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1.
J Thromb Haemost ; 15(4): 774-784, 2017 04.
Article in English | MEDLINE | ID: mdl-28109047

ABSTRACT

Essentials Inhibitors of protein disulfide isomerase (PDI) have been considered a new antithrombotic class. CxxC is a PDI-targeted peptide that has been previously shown to inhibit its reductase activity. CxxC binds to surface PDI and inhibits ADP- and thrombin-evoked platelet activation and aggregation. CxxC binds to Cys400 on CGHC redox motif of PDI a' domain, a site for PDI prothrombotic activity. SUMMARY: Background Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.


Subject(s)
Platelet Aggregation Inhibitors/chemistry , Procollagen-Proline Dioxygenase/chemistry , Protein Disulfide-Isomerases/chemistry , Alanine/chemistry , Amino Acid Motifs , Blood Platelets/metabolism , Catalytic Domain , Cysteine/chemistry , Disulfides , Humans , Oxidation-Reduction , Peptides/chemistry , Platelet Activation , Platelet Aggregation , Protein Binding , Protein Domains , Protein Folding
2.
Arch Biochem Biophys ; 533(1-2): 55-61, 2013 May.
Article in English | MEDLINE | ID: mdl-23500138

ABSTRACT

Platelet activation represents a key event in normal hemostasis as well as during platelet plug formation related to thrombosis. Nitro-fatty acids are novel endogenously produced signaling mediators exerting pluripotent anti-inflammatory actions in cells and tissues. We have recently shown that nitroarachidonic acid inhibits thromboxane synthesis during platelet activation by affecting prostaglandin endoperoxide H synthase (PGHS). Herein, we investigated the regulation of human platelet activation by NO(2)AA and describe a novel mechanism involving protein kinase C (PKC) inhibition. NO(2)AA-mediated antiplatelet effects were characterized using mass spectrometry, confocal microscopy, flow cytometry, western blot and aggregometry. Incubation of NO(2)AA with human platelets caused a significant reduction in platelet sensitivity to thrombin, ADP, arachidonic acid (AA), and phorbol ester (PMA). These effects were cGMP-independent and did not involve Ca(2+) store-dependent mobilization. In contrast, signaling downstream of conventional PKC activation, such as α-granule secretion and extracellular signal regulated kinase 2 activation was strongly inhibited by NO(2)AA. Immunofluorescence confocal microscopy confirmed NO(2)AA-mediated inhibition of PKCα translocation to the membrane. In summary, we demonstrate that NO(2)AA inhibits platelet activation through modulation of PKCα activity as a potential novel mechanism for platelet regulation in vivo.


Subject(s)
Arachidonic Acid/pharmacology , Platelet Activation/drug effects , Protein Kinase C/metabolism , Animals , Arachidonic Acid/metabolism , Biological Transport , Cattle , Humans , Platelet Aggregation/drug effects
3.
Amino Acids ; 32(4): 517-22, 2007.
Article in English | MEDLINE | ID: mdl-17058116

ABSTRACT

Lipid-protein adducts are formed during oxidative and nitrative stress conditions associated with increasing lipid and protein oxidation and nitration. The focus of this review is the analysis of interactions between oxidative-modified lipids and proteins and how lipid nitration can modulate lipid-protein adducts formation. For this, two biologically-relevant models will be analysed: a) human low density lipoprotein, whose oxidation is involved in the early steps of atherogenesis, and b) alpha-synuclein/lipid membranes system, where lipid-protein adducts are being associated with the develop of Parkinson disease and other synucleinopathies.


Subject(s)
Lipoproteins, LDL/metabolism , Nitrates/metabolism , Proteins/metabolism , Reactive Nitrogen Species/metabolism , Tyrosine/analogs & derivatives , alpha-Synuclein/metabolism , Humans , Lipid Metabolism , Lipids , Nitrates/chemistry , Oxidative Stress , Tyrosine/metabolism
4.
Arch Biochem Biophys ; 395(2): 225-32, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11697860

ABSTRACT

Peroxynitrite (PN), the product of the diffusion-limited reaction between nitric oxide (*NO) and superoxide (O*-(2)), represents a relevant mediator of oxidative modifications in low-density lipoprotein (LDL). This work shows for the first time the simultaneous action of low-controlled fluxes of PN and *NO on LDL oxidation in terms of lipid and protein modifications as well as oxidized lipid-protein adduct formation. Fluxes of PN (e.g., 1 microM min(-1)) initiated lipid oxidation in LDL as measured by conjugated dienes and cholesteryl ester hydroperoxides formation. Oxidized-LDL exhibited a characteristic fluorescent emission spectra (lambda(exc) = 365 nm, lambda(max) = 417 nm) in parallel with changes in both the free amino groups content and the relative electrophoretic mobility of the particle. Physiologically relevant fluxes of *NO (80-300 nM min(-1)) potently inhibited these PN-dependent oxidative processes. These results are consistent with PN-induced adduct formation between lipid oxidation products and free amino groups of LDL in a process prevented by the simultaneous presence of *NO. The balance between rates of PN and *NO production in the vascular wall will critically determine the final extent of LDL oxidative modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.


Subject(s)
Lipid Metabolism , Lipoproteins, LDL/metabolism , Nitric Oxide/metabolism , Peroxynitrous Acid/metabolism , Proteins/metabolism , Humans , Liposomes/metabolism , Models, Chemical , Oxygen/metabolism , Protein Binding , Time Factors
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