Extra-platelet low-molecular-mass thiols mediate the inhibitory action of S-nitrosoalbumin on human platelet aggregation via S-transnitrosylation of the platelet surface

Inhibitory Potential

AbstractNitrosylation of sulfhydryl (SH) groups of cysteine (Cys) moieties is an important post-translational modification (PTM), often on a par with phosphorylation. S-Nitrosoalbumin (ALB-Cys34SNO; SNALB) in plasma and S-nitrosohemoglobin (Hb-Cysβ93SNO; HbSNO) in red blood cells are considered the most abundant high-molecular-mass pools of nitric oxide (NO) bioactivity in the human circulation. SNALB per se is not an NO donor. Yet, it acts as a vasodilator and an inhibitor of platelet aggregation. SNALB can be formed by nitrosation of the sole reduced Cys group of albumin (Cys34) by nitrosating species such as nitrous acid (HONO) and nitrous anhydride (N2O3), two unstable intermediates of NO autoxidation. SNALB can also be formed by the transfer (S-transnitrosylation) of the nitrosyl group (NO+) of a low-molecular-mass (LMM) S-nitrosothiol (RSNO) to ALB-Cys34SH. In the present study, the effects of LMM thiols on the inhibitory potential of ALB-Cys34SNO on human washed platelets were investigated. ALB-Cys34SNO was prepared by reacting n-butylnitrite with albumin after selective extraction from plasma of a healthy donor on HiTrapBlue Sepharose cartridges. ALB-Cys34SNO was used in platelet aggregation measurements after extended purification on HiTrapBlue Sepharose and enrichment by ultrafiltration (cutoff, 20 kDa). All tested LMM cysteinyl thiols (R-CysSH) including l-cysteine and L-homocysteine (at 10 µM) were found to mediate the collagen-induced (1 µg/mL) aggregation of human washed platelets by SNALB (range, 0–10 µM) by cGMP-dependent and cGMP-independent mechanisms. The LMM thiols themselves did not affect platelet aggregation. It is assumed that the underlying mechanism involves S-transnitrosylation of SH groups of the platelet surface by LMM RSNO formed through the reaction of SNALB with the thiols: ALB-Cys34SNO + R-CysSH ↔ ALB-Cys34SH + R-CysSNO. Such S-transnitrosylation reactions may be accompanied by release of NO finally resulting in cGMP-dependent and cGMP-independent mechanisms.

Differential Ability of Agonists to Express Distinct Pools of Fibrinogen (Gpllb/llla) Receptors which Can Mediate the Aggregation of Human Platelets

10.1055/s-0038-1651035 ◽

1989 ◽

Vol 62 (03) ◽

pp. 955-961 ◽

Cited By ~ 4

Author(s):

Ian S Watts ◽

Rebecca J Keery ◽

Philip Lumley

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Surface Membrane ◽

Blood Platelet ◽

Human Platelets ◽

Membrane Glycoprotein ◽

Platelet Surface ◽

Differential Ability ◽

Blood Platelet Aggregation

SummaryWe have investigated the effect of two procedures that modify human platelet surface membrane glycoprotein (Gp) IIb and IIIa complexes upon whole blood platelet aggregation to a range of agonists. (A) Irreversible disruption of complexes by temporary (30 min) Ca2+-deprivation with EGTA at 37° C. (B) Binding of a monoclonal antibody M148 to the complex. EGTA exposure abolished aggregation to ADP, adrenaline and PAF. In contrast, full aggregation curves to collagen and U-46619 could still be established. EGTA exposure reduced M148 binding to platelets by 80%. Excess M148 abolished aggregation to ADP, PAF, collagen and U-46619. However, upon removal of unbound antibody from platelets full aggregation curves to collagen and U-46619 but not to ADP and PAF could be re-established. Thus human platelet aggregation to ADP, PAF and adrenaline appears absolutely dependent upon surface membrane GpIIb/IIIa complexes. In contrast, collagen and U-46619 cause expression of an additional distinct pool of Gp complexes inaccessible to EGTA and M148 in unstimulated platelets which is intimately involved in aggregation to these agonists.

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

Blood ◽

10.1182/blood-2018-99-117070 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4971-4971

Author(s):

Yifeng WU ◽

Alan D Michelson ◽

Andrew L Frelinger

Keyword(s):

Nitric Oxide ◽

Platelet Aggregation ◽

Platelet Activation ◽

Plasma Proteins ◽

Research Funding ◽

No Donor ◽

Platelet Surface ◽

Synergistic Inhibition ◽

Dependent Inhibition ◽

Deta Nonoate

Abstract 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.

Direct Inhibitory Mechanisms of Halothane on Human Platelet Aggregation

Anesthesiology ◽

10.1097/00000542-199607000-00014 ◽

1996 ◽

Vol 85 (1) ◽

pp. 96-106 ◽

Cited By ~ 26

Author(s):

Shinji Kohro ◽

Michiaki Yamakage

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Adenosine Monophosphate ◽

Inhibitory Effect ◽

Surface Glycoprotein ◽

Dependent Manner ◽

Glycoprotein Ib ◽

Platelet Surface ◽

Inhibitory Mechanisms

Background Although halothane directly inhibits platelet aggregation, the mechanisms of this effect are still unknown. The current study aimed to clarify the inhibitory mechanisms of halothane on thrombin-induced human platelet aggregation by measuring (1) platelet-surface glycoprotein Ib expression, (2) the concentration of intracellular free Ca2+ ([Ca2+]i) measured simultaneously with aggregation, (3) the concentration of intracellular inositol 1,4,5-triphosphate, and (4) the concentration of intracellular cyclic 3',5'-adenosine monophosphate ([cAMP]i). Methods Washed platelet suspensions, obtained from healthy volunteers, were preincubated with halothane (0-2 mM) for 2 min and then exposed to 0.02 units/ml thrombin for 3 min. The glycoprotein Ib bound to fluorescein-labeled antibody was measured by fluorescence flow cytometry. [Ca2+]i was measured, simultaneously with aggregation, in Fura-2 (Ca2+ indicator)-loaded platelets by use of a fluorometer. Inositol 1,4,5-triphosphate and [cAMP]i were measured by radioimmunoassay. Results Halothane had no effect on glycoprotein Ib expression with or without thrombin. Halothane decreased the thrombin stimulated [Ca2+]i transient and inhibited platelet aggregation in a dose-dependent manner, both in the presence and in the absence of external Ca2+. Isoflurane had no apparent effect on either platelet aggregation or [Ca2+]i in the absence of external Ca2+. Halothane inhibited the increase in inositol 1,4,5-triphosphate induced by thrombin. Halothane moderately but significantly increased [cAMP]i, but the adenylate cyclase activator forskolin (which has the same inhibitory ability on aggregation as halothane) increased [cAMP]i to a much greater extent than did halothane. Conclusions Halothane inhibits thrombin-induced human platelet aggregation by decreasing [Ca2+]i without inhibiting agonist-receptor binding; the inhibitory effect of halothane on [Ca2+]i might be mediated by a decrease in inositol 1,4,5 triphosphate and in part by an increase in [cAMP]i.

Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway

10.1160/th12-09-0636 ◽

2013 ◽

Vol 109 (06) ◽

pp. 1120-1130 ◽

Cited By ~ 14

Author(s):

Wen-Ying Chuang ◽

Po-Hsiung Kung ◽

Chih-Yun Kuo ◽

Chin-Chung Wu

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Thrombus Formation ◽

Critical Factor ◽

Underlying Mechanism ◽

Akt Pathway ◽

Flow Conditions ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

SummarySulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

Chlorobutanol, a Preservative of Desmopressin, Inhibits Human Platelet Aggregation and Release In Vitro

10.1055/s-0038-1647339 ◽

1990 ◽

Vol 64 (03) ◽

pp. 473-477 ◽

Cited By ~ 4

Author(s):

Shih-Luen Chen ◽

Wu-Chang Yang ◽

Tung-Po Huang ◽

Shiang Wann ◽

Che-ming Teng

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Atp Release ◽

Free Calcium ◽

Dependent Manner ◽

Antiplatelet Effect ◽

Acid Pathway ◽

Arachidonic Acid Pathway

SummaryTherapeutic preparations of desmopressin for parenteral use contain the preservative chlorobutanol (5 mg/ml). We show here that chlorobutanol is a potent inhibitor of platelet aggregation and release. It exhibited a significant inhibitory activity toward several aggregation inducers in a concentration- and time-dependent manner. Thromboxane B2 formation, ATP release, and elevation of cytosolic free calcium caused by collagen, ADP, epinephrine, arachidonic acid and thrombin respectively were markedly inhibited by chlorobutanol. Chlorobutanol had no effect on elastase- treated platelets and its antiplatelet effect could be reversed. It is concluded that the antiplatelet effect of chlorobutanol is mainly due to its inhibition on the arachidonic acid pathway but it is unlikely to have a nonspecitic toxic effect. This antiplatelet effect of chlorobutanol suggests that desmopressin, when administered for improving hemostasis, should not contain chlorobutanol as a preservative.

Inhibitory Effect of Staphylokinase on Platelet Aggregation

10.1055/s-0038-1649679 ◽

1993 ◽

Vol 70 (05) ◽

pp. 834-837 ◽

Cited By ~ 6

Author(s):

Akira Suehiro ◽

Yoshio Oura ◽

Motoo Ueda ◽

Eizo Kakishita

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Degradation Products ◽

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Effective Concentration ◽

Inhibit Platelet Aggregation ◽

Platelet Suspension ◽

Washed Platelet ◽

Human Platelet Aggregation

SummaryWe investigated the effect of staphylokinase (SAK), which has specific thrombolytic properties, on human platelet aggregation. Platelet aggregation induced with collagen was observed following preincubation of platelets in platelet-rich plasma (PRP) or washed platelet suspension (WP) with SAK at 37° C for 30 min. SAK inhibited platelet aggregation in PRP only at the highest examined concentration (1 x 10-4 g/ml). Although SAK did not inhibit platelet aggregation in WP which contained fibrinogen, it did when the platelets had been preincubated with SAK and plasminogen. The most effective concentration in WP was 1 x 10-6 g/ml. The effect could be inhibited by adding aprotinin or α2-antiplasmin. The highest generation of plasmin in the same preincubation fluid was detected at 1 x 10-6 g/ml SAK. We concluded that SAK can inhibit platelet aggregation in WP by generating plasmin and/or fibrinogen degradation products, but is only partially effective in PRP because of the existence of α2-antiplasmin.

The Effect of the Phospholipase Inhibitor Mepacrine on Platelet Aggregation, the Platelet Release Reaction and Fibrinogen Binding to the Platelet Surface

10.1055/s-0038-1650183 ◽

1981 ◽

Vol 45 (03) ◽

pp. 257-262 ◽

Cited By ~ 36

Author(s):

P D Winocour ◽

R L Kinlough-Rathbone ◽

J F Mustard

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Surface ◽

Release Reaction ◽

Fibrinogen Binding ◽

Platelet Release

SummaryWe have examined whether inhibition by mepacrine of freeing of arachidonic acid from platelet phospholipids inhibits platelet aggregation to collagen, thrombin or ADP, and the release reaction induced by thrombin or collagen. Loss of arachidonic acid was monitored by measuring the amount of 14 C freed from platelets prelabelled with 14 C-arachidonic acid. Mepacrine inhibited 14 C loss by more than 80% but did not inhibit thrombin-induced platelet aggregation and had a small effect on release. ADP-induced platelet aggregation did not cause 14 C loss. Mepacrine inhibited ADP-induced platelet aggregation by inhibiting the association of fibrinogen with platelets during aggregation. The effect of mepacrine on fibrinogen binding could be considerably decreased by washing the platelets but the inhibition of 14 C loss persisted. Platelets pretreated with mepacrine and then washed show restoration of aggregation to collagen. Thus, mepacrine has two effects; 1. it inhibits phospholipases, 2. it inhibits fibrinogen binding. Freeing of arachidonic acid is not necessary for platelet aggregation or the release reaction.

Synergism Between Prostacyclin (PGI2) and Prazosin on Human Platelet Aggregation

10.1055/s-0038-1657131 ◽

1982 ◽

Vol 47 (01) ◽

pp. 76-76 ◽

Cited By ~ 1

Author(s):

P K Moore

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Human Platelet Aggregation

Heparin Counteracts the Antiaggregating Effect of Prostacyclin by Potentiating Platelet Aggregation

10.1055/s-0038-1657326 ◽

1983 ◽

Vol 49 (02) ◽

pp. 081-083 ◽

Cited By ~ 13

Author(s):

Vittorio Bertelé ◽

Maria Carla Roncaglioni ◽

Maria Benedetta Donati ◽

Giovanni de Gaetano

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Specific Interaction ◽

Inhibitory Effect ◽

Effective Inhibitor ◽

Inhibitory Potency ◽

Human Platelet Aggregation

SummaryIt has recently been reported that heparin neutralizes the inhibitory effect of prostacyclin (PGI2) on human platelet aggregation. The mechanism of this interaction has not yet been unequivocally established. We present here evidence that heparin (Liquemin Roche) does not react directly with PGI2 but counteracts its inhibitory effect by potentiating platelet aggregation. In the absence of heparin, PGI2 was a less effective inhibitor of platelet aggregation induced by the combination of ADP and serotonin than by ADP alone. Moreover, the inhibitory effect of PGI2 was similarly reduced when increasing the concentrations of ADP (in the absence of heparin). The lack of a specific interaction between heparin and PGI2 is supported by the observation that, in the presence of heparin, other prostaglandins such as PGD2 and PGE1, and a non-prostanoid compound such as adenosine also appeared to lose their inhibitory potency. It is concluded that heparin opposes platelet aggregation inhibitory effect of PGI2 by enhancement of platelet aggregation.