The right time to measure anti-Xa activity in critical illness:pharmacokinetics of therapeutic dose nadroparin

BACKGROUND: Peak anti-Xa activity of low-molecular-weight heparin nadroparin is measured 3 to 5 hours after subcutaneous injection. In critically ill patients, physiological changes and medical therapies may result in peak activities before or after this interval, possibly impacting dosing.OBJECTIVES: The primary objective was to determine the percentage of critically ill patients with adequately estimated peak activities drawn 3 to 5 hours after subcutaneous administration of a therapeutic dose of nadroparin. Adequate was defined as a peak activity of ≥80% of the actual peak anti-Xa activity. If ≥80% of patients had adequately estimated peak activities in the 3- to 5-hour interval, measurement in this interval was regarded as acceptable. The secondary objective was to determine the pharmacokinetic profile of nadroparin.METHODS: In this single-center, prospective study, we evaluated anti-Xa activities in patients admitted to a general intensive care unit. After ≥4 equal doses of nadroparin, anti-Xa activity was measured according to a 12- to 24-hour sampling scheme.RESULTS: In 25 patients, anti-Xa activities drawn between 3 and 5 hours after administration ranged 80% to 100% of the actual peak activity. Compared to the threshold level of an adequate estimation in at least 20 patients (≥80%), measuring anti-Xa activities in the 3- to 5-hour interval is an acceptable method (1-tailed binomial test; P &lt; .02). We found a large interindividual variability for nadroparin exposure (mean ± SD area-under-the-curve 0-12h, 10.3 ± 4.8 IU·h/mL) and delayed elimination (t 1/2 range, 4.0-120.9 hours) despite adequate renal function. CONCLUSION: In critically ill patients, measuring anti-Xa activity in a 3- to 5-hour interval after subcutaneous injection of therapeutic nadroparin is an acceptable method to estimate the actual peak anti-Xa activity.</p

Importance of the Hsp70 ATPase Domain in Yeast Prion Propagation

The Saccharomyces cerevisiae non-Mendelian genetic element [PSI(+)] is the prion form of the translation termination factor Sup35p. The ability of [PSI(+)] to propagate efficiently has been shown previously to depend upon the action of protein chaperones. In this article we describe a genetic screen that identifies an array of mutants within the two major cytosolic Hsp70 chaperones of yeast, Ssa1p and Ssa2p, which impair the propagation of [PSI(+)]. All but one of the mutants was located within the ATPase domain of Hsp70, which highlights the important role of regulation of Hsp70–Ssa ATP hydrolysis in prion propagation. A subset of mutants is shown to alter Hsp70 function in a way that is distinct from that of previously characterized Hsp70 mutants that alter [PSI(+)] propagation and supports the importance of interdomain communication and Hsp70 interaction with nucleotide exchange factors in prion propagation. Analysis of the effects of Hsp70 mutants upon propagation of a second yeast prion [URE3] further classifies these mutants as having general or prion-specific inhibitory properties

GTPγS Regulation of a 12-Transmembrane Guanylyl Cyclase Is Retained after Mutation to an Adenylyl Cyclase

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extracellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by guanosine 5'-3-O-(thio)triphosphate (GTPγS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCAkqd in adenylyl cyclase-defective cells restored the phenotype of the mutant. GTPγS stimulated the adenylyl cyclase activity of DdGCAkqd with properties similar to those of the wild-type enzyme (decrease of Km and increase of Vmax), demonstrating that GTPγS stimulation is independent of substrate specificity. Furthermore, GTPγS activation of DdGCAkqd is retained in several null mutants of Gα and Gβ proteins, indicating that GTPγS activation is not mediated by a heterotrimeric G-protein but possibly by a monomeric G-protein.

Chemotaxis: signalling modules join hands at front and tail

Chemotaxis is the result of a refined interplay among various intracellular molecules that process spatial and temporal information. Here we present a modular scheme of the complex interactions between the front and the back of cells that allows them to navigate. First, at the front of the cell, activated Rho-type GTPases induce actin polymerization and pseudopod formation. Second, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) is produced in a patch at the leading edge, where it binds pleckstrin-homology-domain-containing proteins, which enhance actin polymerization and translocation of the pseudopod. Third, in Dictyostelium amoebae, a cyclic-GMP-signalling cascade has been identified that regulates myosin filament formation in the posterior of the cell, thereby inhibiting the formation of lateral pseudopodia that could misdirect the cell

Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches

The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)-triphosphate [PtdIns(3,4,5)P3]. Uniform cyclic AMP (cAMP) concentrations induce persistent translocation of PHCrac-GFP from the cytosol to multiple patches, which are similar to the single patch of PHCrac-GFP at the leading edge in a cAMP gradient. We show that cAMP determines the probability of patch formation (half-maximal effect at 0.5 nM cAMP) but not the size, lifetime or intensity of patches, indicating that patches are self-organizing structures. A pseudopod is extended from the area of the cell with a PHCrac-GFP patch at about 10 seconds after patch formation. Cells treated with the F-actin inhibitor latrunculin A are round without pseudopodia; uniform cAMP still induces localized patches of PHCrac-GFP. Inhibition of phosphoinositide-3-kinase (PI3K) activity with LY294002 inhibits PHCrac-GFP patches and inhibits chemotaxis towards nanomolar cAMP but has no effect at higher cAMP concentrations. Thus, very low cAMP concentrations induce self-organizing PHCrac-GFP patches that serve as a spatial cue for pseudopod formation, which enhances the sensitivity and amplitude of chemotactic movement.

Regulation of Phagocytosis in Dictyostelium by the Inositol 5-Phosphatase OCRL Homolog Dd5P4

Phosphoinositides are involved in endocytosis in both mammalian cells and the amoeba Dictyostelium discoideum. Dd5P4 is the Dictyostelium homolog of human OCRL (oculocerebrorenal syndrome of Lowe); both have a RhoGAP domain and a 5-phosphatase domain that acts on phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Inactivation of Dd5P4 inhibits growth on liquid medium and on bacteria. Dd5p4-null cells are impaired in phagocytosis of yeast cells. In wild-type cells, PI(3,4,5)P3 is formed and converted to PI(3,4)P2 just before closure of the phagocytic cup. In dd5p4-null cells, a phagocytic cup is formed upon contact with the yeast cell, and PI(3,4,5)P3 is still produced, but the phagocytic cup does not close. We suggest that Dd5P4 regulates the conversion of PI(3,4,5)P3 to PI(3,4)P2 and that this conversion is essential for closure of the phagocytic cup. Phylogenetic analysis of OCRL-like 5-phosphatases with RhoGAP domains reveal that D. discoideum Dd5P4 is a surprisingly close homolog of human OCRL, the protein responsible for Lowe syndrome. We expressed human OCRL in dd5p4-null cells. Growth on bacteria and axenic medium is largely restored, whereas the rate of phagocytosis of yeast cells is partly restored, indicating that human OCRL can functionally replace Dictyostelium Dd5P4.

Distinct Roles of PI(3,4,5)P(3) during Chemoattractant Signaling in Dictyostelium: A Quantitative In Vivo Analysis by Inhibition of PI3-Kinase

The role of PI(3,4,5)P(3) in Dictyostelium signal transduction and chemotaxis was investigated using the PI3-kinase inhibitor LY294002 and pi3k-null cells. The increase of PI(3,4,5)P(3) levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 μM LY294002 with half-maximal effect at 5 μM. This correlated well with the inhibition of the membrane translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce cAMP-mediated cGMP production, but significantly reduced the cAMP response up to 75% in wild type and completely in pi3k-null cells. LY294002-treated cells were round, not elongated as control cells. Interestingly, cAMP induced a time and dose-dependent recovery of cell elongation. These elongated LY294002-treated wild-type and pi3k-null cells exhibited chemotactic orientation toward cAMP that is statistically identical to chemotactic orientation of control cells. In control cells, PHcrac-GFP and F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases does not affect the first phase of the actin polymerization at a wide range of chemoattractant concentrations. Our data show that severe inhibition of cAMP-mediated PI(3,4,5)P(3) accumulation leads to inhibition of cAMP relay, cell elongation and cell aggregation, but has no detectable effect on chemotactic orientation, provided that cAMP had sufficient time to induce cell elongation