Snake venom modulators of platelet adhesion receptors and their ligands.

In thrombosis, platelet aggregation is initiated by a specific membrane glycoprotein (GP) Ib-IX-V complex binding to its adhesive ligand, von Willebrand factor, in the matrix of ruptured atherosclerotic plaques or in plasma exposed to high hydrodynamic shear stress. This process closely resembles normal haemostasis at high shear, where GP Ib-IX-V-dependent platelet adhesion to von Willebrand factor in the injured blood vessel wall initiates platelet activation and integrin alphaIIb beta3 (GP IIb-IIIa)-dependent platelet aggregation. At low shear, other receptors such as those that bind collagen, the integrin alpha2beta1 (GP Ia-IIa) or GP VI, mediate platelet adhesion. Recently, snake venom proteins have been identified that selectively modulate platelet function, either promoting or inhibiting platelet aggregation by targeting GP Ib-IX-V, alpha2beta1, GP VI, alphaIIb beta3, or their respective ligands. Interestingly, these venom proteins typically belong to one of two major protein families, the C-type lectin family or the metalloproteinase-disintegrins. This review focuses on recent insights into structure-activity relationships of snake venom proteins that regulate platelet function, and the ways in which these novel probes have contributed in unexpected ways to our understanding of the molecular mechanisms underlying thrombosis.     

The role of protein sulfhydryl groups and protein disulfides of the platelet surface in aggregation processes involving thiol exchange reactions

Blood platelets are central to haemostasis and platelet aggregation is considered to be a direct index of platelet function. Although protein disulfides (PSSP) are structural components of most proteins, current evidence suggests that PSSP work together with protein SH groups (PSH) to activate various platelet functions in dynamic processes involving thiol/disulfide exchange reactions.Based on these assumptions, we performed experiments to demonstrate how PSH and PSSP are involved in platelet aggregation and how modifications of PSH and PSSP concentrations on the platelet surface by N-ethylmaleimide (NEM) (a PSH-blocking reagent) and dithiothreitol (DTT) (a PSSP-reducing reagent), respectively, may condition platelet susceptibility in protein rich plasma and washed platelets and integrin αIIbβ3 conformation. Our data strongly suggest that the PSH blockage and the PSSP reduction of the platelet surface are deeply involved in aggregation processes evoked in protein rich plasma and washed platelets by ADP and collagen; that endogenous thiols (e.g. GSH) may interfere with NEM actions; that NEM and DTT, acting on preexisting PSH and PSSP of active platelets have opposite conformational changes on integrin αIIbβ3 conformation. Although the precise mechanism and the populations of specific PSH and PSSP involved remain unresolved, our data support the notion that PSH and PSSP of the platelet surface are involved in platelet activation by thiol exchange reactions. A plausible molecular mechanism of PSH and PSSP recruitment during thiol exchange reactions is here proposed.     

Effects of TRA-418, a novel TP-receptor antagonist, and IP-receptor agonist, on human platelet activation and aggregation

[4-[2-(1,1-Diphenylethylsulfanyl)-ethyl]-3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxy]-acetic acid N-Methyl-d-glucamine salt (TRA-418) has both thromboxane A2 (TP)-receptor antagonist and prostacyclin (IP)-receptor agonist properties. The present study examined the advantageous effects of TRA-418 based on the dual activities, over an agent having either activity alone and also the difference in the effects of TRA-418 and a glycoprotein alphaIIb/beta3 integrin (GPIIb/IIIa) inhibitor. TRA-418 inhibited platelet GPIIb/IIIa activation as well as P-selectin expression induced by adenosine 5'-diphosphate, thrombin receptor agonist peptide 1-6 (Ser-Phe-Leu-Leu-Arg-Asn-NH2), and U-46619 in the presence of epinephrine (U-46619+ epinephrine). TRA-418 also inhibited platelet aggregation induced by those platelet-stimulants in Ca2+ chelating anticoagulant, citrate and in nonchelating anticoagulant, d-phenylalanyl-l-prolyl-l-arginyl-chloromethyl ketone (PPACK). The TP-receptor antagonist SQ-29548 inhibited only U-46619+epinephrine-induced GPIIb/IIIa activation, P-selectin expression, and platelet aggregation. The IP-receptor agonist beraprost sodium inhibited platelet activation. Beraprost also inhibited platelet aggregation induced by platelet stimulants we tested in citrate and in PPACK. The GPIIb/IIIa inhibitor abciximab blocked GPIIb/IIIa activation and platelet aggregation. However, abciximab showed slight inhibitory effects on P-selectin expression. TRA-418 is more advantageous as an antiplatelet agent than TP-receptor antagonists or IP-receptor agonists separately used. TRA-418 showed a different inhibitory profile from abciximab in the effects on P-selectin expression.     

Protective effect of metallothionein against the toxicity of cadmium and other metals(1)

In the present study, we have investigated the effects of extracellular redox status and metal/thiol interactions on glutathione distribution in HeLa cell cultures. No effects were seen on glutathione distribution after the addition of different thiols, whereas the pro-oxidant copper ions affected glutathione distribution in several ways. The addition of dithiothreitol (DTT) but not the other thiols potentiated the effects of mercury ions on glutathione distribution and cell toxicity. In the presence of DTT, increased intra- and extracellular glutathione concentrations were noted already at 0.05 micromol/l, which was below the previously reported toxicity threshold for mercury ions in blood. Likewise DTT potentiated the effects of copper ions on glutathione distribution and cell toxicity, whereas the addition of DTT to cell cultures with a non-metal thiol reactive agent (hydroquinone) or an oxidative agent (hydrogen peroxide) did not affect glutathione distribution or cell toxicity. Thus, it seems as the synergistic effects between DTT and thiol reactive agents only apply to metal ions.     

Identification and characterization of triosephosphate isomerase that specifically interacts with the integrin alphaIIb cytoplasmic domain

Integrin alphaIIb/beta3 (IIb/IIIa), a platelet fibrinogen receptor, has been shown to play a critical role in thrombosis and hemostasis. However, the mechanisms by which ligands interact with the alphaIIb/beta3 receptor is not very clear at this time. The interaction between the ligand, the receptor and the transmission of extracellular signals may involve the cytoplasmic domains of these integrins. The objective of this investigation was to identify novel proteins that interact with the cytoplasmic tail of alphaIIb. Using alphaIIb cytoplasmic tail as the bait and a yeast two-hybrid system, we have identified three separate clones containing inserts that encoded the same protein with different truncated N-terminals. Sequence analysis showed that the inserts of the three clones encoded a previously identified enzyme: triose phosphate isomerase (TPI). In addition, we demonstrated that TPI failed to interact with the integrin alpha2 tail, beta3 tail and lamin, but showed a weak binding to the alphaV tail which shares the highest homology with alphaIIb tail among the integrin alpha family. Site-directed mutagenesis studies around the homology region indicated that the critical peptide sequence necessary for the interaction between TPI and alphaIIb tail is GFFKRNRPPLEE. Using RT-PCR, we have demonstrated the presence of TPI mRNA in platelets. In addition, experiments were also performed to demonstrate specific binding of TPI to alphaIIb using an ELISA and fusion protein. Taken together, these data suggest that TPI specifically interacts with alphaIIb and may play a critical role in alphaIIb/beta3-mediated platelet function.     

ADP receptor antagonists as antiplatelet therapeutics

With the cloning of the P2Y12 receptor, the molecular basis for ADP-induced platelet aggregation is seemingly complete. Two platelet-bound ADP receptors, P2Y1 and P2Y12, operate through unique pathways to induce and sustain platelet aggregation via the glycoprotein (GP)IIb-IIIa integrin. P2Y1 operates via a glycoprotein q (Gq) pathway, activates phospholipase C, induces platelet shape change and is responsible for intracellular calcium mobilisation. P2Y12 inhibits adenylyl cyclase through a glycoprotein i (Gi)-dependent pathway, and is the target of the clinically used thienopyridines, ticlopidine (Ticlid, F. Hoffman-La Roche) and clopidogrel (Plavix, Bristol-Myers Squibb/Sanofi-Synthelabo). In addition, the receptor is targeted by the ADP analogue AR-C66096, which is currently in Phase IIb clinical trials, as well as other non-nucleoside-based preclinical leads.     

Thrombin-induced conversion of fibrinogen to fibrin results in rapid platelet trapping which is not dependent on platelet activation or GPIb

1. Activation of human platelets by thrombin is mediated by the proteolytic cleavage of two G-protein coupled protease-activated receptors, PAR-1 and PAR-4. However, thrombin also binds specifically to the platelet surface glycoprotein GPIb. It has been claimed that thrombin can induce aggregation of platelets via a novel GPIb-mediated pathway, which is independent of PAR activation and fibrinogen binding to alpha(IIb)beta(3) integrin, but dependent upon polymerizing fibrin and the generation of intracellular signals. 2. In the presence of both fibrinogen and the alpha(IIb)beta(3) receptor antagonist lotrafiban, thrombin induced a biphasic platelet aggregation response. The initial primary response was small but consistent and associated with the release of platelet granules. The delayed secondary response was more substantial and was abolished by the fibrin polymerization blocking peptide GPRP. 3. Cleavage of the extracellular portion of GPIb by mocarhagin partially inhibited thrombin-induced alpha(IIb)beta(3)-dependent aggregation and release, but had no effect on the secondary fibrin-dependent response. 4. Fixing of the platelets abolished alpha(IIb)beta(3)-dependent aggregation and release of adenine nucleotides, whereas the fibrin-dependent response remained, indicating that platelet activation and intracellular signalling are not necessary for this secondary 'aggregation'. 5. In conclusion, the secondary fibrin-dependent 'aggregation' response observed in the presence of fibrinogen and lotrafiban is a platelet trapping phenomenon dependent primarily on the conversion of soluble fibrinogen to polymerizing fibrin by thrombin.     

Effect of the glutathione/glutathione disulfide redox couple on thiopurine methyltransferase

The susceptibility of recombinant human thiopurine methyltransferase (hTPMT) to thiol-disulfide exchange was investigated. The enzyme was incubated in buffers of the redox couple GSH and GSSG. The values of the chosen concentrations and concentration ratios of the redox couple equaled those expected to occur in vivo. Activity measurements of the enzyme over time in these buffers at 30 degrees C indicated that thiol-disulfide exchange may be a part of the posttranslational modulation of hTPMT activity. Activity varied between 5% and 100%, with the lowest activities in buffers of low [GSH]/[GSSG] concentration ratios and of low total concentration of the redox couple. A thiol-disulfide exchange mechanism involving a mixed disulfide was proposed. Titration of the protein thiol groups with Ellmann's reagent (5,5'-dithiobis[2-nitrobenzoic acid]) revealed that at least two protein thiols were readily accessible for conjugation with the reagent, while others were conjugated more slowly. The previous model of hTPMT constructed by our group was in accordance with the experimental results. Inspection of the model indicated that one of the protein thiols subject to slow thiol-disulfide exchange may be situated at the binding site of the co-substrate of the enzyme and thus be responsible for the glutathione/glutathione disulfide modulation of the activity of hTPMT.     

Beta-adrenergic receptors display intramolecular disulfide bridges in situ: analysis by immunoblotting and functional reconstitution

The molecular nature of mammalian beta-adrenergic receptors in situ was probed using immunoblotting and functional reconstitution techniques. Membrane proteins of cells replete with beta-adrenergic receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose and then probed with anti-receptor antibodies. When cell membranes were first treated with agents that cleave disulfides of proteins, immunoblots of these membranes revealed intense immunoreactive bands with electrophoretic mobility similar to that of protein standards of Mr 65,000-67,000, comigrating with purified, reduced, and alkylated beta-adrenergic receptors. However, when cell membranes were prepared under anaerobic conditions, solubilized in the presence of agents that alkylate thiols, and denatured in the absence of added thiols, immunoblotting revealed receptor with Mr 55,000, rather than 65,000. This faster electrophoretic mobility is associated with the presence of intramolecular disulfides in the purified receptor and demonstrates that beta-adrenergic receptors possess intramolecular disulfide bridges in situ. Purified receptors that demonstrate this faster mobility (Mr 55,000 under nonreducing conditions) were co-reconstituted into phospholipid vesicles with the stimulatory GTP-binding protein GS and their ability to catalyze the binding of [35S]guanosine-5'-O-(3-thio)triphosphate to GS was measured. Agonist (isoproterenol) as well as thiol increased the receptor-promoted activation of GS. Taken together, these data demonstrate that native beta-adrenergic receptors possess one or more intramolecular disulfide bridges in situ, reduction of which causes functional activation of the receptor.     

Overview of clinical trials with glycoprotein IIb-IIIa receptor antagonists in the prevention and management of coronary

Platelet aggregation is mediated by the glycoprotein IIb-IIIa receptor, a member of the integrin superfamily of membrane-bound adhesion molecules. In the activated platelet, binding to the major adhesive proteins, fibrinogen and von Willebrand, occurs due to a conformational change of the glycoprotein IIb-IIIa receptor. Glycoprotein IIb-IIIa receptor antagonists effectively block the binding of these adhesive proteins and thus inhibit platelet aggregation. Large-scale clinical trials have demonstrated the benefits of these agents in patients undergoing percutaneous coronary angioplasty and with acute coronary syndromes compared to conventional antiplatelet therapy. Furthermore, trials are in progress in patients with acute myocardial infarction. The beneficial effects of these agents was first demonstrated with abciximab, a monoclonal antibody to the glycoprotein IIb-IIIa receptor, in patients at risk of coronary arterial thrombosis, and was further illustrated in trials with other IIb-IIIa receptor blocking agents, both with synthetic peptide and non-peptide receptor antagonists. This review focuses on the glycoprotein IIb-IIIa receptor antagonists most advanced in clinical development.     

The Role of the Platelet in the Pathogenesis of Atherothrombosis

Platelet adhesion, activation, and aggregation at sites of vascular endothelial disruption caused by atherosclerosis are key events in arterial thrombus formation. Platelet tethering and adhesion to the arterial wall, particularly under high shear forces, are achieved through multiple high-affinity interactions between platelet membrane receptors (integrins) and ligands within the exposed subendothelium, most notably collagen and von Willebrand factor (vWF). Platelet adhesion to collagen occurs both indirectly, via binding of the platelet glycoprotein (GP) Ib-V-IX receptor to circulating vWF, which binds to exposed collagen, and directly, via interaction with the platelet receptors GP VI and GP Ia/IIb. Platelet activation, initiated by exposed collagen and locally generated soluble platelet agonists (primarily thrombin, ADP, and thromboxane A2), provides the stimulus for the release of platelet-derived growth factors, adhesion molecules and coagulation factors, activation of adjacent platelets, and conformational changes in the platelet alpha(IIb)beta3 integrin (GP IIb/IIIa receptor). Platelet aggregation, mediated primarily by interaction between the activated platelet GP IIb/IIIa receptor and its ligands, fibrinogen and vWF, results in the formation of a platelet-rich thrombus. Currently available antiplatelet drugs (aspirin [acetylsalicylic acid], dipyridamole, clopidogrel, ticlopidine, abciximab, eptifibatide, tirofiban) act on specific targets to inhibit platelet activation and aggregation. Elucidation of the multiple mechanisms involved in platelet thrombus formation provides opportunities for selectively inhibiting the pathways most relevant to the pathophysiology of atherothrombosis.     

Fibrinogen interaction of CHO cells expressing chimeric αⅡb/αvβ3 integrin

AIM: The molecular mechanisms of the affinity regulation of alphavbeta3 integrin are important in tumor development, wound repairing, and angiogenesis. It has been established that the cytoplasmic domains of alphavbeta3 integrin play an important role in integrin-ligand affinity regulation. However, the relationship of structure-function within these domains remains unclear. METHODS: The extracellular and transmembrane domain of alphaIIb was fused to the alphav integrin cytoplasmic domain, and the chimeric alpha subunit was coexpressed in Chinese hamster ovary (CHO) cells with the wild-type beta3 subunit or with 3 mutant beta3 sequences bearing truncations at the positions of T741, Y747, and F754, respectively. The CHO cells expressing these recombinant integrins were tested for soluble fibrinogen binding and the cell adhesion and spreading on immobilized fibrinogen. RESULTS: All 4 types of integrins bound soluble fibrinogen in the absence of agonist stimulation, and only the cells expressing the chimeric alpha subunit with the wild-type beta3 subunit, but not those with truncated beta3, could adhere to and spread on immobilized fibrinogen. CONCLUSION: The substitution alphaIIb at the cytoplasmic domain with the alphav cytoplasmic sequence rendered the extracellular alphaIIbbeta3 a constitutively activated conformation for ligands without the need of pinside-outq signals. Our results also indicated that the COOH-terminal sequence of beta3 might play a key role in integrin alphaIIb/alphavbeta3-mediated cell adhesion and spreading on immobilized fibrinogen. The cells expressing alphaIIb/alphavbeta3 have enormous potential for facilitating drug screening for antagonists either to alphavbeta3 intracellular interactions or to alphaIIbbeta3 receptor functions.     

N-acetyl-p-benzoquinone imine-induced protein thiol modification in isolated rat hepatocytes.

Incubation of isolated rat hepatocytes with N-acetyl-p-benzoquinone imine (NAPQI) or 3,5-dimethyl-N-acetyl-p-benzoquinone imine (3,5-Me2-NAPQI) resulted in a concentration-dependent decrease in the protein thiol content of the mitochondrial, cytosolic and microsomal fractions. On a concentration basis, 3,5-Me2-NAPQI induced a more marked depletion of protein thiols than did NAPQI. Sodium dodecyl sulphate-polyacrylamide gel electrophoretic separation of the proteins of each fraction showed that different proteins had different susceptibilities to modification of their cysteine residues by the quinone imines. A few protein bands showed a decreased protein thiol content following incubation with non-toxic concentrations of quinone imines, whereas other proteins were affected by higher concentrations. Concentrations of quinone imines that were highly cytotoxic induced a general loss of protein thiols. NAPQI-induced protein thiol depletion occurred within 5 min and remained essentially unchanged for at least 30 min. In contrast, protein thiol depletion induced by 3,5-Me2-NAPQI increased over the 30-min time course of the experiment. Toxic concentrations of 3,5-Me2-NAPQI caused the formation of high molecular mass aggregates in all three subcellular fractions after 30 min of incubation. The observed crosslinking was not due to protein disulfide formation. However, no aggregate formation was observed after exposure of hepatocytes to NAPQI. One of the major target proteins of quinone imine-induced protein thiol depletion was a 17 kDa microsomal protein that was identified as the microsomal glutathione S-transferase. Exposure of hepatocytes and isolated liver microsomes to the quinone imines resulted in an up to four-fold increase in the specific activity of the microsomal glutathione S-transferase. In conclusion, our results are consistent with the suggestion of a critical role of protein thiol depletion in quinone imine-induced cytotoxicity.     

The mechanism of anti-platelet activity of davallialactone: involvement of intracellular calcium ions, extracellular signal-regulated kinase 2 and p38 mitogen-activated protein kinase

This study was designed to investigate the effect of davallialactone, which was isolated from the mushroom Inonotus xeranticus, on platelet aggregation induced by collagen, thrombin and ADP. We found that davallialactone dose-dependently inhibited platelet aggregation that was stimulated either by collagen (2.5 microg/ml), a potent ligand of integrin alpha2beta1 and glycoprotein VI, or by thrombin (0.1U/ml), a potent agonist of the protease-activated receptors (PARs) PAR1 and PAR3. In addition, davallialactone inhibited platelet aggregation induced by ADP, an agonist of P2Y receptor. To understand the mechanism of anti-platelet activity, we determined whether davallialactone affected the downstream signaling in collagen-activated platelets. Using the fura-2/AM fluorometric assay, we found that davallialactone dose-dependently inhibited intracellular calcium concentration levels ([Ca2+]i). Moreover, davallialactone inhibited the phosphorylation of extracellular signal-regulated protein kinase (ERK)-2 and p38 mitogen-activated protein kinase (MAPK), in a dose-dependent manner. The tyrosine phosphorylation of 60 and 85kDa proteins, which were activated by collagen, were differentially inhibited by davallialactone. Taken together, these data suggest that davallialactone may have potential anti-platelet aggregation activity via suppression of intracellular downstream signaling pathways.     

A redox cycling mechanism of action for 2,3-dichloro-1,4-naphthoquinone with mitochondrial membranes and the role of sulfhydryl groups

The addition of 2,3-dichloro-l,4-naphthoquinone (CNQ) to substrate-depleted, GSH-supplemented rat liver mitochondria resulted in a dose-dependent depletion of readable suflhydryl groups and a concomitant increase in mitochondrial disulfide content at a ratio of 2 thiols depleted/disulfide generated. The molar ratio of thiol depleted/CNQ added approached 20 at low CNQ concentrations and was unity at higher doses. The addition of CNQ to substrate-depleted mitochondrial suspensions resulted in O₂; consumption which increased with increasing concentrations of mitochondria and was sensitive to N-ethylmaleimide (NEM) which establishes the ability of CNQ to interact with mitochondrial thiol redox centers. The CNQ-mediated large amplitude swelling of rat liver mitochondria was exacerbated by thiol oxidizing agents and depressed by disulfide reducing agents. A redox cycling mechanism between mitochondrial thiol groups, CNQ and oxygen was proposed to lower the matrix glutathione pool and make the mitochondria more susceptable to toxic oxygen radicals which induce swelling in isolated mitochondrial suspensions. In support of this mechanism, α-tocopherol was shown to prevent the CNQ-mediated swelling process. Beef heart mitochondrial NADH was oxidized by CNQ in a $\text{1}\text{1}$ molar ratio anaerobically and in a $\text{3}\text{1}$ molar ratio under aerobic conditions, whereas the fully reduced quinone, CNQH₂, oxidized NADH aerobically but not anaerobically. Thus, CNQ is capable of interacting with NADH of the mitochondrial electron transport chain in a redox cycling fashion.     

Advancing the Battle Against Acute Ischemic Syndromes: A Focus on the GP IIb-IIIa Inhibitors

Platelet aggregation and thrombus formation, resulting from disruption of a coronary artery plaque, play a critical role in the pathology of acute coronary syndromes. Currently, aspirin and heparin are administered to decrease platelet aggregation. The discovery of the platelet integrin receptor α_IIbβ₃, also known as the platelet glycoprotein (GP) IIb-IIIa receptor, is a breakthrough in antiplatelet therapy. The GP IIb-IIIa receptor is responsible for the crucial binding of fibrinogen to platelets, leading to cross-links between platelets and further platelet aggregation. Since the introduction of abciximab, the first GP IIb-IIIa-receptor antagonist, several other nonantibody agents have been studied for use in percutaneous transluminal coronary angioplasty and also in stent placement, treatment of unstable angina, and myocardial infarction.     

New perspectives in antiplatelet therapy

Platelet activation is a complex mechanism of response to vascular injury and atherothrombotic disease, leading to thrombus formation. A wide variety of surface receptors -integrins, leucine-rich family receptors, G protein coupled receptors, tyrosine kinase receptors- and intraplatelet molecules support and regulate platelet activation. They are potential targets of antiplatelet therapy for the prevention and treatment of arterial thrombosis. Despite the overall clinical benefit of established antiplatelet drugs targeting cyclooxigenase-1 (COX-1), glycoprotein integrin αIIbβ3, and the purinergic P2Y(12) receptor of adenosine diphosphate, a significant proportion of treated patients continue to experience recurrent ischaemic events. This may be in partly attributed to insufficient inhibition of platelet activation. In addition, it should not be underestimated that these drugs are not immune from bleeding complications. The substantial progress in understating the regulation of platelet activation has played a key role in the development of novel antiplatelet agents. Current examples of drug under development and evaluation include: novel P2Y(12) receptor inhibitors (prasugrel, ticagrelor, cangrelor, and elinogrel), thrombin receptor PAR-1 antagonists (vorapaxar, atopaxar), new integrin glycoprotein IIb/IIIa inhibitors, and inhibitors targeting the thromboxane receptor (TP), phosphodiesterases, the collagen receptor glycoprotein VI, and intraplatelet signalling molecules. This review summarizes the mechanisms of action and current clinical evaluation of these novel antiplatelet agents.     

Thiol/disulfide homeostasis as a marker of oxidative stress in rosacea: a controlled spectrophotometric study

Background: Rosacea is the chronic inflammatory disease of the facial skin. Although its aetiology is not clear yet, inflammatory processes triggered by oxidative stress and oxidation of lipids have been suggested to play a role. While studies on the relationship between inflammation and oxidative stress are ongoing, thiol metabolism and its role in oxidative stress have also begun to be investigated. Thiols are among the key molecules of protein metabolism in the organism and they are the firstly consumed antioxidants in case of oxidative stress. Thiols regulate intracellular redox metabolism and protect keratinocytes against the results of oxidative alterations in the stratum corneum. There is a balance known as dynamic thiol/disulfide homeostasis between thiols and their oxidized forms; disulfides.

Aim: This study aimed to determine the effects of oxidative stress on protein metabolism in rosacea patients by investigating thiol/disulfide homeostasis using a newly developed and fully automated method. Determination of plasma thiol levels provides important clues regarding the extent of free radical-mediated oxidation of proteins causing damage in rosacea.

Methods: The study included 50 rosacea patients who were diagnosed clinically or histopathologically with rosacea and 42 age- and gender-matched healthy controls. Plasma levels of native thiol, total thiol, and disulfide were determined. The following ratios were calculated: disulfide/native thiol ratio, disulfide/total thiol ratio, and native thiol/total thiol ratio.

Results: The mean age was 41.8?±?10.5 in the rosacea patients (35 females) and 42.5?±?10.3?years in the control group (33 females). The mean disulfide level was found to be significantly higher in the rosacea patients than in the control group (23.4?±?5.5?µM/L and 17.3?±?6.2µM/L, respectively; p?p?Conclusion: In rosacea patients, the thiol/disulfide balance was observed to shift towards disulfides, which could be considered an indicator of oxidative stress in rosacea.