Determination of benzene, toluene and xylene concentration in humid air using differential ion mobility spectrometry and partial least squares regression.

Benzene, toluene and xylene (BTX compounds) are chemicals of greatest concern due to their impact on humans and the environment. In many cases, quantitative information about each of these compounds is required. Continuous, fast-response analysis, performed on site would be desired for this purpose. Several methods have been developed to detect and quantify these compounds in this way. Methods vary considerably in sensitivity, accuracy, ease of use and cost-effectiveness. The aim of this work is to show that differential ion mobility spectrometry (DMS) may be applied for determining concentration of BTX compounds in humid air. We demonstrate, this goal is achievable by applying multivariate analysis of the measurement data using partial least squares (PLS) regression. The approach was tested at low concentrations of these compounds in the range of 5-20 ppm and for air humidity in a range 0-12 g/kg. These conditions correspond to the foreseeable application of the developed approach in occupational health and safety measurements. The average concentration assessment error was about 1 ppm for each: benzene, toluene and xylene. We also successfully determined water vapor content in air. The error achieved was 0.2 g/kg. The obtained results are very promising regarding further development of DMS technique as well as its application.

Two polymorphisms of the FVII gene and their impact on the risk of myocardial infarction in poles under 45 years of age.

High levels of coagulation factor VII (FVII) in plasma have been associated with the increased risk of myocardial infarction (MI) in some studies. Both environmental and genetic factors are responsible for different levels of FVII in plasma. In the FVII gene there are two common polymorphisms (-323A1/A2 and IVS7)which are related to the level of FVII. The purpose of this study was to evaluate the influence of these polymorphisms on the risk of acute myocardial infarction in Poles under 45 years of age. We performed a case-control study of 266 patients with the history of MI. All patients had the first incidence of MI before 45 years of age. The control group consisted of 137 healthy individuals older than 45 years. Carriers of the A2 allele (-32341/A2 polymorphism) have a lower risk of MI than noncarriers (OR = 0.40, 95% CI = 0.20 to 0.80). The IVS7 polymorphism was shown not to be related to MI in this study. Our findings suggest that the -323A1/A2 polymorphism of the FVII gene is related to the risk of MI in Polish individuals. We pointed that plasma cholesterol (OR = 1.11, 95% CI = 1.03 to 1.18), arterial hypertension (OR = 3.84, 95% CI = 1.99 to 7.43) and family history (OR = 2.72, 95% CI = 1.57 to 4.73) are significant predictors of acute myocardial infarction.

Differentiated reactivity of whole blood neonatal platelets to various agonists.

Neonatal platelets have been occasionally reported to show a reduced response to various agonists. The molecular mechanism(s) of such a depressed reactivity remains unclear. To further address this problem we studied neonatal platelet activation with thrombin, TRAP (thrombin receptor activating peptide, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) and ADP in 42 healthy 1-2 day old neonates using a whole peripheral blood flow cytometry. The neonates did not show an increased fraction of P-selectin-positive circulating platelets, whereas the expression of GPIb (glycoprotein Ib) in resting neonatal platelets was significantly lower compared to adults. Neonatal platelets were significantly less reactive than adult platelets to thrombin and TRAP, especially at lower agonist concentrations, but not to ADP or when incubated for 1 h at room temperature. Activation of neonatal platelets with agonists resulted in a marked alterations in the expression of P-selectin, whereas the internalization of GPIb was not affected. The reduced neonatal platelet sensitivity to thrombin and TRAP was accompanied by significantly reduced ATIII (antithrombin III) and increased prothrombin fragment F(1+2) in neonatal plasma. We conclude that various receptor systems potentially able to bind thrombin are relatively insensitive in neonatal platelets. The novelty of our work is that neonatal platelet hyposensitivity is not a generalized phenomenon, but concerns only selected agonists and selected receptor systems.

Low doses of aprotinin in aortocoronary bypass surgery--advantages and disadvantages.

INTRODUCTION: Excessive blood loss, as a result of augmented postoperative drainage, is considered one of the most serious cardiosurgical complications. The compounding constitutive anemia seems particularly harmful for patients with coronary artery disease. Aprotinin (Trasylol), a non-specific serine protease inhibitor, is successfully used to reduce excessive postoperative bleeding in such patients. The aim of our study was to verify the hypothesis whether aprotinin used during cardiopulmonary bypass procedure affects hemostatic parameters, which might be crucial for the elevated risk of thromboembolic complications. MATERIAL AND METHODS: The group of 54 patients subjected to coronary artery surgical treatment included 30 patients, who were given intraoperatively 3 million KIU aprotinin each, and 24 subjects non-treated with aprotinin. Aliquots of blood were withdrawn at several time intervals, until the 5th day after the operation. Whole blood platelet activation and reactivity (the expressions of P-selectin and glycoprotein Ib) were monitored by means of flow cytometry. In addition, several plasma parameters, like PAI-1, t-PA, D-dimers, prothrombin fragment F1 + 2, fibrinogen, ATIII activity, troponin I and CK-MB, as well as platelet count were determined at each time point. RESULTS: In this study we confirmed the essential advantage of the use of aprotinin: both the postoperative blood drainage and the blood units to be transfused postoperatively to cardiosurgical patients were vastly reduced in the aprotinin-treated subjects. The enhanced overall frequency of perioperative myocardial infarction events was not attributed to this group of patients, nor the non Q-wave infarctions were observed more often in patients treated with aprotinin. In these patients, fibrinolysis parameters tended to be depressed (with increased PAI-1 dominating over elevated t-PA) on the first day after the operation, and no significant differences with regard to fibrinogen, prothrombin fragment F1 + 2, troponin I and platelet count. There was a continuous rise in D-dimers in all the postoperative patients, which lasted until the third day and tended to reach plateau at the 5th day after the operation. We failed to reveal the preventive effects of aprotinin on platelet function: both platelet activation and reactivity remained apparently unchanged. Overall, our results rather support the reasoning on the advantageous effects of low doses of aprotinin. The use of this inhibitor reduces the risk of postoperative undesirable bleeding and results in a decreased postoperative drainage and reduced transfused blood units. On the other hand, however, a higher incidence of perioperative Q-wave infarction in the aprotinin-treated patients, although purely apparent and not statistically significant, might question the unlimited safety of the use of aprotinin in cardiovascular operations.

Possible mechanisms of the altered platelet volume distribution in type 2 diabetes: does increased platelet activation contribute to platelet size heterogeneity?

A direct consequence of increased platelet sensitivity in diabetes mellitus might be augmented release of platelet granule contents, which, in turn, may lead to the formation of a platelet volume gradient, increased platelet turnover and reduced survival of platelets from diabetic individuals. In this study we addressed the question whether diabetes-induced and lipid fluidity-mediated changes in platelet receptor exposure and accessibility might be part of a general mechanism underlying the increased rate of platelet ageing and reduced platelet survival in diabetes. Diabetic individuals showed higher numbers of platelets of extreme dimensions: very small platelets and larger platelets were more frequent compared to controls ( P(chi(2))< 0.03). The shifts in platelet volume distributions were paralleled by decreased expression of the alpha subunit of glycoprotein Ib (by up to 17%, P < 0.01) in platelet membranes from diabetic patients, increased expression of P-selectin in thrombin-stimulated diabetic platelets (P< 0.02), an increased number of platelet microparticles in diabetic individuals (P< 0.05 or P< 0.03 for resting or stimulated platelets, respectively), and reduced platelet membrane fluidity (by 5.2 +/- 0.6%, P< 0.01). We suggest that the distinct bimodality of platelet distribution in diabetic patients might arise from accelerated thrombopoiesis in diabetic subjects, and this is supported by the demonstration of elevated fractions of reticulated (rich with residual RNA) platelets in diabetic patients (14.6 +/- 5.6% vs 8.1 + 2.1% p(u) < 0.025). Overall, our results point to a fluidity-mediated platelet hypersensitivity and accelerated rate of platelet production in subjects with type 2 diabetes mellitus, which results in a greater number of very large and hypersensitive younger platelets and a more abundant fraction of small exhausted platelets.

Membrane lipid fluidity of blood platelets: a common denominator that underlies the opposing actions of various agents that affect platelet activation in whole blood.

Membrane lipid fluidity (MLF) is thought to play a crucial role in signal transduction and is believed to affect the responsiveness of blood platelets. In a recent study it was demonstrated that EDTA, used as the blood anticoagulant, brought about a significant increase in expression of GMP-140 antigen, and this effect was accompanied by a significant increase in platelet MLF. Moreover, this spontaneous EDTA-driven platelet activation was vastly attenuated in the presence of tissue-type plasminogen activator, which is also known to affect platelet MLF. The hypothesis was raised that the modulation of platelet membrane fluidity by EDTA might underlie platelet spontaneous activation in the presence of EDTA. To further explore the possible molecular mechanism(s) of the EDTA-dependent triggering of signal transduction pathway(s) in human blood platelets, we monitored the extent of spontaneous platelet activation in the presence of EDTA and selected platelet membrane 'fluidizers' and 'rigidizers'. A reduction in the EDTA-dependent platelet release and activation was noted, not only in the presence of rt-PA (by over 50%, P < 0.001), which acted as a rigidizer of platelet membrane fluidity (ESR h+1/h0 ratios of 5-DOXYL-Ste and 12-DOXYL-Ste decreased by 6.2%, P<< 0.0001, and 3.8%, P < 0.02, respectively), but also in the presence of other modulators of MLF, regardless of their fluidizing or rigidizing effects. Both rigidizers (procaine and lidocaine, 5-DOXYL-Ste h+1/h0 reduced by up to 6.5%, 12-DOXYL-Ste h+1/h0- by up to 4.5%, P < 0.02 or less) and fluidizers (benzyl alcohol, ethanol, 12-DOXYL-Ste h+1/h0 increased by 17.8% and 6.1%, respectively, P <<0.0001) of platelet membranes significantly depressed platelet activation (respectively, down to 1.1%, 7.7%, 6.7% and 8.5% vs control EDTA 22.9% of CD62-positive platelets). We suggest that EDTA induces alterations in membrane glycoprotein structure and affect MLF by altering lipid-protein interactions, and thus triggers signal transduction in the course of platelet activation. The resulting displacements in platelet membrane proteins, dislocation of membrane components and/or distortion of lipid-protein interactions could generate an 'outside-in' signalling that is mediated by the altered platelet MLF. Overall, it is likely that interference with the structure and conformation of selected domains of platelet membrane proteins might be the crucial mechanism by which EDTA leads to exaggerated activation of platelets in whole blood.

Molecular insights into the anticoagulant-induced spontaneous activation of platelets in whole blood-various anticoagulants are not equal.

The spontaneous anticoagulant-dependent platelet activation in vitro may potentially interfere with the determination of haemostatic parameters. The effects of various blood anticoagulants on platelet activation were monitored using flow cytometry. Regardless of a blood anticoagulant used (EDTAK2, heparin, citrate or PPACK), platelet activation began immediately after blood withdrawal and was most pronounced in the EDTAK2-anticoagulated blood samples. The progressing expression of GMP-140 antigen was accompanied by the enhanced abundance of the subunit beta 3 of the platelet membrane integrin alpha IIb beta 3 without parallel changes in the fluorescence attributed to the complex form of the integrin alpha IIb beta 3. The increased expression of GMP-140 was paralleled by the enhanced platelet clumping in the samples anticoagulated with either EDTAK2 or heparin, and the raised platelet microparticles in blood withdrawn into citrate. The EDTAK2-induced platelet activation was markedly reduced by methyl 2,5-dihydroxycinnamate, tyrosine kinase inhibitor. The influence of disodium EDTA on platelet membrane dynamics closely mimicked the alterations induced upon the interaction of fibrinogen with platelet GPIIb-IIIa. Thus, the EDTAK2-induced platelet activation might result from an interference with platelet membrane protein structure and conformation and possibly related to an "unspecific" trigerring of a signal transduction pathway. Overall, EDTAK2 and heparin appeared the least suitable anticoagulants, particularly with the regard to the expression of GMP-140 antigen. The failure to recognize the importance of a spontaneous anticoagulant-induced platelet activation may result in misdiagnoses during the monitoring of coagulation parameters.

Diabetes mellitus alters the effect of peptide and protein ligands on membrane fluidity of blood platelets.

The increased nonenzymatic glycosylation of platelet membrane proteins has been suggested to underlie platelet hypersensitivity in diabetes and the relationship of this to the reduced membrane lipid fluidity has been reported. As the modulation in membrane fluidity may determine the degree of accessibility of membrane receptors, the consequent alterations in membrane lipid-protein interactions in diabetes mellitus may also underlie the differentiated effects of various thrombotic and fibrinolytic agents on platelet membrane lipid bilayer. In the present study we employed electron paramagnetic resonance and fluorescence spectroscopy to explore the ligand-induced platelet membrane fluidity changes in diabetic state, i.e. under conditions when the membrane architecture is considerably altered. The yield of the excimer formation of pyrenemaleimide (PM), which depends directly upon the collisional rate and distances between molecules, was elevated in diabetic platelet membranes, thus pointing to the occurrence of some constraints in the structure/conformation of platelet membrane proteins in diabetes mellitus. Such an immobilization of PM was accompanied by the significant elevation in membrane protein glycation in diabetic platelets. The effects of various interacting ligands on platelet membrane fluidity were significantly lower in diabetic platelets, and the differences were much more distinct at the lower depths of a lipid bilayer. Nevertheless, the alterations in membrane lipid fluidity observed upon the interaction of a given ligand occurred with an approximately equal frequency in control and diabetic platelets. Moreover, the probability that these alterations were less profound in diabetic platelets was the same for all types of ligands studied. In diabetic patients the interaction of RGDS and tissue-type plasminogen activator with platelet membranes resulted in much smaller reductions of the h+/h0 parameters in 5-DOXYL-Ste acid-labelled platelets, thus indicating a lesser rigidization of membrane lipid bilayer in diabetes. Likewise, the fluidizing effect of both fibrinogen itself and fibrinogen-derived peptides containing gamma-chain carboxy-terminal sequence H-12-V was less pronounced in diabetic platelet membranes.

Does reduced membrane lipid fluidity underlie the altered thrombin-induced expression of integrin α(IIb)ß(3) and PADGEM-140 in membranes of platelets from diabetic juveniles?

In diabetic patients, where the membrane lipid microviscosity of blood platelets is altered, the availability of platelet membrane receptors may change concomitantly. Platelet hypersensitivity in diabetic subjects was previously hypothesized to result from the nonenzymatic glycosylation-induced loss in platelet membrane fluidity. In our present study juvenile type 1 diabetic subjects were compared with their relevant controls with respect to thrombin-stimulated platelet activation in relation to glycation-induced impairments of platelet membrane dynamics. Our results indicate that: (a) the mean steady-state fluorescence polarization (p) of both 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-anilino-8-naphthalenesulphonate (ANS) in membranes from diabetic subjects were significantly greater than for control subjects, thus indicating reduced membrane lipid fluidity in diabetic platelets in various membrane regions; (b) the significantly higher [(3)H]NaBH(4) reduction, indicating the increased attachment of glucose to protein amino groups, was attributed to the proteins extracted from diabetic platelet membranes; (c) CD62-positive resting platelets were not significantly more abundant in diabetic patients; (d) basically, unaltered amounts of PADGEM-140 membrane antigen (CD62) copies were detected in resting diabetic platelets; (e) significantly higher numbers of membrane glycoprotein ß(3) were found in diabetic platelets; (f) thrombin-induced elevations in the expression of CD61 (ß(3)) and CD62 (PADGEM-140) occurred to much higher extent in platelets of diabetic patients, thus pointing to more profound activation of diabetic platelets by thrombin; (g) the total amounts of platelet membrane glycoprotein ß(3) was significantly reduced in platelet lysates from diabetic subjects. We conclude that glycation-induced rigidization of platelet membranes might hypersensitize diabetic platelets to aggregating agents by rendering platelet membrane receptors more exposed to the external environment. Thus, thrombin may bind more efficiently to the exposed glycoprotein receptors (due to glycation) in diabetic platelets. Such excessive exposure and displacements toward the external environment might favour the accelerated shedding of some membrane proteins in diabetic platelets. We further suggest that their subsequent replacements would render platelet intrinsic storage pools exhausted and thus, might explain the diminished total amount of ß(3) found in platelets of diabetic patients.

The epidermal growth factor-like domain from tissue plasminogen activator. Cloning in E. coli, purification and ESR studies of its interaction with human blood platelets.

To examine whether the epidermal growth factor (EGF)-like domain Pro47-Asp87 is involved in the interaction of tissue plasminogen activator (t-PA) with platelets, we have expressed this domain in E. coli. The peptide fragment was produced from a plasmid expression vector as a fusion protein with beta-galactosidase Met1-Val444 at high yield in eight clones of E. coli. The fusion protein was purified and subjected to mild acid hydrolysis with formic acid, then the peptide Pro47-Asp87, identified by immunoblotting using specific antibodies to t-PA, was isolated by HPLC. After incubation with blood platelets spin labelled with 16-doxylstearic acid or 5-doxylstearic acid, the Pro47-Asp87 peptide fragment reduced fluidity of the membrane lipid bilayer to the same extent as did intact t-PA as indicated by ESR measurements. Our data suggest that the EGF-like domain of t-PA can directly interact with blood platelets and thus it seems to contain those sites of the t-PA molecule that bind the platelet membrane components.

Tissue-type plasminogen activator induces alterations in structure and conformation of membrane proteins upon its interaction with human platelets.

In our very recent ESR study we reported that upon rt-PA binding to platelets the H+1/h0 ratios of 16-doxylstearate and 5-doxylstearate spin labels incorporated into the lipid bilayer of platelet membranes were significantly decreased. It corresponded to the increased rigidity of platelet lipid bilayer. In order to further explore this phenomenon we employed a fluorescence-quenching technique which enabled us to estimate the energy transfer efficiency and the apparent interchromophore distance between membrane protein tryptophan and 1-anilino-8-naphthalenesulphonate (ANS) molecules embedded in the membrane lipid bilayer. As t-PA interacts with the platelet membrane this distance decreases, resulting in the relevant increase of energy transfer efficiency. Thus, the data indicate that upon t-PA binding the membrane tryptophan residues are more exposed to the external environment and the quenchable fraction of membrane tryptophan becomes greater. Furthermore, the spectrum of ANS is slightly shifted towards longer wavelengths, which can be accounted for by an increase in the polarity of the environment. It suggests a diminished contact of membrane tryptophan with phospholipid fatty acids. Based on these observations we concluded that the interaction of rt-PA with platelet membranes might induce conformational changes in the membrane proteins, and consequently result in rearrangements of lipid matrix and the alterations in lipid-protein interactions in platelet membranes.

Do the spectra of maleimide spin-labelled whole blood platelets reflect the structure and conformation of membrane proteins?

The maleimide spin label (4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl, MSL), the commonly used reagent specific for cysteine thiol groups in proteins, penetrates cell membranes and binds to both the membrane and cytoplasmic protein moieties. In order to differentiate the labelling of these two subpopulations of cell proteins, we developed three different methodological approaches varying in the consequent distribution of this label in platelets. (A) The labelling of platelet proteins was negligible when bovine serum albumin was used in the Tyrode's buffer for the isolation of platelets, as the majority of the spin label was bound to the albumin coated on the platelets. (B) Preblocking of the reactive thiol groups in albumin with non-spin maleimide analog, N-ethylmaleimide (NEM), caused a considerable amount of MSL to bind with whole platelets but the impartment of membrane component was below 50%. It suggests that the majority of the spin label penetrated platelets and was bound to the intrinsic platelet proteins. (C) In order to prevent labelling of intrinsic platelet proteins with MSL, platelets were preincubated with N-ethylmaleimide, which was able to penetrate platelets and block the reactive thiol groups inside the cells. Such a treatment resulted in a saturation of the intrinsic protein residues with this non-spin analog. The subsequent incubation of thus-treated albumin-free platelets with MSL was to enhance considerably the likelihood of the attachment of MSL molecules to the thiol groups available in platelet-membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Microenvironment changes in human blood platelet membranes associated with binding of tissue-type plasminogen activator.

Whole washed platelets were labelled with the free radicals [2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinylox y] (16-DOXYL-Ste) or [2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3- oxazolidinyloxy] (5-DOXYL-Ste) and incubated with recombinant tissue-type plasminogen activator (rt-PA). Changes in the membrane fluidity caused by rt-PA were detected by alterations in h+1/h0 calculated from the ESR spectra for 16-DOXYL-Ste and 5-DOXYL-Ste incorporated into the lipid bilayer (h+1 and h0 are the heights of the low-field and middle-field lines of the spectra, respectively). Interaction of rt-PA with both resting and stimulated platelets resulted in increased rigidity of the membrane lipid bilayer as indicated by the reduced value of h+1/h0. This phenomenon can be explained either by conformational changes of membrane receptors caused by the attachment of rt-PA and the subsequent rearrangement of the lipid matrix of platelet membranes, or by the direct association of rt-PA with membrane phospholipids and thus partial embedding of protein molecules into the lipid bilayer restraining lipid mobility.

[Tissue-type plasminogen activator (T-PA). Structure and function]. / Tkankowy aktywator plazminogenu (t-PA). Struktura i funkcja.

The paper describes the results of the studies on structure and function of the tissue-type plasminogen activator collected for the past 10 years. The properties of one- and two-chain t-PA as well as the structure of the particular domains in the t-PA molecule have been characterized and discussed.

Interaction of heterologous fibrinogens with human platelet receptors.

The interaction of ADP-stimulated human platelets with human 125I-fibrinogen as well as with pig and bovine fibrinogens was analysed. It was found that the fibrinogens studied were bound to the same platelet receptors but the affinity of animal preparations was about half the value observed for human fibrinogen (in a homologous system).

The inhibitory effect of UV-light on thrombin-induced pig platelet aggregation.

The effect of ultraviolet radiation on the thrombin-stimulated aggregation of washed pig platelets was studied. It was observed that UV radiation (UV-A, 0.5 W/cm2) inhibited aggregation of pig platelets induced by thrombin. Plasma may have a protective role against the damaging effect of UV light on platelet aggregation.

UV light-induced changes in washed pig platelets.

The effect of ultraviolet radiation (UV-A, 360 nm) on the thrombin-induced aggregation of washed pig platelets as well as on the release of adenine nucleotides and proteins was studied. The level in platelets of adenine nucleotides, mainly ADP and ATP, decreased rapidly following the exposure of platelets to a high dose of UV-A (0.5 W/cm2, 30 min). Through thrombin-induced aggregation of irradiated platelets was inhibited, the release reaction occurred. The amount of the released adenine nucleotides and proteins was, however, dependent on the dose of UV light. These findings suggest that UV-A light can stimulate the platelet release reaction.