets can also function as a storage pool for Tpo.Upon stimulation with various platelet agonists, full-length biologically active Tpo was released by platelets. Platelet fractionation experiments indicated that this Tpo most likely is contained in the granules. When platelets were preincubated with Tpo-peptide mimetic or truncated Tpo prior to maximal activation, a three- to fivefold increment in Tpo release was seen, whereas, the release of other granule proteins such as vWF-propeptide or serotonin remained unchanged. Therefore, the Mpl agonists might compete with Mpl-bound Tpo, thereby releasing Tpo into the platelet supernatant.Intravascular release of Tpo by platelets might occur in patients with massive platelet activation, as occurs in patients with disseminated intravascular coagulation. The Tpo concentration in these patients is elevated (p <0.01) and correlates with markers for thrombin generation, TAT complexes and Fl+2 (rp =0.8 and 0.9; p <0.01). This suggests that the increment in Tpo concentration was attributed to Tpo release by activated platelets in vivo, which might be instrumental in subsequent stimulation of thrombocytopoiesis.
Heparin Induces Synthesis and Secretion of Tissue Factor Pathway Inhibitor from Endothelial Cells
In Vitro
Authors: Svensson Birgit , Olsen Randi , Ezban Mirella , Østerud Bjarne , Paulssen Ruth , Hansen John-Bjarne 
Published: March 2018

SummaryTFPI is a potent inhibitor of the extrinsic coagulation system constitutively synthesized by endothelial cells. A major portion of intravascular TFPI is stored associated with endothelial cells, and administration of unfractionated heparin (UFH) in vivo causes a prompt mobilization of TFPI into the circulation. The present study was conducted to investigate how UFH affected the synthesis, secretion and anticoagulant potency of TFPI in endothelial cells in vitro. A spontaneously transformed immortal endothelial cell line was used (ECV304). Stimulation of ECV304 cells with UFH caused a prompt dose-dependent (0-5 IU UFH/ml) release of TFPI to the medium accompanied by no change of TFPI at the surface membrane assessed by immunocytochemical methods. Northern blot analysis revealed two mRNA transcripts for TFPI with a molecular size of 1.4 kb and 4.4 kb, respectively. Stimulation of ECV304 cells for 24 hrs with various concentrations of UFH caused a dose-dependent increase of TFPI in the medium (6.2-29.6 ng/106 cells within the concentration range 0-10 IU/ml). A similar dose-dependent increase in the expression of both TFPI mRNA species was observed. Long-term incubation of ECV304 cells with 5.0 IU/ml UFH caused a 5-10 fold increase in the TFPI concentration accumulated in the medium over 48 hrs. The increased TFPI mRNA expression induced by UFH appeared already after 10 min, peaked after 2-4 hrs, remained augmented throughout the entire period of UFH exposure, and preceeded the synthesis-dependent increase in TFPI release by 2-4 hrs. The procoagulant activity of the cells was downregulated by 36 % and the contribution of TFPI to the anticoagulant potency of ECV304 cells was moderately increased after 24 hrs heparin stimulation. It is suggested that these mechanisms are of major importance for the anticoagulant function of heparins.
Increased Fibrinolytic Activity during Use of Oral Contraceptives Is Counteracted by an Enhanced Factor XI-independent down Regulation of Fibrinolysis
Authors: Middeldorp Saskia , Tekelenburg Winnie , van den Ende Abraham , Tans Guido , Prins Martin , Rosing Jan , Büller Harry , Bouma Bonno , Meijers Joost 
Published: March 2018

SummaryThe effect of oral contraceptives (OC) on fibrinolytic parameters was investigated in a cycle-controlled cross-over study in which 28 non-OC using women were randomly prescribed either a representative of the so-called second (30 µg ethinylestradiol, 150 µg levonorgestrel) or third generation OC (30 µg ethinylestradiol, 150 µg desogestrel) and who switched OC after a two month wash out period. During the use of OC, the levels of tissue-type plasminogen activator (tPA) activity, plasminogen, plasmin-α2-antiplasmin complexes and D-dimer significantly increased (by 30 to 80%), while the levels of plasminogen activator inhibitor-1 (PAI-1) antigen, PAI-1 activity and tPA antigen significantly decreased (25 to 50%), suggesting an increase in endogenous fibrinolytic activity. These OC-induced changes were not different between the two contraceptive pills. TAFI (thrombin-activatable fibrinolysis inhibitor) levels increased on levonorgestrel, and even further increased on desogestrel. A clot lysis assay that probes both fibrinolytic activity and the efficacy of the coagulation system to generate thrombin necessary to down regulate fibrinolysis via TAFI showed no change of the clot lysis time during OC use. This finding suggests that the OC-induced increase in endogenous fibrinolytic activity is counteracted by an increased capacity of the coagulation system to down regulate fibrinolysis via TAFI. Indeed we observed that during OC use there was a significant increase of F1+2 generation during clot formation. When these assays were performed in the presence of an antibody against factor XI, we observed that the clot lysis time was significantly increased during OC use and that the increase in F1+2 generation during OC therapy was due to a factor XI-independent process, which was significantly higher on desogestrel than on levonorgestrel. These data indicate that the OC-induced inhibition of endogenous fibrinolysis takes place in a factor XI-independent way and is more pronounced on desogestrel than on levonorgestrel-containing OC.
Plasminogen Binding Properties of Macrophage Inflammatory Protein (MIP)-2α
Authors: Arza Begoña , Fábregas Pere , Laroche Yves , Collen Désiré , Lijnen Roger , Félez Jordi 
Published: March 2018

SummaryThe chemokine macrophage inflammatory protein (MIP)-2α was identified as a plasminogen binding protein by phage display analysis. MIP-2α and a truncated form lacking 5 lysine residues in the COOHterminal region (mut-MIP-2α) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2α but not mut-MIP-2α bound specifically to plasminogen, with KA of 3.7×105 M−1 for the interaction of plasminogen with surface-bound MIP-2α. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2α. Activation of plasminogen bound to surface-associated MIP-2α by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency kcat/KM of 0.1 µM−1s−1, as compared to 0.04 µM−1s−1). In contrast, binding of plasminogen to MIP-2α in solution was very weak, as evidenced by the absence of competition of MIP-2α with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2α did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissuetype plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by α2-antiplasmin. Thus, association of MIP-2α with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.
Antiphospholipid and Antiprotein Syndromes in Non-thrombotic, Non-autoimmune Women with Unexplained Recurrent Primary Early Foetal Loss
Authors: Quéré Isabelle , Sanmarco Marielle , Boutiere Brigitte , Mercier Eric , Amiral Jean , Hubert Anne-Michèle , Ripart-Neveu Sylvie , Hoffet Méderic , Tailland Marie-Laure , Rousseau Olivier , Monpeyroux Françoise , Dauzat Michel , Sampol José , Daures Jean-Pierre , Berlan Jacques , Marès Pierre , Gris Jean-Christophe 
Published: March 2018

SummaryVarious antiphospholipid and/or antiprotein antibodies have been suspected to be associated with recurrent early foetal loss in absence of any habitual aetiology. We conducted a hospital-based case control study on women with no antecedent of thromboembolic or autoimmune disease. We studied 3 groups of 518 women: patients with unexplained primary recurrent early foetal loss, patients with explained episodes and mothers with no previous obstetrical accident. Matching the 3 groups was carried out on the basis of age, number or pregnancies and time elapsed since the end of the last pregnancy. Significant biological markers were then prospectively tested.The various antibodies were shown to be dependent on parity and on the presence of previous foetal loss: cut-off values were thus calculated using data obtained from the group of explained accidents, and adjusted for parity. Only anti-phosphatidylethanolamine IgM [odds ratio: 6.0, 95% confidence interval (2.3–15.7), p = 0.0003], anti-β2-glycoprotein I IgG [4.4, (1.6–11.7), p = 0.0035] anti-annexin V IgG antibodies [3.2 (1.2–8.1), p = 0.015] and lupus anticoagulant [3.0, (1.3–6.8), p = 0.009], were found to be independent retrospective risk factors for unexplained early foetal loss. These four markers were subsequently found to be, during the following pregnancy, associated with a significant risk of foetal loss despite a low-dose aspirin treatment.In non-thrombotic, non-auto-immune women with unexplained primary reccurent early foetal loss, subgroups of patients with positive anti-phosphatidylethanolamine IgM antibodies, or positive anti- β2-glycoprotein-I IgG antibodies, or positive anti-annexin V IgG anti-bodies or lupus anticoagulant must be particularised. This should allow therapeutic trials to be carried in well-defined patients.
Modulation of Vascular Human Endothelial and Rat Smooth Muscle Cell Growth by a Fucosylated Chondroitin Sulfate from Echinoderm
Authors: Drouet B. , Matou S. , Mourão P. A. S. , Bros A. , Letourneur D. , Fischer A. M. , Tapon-Bretaudière J. 
Published: March 2018

SummaryFucosylated chondroitin sulfate is a glycosaminoglycan extracted from the sea cucumber Ludwigothurea grisea. This polysaccharide has the same structure as a mammalian chondroitin sulfate but some of the glucuronic acid residues display sulfated fucose branches. Anticoagulant and antithrombotic properties of fucosylated chondroitin sulfate have already been described. In order to further investigate its potential therapeutic use as an antithrombotic agent, we studied its effect on vascular smooth muscle cell (SMC) proliferation and endothelial cell proliferation, migration and Tissue Factor Pathway Inhibitor (TFPI) release. The experiments were performed on SMC from rat thoracic aorta and on human umbilical vein endothelial cell (HUVEC) in culture with or without added fibroblast growth factors (FGF-1 and FGF-2). Our results showed that: (i) fucosylated chondroitin sulfate had a strong inhibitory effect on SMC proliferation (IC50 =10 ± 5 µg/ml) and (ii) no effect on HUVEC proliferation and migration assays, in the absence of exogenous FGF, while heparin had inhibitory effects; (iii) fucosylated chondroitin sulfate (10 µg/ml) enhanced FGF-1 and FGF-2 induced HUVEC proliferation by 45% (145.4 ± 7.2%) and 27% (126.9 ± 4.2%), respectively; (iv) on FGF-induced HUVEC migration, fucosylated chondroitin sulfate (10 µg/ml) had a strong enhancing effect with FGF-1, +122% (222.2 ± 15.8%), three times higher than that of heparin, and a lower enhancing effect with FGF-2, +43% (142.7 ± 4.6%), whereas heparin had no effect; (v) fucosylated chondroitin sulfate stimulated TFPI release, mainly on the free form, +98% (198.2 ± 25.%). In addition, the structural features of the polysaccharide associated with its biological activity were resolved using chemically modified fucosylated chondroitin sulfates. Sulfated fucose branches groups are essential to the potentiating effect of the polysaccharide on HUVEC proliferation and migration. Surprisingly, removal of fucose branches from the fucosylated chondroitin sulfate did not abolish TFPI release. Finally, partial reduction of the glucuronic acid carboxyl groups limited the potentiating effect on HUVEC proliferation and migration but did not affect TFPI release. In conclusion, this fucosylated chondroitin sulfate from invertebrate origin reveals useful properties for an antithrombotic agent: inhibition of SMC proliferation, enhancement of endothelium wound repair and TFPI release. These properties on vascular cells, associated with a low bleeding tendency and an antithrombotic activity, strongly suggest its potential use as a new therapeutic agent in arterial thrombosis and restenosis, with a more favorable effect than heparin.
Human Carotid Artery Smooth Muscle Cells Rarely Express αvβ3 Integrin at Sites of Recent Plaque Rupture
Authors: Yee Karen , Hatsukami Thomas , Schwartz Stephen , Ikari Yuji 
Published: March 2018

SummaryFibrin(ogen) is the major matrix ligand for β3 integrins. If αvβ3 is the major receptor for fibrin(ogen) on intimal smooth muscle cells, we might expect to see this integrin associated with fibrin(ogen). Eightyfour specimens obtained from endarterectomies of 14 patients were studied. Fibrin was frequently observed in carotid intima even at the non-atherosclerotic areas. As for β1 and β3 integrins, β1 was predominant in intima. The αvβ3 integrin expression was less frequent than α5β1, another receptor for fibrin(ogen), in diffuse intimal thickening, fibrous cap and advanced plaques. Furthermore, αvβ3 was generally negative on smooth muscle cells in recent plaque ruptures. In conclusion, we suggest more attention should be paid on abundant fibrin matrix in intima. Histologically, the α5β1 integrin rather than the αvβ3 is the major receptor for fibrin in intimal smooth muscle cells.
Predicting Adverse Outcome in Patients with Acute Pulmonary Embolism: A Risk Score
Authors: Wicki Jacques , Perneger Thomas , Bounameaux Henri , Junod Alain , Perrier Arnaud 
Published: March 2018

SummaryReliable prediction of adverse outcomes in acute pulmonary embolism may help choose between in-hospital and ambulatory treatment. We aimed to identify predictors of adverse events in patients with pulmonary embolism and to generate a simple risk score for use in clinical settings. We prospectively followed 296 consecutive patients with pulmonary embolism admitted through the emergency ward. Logistic regression was used to predict death, recurrent thromboembolic event, or major bleeding at 3 months. Thirty patients (10.1%) had one or more adverse events during the 3-month follow-up period: 25 patients (8.4%) died, thromboembolic events recurred in 10 patients (3.4%), and major bleeding occurred in 5 patients (1.7%). Factors associated with an adverse outcome in multivariate analysis were cancer, heart failure, previous deep vein thrombosis, systolic blood pressure <100 mmHg, arterial PaO2 <8 kPa, and presence of deep vein thrombosis on ultrasound. A risk score was calculated by adding 2 points for cancer and hypotension, and 1 point each for the other predictors. A score of 2 best identified patients at risk of an adverse outcome in a receiver operating characteristic curve analysis. Of 180 low-risk patients (67.2%) (score ≤2), only 4 experienced an adverse outcome (2.2%), compared to 23 (26.1%) of 88 high-risk patients (score ≥3). A simple risk score based on easily available variables can accurately identify patients with pulmonary embolism at low risk of an adverse outcome. Such a score may be useful for selecting patients with pulmonary embolism eligible for outpatient care.
Characterization of Cleaved Plasma Protein S with a Monoclonal Antibody-based Assay
Authors: Morbœuf Olivier , Borgel Delphine , Gaussem Pascale , Vincenot Anne , Pittet Jean-Louis , Aiach Martine , Gandrille Sophie 
Published: March 2018

SummaryA monoclonal antibody (mAb 5A5G2) recognized cleaved plasma protein S (PS) but not uncleaved PS. Interestingly, mAb 5A5G2 did not recognize thrombin-cleaved recombinant PS. Microsequencing of cleaved plasma PS showed a Q-S-T-N amino-terminal sequence, inferring cleavage after the Arg 60 residue. The mAb epitope was located within the sequence encompassing residues 61 to 73, i. e. the carboxyterminal part of the thrombin-sensitive region (TSR). We used this mAb to develop an ELISA assay to quantify in vivo cleaved PS. In plasma from 10 normal subjects, about 10% of PS was cleaved (7.1% to 15.4%), with a more than 2-fold increase in the corresponding sera. We found increased levels of cleaved PS in 8 patients with disseminated intravascular coagulation (DIC) and decreased levels in 22 patients on long-term oral anticoagulant therapy, whereas cleaved PS levels were similar in 8 hemophiliacs and the 10 normal subjects. Cleaved PS levels did not correlate with prothrombin fragment 1+2 levels released after cleavage by FXa in any of the groups, suggesting that circulating FXa is not the main factor involved in the production of cleaved PS in vivo.
Histidine-rich Glycoprotein (HRG) Tokushima 2: Novel HRG Deficiency, Molecular and Cellular Characterization
Authors: Yoshida Hidemasa , Kanagawa Yasuhiko , Satoh Koichi , Wakabayashi Sadao , Matsumoto Toshio , Koide Takehiko , Shigekiyo Toshio 
Published: March 2018

SummaryThe proband, a 76-year-old woman, suffered from dural arteriovenous fistula. Her plasma histidine-rich glycoprotein (HRG) level was 50% of the normal level. A low level of plasma HRG was also found in her third daughter. A single nucleotide substitution of T to C was found at nucleotide position 11,438 in exon 6 of the HRG gene from the proband, converting Cys223 to Arg in the second cystatin-like domain. The same mutation was also identified in her third daughter, but not in the other four family members having normal HRG levels or in 50 unrelated healthy Japanese individuals. Expression studies in BHK cells showed that substantial intracellular degradation of the mutant occurred and only about 40% of the recombinant HRG mutant was secreted. These results indicate that congenital HRG deficiency caused by a substitution of Cys223 to Arg is hereditary in this family.
Third Generation Oral Contraceptives and Heritable Thrombophilia as Risk Factors of Non-fatal Venous Thromboembolism
Authors: Andersen Birthe Søgaard , Olsen Jørn , Nielsen Gunnar Lauge , Steffensen Flemming Hald , Sørensen Henrik Toft , Baech John , Gregersen Henrik 
Published: March 2018

SummaryThird generation oral contraceptives (OCs) are apparently stronger risk factors for venous thromboembolism (VTE) than other OCs, however, the increased risk may be due to confounding by indication related to differences in prescription behaviour.We estimated the risk of VTE associated with use of OCs with and without the presence of Factor V Leiden mutation, protein C-, protein S- or antithrombin deficiency.Sixty-seven cases with VTE were compared with 134 controls. The risk of VTE in the presence of thrombophilia was of the same magnitude for third generation OC users as for users of other OCs; OR: 52.5 (95% CI: 3.7-738.1) and OR: 63.3 (95% CI: 6.2-648.4), respectively.It is unlikely that confounding by indication entirely explains the risk of VTE associated with third generation OCs since the combined effect exceeds what could be explained if this source of error was the only determinant of the association.
D-dimer Strategy in Thrombosis Exclusion
Authors: Trillaud H. , Labrouche S. , Gauthier P. , Javorschi S. , Bernard P. , Grenier N. , Freyburger G. 
Published: March 2018

SummaryDD are now recognized as a valuable tool to screen patients suspected of deep venous thrombosis or pulmonary embolism before carrying out a gold standard radiologic examination. The newest methods available claim to be able to ascertain the absence of thrombosis, but they have yet to prove their efficiency. We compared the performances of 3 reference ELISA methods (D-DI Asserachrom™ Stago, D-dimer Enzygnost™ Behring and Dimertest GOLD EIA™ Agen), 5 recent rapid methods (VIDAS D-Dimer™ bioMérieux, Instant IA™ Stago, Simplired™ Agen, Nycocard D-dimer™ Nycomed and Accuclot D-Dimer™ Sigma Diagnostics) and two routine latex methods (Dimertest™ American Diagnostica and FDP-Slidex™ bioMérieux) in 100 patients. One of the rapid quantitative methods was demonstrated to have a level of efficiency comparable to that of ELISA methods. Finally, the cost and efficiency of different strategies were evaluated, the association of a routine latex method with the VIDAS D-Dimer™ bioMérieux being proven to be the most efficient.