Coagulation and fibrinolytic activity of tenecteplase and alteplase in acute ischemic stroke

Background and Purpose—We compared the fibrinolytic activity of tenecteplase and alteplase in patients with acute ischemic stroke, and explored the association between hypofibrinogenaemia and intracerebral hemorrhage. Methods—Venous blood samples from a subgroup of participants in the Alteplase–Tenecteplase Trial Evaluation for Stroke Thrombolysis (ATTEST) study were obtained at pretreatment, 3 to 12 hours, and 24±3 hours post-intravenous thrombolysis for analyses of plasminogen, plasminogen activator inhibitor-1, D-dimer, factor V, fibrinogen, and fibrin(ogen) degradation products, in addition to routine coagulation assays. Related sample Wilcoxon signed-rank tests were used to test the within-group changes, and independent Mann–Whitney tests for between-group differences. Results—Thirty patients were included (alteplase=14 and tenecteplase=16) with similar baseline demographics. Compared with baseline, alteplase caused significant hypofibrinogenaemia (P=0.002), prolonged prothrombin time (P=0.011), hypoplasminogenaemia (P=0.001), and lower factor V (P=0.002) at 3 to 12 hours after administration with persistent hypofibrinogenaemia at 24 hours (P=0.011), whereas only minor hypoplasminogenaemia (P=0.029) was seen in the tenecteplase group. Tenecteplase consumed less plasminogen (P<0.001) and fibrinogen (P=0.002) compared with alteplase. Conclusions—In patients with acute ischemic stroke, alteplase 0.9 mg/kg caused significant disruption of the fibrinolytic system, whereas tenecteplase 0.25 mg/kg did not, consistent with the trend toward lower intracerebral hemorrhage incidence with tenecteplase in the ATTEST study

The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation

The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

INCENP acts as a protein scaffold that integrates the functions of two crucial mitotic kinases, Aurora B and Polo, at centromeres of mitotic chromosomes

The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

INCENP acts as a protein scaffold that integrates the functions of two crucial mitotic kinases, Aurora B and Polo, at centromeres of mitotic chromosomes

The dual-specificity protein phosphatase DUSP9/MKP-4 is essential for placental function but is not required for normal embryonic development

To elucidate the physiological role(s) of DUSP9 (dual-specificity phosphatase 9), also known as MKP-4 (mitogen-activated protein kinase [MAPK] phosphatase 4), the gene was deleted in mice. Crossing male chimeras with wild-type females resulted in heterozygous (DUSP9(+/−)) females. However, when these animals were crossed with wild-type (DUSP9(+/y)) males none of the progeny carried the targeted DUSP9 allele, indicating that both female heterozygous and male null (DUSP9(−/y)) animals die in utero. The DUSP9 gene is on the X chromosome, and this pattern of embryonic lethality is consistent with the selective inactivation of the paternal X chromosome in the extraembryonic tissues of the mouse, suggesting that DUSP9/MKP4 performs an essential function during placental development. Examination of embryos between 8 and 10.5 days postcoitum confirmed that lethality was due to a failure of labyrinth development, and this correlates exactly with the normal expression pattern of DUSP9/MKP-4 in the trophoblast giant cells and labyrinth of the placenta. Finally, when the placental defect was rescued, male null (DUSP9(−/y)) embryos developed to term, appeared normal, and were fertile. Our results indicate that DUSP9/MKP-4 is essential for placental organogenesis but is otherwise dispensable for mammalian embryonic development and highlights the critical role of dual-specificity MAPK phosphatases in the regulation of developmental outcomes in vertebrates

INCENP and Aurora B Promote Meiotic Sister Chromatid Cohesion through Localization of the Shugoshin MEI-S332 in Drosophila

SummaryThe chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. Here, we show that INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila incenp profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis

The eGFR-C study: accuracy of glomerular filtration rate (GFR) estimation using creatinine and cystatin C and albuminuria for monitoring disease progression in patients with stage 3 chronic kidney disease - prospective longitudinal study in a multiethnic population

Background: Uncertainty exists regarding the optimal method to estimate glomerular filtration rate (GFR) for disease detection and monitoring. Widely used GFR estimates have not been validated in British ethnic minority populations. Methods: Iohexol measured GFR will be the reference against which each estimating equation will be compared. The estimating equations will be based upon serum creatinine and/or cystatin C. The eGFR-C study has 5 components:1)A prospective longitudinal cohort study of 1300 adults with stage 3 chronic kidney disease followed for 3 years with reference (measured) GFR and test (estimated GFR [eGFR] and urinary albumin-to-creatinine ratio) measurements at baseline and 3 years. Test measurements will also be undertaken every 6 months. The study population will include a representative sample of south-Asians and African-Caribbeans. People with diabetes and proteinuria (ACR >=30 mg/mmol) will comprise 20-30% of the study cohort. 2)A sub-study of patterns of disease progression of 375 people (125 each of Caucasian, Asian and African-Caribbean origin; in each case containing subjects at high and low risk of renal progression). Additional reference GFR measurements will be undertaken after 1 and 2 years to enable a model of disease progression and error to be built. 3)A biological variability study to establish reference change values for reference and test measures. 4)A modelling study of the performance of monitoring strategies on detecting progression, utilising estimates of accuracy, patterns of disease progression and estimates of measurement error from studies 1), 2) and 3). 5)A comprehensive cost database for each diagnostic approach will be developed to enable cost-effectiveness modelling of the optimal strategy. The performance of the estimating equations will be evaluated by assessing bias, precision and accuracy. Data will be modelled as a linear function of time utilising all available (maximum 7) time points compared with the difference between baseline and final reference values. The percentage of participants demonstrating large error with the respective estimating equations will be compared. Predictive value of GFR estimates and albumin-to-creatinine ratio will be compared amongst subjects that do or do not show progressive kidney function decline. Discussion: The eGFR-C study will provide evidence to inform the optimal GFR estimate to be used in clinical practice

Aurora B activity is required for the activation of Polo kinase at the inner centromere.

<p>(A) Aurora B phosphorylates Polo kinase in vitro. Bacterially expressed HIS-Polo or HIS-Polo^T182A (which is catalytically inactive and therefore unable to autophosphorylate) were incubated with (or without) Drosophila Aurora B in complex with a fragment of INCENP (residues 654–755) in presence of ³²P-g-ATP, in triplicate. Reaction products were resolved by SDS-PAGE transferred to nitrocellulose and analyzed by autoradiography (AR) and anti-Polo Western blot (WB). Quantitative measurements of signals were obtained (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001250#s4" target="_blank">Materials and Methods</a>), and the ratios were calculated for each reaction (AR/WB, A.U.: arbitrary units). Right, average values for the relative phosphorylatin of Polo^WT and Polo^T182A by Aurora B. Error bars, SEM. (B–D) DMel-2 cells stably expressing Polo-GFP treated with (B) DMSO or (C–D) Binucleine-2, immunostained for INCENP, Polo, and Polo^T182Ph (insets: zoomed images of kinetochores). In (C–D) asterisks indicate centrosomes. Merged images show INCENP/Polo/DNA. Zoomed images in (C–D) insets show examples of kinetochore pairs showing decreased levels of Polo^T182Ph. (E) Dot plot showing the quantification of INCENP/Polo/Polo^T182Ph signal intensity at the kinetochore (<i>t</i> test: *** <i>p</i><0.0001; n.s., not significant; <i>p</i> = 0.4028). Signal intensities for individual kinetochores were measured using the SoftWorx Data Inspector tool; average background was subtracted; data was plotted using KaleidaGraph software. (F) RNAi depletion of Aurora B, but not Aurora A, strongly reduces Polo^T182Ph levels in DMel-2 cells treated with okadaic acid. Cells were transfected with the indicated dsRNAs for 4 d, and 100 nM okadaic acid added for 4 h before immunoblotting to improve visualization of phosphorylated Polo. A dsRNA against the Kanamycin resistance bacterial gene was used as a negative control. Asterisks: non-specific bands. Both bulk Polo and PoloT^182ph appear as doublets. (G) RNAi depletion of Aurora B or INCENP, but not Aurora A, reduces Polo^T182Ph levels at centromeres/kinetochores. Cycling cells were treated with the indicated dsRNAs for 3 d (immunoblots are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001250#pbio.1001250.s006" target="_blank">Figure S6B</a>) and Polo^T182Ph was detected by immunofluorescence. Levels of Polo^T182Ph at centromeres/kinetochores in prometaphase and metaphase cells were measured at individual kinetochores using Image J, subtracting background (Kt-bkd). Asterisks indicate centrosomes. Error bars = S.E.M.</p