bluuebunny/crossref_metadata_2025_split huggingface dataset:
Notch Regulation by the Fbw7/hcdc4/Sel-10 Ubiquitin Ligase.
Authors: Grim Jonathan E. , Sala Olga , Chung Nack Gyun , Radich Jerald , Varnum-Finney Barbara J. , Bernstein Irwin D. , Clurman Bruce 
Published: October 2019

Abstract
T-cell neoplasms frequently sustain mutations in the Notch1 gene, leading to the expression of constitutively active Notch proteins. Such mutations often target the C-terminal PEST domain, which is known to be involved in protein stability. The ubiquitin ligase Fbw7/hdcd4/Sel-10 is a tumor suppressor that negatively regulates Notch function by targeting the Notch protein for ubiquitination and proteasomal degradation. Although the PEST domain is known to be important for Fbw7/Notch interactions, the specific residues that regulate binding of Notch to Fbw7 have not been defined. Based on the structural motifs (known as phosphodegrons) common to known substrates of Fbw7, we have identified two candidate peptide sequences within the Notch protein and have generated a series of mutants in these regions. Using co-immunoprecipitation assays, we show that one potential phosphodegron that is outside of the PEST domain does not appear to influence Notch binding to Fbw7. However, a second potential phosphodegron is present within the PEST domain and contains a conserved threonine residue (T2512) which is central to binding of Fbw7 to Notch. A mutant in which this residue is replaced by alanine (T2512A) shows a prolonged half life when compared to wild type Notch ICD, supporting its role in Notch stability. To evaluate the role of Fbw7 mediated Notch degradation in vitro and in vivo, we used lentiviral vectors to transfect hematopoietic cells with shRNA targeting Fbw7. These studies demonstrate that Fbw7 knockdown leads to phenotypes consistent with increased Notch activity. Because Notch is commonly mutated in human leukemias, we hypothesized that Fbw7 may also sustain mutations that lead to loss of Notch regulation. We evaluated primary human T cell leukemias for mutations in Fbw7 and found that 1 of 23 samples contains a heterozygous mutation in the Fbw7 common region (R505C). We show that this mutant is deficient in binding to Notch, suggesting that Fbw7 mutation may contribute to the deregulation of Notch that is commonly seen in T-cell neoplasms. Together, this work shows that Fbw7 is an important regulator of Notch function whose mutation may be an important step in leukemogenesis.
FLT3-ITD Cooperates with MOZ-TIF2 in Leukemogenesis in a Murine Bone Marrow Transplantation Model.
Authors: Tam Winnie F. , Rocnik Jennifer , Wojiski Sarah , Deguchi Kenji , Okabe Rachel , Lee Ben , Gilliland D. Gary 
Published: October 2019

Abstract
MOZ-TIF2 is expressed as a consequence of the chromosomal inversion inv(8)(p11q13) and is associated with AML FAB subtypes M4 and M5. Mice transplanted with MOZ-TIF2 succumb to monoclonal or oligoclonal leukemias with a long median disease latency, indicating that cooperating mutations are required for MOZ-TIF2 mediated leukemogenesis. The presence of a FLT3-ITD mutation in a patient with inv(8)(p11q13) has previously been reported, suggesting FLT3-ITD as a candidate cooperating mutation. Here we report that FLT3-ITD functionally cooperates with MOZ-TIF2 in co-transduction experiments in both a serial replating and murine bone marrow transplantation assay to induce AML that is transplantable to secondary recipients. At limit dilution, both MOZ-TIF2 and FLT3-ITD retroviruses are present, demonstrating cooperative effect. Moreover, tyrosine to phenylalanine mutations of FLT3-ITD residues 589 and 591, which we have previously reported to abrogate Stat5 signaling, also abolish the ability of FLT3-ITD to cooperate with MOZ-TIF2 both in vivo and in vitro. Furthermore, a constitutively active Stat5 mutant supports factor independent serial replating activity of MOZ-TIF2 in vitro. These data suggest a multi-step transformation model in which constitutive downstream Stat5 signaling by FLT3-ITD cooperates with MOZ-TIF2 in AML induction, and indicate that FLT3-ITD potentiates the properties of self-renewal in hematopoietic progenitors through activation of STAT5.
Association of Complement with Microvascular Endothelial Cell Injury in Thrombotic Microangiopathy In Vivo and in In Vitro Model Systems.
Authors: Di Carlo Brian A. , Stefanescu Radu , Lee Kelvin , Yin Wei , Modaressi Rozbeh , Peerschke Ellinor I.B. , Laurence Jeffrey 
Published: October 2019

Abstract
We have previously shown that plasma from patients with idiopathic as well as HIV and thienopyridine-linked TTP induces apoptosis of microvascular endothelial cells (MVEC) in vitro, with specific sparing of large vessel and pulmonary MVEC, consistent with the clinical syndrome. This correlates with in vivo measures of apoptosis in splenic tissue of TTP patients. We found that osteoprotegrin (OPG), an inhibitor of multiple inducers of MVEC apoptosis, could block TTP plasma-mediated MVEC injury. Utilizing OPG affinity-binding columns and MS-TOFI analysis of column eluates, we subsequently identified complement (C) components and C-regulatory factors as potentially involved in TTP plasma-mediated EC injury. Building on the observation that familial TTP syndromes have been linked to genetic deficiencies of the C-regulatory factors Factor H and MCP, and three cases of sporadic HUS in children have been accompanied by autoantibodies to factor H, we hypothesized that the C-system plays an important role in TTP-induced MVEC injury, facilitated by autoantibody-mediated suppression of C-regulatory proteins. To evaluate the role of the complement system we looked at:
IFA staining of splenic tissue from TTP patients versus controls for C-components in conjunction with CD34+ indentification of MVEC. ELISA-based assays of complement activation/deposition on MVEC exposed to TTP vs. control plasma. Ability of anti-C agents to block TTP plasma-mediated MVEC apoptosis. DNA microarray analysis of TTP plasma MVEC exposed to TTP vs. control plasma. ELISA-based search for autoantibodies against C-regulatory proteins in TTP plasma.
IFA staining demonstrated increased deposition of C-components on MVEC in vivo on splenic tissue of TTP vs. control patients (4/5 TTP, 0/3 control). Increased C-activation was demonstrated on MVEC exposed to plasmas of TTP patients in vitro as evidenced by decreased C3b (p=0.002) and C4d (p=0.0002) deposition. Microarray data revealed increased expression of C-regulatory factors in resistant vs. susceptible MVEC exposed to TTP plasma. C-inhibtion resulted in prevention of TTP-plasma induced MVEC apoptosis by annexin-V staining (P<0.02). The search for autoantibodies against five C-regulatory components: CD46 (MCP), CD55 (DAF), CD59, Factor H, and Factor I by ELISA assay is currently ongoing. In conclusion, the clinical expression of thrombotic microangiopathy likely requires a double-hit of endothelial cell injury and autoantibodies against proteins vital to hemostasis and/or MVEC survival. In some patients this may involve autoantibodies to ADAMTS13; in others, C-system activation and inhibition of C-regulation by C-regulatory protein autoantibodies may be involved. Inhibition of the C-system may be a feasible clinical approach in some patients with thrombotic microangiopathies.
Risk Factors for Umbilical Venous Catheter-Associated Thrombosis in Very Low Birth Weight Infants.
Authors: Narang Shalu , Roy Jason , Stevens Timothy P. , Butler-O’Hara Meggan , Mullen Craig A. , D’Angio Carl T. 
Published: October 2019

Abstract
Background: Thrombosis in neonates is a rare but serious occurrence that is usually associated with central catheterization. Among acquired risk factors, thrombocytosis has often been thought to play a role in neonatal thrombosis, but little evidence exists to support this impression.
Objectives:To investigate the effects of platelet count on catheter-related thrombosis in neonates.To investigate the effects of being small for gestational age (SGA) on catheter-related thrombosis in neonates.
We hypothesized that neonates with catheter-related thrombosis would have relative thrombocytosis and would be SGA.
Methods: The present retrospective study was performed using data from a randomized trial of duration of umbilical venous catheters (UVC) placement among infants <1250 g birth weight (Butler-O’Hara, Pediatrics2006;118:e25–e35). In this study, all subjects had UVC that were left in place for up to 28 days. All subjects were screened biweekly for thrombi with echocardiograms. Twenty-two cases of UVC-associated thrombosis were identified in this sample. The remaining study sample (n=188) served as controls. Data on thrombosis, platelets, gestational age, birth weight, hematocrit, serum sodium (as a measure of dehydration), duration of catheter placement, study group assignment and demographic factors were collected using database and record review.
Results: Among the total subjects (n=210), 112 (53%) were males and 126 (60%) were Caucasians, with mean gestational age of 27.7 ± 2.1 wks (SD) and mean birth weight of 923 ± 195g. Bivariate analysis revealed significant association of thrombosis with hematocrit >55% in the first wk (OR, 5.4; 95% CI, 2.0–14.6; p=0.0003), being small for gestational age (OR, 2.9; 95% CI, 1.2–7.4; p=0.02), lower platelet counts in the first wk (193 ± 57 x 103/uL in infants with thrombus vs. 238 ± 70 x 103/uL in infants without thrombus, p=0.005) and gestational age (27.8 ± 2.5 wks in infants with thrombus vs. 27.6 ± 2.0 wks in infants without thrombus, p=0.02). In multivariate logistic regression analysis, only higher hematocrit was independently associated with thrombus (OR, 3.9; 95% CI 1.3–12.6; p=0.02). There was a trend towards an independent negative association between platelets and thrombosis (OR, 0.93 per 10 x 103/uL platelet rise; 95% CI, 0.85–1.02; p=0.12).
Conclusion: This study demonstrates a significant, independent association of elevated hematocrit and development of UVC-associated thrombosis. We did not observe an increased risk of thrombosis with increased platelet count. Careful monitoring for catheter-associated thrombosis is suggested for neonates with hematocrit >55% in the first wk of life.
Argatroban Anticoagulation for Heparin-Induced Thrombocytopenia in Obese, Versus Non-Obese, Patients.
Authors: Rice Lawrence , Hursting Marcie J. , McCollum David A. 
Published: October 2019

Abstract
Argatroban, a parenteral direct thrombin inhibitor used for prophylaxis or treatment of thrombosis in heparin-induced thrombocytopenia (HIT), is routinely administered in accordance with patient body weight and activated partial thromboplastin time (aPTT) response. The effect of obesity (BMI >30) on argatroban therapy in HIT however is not well described. In this retrospective analysis of a 118-patient, multicenter HIT registry database, we evaluated argatroban doses, aPTTs, and clinical outcomes in obese, vs. non-obese, argatroban-treated adults with clinically diagnosed HIT or history of HIT. Patients lacking sufficient data to calculate BMI, administered argatroban at doses required during angioplasty, or with a baseline (pre-argatroban) platelet count >150x109/L were excluded. Argatroban was infused and adjusted to achieve aPTTs 1.5–3 times baseline, at the treating physician’s discretion. We identified 41 patients for analysis: 21 non-obese (BMI 17.3–28.9, median 22.9; 52% female) and 20 obese (BMI 30.1–50.8, median 34.8; 55% female). Two patients (1 per group) had history of HIT requiring acute anticoagulation; 39 patients had HIT (median baseline platelet count of 85 and 92x109/L, respectively, in non-obese and obese patients), including 9 (5 obese) with baseline HIT-related thrombosis. Indications for heparin therapy, typically cardiac- or surgery-related, were similar between the non-obese and obese groups. Median argatroban doses were 1.0 μg/kg/min initially in each group, and 1.0 and 1.2 μg/kg/min in the non-obese and obese groups, respectively, during therapy (median 5.4 and 6.3 days, respectively). Therapeutic aPTTs occurred within a median 5.6 and 5.0 h, respectively, with median aPTTs during therapy of 60.5 and 58.7 s. By regression analysis, BMI did not significantly affect argatroban dose. Platelet counts recovered (>100x109/L or 1.5 times baseline) within 4.3 and 2.4 days for the non-obese and obese HIT patients, respectively. Within 37 days of argatroban initiation, death occurred in 7 (33%) non-obese and 5 (25%) obese patients (1 in each group while on argatroban); amputation in 0 (0%) non-obese and 1 (5%) obese patient (off argatroban); new thrombosis in 1 (5%) non-obese and 3 (15%) obese patients (2 while on argatroban); and major bleeding in 1 (5%) non-obese (off argatroban) and 0 (0%) obese patient. Cox regression analysis failed to identify any significant risk factors (BMI, obesity, gender, argatroban dose, baseline platelet count, aPTT) for new thrombosis. We conclude that argatroban dose requirements, aPTT responses, and clinical outcomes are generally comparable between obese vs. non-obese patients with HIT or history of HIT. These findings support the use of body weight-adjusted (and aPTT-adjusted) argatroban dosing for safe, effective anticoagulation, irrespective of patient BMI.
Role of Venous Outflow Obstruction and Venous Valvular Incompetence as Mechanisms for Venous Stasis Syndrome Following Deep Vein Thrombosis: A Population-Based Cohort Study.
Authors: Ashrani Aneel A. , Heit John A. , Lahr Brian D. , Petterson Tanya M. , Bailey Kent R. 
Published: October 2019

Abstract
Background: Venous stasis syndrome (VSS) is a relatively common long-term sequelae of deep vein thrombosis (DVT), although it frequently is noted in individuals with no prior history of DVT.
Objective: To evaluate whether: (1) venous stasis syndrome (VSS) is associated with a prior history of DVT; (2a) venous outflow obstruction (VOO) and/or (2b) venous valvular incompetence (VVI) are associated with DVT; and (3) VSS is associated with VVI and/or VOO.
Design: Case-control study nested within a population-based inception cohort study.
Population: 230 residents of Olmstead County, MN (OCM) with a first lifetime VTE over the 25-year period, 1966 – 1990 (cases), and 135 age, gender and year of incident VTE-matched OCM residents without prior history of VTE (controls).
Measurements: Physical examination and patient questionnaire for symptoms or signs of VSS, and strain gauge outflow plethysmography, continuous wave venous Doppler ultrasound, and passive venous drainage and refill testing for VOO and VVI performed between 1996 – 1998.
Results: Of the 365 study participants, 43 (12%) had VOO, 136 (37%) had VVI, and 265 (73%) had VSS. In multivariate logistic regression analyses: (1) age at the follow-up visit [OR Δper 10 years: 1.70 (1.41, 2.04)], prior DVT in the affected limb [OR: 4.03 (2.32, 7.01)], and presence of prior varicose veins [OR: 4.36 (1.84, 10.31)] were significantly associated with VSS; (2a) age at the follow-up visit [OR Δper 10 years (95% CI): 1.84 (1.39, 2.44)] and prior DVT in the affected limb [OR: 5.01 (2.61, 9.63)] were significantly associated with VOO; (2b) prior DVT in the affected limb (OR: 3.91 (2.56, 5.97)], presence of prior varicose veins [OR: 2.19 (1.32, 3.63)] and symptoms of VSS prior to incident DVT [OR: 3.42 (1.46, 8.00)] significantly increased the odds for VVI; and (3) VOO (p=0.004) and VVI (p<0.0001) were highly associated with VSS. Having a DVT in the left leg was associated with a greater odds of developing VOO, VVI or VSS in that leg when compared to their association with right leg DVT (OR: 6.69 vs. 3.65; 4.82 vs. 3.09; 4.71 vs. 3.97, respectively). Interestingly, prior DVT in the opposite leg was associated with an increased odds of subsequent VVI [OR: 2.00 (1.28, 3.10) and VSS [OR: 2.20 (1.31, 3.70)], but not VOO, in the test leg.
Conclusions: Prior DVT imparts an increased risk for subsequent VSS, likely due to VOO and/or VVI. The odds of VOO or VSS increases with age. Presence of varicose veins increases the odds for VVI and VSS. We speculate that the increased odds of left sided VOO, VVI and VSS in patients with prior DVT may be secondary to May-Thurner syndrome. The increased odds of VVI and VSS in the limb opposite to the one affected by prior DVT could reflect occult DVT in the test limb, inferior vena cava thrombosis, or other mechanisms leading for VVI and VSS.
The Interaction of Prothrombin and Its Activation Products with Platelets.
Authors: Samis John A. , Manuel Reginald P. , Nesheim Michael E. 
Published: October 2019

Abstract
The interaction of human 125I-prothrombin and its activation products with unactivated and thrombin-stimulated human platelets was studied. 125I-prothrombin binding to unactivated platelets was found to be a reversible and calcium-dependent process (n=48,000 sites/platelet, Kd=3.0μM). Thrombin stimulation of platelets resulted in increased prothrombin binding both in the presence and absence of calcium (n=84,000 sites/platelet, Kd=6.0μM and n=53,000 sites/platelet, Kd=8.0μM, respectively). Thrombin stimulation of platelets also increased the calcium-independent binding of prethrombin-1 compared with unstimulated cells. Using thrombin-stimulated platelets as a membrane surface for prothrombinase, 125I-prothrombin was converted exclusively to 125I-Fragment 1.2 and 125I-thrombin when the reactions were carried out in the presence of the reversible thrombin inhibitor, 5-dimethylaminonapthalene-1-sulfonylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). 125I-thrombin was the major prothrombin activation product that remained bound to the thrombin-stimulated platelets (50,000 molecules/platelet). Platelet-bound 125I-thrombin either added directly or generated from 125I-prothrombin by the action of prothrombinase was found to be inacessible to human antithrombin (AT)/heparin-induced inactivation. Utilization of phosphatidylcholine/phosphatidylserine (PCPS) vesicles as a membrane surface for prothrombinase in the presence of DAPA demonstrated that 125I-prothrombin activation proceeded initially through a meizothrombin intermediate that was replaced at later times by Fragment 1.2 and the A and B chains of α-thrombin. 125I-Fragment 1.2 was the main prothrombin activation product that remained bound to the PCPS vesicles after Sephadex G-150 gel filtration chromatography. The above results indicate that human prothrombin binds specifically to unstimulated human platelets via its Fragment 1 domain only in the presence of calcium and thrombin-stimulation results in the exposure of additional calcium-dependent and -independent prothrombin binding sites. In addition, thrombin was the major prothrombin activation product that remains associated with the thrombin-stimulated human platelets and this platelet-bound thrombin is protected from AT/heparin- induced inactivation. During prothrombin activation on platelets, large amounts of thrombin remain bound and Fragment 1.2 is released from the platelet surface. In contrast, Fragment 1.2 was the major prothrombin activation product that binds PCPS vesicles. Therefore, although both thrombin-stimulated human platelets and PCPS vesicles may each serve as an effective membrane component of prothrombinase, they appear to differ both in their prothrombin activation pathway as well as their ability to bind the products of prothrombin activation.
Signalling Pathways Have Different Expression Profiles in Human Platelets Isolated from Men and Women.
Authors: Rothwell Stephen W. , Eidelman Ofer , Jozwik Catherine , Mueller Gregory , Poth Merrily , Zeitlin Pamela L. , Guggino William , Jacobowitz David M. , Srivastava Meera , Pollard Harvey B. 
Published: October 2019

Abstract
Background: Cardiovascular diseases (CVD) are the leading causes of death for men and women in the U.S. Platelets play a central role in the inflammation and coagulation mechanisms responsible for these disorders, and anti-platelet drugs are therefore the current mainstays in prevention and therapy. Understanding the signaling pathways involved in normal and abnormal platelet function using proteomic techniques offers a novel strategic approach to the discovery of potential new therapeutic targets.
Hypothesis: The incidence and outcomes of cardiovascular disease are profoundly dependent on gender, and it is possible that differences in platelet signaling mechanisms may contribute to these disparities. We have therefore hypothesized that the platelet signaling proteome is gender-specific.
Methods: Platelets were isolated and purified from four normal adult male and four normal adult female donors under an IRB-approved protocol. Conventional 2D-gel electrophoresis (2DGE) and mass spectrometry were used to profile the high abundance platelet proteome. Antibody microarrays, bearing 507 monoclonal antibodies against low abundance proteins involved in signaling pathways and related functions, were used to interrogate the lower abundance platelet. Western blots were used to validate a subset of the data.
Results: Discovery studies with 2DGE revealed at least nineteen lower abundance proteins which could be significantly distinguished on the basis of gender (P<0.05). Two of these proteins, a thrombopoeitin-activated G-protein component (#1101) and a megakaryocyte-relevant co-factor for DNA synthesis (#1583), were selectively elevated in male platelets. Concurrent studies on the sensitive antibody microarray platform revealed that more than 420 proteins were significantly expressed in a gender-specific manner (P<0.05). Of these, 62 remained significant by the stringent criterion of a False Discovery Rate (FDR) of <10% (SAM Algorithm, 2000 permutations). By rank-ordering the proteins by expression level, we found that platelets from normal male and female donors were significantly and uniquely enriched in different signaling proteins. These gender-specific signaling proteins included IL-1beta, and specific isoforms of iNOS (inducible nitric oxide synthase), PLC (phospholipase C), and PKC (protein kinase C). Western blot analysis validated a subset of the differentially expressed platelet proteome.
Conclusions: These data support the hypothesis that the platelet signaling proteome is gender-specific. We interpret these data to indicate that gender-specific differences in the platelet signaling proteome may contribute to the gender-specific disparity in outcomes for cardiovascular diseases in men and women.