bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Intercellular Junctional Complexes between Human Hematopoietic and Mesenchymal Stem Cells.
Authors: Wuchter Patrick , Saffrich Rainer , Straub Beate , Boda-Heggemann Judit , Miesala Katrin , Diehlmann Anke , Wagner Wolfgang , Seckinger Anja , Krause Ulf , Franke Werner W. , Ho Anthony D. 
Published: April 2020

Abstract
The interaction between hematopoietic stem cells and their niche is essential for the balance between self-renewal and differentiation. We previously demonstrated that intercellular connections in mesenchymal stem cells (MSC) are realized by occasional gap junctions and frequent adherens junctions, comprising specific cadherin-catenin-complexes. Using MSC-feeder-layer as a surrogate model for the hematopoietic stem cell (HSC) niche, we have analyzed the intercellular junctional complexes between HSC and MSC.
MSC were obtained from bone marrow aspirates from healthy voluntary donors. HSC were isolated from umbilical cord blood. Using advanced confocal laser scanning in combination with deconvolution and volume rendering software, we were able to produce 3D-images of intercellular junctions between HSC and MSC. We used a panel of antibodies specific for various components of tight, gap and adherens junctions. Additionally, we compared the data to human and bovine bone marrow tissue in situ.
We could show that intercellular connections between HSC and MSC are mainly realized by podia formation of the HSC linking to the adjacent MSC. These podia vary greatly in length and shape (uropodia, filopodia). Along these podia and especially at the contact zone to the MSC, we have identified the cytoplasmic plaque proteins alpha- and beta-catenin and protein p120ctn, as well as the transmembrane glycoprotein N-cadherin.
This study provided solid evidence for the direct and intimate cellular interaction of HSCs with their niche. Direct cell contact represents a key factor for the regulation of self-renewal versus differentiation. The examination of the specific function of catenins, p120ctn and N-cadherin in this process is concurrently underway.
Metalloproteinase Regulation Determines Integrin αIIbβ3 Function and Enhances the Capacity of ES Cell-Derived Platelets.
Authors: Nishikii Hidekazu , Eto Koji , Kanaji Taisuke , Ware Jerry , Nakauchi Hiromitsu 
Published: April 2020

Abstract
To resolve the lack of sources for products used in blood transfusion therapy, the utilization of embryonic stem cells (ESCs) has been proposed. However, the use of ESC-derived cells has potential drawbacks including, immunological rejection and teratoma formation. Being fully differentiated cells lacking a nucleus, platelets can be irradiated to eliminate passenger ESCs and other lineages before transfusion. We previously established in vitro differentiation system whereby proplatelet-producing megakaryocytes can be derived from murine ESCs on OP9 stroma, but the platelets were generated with a low efficiency and exhibited weak agonist-induced integrin αIIbβ3 activation and spreading on immobilized fibrinogen. Here, we describe an improved method to obtain ESC-derived platelets of potential use for hemostasis and thrombosis in vivo. We generated a novel murine ESC line wherein the GPIbα promoter regulates EGFP expression as a marker (GP-G ESCs). The combination of flow cytometry with GP-G-derived embryoid bodies and OP9 co-culture enabled us to enrich integrin αIIb + c-kit + CD9 + Sca-1 − progenitors on day 5–6 of culture as a major fraction capable of differentiating into GFP+ mature megakaryocytes on day 8–10 in the presence of TPO. This strategy increased platelet production by 10-fold (P&lt;0.01) compared to our previous report (PNAS, 2002, 99:12819). Interestingly, αIIb+platelets included 35–40% GPIbα-negative population besides GPIbα-positive on day 13–15, which was caused by an increased activity of matrix metalloproteinase (MMP) and TACE (ADAM17) to shed GPIbα in culture. Inhibition of MMP and TACE with GM6001 or TAP1 delayed the clearance rate on post transfusion recovery in vivo as transfused into a platelet-depleted mouse model. Maintaining GPIbα expression in the presence of MMP/TACE inhibitors restored the specific fibrinogen-bound to integrin αIIbβ3 and platelet spreading on fibrinogen as evidenced by the generation of filopodia and lamellipodia. Collectively, these results suggest that 1) ESC-derived αIIb+ c-kit + CD9+ Sca-1 − progenitors may contribute to enrich megakaryocytopoiesis at high efficiency; 2) inhibition of metalloproteinase activity to retain GPIbα expression is required for αIIbβ 3 integrin inside-out and outside-in signaling in ESC-derived platelets; and 3) these platelets may be useful alternatives for future clinical application.
The Expression of AML1-ETO9a Isoform in Acute Myeloid Leukemia M2 Subtype and Its Clinical Significance.
Authors: Miao Yu Q. , Chen Zi X. , Cen Jian N. , Yan Ming , He Jun , Qiu Qiao Ch. , Zhang Dong E. 
Published: April 2020

Abstract
AML-ETO (AE) fusion protein caused by t(8;21) is believed to be involved in the development of AML. Most recent studies using a mouse models indicate that expression of a truncated version of AE by alternative splicing, namely AML1/ETO9a (AE9a), may be more closely related to development of leukemia. To validate the hypothesis, we have studied the expression of AE9a in AML-M2 patients and its prognostic value. The Expression of AE fusion gene and AE9a was detected by using RT-PCR in 40 cases of newly diagnosed patients and 30 cases in complete remission. The expression pattern of these fusion genes in M2 patients was compared with that in other subtypes of AML, MDS and healthy controls. In addition, we have also examined the expression level of AE and AE9a in 40 cases of M2 patients during follow-up and analyzed the correlation with clinical manifestations. Our Results demonstrated that 19 of 70 cases of AML-M2 patients simultaneously expressed AE and AE9a while 10 cases expressed only AE; 2 cases expressed AE9a alone; the rest expressed neither. 19 of 40 cases of newly diagnosed patients expressed AE9a (47.5%). Neither 30 AML-M2 cases in complete remission nor the other subtypes of AML, Myelodysplastic syndrome (MDS) and healthy controls expressed AE9a. After treatment by 1–3 course of conventional chemotherapy (MA, DA, DA or AAG regimen), 4 of 19 AE9a positive cases attained complete remission (21%), 7 cases relapsed after relieved (36.8%), 3 cases died after transplantation, 3 cases failed to attained remission and gave up(15.8%), 2 cases lost connection after transplant. While after same conventional chemotherapy, 17 of 21 AE9a negative cases (81%) attained remission, 3 cases(14.3%)relapsed after relieved, 1 case (4.8%) died of giving up treatment after attaining remission. These results confirmed that AE9a was closely related to AML-M2, and it may promote the development of leukemia in combination with AE fusion gene. The remission rate in AML1/ETO9a positive patients is remarkable lower than that in negative’s. Thus AE9a expression may indicate an unfavorable prognosis of AML-M2 patients.
Estradiol Is Concentrated in Human Platelets and Is Released upon Activation.
Authors: Sarabia Stephen F. , Raya Joe L. , Hoogeveen Ron C. , Bray Paul F. 
Published: April 2020

Abstract
Background and Aim: Estrogens are prothrombotic in both normal and atherosclerotic vessels. There is evidence that exogenous estrogen modifies platelet reactivity and signaling pathways, but very little is known about sex hormone metabolism in platelets or whether platelet estrogen stores contribute to atherothrombosis. To begin to characterize the role of sex hormones in platelet biology, we measured estradiol (E2), estrone (E1) and testosterone (T) concentrations within platelets and plasma of normal healthy men and women. Additionally, we tested the hypothesis that platelets have the capacity to release E2 during thrombin-stimulated platelet aggregation and activation.
Methods and Results: Platelets and plasma were prepared from platelet-rich plasma (PRP) samples of 16 male (age 24–63), 13 premenopausal female (age 25–34) and 8 menopausal female (age 39–65) donors; no females were using exogenous hormones. E2, E1 and T were determined by Gas Chromatography-Mass Spectrometry. Hormone content in platelets and plasma was expressed per mL of PRP. Plasma E2, E1 and T levels were within the range of values previously reported in the literature. E2 concentrations were significantly higher in platelets compared to plasma in men (p&lt;0.01) but not in women (premenopausal p=0.33; menopausal p=0.16). The geometric mean and 95% CI for platelet and plasma E2 levels were 199 (114–349) and 55 (37–81) pM for men, 360 (154–831) and 235 (158–357) pM for premenopausal women, and 107 (52–224) and 40 (15–118) pM for menopausal women, respectively. Conversely, T concentrations were significantly lower in platelets than in plasma in both men and women (p&lt;0.01). E1 levels in platelets and plasma were not significantly different within men and within women. In a subset of participants, PRP samples were incubated with 0.5 U/mL thrombin for 10 minutes at 37oC and estradiol levels were measured in the non-clotted plasma+platelet releasate. Compared to no thrombin, thrombin-treated PRP from male subjects (n=7) yielded a 1.6 to 6.7 fold increase in E2 levels (p&lt;0.01). The incubation of PRP from premenopausal (n=5) and menopausal women (n=4) with thrombin did not produce a significant change in E2 release.
Conclusion: Our results show that platelets have the capacity to concentrate E2 and may serve as a vascular vehicle for the delivery of this prothrombotic factor upon platelet activation.
Regulation of Homocysteine Transport in Vascular Cells.
Authors: Jiang Xiaohua , Yang Xiao-feng , Brailoiu Eugen , Jakubowski Hieronim , Schafer Andrew I. , Durante William , Wang Hong 
Published: April 2020

Abstract
Increased levels of plasma homocysteine is an independent risk factor for cardiovascular disease and has cell-type distinct proatherosclerotic effects on vascular cells. In this study, we characterized L- homocysteine transport in cultured human aortic endothelial and aortic smooth muscle cells. L-homocysteine was transported into vascular cells in a time-dependent fashion. L-homocysteine transport activity was about 2-fold higher in aortic smooth muscle cells. In addition, L-homocysteine transport in both cell types was mediated by sodium-dependent and independent carrier systems. Competition studies revealed that the neutral amino acids cysteine, glycine, serine, tyrosine, alanine, leucine, and methionine, and inhibitors of the cysteine transport systems inhibited L-homocysteine uptake in both cell types, but the inhibition was greater in endothelial cells. Eadie-Hofstee plots demonstrated that L-Hcy transport in endothelial cells had a Michaelis constant (Km) of 79mM and a maximum transport velocity (Vmax) of 873 pmol/mg protein/min. In contrast, homocysteine transport in aortic smooth muscle cells had a lower affinity (Km=212mM) but a higher transport capacity (Vmax=4192 pmol/mg protein/min). Interestingly, increases in pH (pH 6.5–8.2) only inhibited L-homocysteine uptake in endothelial cells. Moreover, L-homocysteine transport in endothelial cells was partially inhibited by lysosomal inhibitors. Our studies indicate that L-homocysteine shares transporter systems with cysteine and can be inhibited for transport by multiple neutral amino acids in vascular cells, and that L-homocysteine transport involves lysosomal transport in endothelial cells. The specific lysosomic feature of L-homocystein transport in endothelial cells may contribute to cell type specific growth inhibitory effects and therefore play a role in homocysteine atherogenic potential.
Role of Activated Platelets in the Homing, Maturation, and Differentiation of Endotheleal Progenitor Cells (EPCs) to Vascular Subendotheleal Matrix.
Authors: Lev Eli I. , Dong Jing-fei , Bujak Marcin , Aboulfatova Khatira , Kleiman Neal S. , Harris David , Estrov Zeev 
Published: April 2020

Abstract
We and others have found that platelets play an important role in the recruitment of endothelial progenitor cells to sights of vascular injury. However, it is not clear whether the EPCs mature and differentiate to endothelial cells following recruitment to the vascular injury sites. In addition, there is limited in vivo data to support the role of EPCs in re-endothlialization following vascular injury. We conducted in vitro experiments to investigate the maturation of EPCs on platelet based-media and in vivo experiments to evaluate the recruitment of EPCs following vascular injury. In in vitro experiments human EPCs were isolated from donated buffy coats by magnetic microbeads and flow cytometry cell sorting using CD133 and VEGFR-2, respectively, as cell markers. Isolated viable EPCs (CD133+, VEGFR-2+ cells) were plated on human fibronectin or a monolayer of washed human platelets. Cell colonies were counted 7 days after plating and stained for the endothelial cell markers CD31 (PECAM-1) and CD144 (VE-cadherin). The mean number of colony-forming cells was 35±2.6 colonies/106 cells on platelets, which was significantly higher than 18±4.2 colonies/106 cells on fibronectin (n = 4, P&lt;0.01). Apart from the difference in colony numbers, the EPC colonies grew faster on the platelet substrate, were larger, and had more spindle-shaped cells (Figure 1 - staining of EPC colonies for CD31 and CD144). In the in vivo experiments a model of transluminal injury to mouse femoral arteries was used. Femoral artery denudation was performed by 0.25-mm-diameter angioplasty guidewire. Injured femoral arteries were compared to the contra-lateral controls (uninjured), and were harvested 1.5 hours following the injury and immunostaining performed with an anti-VEGFR-2 antibody. Four experiments showed a markedly higher number of VEGFR-2+ cells in the artery that has undergone denudation. These experiments indicate that a media composed of platelets promotes the maturation and differentiation of EPCs. Furthermore, in vivo, EPCs are recruited early following vascular injury. Thus, homing, maturation, and differentiation of EPCs are mediated by platelets.
Upshaw-Schulman Syndrome Revealed by Pregnancy.
Authors: Seike Masako , Kozuka Teruhiko , Kokame Koichi , Miyata Toshiyuki , Matsuyama Tomomi , Matsumoto Masanori , Fujimura Yoshihiro , Hara Masamichi 
Published: April 2020

Abstract
Background:
Thrombotic thrombocytopenic purpura (TTP) is a rare systemic disease. Its mechanism has been unclear for yeas. Recently, it has been reported that the deficiency of ADAMTS 13 (a disintegrin-like domain, and metalloprotease, with thrombospondin type1 motif 13) activity cause TTP. Congenital TTP (Upshaw-Schulman syndrome: USS) is caused by ADAMTS 13 gene mutation and secondary TTP is caused by anti-ADAMTS 13 antibody. Only five families of USS have been reported in Japan, previously. Here, we report a case of USS patient who has gene mutations.
Case report:
On 23 weeks of gestation, a Japanese pregnant woman was pointed out thrombocytopenia (8.2 x 10E7/mL), and was diagnosed as idiopathic thrombocytopenic purpura. On 31 weeks of gestation, she developed headache, vomiting, high grade fever, disorientation and additional vanishing fetal movement. Unfortunately, intrauterine fatal death was confirmed. The diagnosis of TTP was confirmed, plasma exchange, the administration of methylprednisolone, dipyridamole and high-dose intravenously immunoglobulin was started. She has recovered day by day and the stabilization of platelet count was obtained. Her ADAMTS 13 activity on admission day was lower than detection limit (&lt; 3%), and her father, mother and sister’s low ADAMTS 13 activity (47.4%, 35.8% and 34.8%, respectively) was proved later. Because ADAMTS 13 inhibitor was not detected, she was diagnosed as USS and preventive FFP infusions were started. In accordance with the increase of ADAMTS 13 activity, the platelet count also has a tendency to increase. She has performed regular FFP infusions every two weeks at this time of reporting. This family’s ADAMTS 13 gene mutations were analyzed by DNA sequencing. Patient has compound heterozygote mutations; R193W from father and A606P from mother. Sister has Q606A too, and brother has normal gene.
Conclusion:
Clinical signs of TTP are often masqueraded as a simple thrombocytopenia until 20-30 weeks of gestation. USS is a life threatening disease to both mother and fetus, an early diagnosis is critical. Therefore, pregnant women developed thrombocytopenia need to be determined ADAMTS 13 activity routinely.
Childhood ITP: A Single Institution Review of Treatment and Outcomes.
Authors: Houser Laura , Luchtman-Jones Lori 
Published: April 2020

Abstract
Immune thrombocytopenia purpura (ITP) is diagnosed in approximately 50 per 1 million children per year. Although thrombocytopenia can be associated with serious, life-threatening bleeding, the outcome for most patients with ITP is excellent. Approximately 80% of children with ITP will recover by 6 – 12 months from diagnosis. Large randomized, controlled trials have not been done to demonstrate the optimal management strategy, either for safety or efficacy. Some patients are managed with observation, given the benign ultimate outcome of most cases, but, because of existing or potential bleeding, the majority of patients with severe thrombocytopenia receive treatment with glucocorticoids, IVIG, or anti-D globulin. At our institution, we do not have a standardized strategy for management of patients with childhood ITP. We performed a 10-year retrospective review of our institutional experience with childhood ITP. The approximately 400 eligible patients were aged 0.5 to 18 years, and were managed between January 1996 and January 2006 at St. Louis Children’s Hospital or Washington University Pediatric Hematology/Oncology Clinic. Data from the medical records of these patients was collected and will be analyzed to determine the following primary endpoints:
Time to reach target platelet count, stratified by initial therapy type; Rate of re-treatment and/or addition of second therapy over the first 28 days, stratified by initial therapy type and dosage; Incidence of treatment-related toxicities, Incidence of hemorrhage, anemia and/or transfusion, stratified by therapy choice, Rates and duration of hospitalization, Time to resolution of normal platelet count, stratified by initial therapy.
The management and outcome of patients without platelet recovery by 6 and 12 months (i.e. chronic ITP) and will be detailed. Results from this study will be used as a framework for developing institutional guidelines for management of childhood ITP.
The Role of GBPIalpha in Phagocytosis of Platelets by Macrophages.
Authors: Badlou Bahram Alamdary , Spierenburg Gerit , Ulricht Hans , Deckmyn Hans , Smid W. Martin , Akkerman J.W.N. , Nieuwenhuis H. Karel 
Published: April 2020

Abstract
Platelet storage at 04 C suppresses bacterial multiplication, but induces clusters of glycoprotein (GP) Ibalpha that trigger their phagocytosis by macrophages and reduce their survival after transfusion. We searched for a method that detects cold-induced changes in GPIbalpha involved in phagocytosis.
STUDY DESIGN AND METHODS: Human platelets were isolated and stored for up to 48 hrs at 0C. Binding of a PE-labeled antibody directed against amino acids 1–35 on GPIbalpha (AN51-PE) was compared with phagocytosis of platelets by matured monocytic THP-1 cells, analyzed by FACS.
RESULTS: Freshly isolated platelets were detected as a single population of AN51-PE positive particles and showed &lt; 5% phagocytosis. Cold storage led to a decrease in AN51-PE binding and an increase in phagocytosis. N-acetylglucosamine (GlcNAc), known to interfere with macrophage recognition of GPIbalpha clusters, restored normal AN51-PE binding to cold-stored platelets and suppressed phagocytosis.
CONCLUSIONS: We conclude that binding of an antibody against AA 1–35 on GPIbalpha reflects changes in GPIbalpha that make platelets targets for phagocytosis by macrophages.
Double Knockouts Reveal That Protein Tyrosine Phosphatase 1B Is a Physiological Substrate of Calpain-1 in Platelets.
Authors: Kuchay Shafi M. , Fay William P. , Chishti Athar H. 
Published: April 2020

Abstract
Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Previously we used gene-targeting to evaluate the physiological function of mouse calpain-1, and established that its inactivation results in reduced platelet aggregation and clot retraction, potentially by causing dephosphorylation of platelet proteins. Here, we present data showing that calpain-1 null platelets accumulate protein tyrosine phosphatase 1B (PTP1B) that correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates in platelets. Using antibodies specific for phosphotyrosines 747 and 759 of the b3 subunit of αIibβ3 integrin, we show that the tyrosine phosphorylation of both tyrosine residues at positions 747 and 759 in the cytoplasmic domain of b3 subunit is reduced by approximately 60–70% in the calpain-1 null platelets. Treatment of calpain-1 null platelets with DMHV, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. Importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Consistent with this paradigm, treatment of wild type mouse platelets as well as human platelets with the tyrosine phosphatase inhibitor DMHV enhanced their aggregation at low doses of thrombin. Conversely, MDL, a cell permeable inhibitor of calpains, potently inhibited aggregation of wild type mouse platelets in a dose-dependent manner upon thrombin activation. Further evaluation of mutant mice by ferric chloride induced arterial injury model suggests that the calpain-1 null mice are relatively resistant to thrombosis in vivo. Finally, the calpain-1 mediated regulation of PTP1B appears to be a systemic event as evident by the enhanced tyrosine dephosphorylation of B lymphocytes and their resistance to apoptosis in calpain-1 null mice. Together, our results demonstrate that PTP1B is a physiological substrate of calpain-1 and suggest that a similar mechanism may regulate calpain-1 mediated tyrosine dephosphorylation in other cells.