bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mice Engineered To Express a Form of Thrombin Favoring Protein C Are Resistant to S. aureus-Induced Sepsis.
Authors: Flick Matthew J. , Chauhan Anil K. , Welch Sara , Shaw Maureen A. , Talmage Kathryn E. , Mullins Eric S. , Palumbo Joseph S. , Thornton Sherry , Wagner Denisa D. , Degen Jay L. 
Published: April 2020

Abstract
Thrombin is central in thrombus formation as both a positive mediator of thrombus formation through the proteolytic activation of PARs, fibrinogen, fXI and other prothrombotic substrates, and a negative modulator of the coagulation cascade through the activation of protein C. Detailed structure-function studies have revealed that thrombin can be redesigned to favor either procoagulant or anticoagulant substrates. The introduction of W215A/E217A substitutions in the murine thrombin active site (fIIWE) results in a pronounced “specificity switch” that reduces catalytic efficiency with fibrinogen by at least 3-orders-magnitude while only modestly reducing activity for protein C activation. To evaluate the effects of fIIWE activity in vivo, we have used a gene-targeting strategy to generate mice carrying the W215A/E217A mutations in the endogenous murine prothrombin gene. The mutant allele was transmitted through the germline and was found to support the expression of normal levels of hepatic fII mRNA and plasma fII in both heterozygous and homozygous neonates. Unlike fII knockout mice, homozygous fIIWE mice were observed at term with the expected Mendelian frequency. Nevertheless, homozygous fIIWE offspring uniformly succumbed to spontaneous bleeding events within days of birth. Heterozygous fIIWE/WT animals generally survived to adulthood, were capable of carrying multiple liters to term, and unchallenged mice displayed a hematological profile similar to wildtype mice. However, consistent with a predicted anticoagulant phenotype, adult fIIWE/WT heterozygotes exhibited significantly delayed thrombus formation following ferric chloride injury of mesenteric arterioles and extended bleeding times following tail tip excision relative to control mice expressing wildtype fII. Given that activated protein C has been shown to be efficacious in the treatment of sepsis, we explored whether the shift in thrombin specificity in heterozygous fIIWE/WT mice would confer the benefit of rendering animals tolerant to acute septic challenges. Kaplan-Meier analyses following intravenous administration of S. aureus revealed that fIIWE/WT mice exhibited a significant survival advantage over littermate wildtype animals challenged in parallel and tracked over a 7-day observation period. Notably, extended thrombus formation and bleeding times as well as resistance to sepsis was not simply a function of half normal wildtype fII expression. When these analyses were performed in animals carrying one wildtype allele and one null mutation allele, results were similar to wiltype. These studies further underscore the interplay between the hemostatic and inflammatory systems in vivo and highlight the possible therapeutic utility of recombinant (pro)thrombin derivatives with selected alterations in substrate specificity.
Subpopulations with Iron Deficiency, Liver Disease, or HFE Mutations Revealed by Statistical Mixture Modeling of Transferrin Saturation and Serum Ferritin Concentration in Asians, African Americans, Hispanics, and Whites.
Authors: McLaren Christine E. , Gordeuk Victor R. , Chen Wen-Pin , Barton James C. , Acton Ronald T. , Speechley Mark , Castro Oswaldo , Adams Paul C. , Snively Beverly M. , Harris Emily L. , Reboussin David M. , McLachlan Geoffrey J. , Bean Richard , McLaren Gordon D. 
Published: April 2020

Abstract
In previous investigations, we modeled the distribution of transferrin saturation (TS) in Caucasians and demonstrated a strong association between HFE genotype and TS subpopulations. Extending this approach, we now have analyzed joint population distributions of TS and serum ferritin concentration (SF) measured in the multi-ethnic Hemochromatosis and Iron Overload Screening (HEIRS) Study and examined the association of these distributions with the presence of HFE C282Y and H63D mutations, self-reported liver disease, and iron deficiency (defined as SF &lt;15 μg/L). Based on separate models for each race/ethnicity by gender, four components with successively increasing age-adjusted means for TS and SF were identified in data from 26,832 African Americans, 12,620 Asians, 12,264 Hispanics, and 43,254 Whites. Fig. 1 illustrates age-adjusted values from 16,662 White men. Superimposed 95% confidence ellipses reflect component probability densities and show separation of the 1st and 4th components that had the lowest and highest means for TS and SF, respectively. Table 1 presents the range of estimates from individual models and indicates that the 2nd (largest) component had TS means of 22–26% for women (29–30% for men) and SF means of 43–82 μg/L for women (165–242 μg/L for men). The 3rd and 4th components had progressively smaller proportions and higher mean values of TS and SF, while the 1st component in each model had mean TS &lt;16% for women (&lt;20% for men), and mean SF &lt;28 μg/L for women (&lt;47 μg/L for men). Compared to the 2nd component:
adjusted odds of iron deficiency were significantly higher in the 1st component (15–48 for women, 61–3530 for men); adjusted odds of self-reported liver disease were significantly higher in the 3rd and 4th components for African-American women and all men; and adjusted odds of any HFE mutation were increased in the 3rd component (1.4–1.8 for women, 1.2–1.9 for men) and in the 4th component for Hispanic and White women (1.5, 5.2, respectively) and men (2.8, 4.7, respectively).
Joint mixture modeling identifies one component with lower mean SF and TS at risk for iron deficiency and two components with higher mean SF and TS at risk for liver disorders and HFE mutations. This approach permits characterization of the aggregate effects of hereditary or acquired factors that influence these serum iron measures in populations, and complements and enhances genetic and phenotypic testing for assessment of disease characteristics.
Table 1 Range of estimates from models.
Screening for Hemochromatosis by Measuring Ferritin Levels: A More Effective Approach.
Authors: Waalen Jill , Beutler Ernest 
Published: April 2020

Abstract
Population screening for hereditary hemochromatosis has traditionally been performed by determining the serum transferrin saturation. With the recognition that the penetrance of HFE hemochromatosis is extremely low, it has become apparent that population screening with transferrin saturation is unlikely to be cost effective as the vast majority of individuals detected neither have clinical disease nor are likely to develop it. Although the factors that cause a subset of patients to develop the severe disease phenotype have not been identified, it is clear from at least three independent studies that only patients with serum ferritin concentrations of over 1000 ng/ml are at risk. We have utilized data from the Scripps-Kaiser hemochromatosis study to model results of a screening program using this ferritin cut-off value. Among 29,699 white subjects screened, only 59 had serum ferritin levels of greater than 1000 ng/ml. Twenty-four of these had homozygous mutant or compound heterozygous mutant HFE genotypes that could account for the ferritin levels. Among the remaining patients, the main causes of elevated ferritin levels were excessive alcohol intake, cancer, or liver disease. Causes for the hyperferritinemia were found in all but 8% of the 59 patients. We conclude that screening for hemochromatosis with serum ferritin levels will detect the vast majority of patients who will be clinically affected, and may detect other clinically significant disease in patients who do not have hemochromatosis phenotypes. Since it is clear from other longitudinal studies that the ferritin level of the vast majority of adult homozygotes for HFE mutations does not rise over long periods of time, excluding subjects with serum ferritin levels &lt;1000 ng/ml should not result in missed opportunities for early treatment of patients who could benefit.
Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.
Authors: Soeda Tetsuhiro , Nogami Keiji , Takeyama Masahiro , Ogiwara Kenichi , Tomokiyo Kazuhiko , Sakata Yoichi , Yoshioka Akira , Shima Midori 
Published: April 2020

Abstract
Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.
P2 and P2′ Residues Are Important for the Efficient Proteolysis of Factor VIIIa at Arg336 Catalyzed by Activated Protein C.
Authors: Varfaj Fatbardha , DeAngelis Jennifer , Wakabayashi Hironao , Fay Philip J. 
Published: April 2020

Abstract
Activated Protein C (APC) is an anticoagulant serine protease that proteolytically inactivates the coagulation cofactors, factors (F) Va and VIIIa. FVIIIa is a non-covalent heterotrimer consisting of A1, A2 and A3-C1-C2 subunits. APC-catalyzed inactivation of FVIIIa results from proteolysis at the P1 residues Arg336 and Arg562 within the A1 and A2 subunits, respectively, with cleavage at Arg336 representing the dominant reaction. We recently showed that replacement of the P4-P3′ residues surrounding the Arg336 site with the corresponding residues flanking Arg562 resulted in an ∼100-fold reduction in cleavage rate at Arg336 (Varfaj et al, J. Biol. Chem.2007). Comparison of the P4-P3′ residues at the slow-reacting site in FVIIIa (Arg562) with the corresponding residues at the fast-reacting site in FVa (Arg506) revealed that these sequences differ primarily at the P2 and P2′ positions. This observation suggested an important contribution by these residues to cleavage efficiency. The role of the P2 and P2′ residues in the proteolysis of FVIIIa by APC was investigated by preparing recombinant FVIII proteins possessing mutations at Leu335 (P2) and Lys338 (P2′) flanking the P1 Arg336. B-domainless FVIII proteins were stably expressed in BHK cells and purified. Leu335Arg and Leu335Gln mutants were constructed based upon the P2 residues preceding Arg506 in FVa and Arg562 in FVIIIa, respectively. The Lys338Ile mutant was based upon the P2′ residue following Arg506 in FVa. Specific activity values for all FVIII variants were similar to wild type (WT) FVIII. APC-catalyzed inactivation rates for FVIIIa were determined using a FXa generation assay, and rates for proteolysis at the scissile bonds within the A1 and A2 subunits were determined by SDS-PAGE and Western blotting. Rates for APC-catalyzed cleavage of the A1 subunit for the FVIIIa variants Leu335Arg and Leu335Gln were reduced ∼2- and ∼4-fold, respectively, as compared to WT. However, the rate of cleavage of the A1 subunit in the Lys338Ile FVIIIa variant was enhanced ∼3-fold compared to WT. Cleavage rates at Arg562 in all the variants were unaffected by mutation at either residue 335 or 338. These relative values for rates of proteolysis paralleled the observed rates for inactivation of the FVIIIa forms. Overall, these results suggest that both P2 and P2′ residues are important in the efficient proteolysis at Arg336 in FVIIIa. Furthermore, Leu appears more optimal than either Arg or Gln at the P2 position, whereas Ile is preferred over Lys at the P2′ position in this macromolecular substrate for APC.
Inhibition of the Thrombin-Thrombomodulin by a Covalent Antithrombin-Heparin.
Authors: Van Walderveen Maria C. , Berry Leslie R. , Chan Anthony K.C. 
Published: April 2020

Abstract
Endothelial thrombin·thrombomodulin (IIa-TM) complex is capable of activating protein C anticoagulant. In vivo, TM can exist in two forms: one with a chondroitin sulfate (CS) (acidic) and one without (non-acidic). TM binding has been shown to reduce the rate of IIa inhibition by antithrombin (AT) in the presence of unfractionated heparin (UFH), especially when the TM possesses a glycosaminoglycan chain. We have recently developed a covalent antithrombin-heparin (ATH) with high potency for inhibition of IIa bound to surfaces. In the present study we examined the role of the CS chain in the inhibition of IIa-TM complex by the novel anticoagulant, ATH, in comparison to AT + UFH. For this study, discontinuous second order rate constant (k2) inhibition assays of IIa-TM were performed at 37°C. IIa was incubated with TM for 30 min and inhibited using either 20nM AT + varying concentrations (0–6000nM) of UFH or a constant concentration of 10nM ATH. Residual IIa activity was measured at 405nm after stopping the reaction using a solution of polybrene and chromogenic substrate (S-2238). Second order rate constants (mean +/− SEM; n≥5) were calculated from plots of IIa-TM activity vs. time and differences determined using two-sample t-tests. Comparison of the second order rate constants for inhibition of IIa, IIa-TM(−CS), or IIa-TM(+CS) by AT + UFH (2.015 × 108 +/− 7.079 × 106 M−1min−1, 1.633 × 108 +/− 8.922 × 106 M−1min−1, and 5.248 × 107 +/− 5.693 × 106 M−1min−1, respectively) showed that the presence of TM reduced the rate of inhibition (with respect to IIa alone; p=0.012, p&lt;0.001, respectively). Moreover, the presence of a CS on the TM inhibited IIa neutralization to a greater degree than when the CS is absent (p&lt;0.001). Furthermore, ATH was a much faster inhibitor of IIa, IIa-TM(−CS), and TM(+CS) (1.486 × 109 +/− 1.193 × 108 M−1min−1, 1.370 × 109 +/− 1.246 × 108 M−1min−1, and 8.869 × 108 +/− 1.246 × 108 M−1min−1, respectively) compared to AT + UFH (p&lt;0.001). CS removal by chondroitin ABC lyase (ABCase) showed that the rate of IIa-TM(+CS) inhibition significantly increased for both AT + UFH and ATH, while no significant change due to ABCase treatment was found for the rate of inhibition of IIa-TM(−CS) by either AT + UFH or ATH. We speculate that during IIa-TM(+CS) inhibition by ATH, the covalently linked heparin moiety may be able to repel (rotate) the CS chain in order for ATH to interact with the IIa-TM complex. In the case of AT + UFH, lack of permanent UFH linkage to AT may allow the CS to displace the UFH chain from binding both AT and IIa. Steric hindrance by CS chains remains a significant factor since the k2 values were lower for the AT + UFH or ATH reactions with IIa-TM(+CS) compared to those with IIa-TM(−CS). Given the rapid reaction rate with free or bound IIa, ATH significantly limits availability of IIa for coagulant or anticoagulant functions.
Mapping of Allosteric Modulator Effects in the Active Site of Factor IXa.
Authors: Villegas Karla , Baker-Deadmond Kimberly , Neuenschwander Pierre F. 
Published: April 2020

Abstract
Activated factor IX (fIXa) is a vitamin K-dependent blood coagulation serine protease involved in propagation of the coagulant response through activation of fX. Maximal enzymatic and procoagulant activity of fIXa requires the presence of several cofactors; one of which is ionic calcium, which is known to bind to a site in the protease domain of fIXa as well as several sites within the light chain Gla domain region. One of the roles of calcium appears to be allosteric modulation of the fIXa active site as evidenced by an increase in enzymatic activity towards small peptidyl substrates. We and others have additionally found that certain small hygroscopic molecules can also enhance fIXa amidolytic activity. The molecular details involved in either of these effects are not well understood. Previous studies by us have shown that a pentapeptide substrate (AGRSL; the reactive site sequence of antithrombin) is hydrolyzed by fIXa in the absence of cofactors or modulators. This hydrolysis is enhanced in the presence of ionic calcium, ethylene glycol or low molecular weight heparin suggesting effects of these molecules on the immediate active site vicinity of fIXa. In order to gain insight into the potential allosteric modulation that each of these effectors may affect in fIXa, we examined the hydrolysis of four peptide libraries based on the AGRSL pentapeptide sequence, in the presence and absence of various fIXa modulators. The four libraries synthesized were XGRSL, AXRSL, AGRXL and AGRSX; where X denotes any of the possible 20 amino acids. Each of these libraries were screened for hydrolysis by fIXa under various conditions with substrates and products being identified en masse using MALDI-TOF mass spectrometry. The results suggest that ionic calcium enhances fIXa reactivity in part by modulation of the S2 subsite in fIXa. In contrast, ethylene glycol enhances fIXa activity via modulation of the S3 subsite and heparin was found to effect the overall active site region.
Phase II Evaluation of Sunitinib Malate, a Multi-Targeted Inhibitor of Receptor Tyrosine Kinases, in Patients with Myelofibrosis.
Authors: Tam Constantine S. , Thomas Deborah A. , Ravandi Farhad , Richardson Catherine D. , Borthakur Gautam , Burger Jan A. , Estey Elihu , Kantarjian Hagop , Verstovsek Srdan 
Published: April 2020

Abstract
BACKGROUND: Sunitinib Malate is a small molecule inhibitor of receptor tyrosine kinases (TK) with a broad spectrum of activity against VEGFR-1, -2, and -3, PDGFR-α and -β, c-kit, the TK encoded by the ret oncogene, and Flt-3. As TK-mediated angiogenesis may be an integral element in the pathogenesis and progression of myelofibrosis (MF), we investigated the use of sunitinib in MF patients.
METHODS: 14 patients (pt) have been treated with sunitinib 37.5mg orally daily in six-week cycles, with response assessment after 2 and 4 cycles. Median baseline characteristics (range): age 65 years (50–79); 2 previous therapies (0–4); B-symptoms 64%; Hb &lt; 8g/dL or transfusion-dependent 50%; JAK-2 mutated 57%.
RESULTS: Pt received sunitinib for a median of 101 days (23–173). Six stopped study drug prior to the planned four cycles because of toxicity (5) or disease progression (1); four stopped at four cycles because of toxicity (1) or lack of response (3); one stopped at five cycles because of lack of response; and three patients still on therapy had shown no responses at 3, 3 and 5 cycles. No responses were observed. One patient had 50% reduction in splenomegaly but became transfusion-dependent, and was considered a non-response. Grade 1 or 2 hypertension developed in 7 (50%) patients and was more common in patients with a pre-existing history of hypertension (5/7) than patients without a history of hypertension (2/7); in six patients, hypertension led to interruption and/or dose reduction in study medication. Common toxicities were constitutional (fatigue 86%, Grade 3=21%), gastrointestinal (abdominal pain, nausea or diarrhea 79%, Grade 3=14%) or neurologic (pain or numbness in extremity 43%, Grade 3=0%). Grade 3 bleeding (1 epistaxis, 1 upper GI) occurred in 2 patients, and grade 3 optic disc edema in 1. Toxicities were cumulative and dose-related, with dose reductions being required in 7/10 patients who continued beyond 2 cycles.
CONCLUSIONS: At the first planned interim analysis, no responses were recorded in 14 patients treated with sunitinib. This fulfiled the early stopping rule in the Gehan two-stage study design, and the study has been closed to new patient accrual because of inadequate response.
Identification of Critical Phosporylation Sites within NPM-ALK That Regulate JUNB mRNA Translation.
Authors: Fuchs Claudia , Vesely Paul , Bambach Isabella , Schauer Silvia , Linkesch Werner , Sternberg David W. , Hoefler Gerald , Staber Philipp B. 
Published: April 2020

Abstract
Anaplastic large cell lymphoma (ALCL) accounts for approximately 30% of childhood lymphomas and 3% of adult non-Hodgkin lymphomas. The nucleophosmin - anaplastic lymphoma kinase (NPM-ALK) fusion which is the product of a t(2;5)(p23;q35) chromosomal translocation is present in about half of nodal ALCL. Expression of this fusion kinase results in induction of the AP-1 transcription factor JunB and IL-3 independent outgrow of murine hematopoietic Ba/F3 cells. We demonstrated that wild type NPM-ALK increases the amount of ribosomes bound to JUNB mRNA resulting in its more effective translation in large polysomes. The NPM-ALK fusion tyrosine kinase has 20 potential tyrosine residues available for autophosphorylation and phosphorylation by other protein tyrosine kinases. Here we used series of Y-to-F-substituted mutants of NPM-ALK to identify tyrosine residues that are required to regulate the segregation of JUNB mRNAs between polysomes and monosomes as well as ribonucleic particles (RNPs). Neither JUNB transcription nor JunB translation was altered in Ba/F3 cells expressing NPM-ALK mutants Y17F/Y29F/Y67F Y138F/Y152F Y156F/Y191F/Y299F Y378F/Y418F/Y445F and Y646F/Y664F compared to NPM-ALK wild type. Conversely, in NPM-ALK Y567F/Y461F/Y644F mutant cells proliferation was markedly decreased. These cells demonstrated active MEK-ERK pathway, while AKT, mTOR, and rpS6 phosphorylation was impaired. Moreover a shift of JUNB mRNA from the polysomic to the monosomic/mRNP fraction could be observed. In conclusion, we identified specific NPM-ALK phosphorylation sites required to mediate the effect of NPM-ALK on the JUNB translational regulation and therefore provide further insights in the transforming mechanisms of the oncoprotein NPM-ALK.