bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of Recombinant Human Protein C In Vitro and In Vivo by Muscle Cells
Authors: Türkay Ataç , Wang Jian-Min , Kurachi Kotoku 
Published: March 2018

SummaryProtein C plays a key role in a natural anticoagulation mechanism, and is also implicated in fibrinolytic and anti-inflammatory functions. Here we describe the production of biologically active human protein C by muscle-targeted gene transfer. Human protein C expression vectors were designed and constructed to produce human protein C in skeletal muscle cells. These vectors were tested in transient and stable transfections of SCID mice myoblasts. Stably transfected cells produced as high as 2.27 μg/106 cells/day. Human protein C produced had a relative activity of 92 ± 8% compared to the plasma derived human protein C, and was composed of α and β forms, 69% and 31%, respectively. After implantation of stably transfected myoblasts into the hind limb muscles of SCID mice, systemic stable production of human protein C in a range of 31-116 ng/ml serum was obtained up to at least 2.5 months.Current address: J. Wang, Department of Hematology, Changai Hospital, 174 Changai Road, Yangpu District, Shangai 200433, China
Plasmodium Falciparum-infected Erythrocytes: A Mutational Analysis of Cytoadherence via Murine Thrombomodulin
Authors: Rabhi-Sabile Samia , Steiner-Mosonyi Marta , Pollefeyt Saskia , Collen Desiré , Pouvelle B. , Gysin Jurg , Boffa Marie-Claire , Conway Edward 
Published: March 2018

SummaryThe pathophysiologic events leading to organ damage in Plasmo-dium falciparum malaria infections involve adhesion and sequestration of parasite-infected erythrocytes (PRBC) to the vascular endothelium and syncytiotrophoblast. Several potential receptors to which the PRBCs may bind have recently been identified, one of which is thrombomodulin (TM). TM has been implicated particularly in mediating sequestration of P. falciparum-infected erythrocytes in the placenta and brain, two sites of disease associated with high morbidity. In order to establish that binding of parasite-infected red blood cells to TM is dependent on its containing chondroitin-4-sulfate (CSA), we have mutated the CSA-attachment site of murine TM, and expressed this mutant form (TMser-gly) in COS-7 cells. In cytoadhesion assays, we demonstrate that, in contrast to wild-type TM which contains CSA and supports the adhesion of 1466 PRBCs/mm2, TMser-gly does not contain CSA and adhesion of PRBCs to those cells expressing TMser-gly is entirely abrogated (200 PRBCs/mm2). These studies further confirm that the CSA of TM may play a role in the pathophysiology of malaria by providing a binding site for PRBCs.
DX-9065a, an Orally Active Factor Xa Inhibitor, Does not Facilitate Haemorrhage Induced by Tail Transection or Gastric Ulcer at the Effective Doses in Rat Thrombosis Model
Authors: Tanabe Kiyoshi , Morishima Yoshiyuki , Shibutani Tomoko , Terada Yasuko , Hara Tsuyoshi , Shinohara Yasutaka , Aoyagi Kazuharu , Kondo Takashi , Kunitada Satoshi 
Published: March 2018

SummaryDX-9065a is an antithrombin III (AT III)-independent and selective inhibitor of activated blood coagulation factor X (FXa). We evaluated the effects of DX-9065a and warfarin on bleeding time and blood loss in rat tail transection model and on blood loss in hydrochloride (HCl)-induced rat gastrointestinal haemorrhage model. The blood loss was determined by measuring the haemoglobin content in saline immersed with transected tail or hematin chloride content in the gaster after HCl administration. DX-9065a or warfarin was administered orally at 1 h or 15-21 h before the haemorrhagic stimuli, respectively. The dose required for 50% inhibition of thrombus formation (ID50) was 21 mg/kg for DX-9065a and 0.75 mg/kg for warfarin in a copper wire-inserted arteriovenous (AV) shunt model. In contrast to DX-9065a (10 or 30 mg/kg), warfarin (0.75 mg/kg) significantly prolonged the bleeding time. In rat tail transection model, the blood loss for the control group was 102 ± 41 μl at 20 min after the transection. While warfarin (0.75 mg/kg) facilitated the blood loss about 5 times as much as the control, DX-9065a (10 or 30 mg/kg) did not. In rat gastrointestinal model, the blood loss for the control group was 15.9 ± 5.6 μl at 15 min after HCl administration. In contrast to DX-9065a (10 or 30 mg/kg), warfarin (0.75 mg/kg) increased the blood loss about twice as much as the control. Thus, compared with warfarin, DX-9065a only increased bleeding time or blood loss to a minor extent in the doses tested. These observations suggest that direct inhibition of FXa could be preferable to warfarin in the suppression of thrombosis without haemorrhagic complications.
Procedure-dependence and Tissue Factor-independence of Hypercoagulability during Orthopaedic Surgery
Authors: Gori A. M. , Falciani M. , Cellai A. P. , Aglietti P. , Baldini A. , Vena L. M. , Prisco D. , Abbate R. , Gensini G. F. , Fedi S. 
Published: March 2018

SummaryThe increased risk for deep vein thrombosis (DVT) after orthopaedic surgery has been well documented as well as hypercoagulable state during both total hip arthroplasty (THA) and total knee replacement (TKR). To investigate the influence of the surgical procedure [postero-lateral (PL) or lateral (L) approach for THA, use of tourniquet (TQ) or not use of TQ for TKR] on the hypercoagulability and the role of extrinsic pathway activation and endothelial stimulation during orthopaedic surgery we have examined 40 patients (20 patients undergoing primary THA – 10 with PL approach and 10 with L approach – and 20 patients undergoing TKR – 10 with TQ application and 10 without TQ). Thrombin-antithrombin complexes (TAT), tissue factor (TF), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM) and von Willebrand factor antigen (vWF:Ag) were analyzed before and during the orthopaedic surgery. During THA, TAT plasma levels increased more markedly in patients assigned to the L than PL approach (p <0.05); during TKR an elevation of TAT of higher degree (p <0.05) was observed when TQ was not applicated. Blood clotting activation was significantly (p <0.001) more relevant during THA than TKR. No changes in TF and vWF:Ag plasma levels were observed in all patients undergoing THA and TKR. TFPI plasma levels significantly (p <0.05) decreased 1 h after the end of the THA in group PL and group L, whereas they remained unaffected in the two groups of patients undergoing TKR. Similarly TM plasma levels significantly decreased during THA, but not during TKR. In conclusion, these results show that: 1) the site of surgical procedures and the type of approach affect the degree of hypercoagulability, 2) the blood clotting activation takes place in the early phases of orthopaedic surgery, without signs of extrinsic pathway and endothelial activation.
A New Type of Ser Substitution for γ Arg-275 in Fibrinogen Kamogawa I Characterized by Impaired Fibrin Assembly
Authors: Mimuro Jun , Kawata Yoichi , Niwa Kazuki , Muramatsu Shin-ichi , Madoiwa Seiji , Takano Hiroshi , Sugo Teruko , Sakata Yoichi , Sugimoto Tomoyoshi , Nose Koichiro , Matsuda Michio 
Published: March 2018

SummaryA new type of substitution, Arg to Ser at γ275, has been found in a heterozygous dysfibrinogen derived from a 23-year-old woman with no major bleeding or thrombosis. By sequence analyses of the affected γ-chain and its gene, we found a single amino acid substitution of γ Arg-275 to Ser in an aberrant γ (274-302) residue peptide isolated from lysyl endopeptidase-digests of the patient’s fibrinogen. In agreement with this amino acid substitution, we identified a single nucleotide exchange of A for C at position 5728 in the γ-chain gene creating a codon (AGC) encoding Ser instead of the codon (CGC) encoding Arg at position γ 275. Like two other known types of mutants with a His or Cys substitution at this position, the functional abnormality was characterized by delayed fibrin polymerization, most likely due to impaired abutting of two D domains of adjacent fibrin monomers in the same strand of fibrin protofibrils. The structural derangement that affects the D:D association may not be so severe as compared with those of Cys and His mutants, possessing an additional disulfide-linked Cys molecule and an imidazole ring at the mutation site, respectively.
Effect of Clopidogrel on Thrombin Generation in Platelet-Rich Plasma in the Rat
Authors: Hérault J.-P. , Dol F. , Gaich C. , Bernat A. , Herbert J.-M. 
Published: March 2018

SummaryClopidogrel (25 mg/kg, p.o.), a potent and selective inhibitor of ADP-induced platelet aggregation, significantly inhibited, in the presence of platelets, ex vivo thrombin generation triggered by low concentrations of tissue factor. Clopidogrel reduced the area under the curve (23%, p <0.05) and the thrombin peak concentration (35%, p <0.05) but did not affect the lag phase of thrombin generation. Under the same experimental conditions, heparin (100 μg/ml) inhibited thrombin generation mostly by delaying and by reducing the burst of thrombin. In a stasis-induced venous thrombosis model in rats under low thrombogenic challenge, clopidogrel inhibited thrombus formation (ED50 = 7.9 ± 1.5 mg/kg, p.o. – n = 10), confirming the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation and venous thrombosis.
Characterization of the Genetic Defects in Recessive Type 1 and Type 3 von Willebrand Disease Patients of Italian Origin
Authors: Castaman Giancarlo , Vos Hans , Bertina Rogier , Rodeghiero Francesco , Eikenboom Jeroen 
Published: March 2018

SummaryThe genetic defects causing recessive type 1 and type 3 von Wille-brand disease (VWD) in eight families from the northern part of Italy have been investigated. Mutations were identified in 14 of the 16 disease-associated von Willebrand factor (VWF) genes. Only one mutation, a stop codon in exon 45, was previously reported. Several new mutations were identified: one cytosine insertion in exon 42, one guanine deletion in exon 28, one probably complete VWF gene deletion, one substitution in the 3’ splice site of intron 13, one possible gene conversion, and three candidate missense mutations. One missense mutation, the substitution of a cysteine in exon 42, was identified in all type 3 VWD patients that were previously characterized as a subgroup with significant increase of factor VIII procoagulant activity after desmopressin infusion. This paper demonstrates again that the molecular defects of quantitative VWD are diverse and located throughout the entire VWF gene.
Polymorphism of Platelet Membrane Glycoprotein IIIa: Human Platelet Antigen 1b (HPA-1b/PlA2) Is an Inherited Risk Factor for Premature Myocardial Infarction in Coronary Artery Disease
Authors: Zotz Rainer , Winkelmann Bernhard , Nauck Markus , Giers Günther , Maruhn-Debowski Beate , März Winfried , Scharf Rüdiger E. 
Published: March 2018

SummaryConflicting results of an association between the human platelet antigen 1b (HPA-1b or PlA2) allele and the risk of myocardial infarction and coronary artery disease have been reported. To assess the reason for this discrepancy, we determined the HPA-1 genotype in 298 men who had undergone coronary angiography, including 124 individuals with myocardial infarction, 83 individuals with coronary artery disease but no history of myocardial infarction, and 91 control patients. Among patients with acute or recent onset myocardial infarction (<1 year), the prevalence of HPA-1b was higher than among patients with coronary artery disease but without myocardial infarction (33 percent vs. 14 percent, p = 0.016). In patients under 60 years of age this difference was even more pronounced (45 percent vs. 15 percent, p = 0.003). Unlike conventional risk factors HPA-1b does not represent a risk factor for coronary artery disease itself but appears to be associated with increased platelet thrombogenicity.
Relations of Platelet Ca2+ Handling and Membrane Microviscosity to Vascular Tone and Restenosis after Angioplasty in Human Coronary Artery
Authors: Feuvre Claude Le , Brunet Annie , Catuli Didier , Metzger Jean-Philippe , Vacheron André , Devynck Marie-Aude , Sang Kim-Hanh Le Quan 
Published: March 2018

SummaryThis study was designed to assess whether platelet Ca2+ handling or membrane microviscosity could be considered as indexes of vascular tone, or could help to predict an increased risk of restenosis after coronary angioplasty. Vascular tone was quantified in 21 patients with stable angina by the vasodilator response to sin-1 intracoronary injection in the reference coronary segment and by the importance of the acute recoil after angioplasty in the narrowed segment. The degree of restenosis was quantified by coronary angiography 6 months later.Individual values of relative sin-1-induced changes in the reference coronary diameter were positively correlated with cytosolic Ca2+ concentration in unstimulated platelets, irrespective of the extracellular Ca2+ concentration (p <0.01). This relationship was also observed with the thrombin-evoked Ca2+ changes, measured in the absence of a Ca2+ influx (p = 0.01). No relationship was found between sin-1-induced coronary changes and membrane microviscosity evaluated by TMADPH and DPH anisotropies or platelet volume, or between degree of acute recoil and platelet characteristics.In conclusion, platelet Ca2+ reflects the vasodilating efficacy in response to sin-1, but cannot help to predict restenosis after coronary angioplasty.
Effect of SR121566A, a Potent GP IIb-IIIa Antagonist, on the HIT Serum/heparin-Induced Platelet Mediated Activation of Human Endothelial Cells
Authors: Savi Pierre , Jeske Walter , Walenga Jeanine , Herbert Jean-Marc 
Published: March 2018

SummaryHeparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1β, IL6, TNFα and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-IIIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPγS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPγS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs.Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.
Hemostatic Effects of Oral Contraceptives in Women who Developed Deep-vein Thrombosis while Using Oral Contraceptives
Authors: Bloemenkamp Kitty , Helmerhorst Frans , Koster Ted , Bertina Rogier , Vandenbroucke Jan , Rosendaal Frits 
Published: March 2018

SummaryObjective: Comparison of the effect of oral contraceptives on hemostatic variables in venous thrombosis patients (thrombosis while using oral contraceptives) with the effect in healthy control subjects. Our aim was to assess whether some of these effects were more pronounced in women who had suffered thrombosis, i.e., whether these were “hemostatic hyperresponders”. Study Design: A population-based case-control study, the Leiden Thrombophilia Study. Materials and Methods: We investigated 99 pre-menopausal women, age 15-49 years, who had used oral contraceptives at the time of a first, objectively confirmed episode of deep-vein thrombosis. They were not pregnant, nor in puerperium, nor had had a recent miscarriage, and were not using injectable progestogens, nor suffering from inherited coagulation defects. The median time between occurrence of deep-vein thrombosis and venepuncture was 18 months, and 30 of the 99 women were still using oral contraceptives, while 69 had discontinued oral contraceptive use. In addition, a group of 153 control women (54 of them were oral contraceptive users and 99 were non-users) were studied. The following hemostatic variables were measured: APTT, factor VII, factor VIII, factor XII, fibrinogen, prothrombin, total antithrombin, normalised activated protein C sensitivity ratio (n-APC-sr), protein C, protein S and free protein S. Results: We found marked and significant effects of oral contraceptive use on the levels of several clotting factors, with an increase in factor VII, factor XII, protein C and a decrease in antithrombin, n-APC-sr and protein S. Less marked effects that were non-significant or only significant in either patients or controls, were an increase in factor VIII, fibrinogen and prothrombin and a decrease in the APTT and free protein S. In the former thrombosis patients several of these effects of oral contraceptives were more pronounced than in healthy women: specifically on factor VII, antithrombin, n-APC-sr and protein C. Conclusions: Our results of the effects of oral contraceptives generally confirm previous reports in healthy volunteers. Our data also show that in former deep-vein thrombosis patients these effects are more pronounced. Apparently some women become “high hemostatic responders” when exposed to oral contraceptives, and they may be the women most vulnerable to its thrombogenic effects.
Molecular Bases of Pseudo-homozygous APC Resistance: The Compound Heterozygosity for FV R506Q and a FV Null Mutation Results in the Exclusive Presence of FV Leiden Molecules in Plasma
Authors: Castoldi Elisabetta , Kalafatis Michael , Lunghi Barbara , Simioni Paolo , Ioannou Panayiotis , Petio Margherita , Girolami Antonio , Mann Kenneth , Bernardi Francesco 
Published: March 2018

SummaryPseudo-homozygous APC resistance, the condition resulting from compound heterozygosity for FV R506Q (FV Leiden) and quantitative FV deficiency, provides a natural model to study the interaction between procoagulant and anticoagulant defects. This paper reports a complete FV characterization of a pseudo-homozygous APC resistant thrombotic patient. The expression of the patient’s non-Leiden gene was found to be severely impaired both at the mRNA and protein levels. In particular, only FV Leiden molecules were detected in the patient’s plasma by immunoblotting, which accounts for the observed marked APC resistance. Analysis of the FV cDNA obtained by reverse transcription of platelet RNA revealed that the mRNA of the non-Leiden gene was extremely reduced in amount. A PAC clone containing the whole FV gene was used to design primers for a complete FV exon scanning. A 2-bp insertion at nucleotide 3706 in the large exon 13 of the non-Leiden gene, predicting a frame-shift and premature termination of protein synthesis, was identified as responsible for the FV defect. Failure to find any case of pseudo-homozygous APC resistance in a large sample (6,804) of blood donors suggests that this condition is extremely rare among normal controls and that its detection is favoured by the thrombotic risk that it may confer.
Thrombolysis in Arterial Occlusion
Authors: Verhaeghe Raymond 
Published: March 2018

SummaryIntra-arterial thrombolytic therapy has replaced systemic intravenous infusion of thrombolytic agents as a treatment modality for arterial occlusion in the limbs. Several catheter-guided techniques and various infusion methods and schemes have been developed. At present there is no scientific proof of definite superiority of any agent in terms of efficacy or safety but clinical practice favours the use of urokinase or alteplase. Studies which compared thrombolysis to surgical intervention suggest that thrombolytic therapy is an appropriate initial management in patients with acute occlusion of a native leg artery or a bypass graft. Underlying causative lesions are treated in a second step by endovascular or open surgical techniques. Severe bleeding is the most feared complication: the risk of hemorrhagic stroke is 1-2%.
Generation of a Humanized, High Affinity Anti-tissue Factor Antibody for Use as a Novel Antithrombotic Therapeutic
Authors: Sims Paul , Meng Y. Gloria , Moran Paul , Bullens Sherron , Bunting Stuart , Schoenfeld Jill , Lowe David , Lai Jadine , Rancatore Patricia , Iverson Mark , Lim Amy , Chisholm Vanessa , Kelley Robert , Riederer Markus , Kirchhofer Daniel , Presta Leonard 
Published: March 2018

SummaryBlocking the cofactor function of human tissue factor may be beneficial in various coagulation-mediated diseases. The murine antibody D3 binds to the membrane proximal substrate interaction region of human tissue factor and blocks tissue factor function even in the presence of bound factor VIIa. The cloned murine D3 antibody was humanized and affinity matured by exchanging amino acids in the complementarity determining regions as well as in the antibody framework. The humanized antibody, D3H44, bound to tissue factor with a 100-fold increased affinity (KD 0.1 nM) as compared to the original murine and chimeric versions. Depending on the particular disease, different pharmacokinetic properties of the antibody may be required and, therefore, several antibody variants – F(ab), F(ab’)2, IgG2, IgG4 and IgG4b – were generated. In vitro, the humanized D3 antibodies displayed potent inhibition of plasma clotting and tissue factor: factor VIIa-mediated activation of factors IX and X (e. g. D3H44-F(ab’)2, IC50 (F.X) 47 pM). In addition, D3H44-F(ab’)2 completely prevented fibrin deposition in a human ex vivo thrombosis model under venous blood flow conditions (IC50 37 nM). The humanized D3 antibodies may be utilized for treatment of cardiovascular diseases which involve tissue factor activity, e. g. acute coronary syndrome and venous thrombosis.