The effect of the nitroxide pirolin on oxidative stress induced by doxorubicin and taxanes in the rat brain.

The anticancer drugs doxorubicin (DOX), paclitaxel (PTX) and docetaxel (DTX) have been proven to induce oxidative stress (OS)-dependent side-effects in non-targeted tissues. In normal conditions, the blood-brain barrier (BBB) prevents these drugs from penetrating into the brain. However, some studies have demonstrated that small amounts of DOX can penetrate the brain via an oxidatively impaired BBB and cause damage, which suggests that including antioxidants in chemotherapy could possibly protect the brain against the toxicity of anticancer drugs. We investigated whether DOX, DTX and PTX can induce oxidative damage in rat brains in vivo and whether inclusion of the nitroxyl antioxidant Pirolin (PL) to DOX/taxane chemotherapy can protect the brain from the OS toxicity of these drugs. Wistar rats received i.p. a single dose (10 mg/kg b.w.) of DOX, DTX, PTX or PL alone or a combination of a drug + PL. After four days, the rats were anesthetized, the brains were excised, homogenized and used for the measurements of lipid peroxidation (LPO), thiol groups, activities of antioxidant enzymes, DNA damage and tumor necrosis factor-α (TNF-α), neuronal nitric oxide synthase (nNOS) and poly (ADP-ribose) polymerase-1 (PARP-1) expression. The results were analyzed using the Kruskal-Wallis and Conover-Inman tests or ANOVA and the Tukey-Kramer test. Doxorubicin, PTX and DTX induced OS, DNA damage and changes in expression of TNF-α, nNOS and PARP-1 in the rat brain. Pirolin alone increased LPO, manganese superoxide dismutase (MnSOD) and catalase (CAT) activities and the expression of PARP-1 but decreased TNF-α expression. PL, in combination with anticancer drugs, partially protected the rat brain against the toxic effects of DOX and taxanes. The best protective effects of PL were obtained with PTX. Pirolin partially attenuated brain damage caused by DOX/taxanes, highlighting its potential application in protecting the brain against DOX-, DTX- and PTX-evoked OS.

Erythrocytes properties in varicose veins patients.

Varicose veins (VV) are enlarged veins of the subcutaneous tissue, usually caused by faulty or damaged venous valves leading to impaired blood flow. Blood stasis, excessive clotting disorder and alterations in the vein walls are symptoms of Virchow's triad which may affect the morphotic elements of blood, including erythrocytes. The aim of this study was to investigate alterations in the properties of the erythrocytes taken from varicose veins in comparison to those from antecubital vein of patients with chronic venous disease. The investigation was conducted on whole erythrocytes using spin labeling method in EPR spectroscopy and flow cytometry. The internal viscosity of cells was determined by Tempamine. The conformation state of internal proteins, mainly hemoglobin and membrane proteins was determined by maleimide spin label (MSL, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl). The plasma membrane fluidity was measured using two spin labeled fatty acids (5- and 16-doxylstearic acid), while conformational state of membrane protein was measured using two covalently bound spin labels MSL and ISL [4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl]. The osmotic fragility and the shape and size of the erythrocytes were also determined. A decrease in internal viscosity of the erythrocytes from varicose vein was observed. A significant decrease in lipid membrane fluidity indicated by 5-DS, which is located at the polar region of lipid layer was found in the erythrocytes from varicose vein in comparison to normal vein. A significant decrease in the motion of MSL and ISL attached to erythrocyte membrane proteins from varicose vein was found. Changes in the plasma membrane of the erythrocytes from varicose vein were also confirmed by measuring osmotic fragility. These cells were more sensitive to hemolysis than red blood cells from the peripheral blood vein. Meanwhile, no significant differences in size and shape were observed between the erythrocytes taken from varicose veins and those from peripheral veins. In conclusion, the erythrocytes from varicose veins exhibited decreased intracellular viscosity and decreased plasma membrane fluidity. At the same time, conformational changes of membrane proteins and higher osmotic fragility of these cells were found in comparison to the erythrocytes obtained from peripheral veins in the same patients with chronic venous disease. Our findings strongly suggest that presented abnormalities in the erythrocyte plasma membrane may have significant pathophysiological implications, including shortened cell survival and alterations in the hemorheology of the varicose vein blood.

Oxidative stress induced in rat liver by anticancer drugs doxorubicin, paclitaxel and docetaxel.

PURPOSE: Oxidative stress generated by anticancer drugs in non-targeted tissues, is considered as a significant factor responsible for their severe side effects, e.g. cardiotoxicity, neurotoxicity and hepatotoxicity. Lack of data on the effect of concurrent administration of commonly used anticancer drugs: doxorubicin (DOX), paclitaxel (PTX) and docetaxel (DTX) on normal tissue, prompted us to examine the markers of oxidative stress in the liver of rats treated with these drugs. MATERIAL/METHODS: Male Wistar rats of average weight 200 g were injected intraperitoneally (i.p.) with 10 mg/kg of body weight (b.w.) of DOX, PTX and DTX. The drugs were given alone or in combinations DOX+taxane. The activities of superoxide dismutase (SOD), catalase (CAT), low molecular weight and total thiols and thiobarbituric acid-reactive substances (TBARS) were estimated. RESULTS: Combination of two drugs generated greater changes than single agents. Concurrent administration of DOX and PTX increased SOD activity and TBARS, decreased the amount of low molecular weight and total thiols, but did not cause any changes in the activity of catalase. Combination of DOX and DTX induced similar changes except for the activity of catalase, which decreased after the treatment. Of the three drugs only DTX significantly decreased the activity of SOD. However, both taxanes increased the activity of catalase. Although a decrease in concentration of -SH groups, depletion of glutathione and an increase of TBARS were observed after treatment with single drugs, the changes were not statistically significant. CONCLUSION: Concurrent administration of DOX and taxane induced enhanced oxidative stress in comparison to single drugs, which suggests their synergistic prooxidant mode of action in liver.

Nitroxide pirolin reduces oxidative stress generated by doxorubicin and docetaxel in blood plasma of rats bearing mammary tumor.

Combination of doxorubicin (DOX) and docetaxel (DTX) is clinically effective against many drug-refractory cancers, nevertheless, enhanced side effects, e.g. cardiotoxicity related to oxidative damage of tissue macromolecules is observed. Nitroxides represent an attractive class of synthetic compounds to ameliorate DOX-DTX toxicity in non-targeted tissues due to their antioxidant and iron-oxidizing properties. The aim of the study was to define the ability of 3-carbamoylpyrroline nitroxyl derivative pirolin (PL) to mitigate oxidative damage to blood plasma proteins and lipids induced by DOX-DTX chemotherapy in Sprague-Dawley rats bearing DMBA-induced mammary tumor. Additionally we also evaluated: i) pro-oxidant and antioxidant activity of pirolin administered as a single agent according to different regimens and ii) differences in biomarkers of the oxidative stress between healthy rats and rats with DMBA-induced mammary tumors. The extent of oxidative stress was evaluated on the basis of its foremost biomarkers: thiol and carbonyl groups, lipid peroxidation products (hydroperoxides, TBARS), activity of antioxidant defense enzyme superoxide dismutase (SOD) and non-enzymatic antioxidant capacity (NEAC). We have found that pirolin alone displayed dual, antioxidant and pro-oxidant activity depending on the regimen of treatment. Daily treatment for 2 weeks increased the amount of thiols, and decreased the protein carbonyl groups. Three administrations of pirolin at 3-week intervals did not influence thiol content but increased hydroperoxides, TBARS and carbonyl groups. Chemotherapy employing DOX-DTX combination caused considerable oxidative stress in the plasma. Significant and dose-dependent oxidative damage to lipids and proteins with concomitant thiol depletion were evident in treated animals. Drugs also increased SOD activity and NEAC. Association of pirolin with DOX-DTX chemotherapy resulted in a partial amelioration of oxidative stress generated by anticancer drugs. This study indicates that a nitroxyl compound pirolin applied as a single agent in vivo can display both antioxidant and pro-oxidant properties but in conjunction with DOX-DTX it is able to protect partially blood plasma against oxidative stress generated by chemotherapy. The outcome, however, seems to be highly dependent on the ratio between the doses of employed anticancer drugs and the nitroxide.

Different effectiveness of piperidine nitroxides against oxidative stress induced by doxorubicin and hydrogen peroxide.

The piperidine nitroxides Tempamine and Tempace have been studied for their effect on doxorubicin (DOX) and hydrogen peroxide (H(2)O(2)) cytotoxicity in immortalized B14 cells, a model for neoplastic phenotype. The significance for nitroxide performance of the substituent in position 4 of the piperidine ring was evaluated. The cells were exposed to DOX/H(2)O(2) alone or in combination with the nitroxides Tempamine or Tempace. Two other piperidine nitroxides, Tempo and Tempol, were used for comparison. All the nitroxides except Tempamine modestly reduced DOX cytotoxicity. Tempamine evoked a biphasic response: at concentrations lower than 200 micromol/L the nitroxide decreased DOX cytotoxicity, while at concentrations higher than 200 micromol/L, it enhanced DOX cytotoxicity. In contrast to Tempo and Tempol, Tempamine and Tempace ameliorated hydrogen peroxide cytotoxicity, but none of the nitroxides influenced TBARS stimulated by hydrogen peroxide. The cytoprotective effect of Tempace, Tempo and Tempol in DOX-treated cells correlated with the inhibition of DOX-induced lipid peroxidation. The bioreduction rates of the investigated nitroxides differed significantly and were variously affected by DOX depending on the nitroxide substituent. In combination with DOX, Tempo and Tempol were reduced significantly more slowly, while no influence of DOX on Tempamine and Tempace bioreduction was observed. Our results suggest that the structure of the 4-position substituent is an important factor for biological activity of piperidine nitroxides. Among the investigated nitroxides, Tempace displayed the best protective properties in vitro but Tempamine was the only nitroxide that potentiated cytotoxicity of DOX and did not influence DOX-induced lipid peroxidation. However, this nitroxide showed different performance depending on its concentration and conditions of oxidative stress.

Structure-activity relationship studies of protective function of nitroxides in Fenton system.

The aim of this study was to evaluate the effect of piperidine nitroxides and their amine precursors on deoxyribose oxidation in the Fenton system. Protecting activity of nitroxides was found to be concentration-dependent and strongly influenced by ring substituents, while secondary amines did not provide any protection. The reported results suggest a mechanism of nitroxide action through iron oxidation rather than through direct scavenging of hydroxyl radicals. Moreover, presented data point to the danger of interference of nitroxides during the TBARS assay procedure.

Tempicol-3, a novel piperidine-N-oxide stable radical and antioxidant, with low toxicity acts as apoptosis inducer and cell proliferation modifier of Yoshida Sarcoma cells in vivo.

The novel nitroxyl, Tempicol-3 (nitroxide-N-oxide) was synthesized and its capacity to act as a scavenger of hydroxyl radicals was tested. The concentration-dependent reducibility of this novel compound was also examined and compared with those of previously characterized nitroxides, Tempo and Tempace. The cytotoxicity of Tempicol-3 in vitro was measured by the modified tetrazolium assay (MTT), using, model cells for neoplastic phenotype (mouse NIH 3T3 fibroblast line). The ability of Tempicol-3 to act as an antitumor agent in vivo was also investigated in a pharmacological test, using rats bearing 3-day old Yoshida Sarcoma (promotion phase of the disease). Our results clearly indicated that Tempicol-3 acts as an effective and promising hydroxyl radical scavenger-antioxidant. Structure- and concentration-dependent bioreduction of Tempicol-3 by ascorbic acid may account for some of its biological effects, causing modulation of the antioxidant status of cells. The presence of one nitrone moiety per molecule of Tempicol-3 caused a significant decrease in nitroxide cytotoxicity as compared with Tempo, in vitro. The results clearly confirmed that the toxic effect could result either from the presence or structure of substituent(s) at position 4 of the free radical piperidine moiety. It can be stated that Tempicol-3 is a lowtoxicity nitroxide, which could be effective in providing antioxidative activity. We have also observed that lowtoxic Tempicol-3, at m.e.d. (minimal effective dose) suppressed tumorigenesis, acting as a cell proliferation modifier and apoptosis inducer in vivo. This work provides the base for further investigations on nitroxide-N-oxide derivatives since the serious question remains to be solved as to what is the molecular mechanism of action of the nitroxide-N-oxides.

Response of human blood platelet membrane to sodium selenite.

It was demonstrated that incubation of blood platelets with sodium selenite (1-100 microM) resulted in a dose- and time-dependent loss of platelet thiols (both glutathione and protein -SH groups). The effects of sodium selenite on platelet membrane lipid fluidity by the EPR spin-labelling method was also investigated. We showed there were no alterations in membrane fluidity at the deeper regions (12-DOXYL-Ste) in lipid bilayer, a slight increase (approx. 7%, p < 0.03) of h +1/h0 for spin probe 5-DOXYL-Ste was monitored. The amount of Triton-insoluble protein fraction isolated from platelets after incubation (60 min) with selenite was significantly elevated (p < 0.006). It has been suggested that limited increase in lipid fluidity at the surface regions in the lipid bilayer of the platelet membrane in selenite-treated platelets may be the result of alteration in lipid-protein interactions caused by protein conformational changes.

[Platelet membrane fluidity and receptor exposition in patients with Alzheimer's disease]. / Plynnosc blon plytek krwi i ekspozycja receptorów blonowych u pacjentów z choroba Alzheimera.

The majority of reports indicate that blood platelet membrane fluidity is increased in Alzheimer's disease (AD) patients. The increased membrane fluidity implies that platelet membrane receptors are less exposed to the external environment. To verify this hypothesis we tried to estimate platelet membrane fluidity at its two different depths and receptors' exposure after platelet activation in 12 AD sufferers. Platelet membrane fluidity was measured by the EPR with the use of 2 spin-labelled markers. In AD patients both "near surface" (p < 0.04) and "deeper depth" (p < 0.005) fluidity was significantly increased. The exposure of platelet granule membrane protein--P-selectin and membrane glycoprotein receptors: alpha subunit of glycoprotein Ib and beta3 subunit of GP complex IIb/IIIa were measured by flow cytometry with the use of human platelet monoclonal antibodies labelled with fluorescein and ficoerithrin. The exposure of GPIb alpha subunit was significantly decreased both in resting state (p < 0.0001) and after thrombin activation (p < 0.005). In thrombin-activated platelets the expression of P-selectin and beta3 subunit of fibrinogen receptor were also significantly decreased (p < 0.00001 and p < 0.04, respectively). The authors conclude that both platelet membrane fluidity and receptor exposure might serve as an adjunct marker of in vivo AD diagnosis.

Changes in plasma membrane fluidity of immortal rodent cells induced by anticancer drugs doxorubicin, aclarubicin and mitoxantrone.

The aim of this study was to examine the effect of three structurally different anticancer drugs-the pro-oxidative anthracyclines doxorubicin (DOX) and aclarubicin (ACL), and antioxidative anthraquinone mitoxantrone (MTX) on the fluidity of plasma membrane of immortalized rodent fibroblasts using fluorescence spectroscopy and electron spin resonance (ESR) techniques. Two kinds of fluorescent probes (TMA-DPH and 12-AS) and spin labels (5-DS and methyl-12-DS) were used to monitor fluidity in the hydrophobic core and in the polar headgroup region of the lipid bilayer. Immortalized hamster B14 and NIH 3T3 mouse fibroblasts were exposed to DOX, ACL and MTX. We demonstrate that these drugs influence predominantly the hydrophobic core of the lipid bilayer, inducing significant decrease in its fluidity at low concentrations (2-5 microM). A decreased membrane fluidity at the surface of the lipid bilayer was observed only at a higher concentration (20 microM) of the drugs, which indicates that DOX, ACL and MTX intercalate mainly into the hydrophobic core of the membrane, thereby perturbing its structure.

Tempicol-2 (4-hydroxy-4-(2-picolyl)-2,2,6,6-tetramethylpiperidine-1-oxyl), a stable free radical, is a novel member of nitroxide class of antioxidants and anticancer agents.

As a part of our studies on the chemical, biochemical and pharmacological characteristics of the newly synthesized antioxidants, nitroxide derivatives, we designed a novel nitroxide, named Tempicol-2. Its capacity to act as antioxidant of potential pharmacological application was tested in three model systems: xanthine/xanthine oxidase, iron- and ascorbate Fenton reaction(s) and gamma-radiolysis. The antioxidant properties of Tempicol-2 as a function of concentration were compared with those previously characterized nitroxide derivatives Tempace and Rutoxyl which we had synthesized. The possibility of one-electron reduction of the novel substance by ascorbic acid was also examined and compared. The ability of Tempicol-2 to act as anticancer agent in vivo was also investigated in pharmacologic tests. The administration of Tempicol-2 to rats bearing 3 day-old Yoshida Sarcoma (promotion phase) led to both growth inhibition and the induction of apoptotic cells(s) death, comparable to the effects of Tempace and Rutoxyl under the same experimental conditions. Our results confirmed the suggested involvement of free radicals in the pathogenesis of model. Yoshida Sarcoma, thus indicating that anticancer activity of the investigated nitroxides may indirectly involve an antioxidant mechanism. The results reported here are encouraging as we find a limited correlation between the molecular redox properties, structure of nitroxides and their antitumor action. Tempicol-2, similarly to Tempace and Rutoxyl, is a promising antioxidant which can induce apoptosis, thus providing the basis for further investigations of the concentration and phase-dependent effects and the exact mechanisms of nitroxide(s) apoptotic action using cell line(s) model.

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.

Changes in red blood cell membrane structure in patients with chronic renal failure.

The properties of red blood cell membranes in patients with chronic renal failure were investigated using electron paramagnetic resonance spectroscopy. Using spin traps, 5,5-dimethylpirroline-1 oxide and N-tert-butyl-alpha-phenylnitrone, we found generation of hydroxyl radicals in the blood of patients with chronic renal failure after 20 min of regular hemodialysis. The physical state of membrane proteins and membrane osmotic fragility and reductive properties of red blood cells were studied. The increase in the relative correlation time of 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1 oxyl indicates the immobilization of membrane protein molecules in erythrocytes of chronic renal failure patients. The decrease in membrane protein mobility was observed in whole blood incubated with tert-butylhydroperoxide, regardless of the presence of iron. We found that the addition of ferrous ions did not aggravate profound changes in membrane proteins induced with tert-butylhydroperoxide. We also demonstrated higher osmotic fragility of erythrocytes in the patients with renal failure as compared to normal subjects. These alterations in membrane structure of red blood cells in hemodialysed patients suggest that hydroxyl radicals generated during hemodialysis can play an important role in the oxidative mechanism of erythrocyte damage.

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients.

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy on order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure on conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules ( the increased h+1/h0 parameter of maleimide spin label, MSL; 0.277 +/- 0.021 in diabetics vs 0.338 +/- 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (tau c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 +/- 0.42 in diabetics, n = 21 vs 4.79 +/- 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 +/- 1.08, n = 28 vs 7.31 +/- 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h+1/h0 parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.

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.

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.

Effect of aspirin on conformation and dynamics of membrane proteins in platelets and erythrocytes.

The effect of the chemical modifications induced by aspirin (acetylsalicylic acid), acetyl chloride or salicylate on platelet membranes and erythrocyte ghosts has been investigated by means of fluorescence quenching and ESR spectroscopy in relation to our earlier findings of acetylation-induced reduction of platelet and erythrocyte membrane lipid fluidity. Only aspirin was found to induce disorders in the lipid-protein matrix and membrane protein conformation. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules was decreased after aspirin action in both platelet and erythrocyte membranes. This resulted in a significant increase in the maximum energy transfer efficiency. The decrease in the ratio of the amplitudes of low-field peaks of weakly to strongly immobilized fractions of maleimide spin label (4-maleimido-2,2,6-6-tetramethylpiperidine-1-oxyl) and the rise in the relative rotational correlation time of iodoacetamide spin label [4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl] indicate that aspirin effectively immobilizes membrane proteins in the plane of the lipid bilayer, whereas neither acetyl chloride or salicylate gave rise to detectable effects. We conclude that aspirin-induced alterations in membrane protein structure induce a reorganization of lipid assembly as well as rearrangements in the membrane protein pattern, and consequently alterations in lipid-protein interactions. Thus, the interaction of aspirin with platelet and erythrocyte membranes may induce local conformational changes in membranes, which are discussed in connection with impairment of platelet function. A new mode of protein chemical modification by aspirin is suggested which involves the generation of reactive salicylic residue during the fast degradation of aspirin under physiological conditions.