The Total Thrombus Formation (T-TAS) platelet (PL) assay, a novel test that evaluates whole blood platelet thrombus formation under physiological conditions.

Dual antiplatelet therapy (DAPT, aspirin, and a P2Y12 inhibitor) reduces thrombotic events in patients with coronary artery disease (CAD). The T-TAS PL assay uses arterial shear flow over collagen surface, better mimicking in vivo conditions compared to conventional agonist-based platelet function assays, to evaluate platelet function. Here, the platelet function in patients taking DAPT is evaluated with the T-TAS PL assay. In 57 patients with CAD, taking DAPT ≥7 days (n = 22 for clopidogrel, n = 15 for prasugrel, n = 20 for ticagrelor), T-TAS PL assessments were performed in duplicate, and expressed as area under the flow pressure curve within a 10-minute period (AUC10). The duplicate measurements were strongly correlated (r = 0.90, p < .001), with an intra-assay coefficient of variation (CV) of 11,5%. For clopidogrel, the median AUC10 was 11.5 (IQR5.9-41.8), for prasugrel 28.8 (IQR10.3-37.6), and for ticagrelor 8.9 (IQR 6.4-10.9). All measurements were below the AUC10 cutoff of 260 measured in healthy volunteers, suggesting excellent discrimination of DAPT-treated and untreated persons. The new T-TAS PL assay demonstrated complete discrimination of platelet function in patients on DAPT based on an established cutoff. Ticagrelor showed lower levels of platelet function and a more uniform response compared to prasugrel and clopidogrel.

Evaluating the clinical usefulness of platelet function testing: considerations for the proper application and interpretation of performance measures.

Various diagnostic and prognostic performance measures have been used to describe the clinical usefulness of platelet function testing in the evaluation and management of patients taking P2Y12 inhibitors, which reduce the risk for thrombosis due to their action on the platelet P2Y12 receptor. Platelet function tests are used to confirm the presence of an antiplatelet effect of a P2Y12 inhibitor, and confirmation that the pharmacodynamic effect is associated with a reduction in the rate of thrombosis. Despite this clear association, enthusiasm for the clinical usefulness of platelet function testing has been tempered based on observed sensitivity, specificity, and positive predictive value for the detection of future thrombotic events. However, evaluating the prognostic utility of a test based on diagnostic performance indicators is not appropriate because prognostic tests are not used to diagnose which patients will have events; instead, they are used to assist in risk stratification. Therefore, when evaluating the usefulness of platelet function testing, diagnostic performance measures such as sensitivity, specificity, and predictive values should focus on diagnostic performance in identifying a pharmacodynamic effect, and prognostic performance should be evaluated using prognostic performance measures such as hazard ratios and net reclassification improvement, which are comparable to other well-established risk factors for cardiovascular events.

Determination of nitrate and other water quality parameters in groundwater from UV/Vis spectra employing partial least squares regression.

The use of UV/Vis spectroscopy in combination with partial least squares (PLS) regression for the simultaneous prediction of nitrate and non-purgeable organic carbon (NPOC) in groundwaters was evaluated. A model of high quality was obtained using first order derivative spectra in the range 200-300 nm. Inclusion of non-UV-absorbing constituents in the modeling procedure, i.e., chloride, sulfate, fluoride, total carbon (TC), inorganic carbon (IC), alkalinity, pH and conductivity was also evaluated. This model seemed to be useful for prediction of chloride, TC, IC, alkalinity and conductivity, while its ability to predict sulfate, fluoride and pH was poor. In conclusion, application of PLS regression, which requires neither filtration of samples nor addition of chemicals, is a promising alternative for fast interpretation of geochemical patterns of groundwater quality.

Analysis of low molecular weight organic acids in water with capillary zone electrophoresis employing indirect photometric detection.

A capillary zone electrophoretic method for the analysis of low molecular weight carboxylic acids in water was developed. The application uses the chromophore trimellitic acid that allows for indirect photometric detection at 254 nm. Selectivity control by the addition of Ca2+ to the carrier electrolyte made possible a baseline resolved separation of 14 carboxylic acids. Good linearity and repeatability were obtained for both electrokinetic and hydrostatic injection modes. The usefulness of the method is demonstrated by the analysis of a snow sample.

Inhibition of neutrophil elastase by recombinant human proteinase inhibitor 9.

Proteinase inhibitor PI9 (PI9) is an intracellular 42-kDa member of the ovalbumin family of serpins that is found primarily in placenta, lung and lymphocytes. PI9 has been shown to be a fast-acting inhibitor of granzyme B in vitro, presumably through the utilization of Glu(340) as the P(1) inhibitory residue in its reactive site loop. In this report, we describe the inhibition of human neutrophil elastase by recombinant human PI9. Inhibition occurred with an overall K(i)' of 221 pM and a second-order association rate constant of 1.5 x 10(5) M(-1) s(-1), indicating that PI9 is a potent inhibitor of this serine proteinase in vitro. In addition, incubation of recombinant PI9 with native neutrophil elastase resulted in the formation of an SDS-resistant 62-kDa complex. Amino-terminal sequence analyses provided evidence that inhibition of elastase occurred through the use of Cys(342) as the reactive P(1) amino acid residue in the PI9 reactive site loop. Thus, PI9 joins its close relatives PI6 and PI8 as having the ability to utilize multiple reactive site loop residues as the inhibitory P(1) residue to expand its inhibitory spectrum.

Caspase-1 (interleukin-1beta-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9.

The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1beta-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7x10(2) M(-1).s(-1). The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases.

Determination of group parameters for organically bound chlorine, bromine and iodine in precipitation

The presence of organically bound halogens in precipitation was studied by first adsorbing organics on activated carbon and then analysing the chloride, bromide and iodide formed during combustion of the carbon. A standard instrument for the group parameter AOX (adsorbable organic halogens) was used for the combustion. The halides formed were trapped in an alkaline solution and analysed by capillary zone electrophoresis. The method described enabled determination of sub-ppb concentrations of the group parameters AOCl, AOBr and AOI (adsorbable organic chlorine, bromine and iodine, respectively). Analysis of rain and snow collected at different sites in Europe showed that organochlorines were responsible for the major part of the AOX content in all samples collected. Organically bound bromine was found in sub-ppb concentrations in all of the samples, whereas organic iodine was detected in only two of the samples.

Determination of the molecular weight of fulvic acids by UV/VIS spectroscopy

Partial least squares (PLS) modeling was applied to investigate number-average molecular weights (Mn) and weight-average molecular weights (Mw) of fulvic acids (FAs) in relation to the corresponding UV/VIS spectra. The Mn and Mw values were determined by size exclusion chromatography (SEC). The impact of pH control, wavelength range and density as well as smoothing and derivation of spectra were tested. It was found that PLS models based on absorbance spectra can be a fast and powerful complement to existing techniques employed for determination of molecular weights of FAs. Control of pH of the FA solutions is important for the performance of the models. The models were also compared with the best univariate alternatives.

Determination of low molecular weight organic acids in soil solution by HPLC.

An HPLC method employing an ion exclusion column was developed for the determination of low molecular weight organic acids in soil solution. The method includes extensive sample pretreatment using ultrafiltration and cation exchange. The method showed linear calibration graphs (r>0.99) and the limits of detection in the range 0.1-26 muM. The recovery of eleven added acids ranged from 89 to 102%. Soil solutions of five horizons of a podzolised soil were analysed. The results showed that these compounds made up 1-3% of the dissolved organic carbon and 0-14% of the acidity. Identification of the major acids was also carried out by capillary zone electrophoresis.

The inhibitory specificity of human proteinase inhibitor 8 is expanded through the use of multiple reactive site residues.

Serine proteinase inhibitors function as regulators of serine proteinase activity in a variety of physiological processes. Proteinase inhibitor 8 (PI8) is a 45 kDa member of the ovalbumin family of serpins that is an inhibitor of trypsin-like proteinases through the use of Arg339 as the inhibitory P1 amino acid residue in its reactive site loop. In this study, we have described the inhibitory mechanism of recombinant human PI8 towards chymotrypsin. PI8 formed an SDS-stable complex with and inhibited the amidolytic activity of chymotrypsin via a two-step mechanism with an overall equilibrium inhibition constant of 1.7 nM and an overall second-order association rate constant of 1.0 x 10(4) M-1s-1, utilizing Ser341 as the P1 residue. The use of separate reactive site loop residues by PI8 to inhibit distinctly different classes of proteinases not only supports the hypothesis of the existence of the serpin reactive site as a highly mobile and flexible loop, but also suggests an evolved function in which separate amino acid residues can be used to broaden the inhibitory specificity of PI8.

Inhibition of soluble recombinant furin by human proteinase inhibitor 8.

Furin is a ubiquitous prototypical mammalian kexin/subtilisin-like endoproteinase that is involved in the proteolytic processing of a variety of proteins in the exocytic and endocytic pathways, with cleavage occurring at the C terminus of the minimal consensus furin recognition sequence Arg-Xaa-Xaa-Arg. In this study, human proteinase inhibitor 8 (PI8), a widely expressed 45-kDa ovalbumin-type serpin that contains two sequences homologous to the minimal sequence for recognition by furin in its reactive site loop, was tested for its ability to inhibit a recombinant soluble form of human furin. PI8 formed an SDS-stable complex with furin and inhibited its amidolytic activity via a two-step mechanism with a kappa assoc of 6.5 x 10(5) M-1 S-1 and an overall Ki of 53.8 pM. Thus, PI8 inhibits furin in a rapid, tight binding manner that is characteristic of physiological serpin-proteinase interactions. PI8 is not only the first human ovalbumin-type serpin to demonstrate inhibitory activity toward furin, but it is also the first significant inhibitor of furin identified that is not a serpin reactive site loop mutant, either naturally occurring or engineered.

Expression, purification, and inhibitory properties of human proteinase inhibitor.

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.

Human proteinase inhibitor 9 (PI9) is a potent inhibitor of subtilisin A.

Serine proteinase inhibitors function as regulators of serine proteinase activity in a variety of physiological processes. Proteinase inhibitor 9 (PI9) is a 42 kDa member of the ovalbumin family of serpins that is expressed in placenta, lung, and cytotoxic lymphocytes. In this study, we have described the inhibitory mechanism of recombinant human PI9 towards the bacterial endoproteinase subtilisin A. PI9 inhibited the amidolytic activity of subtilisin A via a rapid, single step mechanism with an equilibrium inhibition constant of 3.6 pM and an overall second-order association rate constant of 2.4 x 10(6) M-1s-1, which is the strongest inhibitory mechanism of PI9 that has been described. The inhibitory action of PI9 towards subtilisin as a model proteinase may yield some indication of potential proteinases that may be regulated by PI9 in vivo.

Molecular cloning, expression, and partial characterization of two novel members of the ovalbumin family of serine proteinase inhibitors.

A human placental lambda gt11 cDNA library was screened for sequences encoding proteins related to human proteinase inhibitor 6 (PI6), and two plaques were identified that displayed weak hybridization at high stringency. Isolation and characterization of the DNA inserts revealed two novel sequences encoding proteins composed of 376 and 374 amino acids with predicted molecular masses of approximately 42 kDa. The novel proteins displayed all of the structural features unique to the ovalbumin family of intracellular serpins including the apparent absence of a cleavable N-terminal signal sequence. The degree of amino acid sequence identity between the novel serpins and PI6 (63-68%) significantly exceeds that of any other combination of known intracellular serpins. The two novel serpins encoded by the two novel cDNA sequences have been designated as proteinase inhibitor 8 (PI8) and proteinase inhibitor 9 (PI9). The putative reactive center P1-P1' residues for PI8 and PI9 were identified as Arg339-Cys340 and Glu340-Cys341, respectively. PI9 appears to be unique in that it is the first human serpin identified with an acidic residue in the reactive center P1 position. In addition, the reactive center loop of PI9 exhibits 54% identity with residues found in the reactive center loop of the cowpox virus CrmA serpin. Two PI8 transcripts of 1.4 kilobases (kb) and 3.8 kb were detected by Northern analysis in equal and greatest abundance in liver and lung, while the 1.4-kb mRNA was in excess over the 3.8-kb mRNA in skeletal muscle and heart. Two PI9 transcripts of 3.4 and 4.4 kb were detected in equal and greatest abundance in lung and placenta and were weakly detected in all other tissues. PI8 and PI9 were expressed in baby hamster kidney and yeast cells, respectively. Immunoblot analyses using rabbit anti-PI6 IgG indicated the presence of PI8 in the cytosolic fraction of stably transfected cells that formed an SDS-stable 67-kDa complex with human thrombin. PI9 was purified to homogeneity from the yeast cell lysate by a combination of heparin-agarose chromatography and Mono Q fast protein liquid chromatography and migrated as a single band in SDS-polyacrylamide gel electrophoresis with an apparent molecular mass of 42 kDa. Purified recombinant PI9 failed to inhibit the amidolytic activities of trypsin, papain, thrombin, or Staphylococcus aureus endoproteinase Glu-C and did not form an SDS-stable complex when incubated with thrombin. The cognate intracellular proteinases that interact with PI8 and PI9 are unknown.

Complementary functioning of two components from nitrogen-fixing bacteria.

Use of alternate substrates (acetylene, azide, cyanide) for estimating N(2) fixation confirmed that recombined fractions from different bacteria are functional but restricted in complementarity.