Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of,and Correlations and Differences between aPTT,Anti-Factor Xa and Thrombin Generation Assays

Background

Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin.

Aim

To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance.

Method

Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents.

Results

Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (R_s0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (R_s0.80–0.92).

Conclusions

aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is a promising method for monitoring LMWH’s.     

Siaα2-3Galβ1- Receptor Genetic Variants Are Associated with Influenza A(H1N1)pdm09 Severity

Different host genetic variants may be related to the virulence and transmissibility of pandemic Influenza A(H1N1)pdm09, influencing events such as binding of the virus to the entry receptor on the cell of infected individuals and the host immune response. In the present study, two genetic variants of the ST3GAL1 gene, which encodes the Siaα2-3Galβ1- receptor to which influenza A(H1N1)pdm09 virus binds for entry into the host cell, were investigated in an admixed Brazilian population. First, the six exons encoding the ST3GAL1 gene were sequenced in 68 patients infected with strain A(H1N1)pdm09. In a second phase of the study, the rs113350588 and rs1048479 polymorphisms identified in this sample were genotyped in a sample of 356 subjects from the northern and northeastern regions of Brazil with a diagnosis of pandemic influenza. Functional analysis of the polymorphisms was performed in silico and the influence of these variants on the severity of infection was evaluated. The results suggest that rs113350588 and rs1048479 may alter the function of ST3GAL1 either directly through splicing regulation alteration and/or indirectly through LD with SNP with regulatory function. In the study the rs113350588 and rs1048479 polymorphisms were in linkage disequilibrium in the population studied (D’ = 0.65). The GC haplotype was associated with an increased risk of death in subjects with influenza (OR = 4.632, 95% CI = 2.10;1.21). The AT haplotype was associated with an increased risk of severe disease and death (OR = 1.993, 95% CI = 1.09;3.61 and OR 4.476, 95% CI = 2.37;8.44, respectively). This study demonstrated for the first time the association of ST3GAL1 gene haplotypes on the risk of more severe disease and death in patients infected with Influenza A(H1N1)pdm09 virus.     

Ontology Alignment Repair through Modularization and Confidence-Based Heuristics

Ontology Matching aims at identifying a set of semantic correspondences, called an alignment, between related ontologies. In recent years, there has been a growing interest in efficient and effective matching methods for large ontologies. However, alignments produced for large ontologies are often logically incoherent. It was only recently that the use of repair techniques to improve the coherence of ontology alignments began to be explored. This paper presents a novel modularization technique for ontology alignment repair which extracts fragments of the input ontologies that only contain the necessary classes and relations to resolve all detectable incoherences. The paper presents also an alignment repair algorithm that uses a global repair strategy to minimize both the degree of incoherence and the number of mappings removed from the alignment, while overcoming the scalability problem by employing the proposed modularization technique. Our evaluation shows that our modularization technique produces significantly small fragments of the ontologies and that our repair algorithm produces more complete alignments than other current alignment repair systems, while obtaining an equivalent degree of incoherence. Additionally, we also present a variant of our repair algorithm that makes use of the confidence values of the mappings to improve alignment repair. Our repair algorithm was implemented as part of AgreementMakerLight, a free and open-source ontology matching system.     

Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO₂⁻ binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO₂⁻ concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO₂−binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.     

Actinomyces israelii endocarditis misidentified as “Diptheroids”

Actinomyces is a rare cause of endocarditis, however misidentification as Cornyebacteria often hampers the diagnosis.     

The plasma membrane Ca2+ pump isoform 4a differs from isoform 4b in the mechanism of calmodulin binding and activation kinetics: implications for Ca2+ signaling

The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., T?r?k, K., and Caride, A. J. (2003) Biochemistry 41, 12115-12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500-24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.     

Cruciform extrusion propensity of human translocation-mediating palindromic AT-rich repeats

There is an emerging consensus that secondary structures of DNA have the potential for genomic instability. Palindromic AT-rich repeats (PATRRs) are a characteristic sequence identified at each breakpoint of the recurrent constitutional t(11;22) and t(17;22) translocations in humans, named PATRR22 (~600bp), PATRR11 (~450bp) and PATRR17 (~190bp). The secondary structure-forming propensity in vitro and the instability in vivo have been experimentally evaluated for various PATRRs that differ regarding their size and symmetry. At physiological ionic strength, a cruciform structure is most frequently observed for the symmetric PATRR22, less often for the symmetric PATRR11, but not for the other PATRRs. In wild-type E. coli, only these two PATRRs undergo extensive instability, consistent with the relatively high incidence of the t(11;22) in humans. The resultant deletions are putatively mediated by central cleavage by the structure-specific endonuclease SbcCD, indicating the possibility of a cruciform conformation in vivo. Insertion of a short spacer at the centre of the PATRR22 greatly reduces both its cruciform extrusion in vitro and instability in vivo. Taken together, cruciform extrusion propensity depends on the length and central symmetry of the PATRR, and is likely to determine the instability that leads to recurrent translocations in humans.     

Synthesis of 3-(1H-benzimidazol-2-yl)-5-isoquinolin-4-ylpyrazolo[1,2-b]pyridine, a potent cyclin dependent kinase 1 (CDK1) inhibitor

The novel compound 3-(1H-benzimidazol-2-yl)-5-isoquinolin-4-ylpyrazolo[1,2-b]pyridine was discovered to be a potent CDK1 inhibitor. Described here is the chemistry for its synthesis, including Pd(II) catalyzed Stille coupling reaction and sulfur(0) induced benzimidazole formation. Its effects on VEGFR-2 kinase activity and tumour cell proliferation are also described.     

Synthesis and evaluation of pyrazolo[3,4-b]pyridine CDK1 inhibitors as anti-tumor agents

A series of 3,5-disubstituted pyrazolo[3,4-b]pyridine cyclin-dependent kinase (CDK) inhibitors was synthesized. These compounds showed potent and selective CDK inhibitory activities and inhibited in vitro cellular proliferation in cultured human tumor cells. Selected compounds were evaluated in an in vivo tumor xenograft model. The synthesis and biological evaluation of these pyrazolo[3,4-b]pyridines and related compounds are reported.