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Decreased S-nitrosation of peptide thiols in the membrane interior.

ABSTRACT: It has been proposed that autoxidation of nitric oxide (()NO) stimulates S-nitrosation of thiols located in the hydrophobic milieu. We tested whether thiols located in hydrophobic membranes undergo enhanced S-nitrosation in the presence of ()NO/O(2). The transmembrane cysteinyl peptides C(4) (AcNH-KKACALA(LA)(6)KK-CONH(2)) and C(8) (AcNH-KKALALACALA(LA)(3)KK-CONH(2)) were incorporated into dilauroylphosphatidylcholine bilayers; their location in the membrane was determined by EPR spin labeling. The peptides, C(8) and C(4), and glutathione (GSH; 300 microM) were treated with a ()NO donor, DEA-NONOate, and nitrosothiol formation was determined under various O(2) levels. Surprisingly, the more hydrophobic cysteinyl peptide, C(8), did not yield any S-nitrosated product compared to GSH in the aqueous phase or C(4) peptide in the liposomes in the presence of ()NO/O(2). These data suggest that thiols located deeply in the hydrophobic core of the membrane may be less likely to undergo S-nitrosation in the presence of ()NO/O(2).

SUBMITTER: Zhang H

PROVIDER: S-EPMC2874255 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Decreased S-nitrosation of peptide thiols in the membrane interior.

Zhang Hao H Andrekopoulos Christopher C Xu Yingkai Y Joseph Joy J Hogg Neil N Feix Jimmy J Kalyanaraman B B

Free radical biology & medicine 20090630 7

It has been proposed that autoxidation of nitric oxide (()NO) stimulates S-nitrosation of thiols located in the hydrophobic milieu. We tested whether thiols located in hydrophobic membranes undergo enhanced S-nitrosation in the presence of ()NO/O(2). The transmembrane cysteinyl peptides C(4) (AcNH-KKACALA(LA)(6)KK-CONH(2)) and C(8) (AcNH-KKALALACALA(LA)(3)KK-CONH(2)) were incorporated into dilauroylphosphatidylcholine bilayers; their location in the membrane was determined by EPR spin labeling. ...[more]

PMID: 19573593

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Project description:Essentials Biological activity of human neutrophil peptide (HNP)-1 in hemostasis under physiological conditions is not fully understood. HNP-1 inhibits the adhesion/aggregation of murine platelets on a fibrillar collagen surface or an activated endothelial cell surface under flow. The anti-adhesion activity appears to depend on the terminal free thiols of HNP-1, which may inhibit VWF-VWF lateral associations. Our results suggest a protective role and potential novel therapeutic use of HNP-1 for arterial thrombosis. SUMMARY: Background Human neutrophil peptides (HNPs), also known as α-defensins, are released from degranulated neutrophils and play an important role in innate immunity. However, their biological roles in hemostasis under flow are not fully explored. Objective This study aims to determine the role of HNP-1 on platelet adhesion and aggregation on a collagen surface or ultra large von Willebrand factor (ULVWF) on endothelium under flow and elucidate the structural elements required for its activity. Methods Anticoagulated whole blood from wild-type or Adamts13-/- mice was incubated with a fluorescein-conjugated anti-human CD41 in the presence of increasing concentrations of a synthetic HNP-1 and perfused over a collagen surface or a tumor necrosis factor (TNF)-α activated murine endothelial cell surface under arterial flow. The rate of accumulation and the final surface coverage of fluoresceinated murine platelets or the rate of forming platelet-decorated ULVWF strings were determined using the BioFlux microfluidic system. Results HNP-1 inhibited the rate and final coverage of fluorescein-labeled murine platelets on a fibrillar collagen surface under flow (100 dyne/cm2 ) in a concentration-dependent manner and the anti-adhesive activity of HNP-1 depended on its terminal free cysteine thiols. HNP-1 (20 μM) also dramatically inhibited the formation of platelets-decorated ULVWF strings on TNF-α activated murine endothelial surface under arterial flow. Conclusions Our results demonstrate for the first time an antiplatelet adhesion or antithrombotic activity of HNP-1; this activity depends on its terminal free thiols, likely affecting VWF-VWF lateral associations. These findings may suggest a potential novel therapeutic strategy for arterial thrombosis.

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Decreased S-nitrosation of peptide thiols in the membrane interior.

Publications

Decreased S-nitrosation of peptide thiols in the membrane interior.

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