Increased thromboxane biosynthesis in a human preparation of platelet activation: biochemical and functional consequences of selective inhibition of thromboxane synthase.

Abstract Genomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs.

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Quantification Of Malondialdehyde Adducts In Platelet Activation As An Indicator Of Proinflammatory and Prothombotic State

Blood ◽

10.1182/blood.v122.21.4735.4735 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4735-4735

Author(s):

Irene Zagol-Ikapite ◽

Iberia Romina Sosa ◽

Audra M Judd ◽

Olivier Boutaud ◽

John A Oates

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Primary Amines ◽

Specific Marker ◽

Medical Conditions ◽

Thrombotic Risk ◽

Thromboxane Synthase ◽

Reticulated Platelets ◽

Platelet Proteins

Background The formation of malondialdehyde (MDA) has been previously described as a product of the thromboxane synthase. However, the reported approaches for its quantification have not been reliable, stymieing its use in research. As a reactive di-carbonyl, MDA reacts with primary amines, notably lysines on proteins, to form covalent adducts of several types. Three of the products of the reaction of MDA with lysine are an N-propenal adduct, a dihydropyridine ring adduct (N-lysyl-4-methyl-2, 6-dihydropyridine-3, 5-dicarbaldehyde), and a lysyl-MDA crosslink. Measurement of platelet protein modifications, such as MDA adducts, could provide a specific marker of in vivo activation of platelets, since these modifications accumulate over the lifespan of the platelet. Methods and Results To investigate thromboxane synthase-dependent formation of MDA adducts on platelet proteins, we developed an LC/MS/MS method for analysis of one of the MDA adducts, the lysyl-MDA crosslink, employing a [13C12] labeled internal standard. We demonstrated that levels of lysyl-MDA crosslink in human platelets are increased following its activation with arachidonic acid. This increase is inhibited by aspirin, the thromboxane synthase inhibitor, ozagrel and by γ-ketoaldehyde specific scavengers: 3-Methoxysalicylamine (3-MOSA) and Salicylamine (SA). To determine whether lysyl-MDA crosslinks reflect in vivo platelet activation, we analyzed samples from patients with medical conditions known to be associated with increased platelet activation. We employed traditional methods of measuring platelet activation: flow cytometry of p-selectin and reticulated platelets, and serum thromboxane, to measure platelet activation in patients with metabolic syndrome and sickle cell disease. These assays were compared with the levels of lysyl-MDA-crosslinks. In both populations, the levels of MDA-lysine-crosslink are increased by 2.5 fold compared to healthy volunteers and provide greater discrimination between groups than p-selectin expression and reticulated platelets. The inhibition of the lysyl-MDA crosslink adduct in patients taking NSAIDs further confirms the specificity for thromboxane synthase-dependent MDA modifications on platelet proteins. Discussion The results of this study provide compelling evidence that MDA-protein adducts in platelets may be a useful marker of in vivo platelet activation in humans and potentially helpful in predicting thrombotic risk and the benefit of antiplatelet therapy in patients with medical conditions associated with platelet hyperactivity. Disclosures: No relevant conflicts of interest to declare.

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Effects of S-nitroso-glutathione in the human forearm circulation: evidence for selective inhibition of platelet activation

Cardiovascular Research ◽

10.1093/cvr/28.5.691 ◽

1994 ◽

Vol 28 (5) ◽

pp. 691-694 ◽

Cited By ~ 125

Author(s):

A. J d. Belder ◽

R. MacAllister ◽

M. W Radomski ◽

S. Moncada ◽

P. J T Vallance

Keyword(s):

Platelet Activation ◽

Selective Inhibition ◽

Human Forearm ◽

Forearm Circulation

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A new role for nicotine: selective inhibition of thromboxane formation by direct interaction with thromboxane synthase in human promyelocytic leukaemia cells differentiating into macrophages

The Clinical Investigator ◽

10.1007/bf00184657 ◽

1992 ◽

Vol 70-70 (3-4) ◽

Cited By ~ 6

Author(s):

M. Goerig ◽

V. Ullrich ◽

G. Schettler ◽

C. Foltis ◽

A. Habenicht

Keyword(s):

Direct Interaction ◽

Selective Inhibition ◽

Promyelocytic Leukaemia ◽

Thromboxane Synthase ◽

Thromboxane Formation

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Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A

Blood ◽

10.1182/blood-2006-10-052522 ◽

2007 ◽

Vol 110 (5) ◽

pp. 1475-1482 ◽

Cited By ~ 51

Author(s):

Wei Zhang ◽

Robert W. Colman

Keyword(s):

Cyclic Amp ◽

Platelet Activation ◽

Selective Inhibition ◽

Human Platelets ◽

Intracellular Camp ◽

Akt Inhibitor ◽

Camp Content ◽

Fibrinogen Receptor ◽

Thrombin Activation ◽

Camp Hydrolysis

Abstract Thrombin-induced cyclic AMP (cAMP) reduction potentates several steps in platelet activation, including Ca++ mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not 2 different PDE2 inhibitors. Selective inhibition of PDE3A resulted in reversal of thrombin-induced cAMP reduction, indicating that thrombin activated PDE3A. In synergy with inhibition of adenylate cyclase by thrombin, activated PDE3A accelerates cAMP hydrolysis and maximally reduces the cAMP content. Thrombin-induced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y12 inhibitor also reduced thrombin-induced cAMP reduction. The combination of both reversed cAMP decrease by thrombin. Thrombin-mediated phosphorylated PDE3A was isolated by liquid chromatography, detected by a monoclonal antibody against Akt-phosphorylated substrate, and verified by immunoprecipitation study. The predominant isoform phosphorylated by Akt was the 136-kDa species. We suggest that activation/phosphorylation of PDE3A via Akt signaling pathway participates in regulating cAMP during thrombin activation of platelets.

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Functional Consequences of Cellular Interactions with Heparin-PF4 Antibodies in Heparin-Induced Thrombocytopenia.

Blood ◽

10.1182/blood.v104.11.3498.3498 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3498-3498 ◽

Cited By ~ 1

Author(s):

Sarfraz Ahmad ◽

Ali Amirkhosravi ◽

Jamie M. Walker ◽

Hina Desai ◽

Mildred Amaya ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Platelet Activation ◽

Procoagulant Activity ◽

Antibody Detection ◽

Cellular Interactions ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Functional Consequences ◽

Clot Time

Abstract Heparin-induced thrombocytopenia (HIT) is a potentially devastating complication of heparin therapy. The condition is due to formation of antibodies to the heparin-platelet factor 4 (HPF4) complex that activate platelets and/or endothelial cells by binding to FcγIIa (CD32) receptors. It is not clear why only a sub-population of patients that form HPF4 antibodies also develops clinical HIT, and why only a fraction of those develop thrombosis. We hypothesized that some functionally active HPF4 antibodies (i.e. that activate platelets) may also activate leukocytes and endothelial cells by upregulating procoagulant activity; a property not shared by non-functional HPF4 antibodies. Citrated plasmas were obtained from patients clinically suspected of HIT. A commercially available ELISA (GTI, Brookfield, WI) combined with a 14C-serotonin release assay (SRA) was used to stratify patient plasmas into functional (F) and non-functional (NF) HPF4 antibody groups. Pooled normal plasma (NP) served as negative control. Some (n=20) GTI-ELISA positive samples (both F and NF) were also tested by flow cytometry using HPF4 coated microspheres (DiaMed-Caribbean, Inc., Miami, FL) for immunological antibody detection, and fluorescent annexin V binding to detect functional activity. The ability of HPF4 antibodies to cause platelet-monocyte aggregate (conjugate) formation was evaluated by two-color flow cytometry. The functional consequences of HPF4 antibody interaction with EaHy926 hybridoma endothelial cells were studied in the presence/absence of washed platelets and therapeutic heparin levels. Cell surface procoagulant activity (PCA) was measured by a single-stage clotting assay. Approximately 25% of ELISA positive plasmas were also positive by SRA. Although most ELISA positive samples were strongly positive in the microsphere flow cytometry assay (5–20 fold increase in mean fluorescence intensity [MFI] compared to NP) there was no direct correlation between the ELISA OD values and MFI. In the functional flow cytometric assay, not all the SRA positive samples were able to cause platelet activation. Platelet-monocyte aggregate formation in the presence of functional HPF4 antibodies was increased by 2–6 fold (MFI increase 50–300%). This was significantly higher than seen with the NF (p<0.05, n=6) or NP (p<0.01, n=6) plasma pools. Exposure of confluent EaHy926 cells to functional HPF4 antibodies and washed platelets caused a significant and time-dependent increase (20–35%) in cellular PCA as evidenced by the shortening of plasma clot time (235±15 vs. 330±12 sec, p<0.05). The clot time was not significantly different in samples incubated with NP or with HPF4 positive samples incubated without platelets. These observations suggest that in addition to platelet activation, a subset of functional HPF4 antibodies can cause platelet-monocyte aggregation and platelet-dependent endothelial cell interaction. This may lead to upregulation of cellular PCA (tissue factor) and thereby contribute to the pathogenesis of HIT. Non-functional HPF4 antibodies, even at comparable titer, appear to be devoid of such cellular effects. A prospective study to relate these observations to clinical findings in HIT patients is currently underway.

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