bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct Activation of Platelets by Tissue Factor Is Amplified by FVIIa and Potentiated by Serotonin: Influence of Shear Rate.
Authors: Diaz-Ricart Maribel , Lopez-Vilchez Irene , Galan Ana M. , Tonda Raul , Ordinas Antonio , Escolar Gines 
Published: October 2019

Abstract
Although exposure of tissue factor (TF) at sites of disrupted atherosclerotic plaques plays a critical role in platelet-triggered ischemic complications, a direct interaction of TF with platelets has not been yet demonstrated. We have investigated the effects of TF on platelets under different shear rate conditions. Exposure of isolated washed platelets to TF derived from human placenta resulted in reversible aggregation (35-50% max at 1 min) using standard platelet aggregometry. This response was αIIb-β3 dependent and occurred in the presence of residual concentrations of calcium. TF-induced reversible aggregation was enhanced and made irreversible to 90–100% by the addition of FVIIa (5μg/ml). This second phase of activation was blocked by the presence of serine-protease inhibitors. A combination of ultrastructural and flow cytometry studies revealed that although TF induced shape change and moderate expression of P-selectin an ANV positive phospholipids, internal contraction and full platelet degranulation only occurred after addition of FVIIa. All these transformations occurred together with tyrosine phosphorylation of proteins. Platelets adhered, spread and aggregated onto synthetic membranes coated exclusively with TF that had been exposed to circulating blood. Addition of FVIIa caused a 50% enhancement on platelet interactions and a two-fold increase in fibrin deposition. Serotonin enhanced overall TF induced platelet interactions under the different experimental approaches. A selective serotonin reuptake inhibitor (citalopram) reduced the intensity and extent of the effects of TF in the two experimental settings used in our investigations. Our results are consistent with a direct role of TF on platelet-triggered ischemic complications. We demonstrate that a lipidated from of TF activates isolated platelets in suspension. In studies conducted with flowing whole blood we show that TF firmly attached to a surface acts itself as an adhesive substrate, and promotes formation of aggregates. These TF-platelet interactions are enhanced through serotoninergic mechanism and markedly potentiated in the presence of FVIIa. The possible synergism between FVIIa and serotoninergic mechanisms may well contribute to the development of severe ischemic complications triggered on ruptured atherosclerotic plaques.
Isolation of CD34+AC133+Flk1+ Cells Does Not Identify the Most Primitive Endothelial Progenitor Cells.
Authors: Ingram David A. , Fenoglio Amy , Mead Laura E. , Yoder Mervin C. 
Published: October 2019

Abstract
Human EPCs are currently defined as a CD133+/Flk1+ subpopulation of CD34+ cells that can be isolated from peripheral blood, bone marrow, and fetal liver (Nature Medicine, 2003). While these cells are widely considered to be EPCs and subsequently used for angiogenic therapies and biomarkers of cardiovascular disease, their proliferative and clonogenic potential has not been compared to other populations of EPCs, which do not express the cell surface antigens, CD34 and AC133. Analogous to a paradigm established in the hematopoietic cell system, we recently developed a single cell clonogenic assay to reproducibly identify the following EPCs: (1) high proliferative potential - endothelial colony forming cells (HPP-ECFC), which form macroscopic colonies that form secondary and tertiary colonies upon replating, (2) low proliferative potential - endothelial colony forming cells (LPP-ECFC), which form colonies greater than 50 cells, but do not form secondary colonies upon replating, (3) endothelial cell clusters (EC-clusters) that contain less than 50 cells, and (4) mature terminally differentiated endothelial cells (EC), which do not divide (Blood, 2004). Utilizing this assay and EPCs derived from human umbilical cord blood mononuclear cells, we compared the clonogenic and proliferative potential of 1,000 single CD34+AC133+Flk1+ and CD34-AC133−Flk1+ umbilical cord blood derived endothelial cells. We conducted four independent experiments. We demonstrate that a complete hierarchy of EPCs can be identified in a population of CD34−AC133−Flk1+ cord blood derived ECs (Table I, n=4). Remarkably, we further show that CD34−AC133−Flk1+ cord blood derived ECs contain more proliferative EPCs (LPP-ECFCs and HPP-ECFCs) compared to CD134+AC133+Flk1+ cord blood derived ECs (Table I, n=4, *p<0.05). In fact, preliminary experiments suggest that CD34−AC133−Flk1+ cells mature into CD34+AC133+Flk1+ cells. Finally, some individual single CD34−AC133−Flk1+ HPP-ECFCs can expand rapidly to 107 cells in ex vivo culture. Retroviral marking of the cell progeny derived from the single cell confirmed that they were derived from the parent cell. Thus, these studies describe the use of a clonogenic assay to identify a novel population of CD34−AC133−Flk1+ EPCs, and preliminary experiments demonstrate that CD34 and AC133 are not universal cell surface markers of the most primitive EPCs.
Percent of 1,000 Single Cells Plated
Mature EC EC-Cluster LPP-ECFC HPP-ECFC CD34+AC133+Flk1+ ECs 70±11 16±5 10±4 3±1 CD34-AC133-Flk1+ ECs 52±12 17±6 20±3* 10±4*
Vascular Endothelial Growth Factor (VEGF) Induces Cell Survival Via an Additional Urokinase (uPA)-Dependent Mechanism.
Authors: Prager Gerald W. , Koshelnick Yuri , Mihaly Judit , Brunner Patrick , Binder Bernd R. 
Published: October 2019

Abstract
VEGF, the most important angiogenic growth factor, is known to induce cell-survival mainly via phosphorylation of the pro-apoptotic proteins BAD, PED/PEA-2 or pro-caspase-9 or inhibition of SAPKs (stress activated kinases). These mechanisms are all dependent on the PI3-kinase/Akt pathway. We could show recently that VEGF also induces pro-uPA activation via the PI3-kinase signaling pathway beside and independent of the transcriptional upregulation of uPA. Active uPA does not only contribute to angiogenesis via its proteolytic properties, but also effectuates itself pro-angiogenic signalling by the induction of endothelial cell migration, proliferation and differentiation. We were interested whether generation of uPA upon VEGF is inducing an additional effect on endothelial cell survival. First, we compared VEGF with urokinase in respect to cell survival in apoptosis assays and observed a pivotal anti-apoptotic effect of both stimuli, dose dependently and dependent on the type of matrix used. In addition, cell survival effects were additive, when both stimuli were added simultaneously. While VEGF-induced cell survival was PI3-kinase dependent, because it could be inhibited by the specific PI3-kinase inhibitor LY294002, the uPA-induced cell survival was PI3-kinase independent. Furthermore, uPA was able to rescue apoptosis induced by PI3-kinase inhibition in VEGF stimulated endothelial cells. From these data we conclude that uPA is indeed inducing an additional - PI3-kinase independent - cell survival mechanism. While VEGF led to a PI3-kinase dependent phosphorylation of Akt, which resulted in CDC42 activation, uPA activated CDC42 and its downstream effectors PAK leading to IKK-1 phosphorylation in a PI3-kinase/Akt independent manner. This indicates that the anti-apoptotic properties of uPA are not Akt, but NF-kappaB mediated. Indeed, when we used adenovirus overexpressing I-kappaB to block the NF-kappaB pathway, uPA was ineffective to support cell survival. In addition VEGF and uPA, both induced a transcriptional upregulation of inhibitor of apoptosis proteins (IAPs) in an NF-kappaB-dependent manner, among them most significantly the X-linked inhibitor of apoptosis protein (XIAP); again VEGF in a PI3-kinase dependent, uPA in a PI3-kinase independent manner.
From these data we conclude that VEGF is inducing cell survival in a strictly PI3-kinase dependent manner, on the one hand via its known mitochondrial pathway, but also via the PI3-kinase dependent pro-uPA activation leading to an NFkappa B dependent upregulation of inhibitor of antiapoptosis proteins.
Sulfur- and Selenium-Containing Compounds Derived from Natural and Synthetic Sources in Angiogenesis Modulation.
Authors: Mousa Shaker A. , Mohamed Seema , O’Connor Laura , Block Eric 
Published: October 2019

Abstract
Increases in serum sulfate may explain some of the therapeutic effects of methylsulfonyl methane (MSM), dimethyl sulfoxide, and glucosamine sulfate. Organic sulfur, as sulfur-containing amino acids, can be used to increase synthesis of S-adenosylmethionine, glutathione, taurine, and N-acetylcysteine. MSM may be effective for the treatment of allergy, pain syndromes, athletic injuries, and bladder disorders. Epidemiological studies have suggested that brassica vegetables are protective against cancers of the lungs and alimentary tract. Cruciferous vegetables are the dietary source of glucosinolates, a large group of sulfur-containing glycosides. However, dosages, mechanisms of action, and rationales for the use of various sulfur-containing compounds from natural or food products need to be better defined. The low toxicological profiles of these sulfur compounds, combined with promising therapeutic effects, warrant continued preclinical and clinical investigations. In this current study the angiostatic (antiangiogenesis) efficacy for individual sulfur-containing compounds derived from natural or synthetic sources was determined using in vitro (endothelial cell [EC] tube formation assay) and in vivo (chick chorioallantoic membrane [CAM] model). In vitro EC tube formation studies demonstrated significant inhibition of fibroblast growth factor-2 (FGF-2) stimulated EC tube formation by the different sulfur- and selenium-containing compounds, with different potency depending on the oxidation state of the sulfur or selenium. Furthermore, utilizing the CAM model, similar inhibitory efficacy of FGF-2 induced angiogenesis. These data suggest that sulfur and selenium compounds derived from natural sources might be a useful therapy for the inhibition of angiogenesis associated with human tumor growth and other pathological angiogenesis-mediated disorders, such as ocular and inflammatory diseases.
Antiangiogenesis Efficacy of Sulfone/Sulfoxide and Selenone/Selenoxide in the Chick Chorioallantoic Membrane Model
Treatment Branch Points ± SEM % Inhibition ± SEM FGF = fibroblast growth factor; PBS = phosphate-buffered saline. PBS control 64.67 ± 3.00 ----- FGF-2 (1.5 ug/ml) 178.20 ± 5.32 ----- FGF-2 + diphenyl sulfoxide (100 uM) 145.65 ± 11.10 34.12 ± 4.8 FGF-2 + diphenyl sulfone (3 uM) 82.56 ± 4.4 84.25 ± 3.6 FGF-2 + dimethyl selenoxide (30 uM) 120.86 ± 3.1 50.74 ± 2.7 FGF-2 + dimethyl selenone (3 uM) 71.63 ± 3.8 93.87 ± 3.3
Heightened Proteolysis of Plasma ADAMTS13 Antigen in Patients with Thrombotic Thrombocytopenic Purpura.
Authors: Yagi Hideo , Matsumoto Masanori , Iwamoto Taka-aki , Ishizachi Hiromichi , Nishimura Hitomi , Isonishi Ayami , Soejima Kenji , Nakagaki Tomohiro , Mori Toshio , Fujimura Yoshihiro 
Published: October 2019

Abstract
A total of 140 subjects with thrombotic thrombocytopenic purpura (TTP)/hemolytic-uremic syndrome (HUS), in which 25 were Upshaw-Schulman syndrome (USS, belonged to 20 families), 107 were acquired TTP (96 idiopathic and 11 drug-induced), and 8 were acquired O157:H7-associated HUS, were analyzed on their plasma ADAMTS13 antigen (:AGN), using 3 novel anti-ADAMTS13 murine monoclonal IgG antibodies (A10, C7, and WH2-1–11). Epitope of these antibodies resided on the disintegrin-like, 7th/8th thrombospondin-1, and 4th thrombospondin-1 domains, respectively. The WH2-11–1 alone was immunoreactive on western blot (WB) under reducing conditions. Further, in a static assay system, A10 totally and C7 partially inhibited ADAMTS13 activity (:ACT), but WH2-11–1 did not. A sandwich ELISA using C7 as the 1st antibody and biotinylated A10 as the 2nd antibody revealed a significant reduction of plasma ADAMTS13:AGN level in patients with acquired TTP, but a considerable variation in those with USS, suggesting that the quantitative ELISA has a limited diagnostic value.
A quantitative and semi-qualitative WB analysis using A10 and WH2-11-1, however, demonstrated that normal plasmas had a 190/180 kD-doublet band under non-reducing conditions. A size exclusion chromatography of normal plasma was able to separate a 190 kD-protein from a 180 kD-band, and the former turned to be a single 200 kD-band on WB under reducing conditions, but in the latter such protein band was completely invisible, indicating that the 180 kD-band had undergone proteolytic modification. All the patient plasmas with USS and a roughly half of those with acquired TTP had a broad and fuzzy ~190/180 kD-band on WB before reduction, but it almost entirely disappeared after reduction. In patient plasmas with acquired HUS, however, the pattern of WB analysis was indistinguishable from that of the normal subjects.
Aforementioned WB analysis was then performed on a girl of acquired TTP with high-titer inhibitors against ADAMTS13, using the sequential plasma samples obtained during the past 3 years, and confirmed complete normalization of the abnormal ADAMTS13:AGN patterns in accord to the increase of plasma ADAMTS13:ACT, together with her clinical improvements. Protease(s) responsible for this heightened cleavage is currently under investigation.
Generation of Floxed Tissue Factor Mice.
Authors: Pawlinski Rafal , Pedersen Brian , Tencati Michael , Mackman Nigel 
Published: October 2019

Abstract
Tissue factor (TF) is the primary cellular initiator of blood coagulation. Since TF Deficiency leads to embryonic lethality, we generated floxed TF mice to allow analysis of the effect of deleting the TF gene in different cell types. A targeting vector was constructed containing two loxP sites spanning the promoter and intron 1 together with a neomycin resistance gene (NEO) in intron 1 that is flanked by two frt sites. The floxed TF gene targeting plasmid was transfected into ES cells and clones were selected using G418 and gancyclovir. We screened 144 clones by Southern blotting and found six targeted clones. Three clones were injected into blastocysts to generate chimeric mice. Only one chimera demonstrated germline transmission when bred with C57BL/6 mice. TFflox/flox mice were generated at the expected frequency (15 out of 58; 25.8%) from TF+/flox intercrosses, indicating that the floxed TF allele is functional. To detemine if the DNA between the loxP site can be efficiently excised from the floxed TF allele to create an inactive gene, we crossed TFflox/flox mice with mice expressing the Cre transgene under the control of the adenovirus EIIa promoter. This transgene is expressed in one cell zygotes and results in deletion of DNA at the earliest stage of development. From this breeding, we generated TF+/flox− offspring and intercrossed them. To date, analysis of 14 mice at wean has not identified a TFflox−/flox− mouse, suggesting efficient inactivation of the TF gene. To remove the NEO cassette from intron 1 of the floxed TF allele, we crossed TFflox/flox with transgenic mice expressing flipase under the control of the human β-actin promoter (FLP). TF+/flox/FLP+ mice were intercrossed. To date, we have successfully generated 9 TFflox/flox/NEO− mice. These mice will be crossed with tissue-specific deleter strains to investigate the contribution of TF expression in various cell types to a variety of biological processes, including hemostasis, inflammation, angiogenesis, metastasis and cell migration.
F VIII Inhibitors in Haemophilia A Patients who Are Considered as Previously Treated Patients.
Authors: von Auer Ch. , Krause M. , Miesbach W. , Scharrer I. , Asmelash G. , Stier-Brueck I. ,   PTP Study Group in Germany
Published: October 2019

Abstract
Previously treated patients (PTP), who have not developed an inhibitor (inh) so far, are considered to be tolerant to factor VIII and at low risk for inh development. Therefore inh detection in a PTP should raise concerns about the concomitant variables such as product neo-antigenicity or way of application.
In our own center we recently detected the new development of a high responding inh to factor VIII in a 58 year old patient with severe haemophilia A. To find out about the current situation regarding inh development in PTP in Germany, we conducted a retrospective study.
A questionnaire was sent to 99 haemophilia treating physicians, so far 46 of them answered.
24 PTP-inh were registered during the last 5 years. Patients had at least 20 ED and/or one change of factor concentrate. Age (9 months to 70 years, median 35), severity of haemophilia A (16 severe, 2 moderate, 6 mild), exposure days (ED 6 to >1500, median 37) and genotype (4 intron-22-inversions, 3 large deletions, 2 missense mutations, 1 stop mutation, 1 insertion, 1 small deletion, 11 unknown) were recorded.
8 different factor VIII concentrates were given during inh development (5 plasma derived, 3 recombinant). Way of application (16 bolus infusion, 3 continuous infusion, 5 times both), infused amount until inh development (3000 IU to >1 mio IU), inh characteristic (15 HR, 9 LR), concomitant diseases and medication were registered.
In conclusion it became obvious that inh in PTP are still a serious and underestimated problem in haemophilia treatment today.
Our patient numbers are still too small to draw conclusions concerning given F VIII products or way of application. Secondly data showed that there is a variety of PTP definitions in Germany, referring to age of pat, number of ED and former change of product. A definition from the SSC of the ISTH for PTP would be helpful. The continuous use of the German register for drug side effects would make it easier to evaluate data in the future. A prospective, not product related study should be conducted.
Nonpuerperal Ovarian Vein Thrombosis: A Report of Seven Cases.
Authors: Ferman Debra , Bradley Thomas P. , Warshawsky Robin , Allen Steven L. 
Published: October 2019

Abstract
Background: Nonpuerperal ovarian vein thrombosis (OVT) is a rare clinical entity. Therapy is not well defined. OVT is usually asymptomatic. Improved CT and MR imaging technology enables OVT to be diagnosed with greater frequency. Nonpuerperal OVT may be a distinct clinical entity.
Methods: The medical records of women with nonpuerperal OVT were reviewed and their clinical course and treatment recorded. Cases were identified by scanning a CT computerized database over the past 3 years with the key words ovarian vein thrombosis.
Results: 7 patients (pts) were identified. Age range was 38–61, median 51 years. 3 pts had OVT on the left and 4 on the right. OVT followed a procedure in 2 pts. 2 pts with breast cancer developed OVT, 1 during adjuvant tamoxifen and 1 receiving chemotherapy for metastatic disease. 1 pt had AML in CR. 3 pts presented with lower abdominal pain on the involved side and 1 pt had lower abdominal pain on the opposite side. The 3 pts with cancer were asymptomatic. 4 pts had uterine fibroids. 1 developed OVT in the setting of an acute diarrheal illness. 1 pt had prior DVT and 1 a family history of DVT. Only 1 pt had a hypercoagulable evaluation; negative. 2 pts had clot extending to the junction with the inferior vena cava (IVC) and both were anticoagulated with enoxaparin followed by warfarin. 1 pt was anticoagulated with enoxaparin alone. No embolic complications occurred.
Conclusion: 4 pts with OVT were symptomatic and 3 were detected incidentally. 2 developed OVT following procedures, 3 had predisposing underlying conditions, and 2 had a personal or family history of thrombosis. Optimum therapy is not defined. Symptomatic disease alone is not an indication for anticoagulation. Anticoagulation may be indicated if clot extends to the IVC.
Hyperhomocysteinemia in Cancer Patients with Thrombosis Is Independent of Methylene Tetrahydrofolate Reductase Gene Mutation.
Authors: Fareed Jawed , Tobu Mahmut , Hoppensteadt Debra A. , Cunanan Josephine , Iqbal Omer , Demir Muzaffer , Deitcher Steven 
Published: October 2019

Abstract
Plasma homocysteine levels are considered to be an important surrogate marker of vasculopathy in thrombotic disorders. Methylene tetrahydrofolate reductase (MTHFR) gene mutations, nutritional factors (folic acid/ B12) and anticancer drugs are primarily responsible for the observed increase in this surrogate marker. In addition, it is well known that cancer patients exhibit increased prevalence of thrombotic events. The aim of these studies was to demonstrate that an upregulation of homocysteine plays a role in cancer associated thrombosis. However, initial screening of cancer patients showed that the malignancy related thrombotic state was independent of molecular defects in Factor V Leiden, Prothrombin 20210 and MTHFR ( 677 and 1298) variants (Hoppensteadt et al. Proceedings ASCO2003. 22:346;861). In a group of 102 patients who were diagnosed with cancer and thrombosis and in a group of medical patients (cancer free) with thrombosis enrolled in a treatment study with low molecular weight heparin (CORTES, n=50), blood levels of homocysteine were measured using a highly specific ELISA method (Diazyme Laboratories, San Diego, CA). In addition, molecular methods utilizing PCR were used to measure the MTHFR (677 and 1298) variants. On a cumulative basis the cancer patients with thrombosis exhibited a relatively higher level of homocysteine (11.8± 4.7 uM/L) in comparison to normal (n=140, 4.6± 2.9 uM/L) and medical patients with thrombosis (5.3± 3.1 uM/L). Of the 102 cancer patients profiled for the molecular analysis 18% were positive for the 677 variant and 12% were positive for the 1298 variant. Similar results were obtained in the medical patients and normal volunteers. These results clearly suggest that, cancer patients with thrombosis exhibited a relatively higher level of circulating homocysteine levels which are unrelated to a molecular defect in MTHFR. Medical (cancer free) patients with thrombosis do not exhibit this increase in circulating homocysteine levels. Thus, other relevant factors such as the use of chemotherapeutic agents like methotrexate and purine analogs may contribute to this apparently acquired upregulation of homocysteine. This observation warrants periodic monitoring of homocysteine in patients with thrombosis and cancer. Nutritional supplementation of folic acid and B12 may be helpful in the overall management of these patients including those who are treated with methotrexate and purine analogues.