Synergistic Platelet Inhibitory Effects of Riociguat and Nitric Oxide Are Decreased By Protein Binding

Introduction Nitric oxide (NO) released by endothelial cells interacts with platelets in which it stimulates soluble guanylate cyclase (sGC), thereby increasing platelet cyclic guanosine monophosphate (cGMP) and inhibiting platelet activation. Stimulation of sGC in other cells has been suggested as an attractive target for intervention in a range of diseases including pulmonary arterial hypertension, heart failure, and diabetes mellitus. Riociguat, the first FDA-approved sGC stimulator, potently increases platelet cGMP and inhibits platelet aggregation in washed platelets. Because riociguat binds to plasma proteins, higher concentrations of riociguat are required to inhibit platelet function in whole blood. However the potential synergistic inhibition of platelet function by riociguat and NO has not been well studied. Goal To investigate the possible synergistic effects of riociguat and NO on platelet inhibition and to determine the effects of protein binding. Methods Platelet-rich plasma (PRP) was prepared from citrate (3.2%) anticoagulated whole blood collected from healthy donors following informed consent. Riociguat (10 mM) in DMSO and DETA-NONOate 10 mM (an NO donor) in 10 mM NaOH were stored at -80°C until use. PRP was diluted 10-fold in either HEPES-Tyrode's buffer or platelet poor plasma (PPP), then incubated with vehicle or riociguat 1, 10, or 100 µM, alone or in combination with DETA-NONOate 16, 31, or 250 µM for 30 minutes, then analyzed by flow cytometry. Platelet surface activated GPIIb-IIIa (detected by monoclonal antibody PAC1) and platelet surface P-selectin were measured with and without activation by ADP 5 µM or thrombin receptor activating peptide (TRAP) 5 µM. For light transmission platelet aggregation (LTA) and 96-well platelet aggregation, PRP was used without dilution. Results For PRP diluted in buffer, riociguat and DETA-NONOate each produced concentration-dependent inhibition of ADP- and TRAP-stimulated platelet activation, as reported by platelet surface activated GPIIb-IIIa (Figure A) and P-selectin, and a synergistic inhibitory effect was observed when riociguat and DETA-NONOate were combined (for platelet surface activated GPIIb-IIIa, 40% inhibition with 1 µM riociguat alone; 30% inhibition with 31 µM DETA-NONOate alone; 90% inhibition with 1 µM riociguat and 31 µM DETA-NONOate combined). In contrast, when PRP was diluted in PPP, the concentrations of riociguat alone and DETA-NONOate alone needed to inhibit activation were dramatically increased and the combination of 1 µM riociguat and 31 µM DETA-NONOate produced less than 10% inhibition of platelet surface activated GPIIb-IIIa (Figure B). Synergistic inhibition in plasma was observed when DETA-NONOate was increased to 250 µM. Based on these results, a sub-threshold concentration of DETA-NONOate was chosen for investigation of the effects of riociguat on platelet aggregation. Using ADP 5 µM, TRAP, 2 µM, or collagen 2 µg/mL, riociguat alone at 10 µM (Figure C) and DETA-NONOate alone at 31 µM showed no inhibition of platelet aggregation. However, in the presence of 31 µM DETA-NONOate, riociguat showed a concentration-dependent inhibition of aggregation by each agonist (Figure C). Conclusions Platelets exposed to riociguat in combination with sub-threshold concentrations of NO, such as may occur in microvessels adjacent to the endothelial layer, are inhibited from undergoing platelet activation and aggregation. The presence of plasma proteins blunts the effects of riociguat and has even larger effects on the NO donor, DETA-NONOate. Taken together, these data suggest that NO potently synergizes with riociguat to inhibit platelet activation and aggregation, but in vivo this effect likely only occurs immediately adjacent to endothelial cells where NO concentrations are highest. Figure. Figure. Disclosures Michelson: Alnylam, Instrumentation Laboratory, Surface Oncology: Consultancy; AstraZeneca, Chiesi, Dova, Janssen, LightIntegra, Megakaryon, Remora: Other: Scientific Advisory Board; Baxalta, Cellular Preservation Technologies, Ionis, Ironwood, Medtronic, Megakaryon, Pfizer, Sysmex: Research Funding. Frelinger:Surface Oncology: Consultancy; Cellular Preservation Technologies, Ironwood, Ionis, Medtronic, Megakaryon, Pfizer and Sysmex: Research Funding.

Nitric oxide sensing by chlorophylla

Nitric oxide (NO) acts as a signalling molecule that has direct and indirect regulatory roles in various functional processes in biology, though in plant kingdom its role is relatively unexplored. One reason for this is the fact that sensing of NO is always challenging. There are very few probes that can classify the different NO species. The present paper proposes a simple but straightforward way for sensing different NO species using chlorophyll, the source of inspiration being hemoglobin that serves as a NO sink in most mamalian system. The proposed method is able to classify NO from DETA-NONOate or (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate, nitrite, nitrate and S-nitrosothiol or SNO. This discrimination is carried out by chlorophylla(chl a) at nano molar (nM) order of sensitivity and at 293K to 310K. Molecular docking reveals the differential binding behaviour of NO and SNO with chlorophyll, the predicted binding affinity matching with the experimental observation. Additional expreiments with diverse range of cyanobacteria reveals that apart from spectroscopic approach the proposed sensing module can be used in microscopic inspection of NO speices. Binding of NO is sensitive to tempertaure and static magnetic field. This provides additional support to the involvement of the porphyrin ring structure to the NO sensing process. This also broadens the scope of the sensing methods as hinted in the text.

Endothelium-Independent Hypoxic Contraction Is Prevented Specifically by Nitroglycerin via Inhibition of Akt Kinase in Porcine Coronary Artery

Objective. Hypoxia-induced sustained contraction of porcine coronary artery is endothelium-independent and mediated by PI3K/Akt/Rho kinase. Nitroglycerin (NTG) is a vasodilator used to treat angina pectoris and acute heart failure. The present study was to determine the role of NTG in hypoxia-induced endothelium-independent contraction and the underlying mechanism.Methods and Results. Organ chamber technique was used to measure the isometric vessel tension of isolated porcine coronary arteries. Protein levels of phosphorylated and total Akt were determined by western blot. A sustained contraction of porcine coronary arteries induced by hypoxia was significantly reduced by NTG but not by isoproterenol. This contraction was also inhibited by DETA NONOate, 8-Br-cGMP, which can be reversed by ODQ, and Rp-8-Br-PET-cGMPS. The restored contraction was blocked by LY294002. The reduction of Akt-p at Ser-473 by NTG, DETA NONOate, and 8-Br-cGMP was significantly inhibited by ODQ, PKG-I. The decrease in Akt-p level by NTG and 8-Br-cGMP was prevented by calyculin A but not by okadaic acid.Conclusions. These results demonstrated that the endothelium-independent sustained hypoxic vasoconstriction can be prevented by NTG and that the inhibition of PI3K/Akt signaling pathway may be involved.

Progesterone secretion by ovine granulosa cells: effects of nitric oxide and plane of nutrition

The aim was to evaluate the effects of nutritional plane on in vitro progesterone (P4) secretion by granulosa (G) cells cultured in the presence or absence of effectors of the nitric oxide (NO) system. Ewes were randomly assigned into three nutritional groups: control (C), overfed (O; 2 × C), or underfed (U; 0.6 × C). Follicular development was induced by FSH injections. On day 15 of the estrous cycle, G cells were isolated and cultured with or without DETA-NONOate (NO donor), L-NAME (NO synthase [S] inhibitor), Arg and (or) LH for 8 h. DETA-NONOate decreased basal and LH-stimulated P4 secretion, and L-NAME increased basal P4 secretion in all groups. In U, Arg decreased LH-stimulated P4 secretion. These data demonstrate that (i) plane of nutrition affects basal P4 secretion by G cells, (ii) the NO donor decreases, NOS inhibitor increases but Arg does not affect basal P4 secretion, and (iii) effects of Arg on LH-stimulated P4 secretion are affected by plane of nutrition in FSH-treated sheep. Thus, plane of nutrition affects G cell function, and the NO system is involved in the regulation of basal and LH-stimulated P4 secretion. The mechanism of the NO system effects on secretory activity of G cells remains to be elucidated.

cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries

cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3′,5′-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (Po2: 25–30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5′-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr853, which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK.

Nitric oxide inhibits succinate dehydrogenase-driven oxygen consumption in potato tuber mitochondria in an oxygen tension-independent manner

NO (nitric oxide) is described as an inhibitor of plant and mammalian respiratory chains owing to its high affinity for COX (cytochrome c oxidase), which hinders the reduction of oxygen to water. In the present study we show that in plant mitochondria NO may interfere with other respiratory complexes as well. We analysed oxygen consumption supported by complex I and/or complex II and/or external NADH dehydrogenase in Percoll-isolated potato tuber (Solanum tuberosum) mitochondria. When mitochondrial respiration was stimulated by succinate, adding the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) or DETA-NONOate caused a 70% reduction in oxygen consumption rate in state 3 (stimulated with 1 mM of ADP). This inhibition was followed by a significant increase in the Km value of SDH (succinate dehydrogenase) for succinate (Km of 0.77±0.19 to 34.3±5.9 mM, in the presence of NO). When mitochondrial respiration was stimulated by external NADH dehydrogenase or complex I, NO had no effect on respiration. NO itself and DETA-NONOate had similar effects to SNAP. No significant inhibition of respiration was observed in the absence of ADP. More importantly, SNAP inhibited PTM (potato tuber mitochondria) respiration independently of oxygen tensions, indicating a different kinetic mechanism from that observed in mammalian mitochondria. We also observed, in an FAD reduction assay, that SNAP blocked the intrinsic SDH electron flow in much the same way as TTFA (thenoyltrifluoroacetone), a non-competitive SDH inhibitor. We suggest that NO inhibits SDH in its ubiquinone site or its Fe–S centres. These data indicate that SDH has an alternative site of NO action in plant mitochondria.