Inhibition of TLR-4 Prevents From Sepsis-Related eNOS/NO Disturbance in Human Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5264-5264
Author(s):  
Liping Ma ◽  
Qiuhong Yang ◽  
Jianxing Chang ◽  
Yiqing Li ◽  
Liping Zhang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Healthy Volunteers ◽  
Conflicts Of Interest ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Serine Phosphorylation ◽  
No Production ◽  
Platelet Suspension ◽  
Lps Challenge

Abstract Abstract 5264 Objective Toll-like receptor 4 (TLR-4) has the ability to activate platelet and involve in intravascular coagulation, inflammatory cytokine and oxidative stressors release in sepsis. Nitric oxide (NO) synthesis in platelets is adjusted by iNOS and/or eNOS and contributes to platelet aggregation and adhesion. Our previous study has found that increased platelet aggregation in patients with sepsis was associated with the expression of TLR4 on platelets and NO synthesis in platelets. This study was to investigate whether inhibition of TLR-4 activation on platelets decreases eNOS(endothelial NO synthase)/NO disturbance-related platelet aggregation. Methods Blood samples were collected from 10 patients with severe sepsis and 10 healthy volunteers as controls. PRP(platelet-rich plasma) and PPP (platelet-poor plasma) were prepared for platelet aggregation, and platelet suspension (at a concentration of 2×108/mL platelets) for next experimemts. After the platelet suspension from healthy volunteers was incubated with L-Arginine (LA, 10 mM) alone, and LA and L-NAME (NΩ-Nitro-L-arginine methyl ester hydrochloride, 25mg/mL, a nonselective NOS inhibitor) for 1 hour, LPS(10.0μg/ml) was respectively added them. In separate experiments, the platelet suspension was preincubated with anti-TLR-4 Abs (10μg/mL) and then repeat above experiments. The levels of serine phosphorylation in eNOS (p-eNOS) and NO production, and platelet aggregation were determined with Western blotting, nitrate concentration analysis and platelet aggregometer, respectively. Results The protein levels of p-eNOS and NO production had 2.2-fold and 1.8-fold of increases in platelets from septic patients, and in vitro had 1.8-fold and 1.7-fold of increases in LA-incubated platelets stimulated with LPS as compared to controls. The p-eNOS expression and NO production were inhibited in the presence of L-NAME. Furthermore, thrombin-induced platelet aggregation was markedly promoted by LPS and L-NAME [(61 □ ‘98) %, (80□ ‘100)% versus (40□ ‘72)% of control) ], and the effect of LPS was abolished by LA pretreatment [(54□ ‘76)% ]. Blockade of TLR-4 didn't alter the elevated levels of p-eNOS and NO under LPS challenge, but the inhibition of L-NAME on p-eNOS and NO was significantly decreased. Importantly, blockade of TLR-4 significantly decreased platelet aggregation induced by LPS, and both LPS and L-NAME, but had no effects on LA pretreatment. Conclusion These data suggest that inhibition of TLR-4 may play a role in prevention from sepsis-induced platelet aggregation and maintaining platelet eNOS activity in sepsis. Disclosures: No relevant conflicts of interest to declare.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Interactions of Staphylokinase with Human Platelets

1995 ◽  
Vol 73 (03) ◽  
pp. 472-477 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Normal Plasma ◽  
Atp Secretion ◽  
Platelet Disaggregation ◽  
Activation Rate

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

Arginine vasopressin release from human platelets after irreversible aggregation

1990 ◽  
Vol 78 (1) ◽  
pp. 113-116 ◽  
Author(s):  
Giovanni Anfossi ◽  
Elena Mularoni ◽  
Mariella Trovati ◽  
Paola Massucco ◽  
Luigi Mattiello ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Arginine Vasopressin ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Vasopressin Release ◽  
Irreversible Aggregation ◽  
Local Release

1. The release of arginine vasopressin from human platelets was investigated in platelet-rich plasma after irreversible aggregation induced by adenosine 5′-pyrophosphate, collagen, sodium arachidonate, thrombin and adrenaline in vitro. 2. Arginine vasopressin levels were significantly higher in the supernatant from stimulated platelet-rich plasma than from unstimulated samples, reaching 3.5 × 10−12 (range 1.6–12.5 × 10−12) mol/l in the absence of an aggregating agent, 8.8 × 10−12 (range 4.2–17.5 × 10−12) mol/l after adenosine 5′-pyrophosphate, 13.7 × 10−12 (2.2–63.2 × 10−12) mol/l after collagen, 7.8 × 10−12 (2.2–14.6 × 10−12) mol/l after sodium arachidonate, 7.8 × 10−12 (2.2–16.3 × 10−12) mol/l after thrombin and 12.2 × 10−12 (4.8–32.1 × 10−12) mol/l after adrenaline. 3. An arginine vasopressin level of 18 × 10−12 mol/l, which can be achieved physiologically, increased the sensitivity of platelets to adenosine 5′-pyrophosphate and collagen in vitro; the same concentration of arginine vasopressin caused a potentiation of the effect of catecholamines on the response of platelets to sodium arachidonate. 4. These results indicate that intraplatelet arginine vasopressin is released during aggregation and suggest that a local release of arginine vasopressin could occur after complete platelet aggregation in vivo.

Endotoxin-Induced Platelet Activation in Human Whole Blood In Vitro

1988 ◽  
Vol 59 (03) ◽  
pp. 378-382 ◽  
Author(s):  
Gyorgy Csako ◽  
Eva A Suba ◽  
Ronald J Elin
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Atp Release ◽  
Human Platelets ◽  
Lag Phase ◽  
Human Whole Blood ◽  
Dose Dependent

SummaryThe effect of purified bacterial endotoxin was studied on human platelets in vitro. In adding up to 1 μg/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma. On the other hand, endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size. Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP. The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation. Thus, the activation of human platelets by “solubilized” endotoxin in plasma requires the presence of other blood cells. We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin.

Effect of Heparin on Aggregation, Release Reaction and Thromboxane A2synthesis in Human Platelets

1979 ◽  
Author(s):  
H.Y.K. Chuang ◽  
S.F. Mohammad ◽  
R.G. Mason
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Thromboxane A2 ◽  
Rabbit Aorta ◽  
Human Platelets ◽  
Appreciable Effect ◽  
Layer Chromatography ◽  
Aggregation Of Platelets ◽  
Platelet Functions

Studies on the effect of heparin on platelet functions have resulted in conflicting observations: heparin has been reported to cause aggregation of platelets, potentiate aggregation induced by various aggregating agents, or cause inhibition of aggregation. Using paritally purified heparin (beef lung or porcine mucosa) we observed that addition of heparin to citrated platelet rich plasma(C-PRP)potentiated the aggregation of platelets induced by ADP, epinephrine, or arachidonic acid. Presence of heparin in C-PRP results in complete inhibition of thrombin induced effects and partial inhibition of platelet aggregation induced by collagen. Presence of heparin in C-PRP also resulted in release of significantly higher concentrations of 14C-serotonin when platelets were challenged by appropriate aggregating agents. Those concentrations of heparin that resulted in potentiation of aggregation had no appreciable effect on c-AiMP or c-GMP levels of platelets. However, the presence of heparin results in a significant elevation of thromboxane A2 as determined by contraction of rabbit aorta or after conversion to thromboxane B2 by thin layer chromatography. These observations are of interest since increased production of thromboxane A2 in the presence of heparin may explain in part, the potentiation of platelet aggregation in vitro or thrombocytopenia observed frequently in patients receiving heparin intravenously Supported in part by grants HL22583 & 20679 from NHLBI of NIH.

Effects of Lead Acetate on Platelet Aggregation, Ultrastructure and Serotonin Release

1971 ◽  
Vol 26 (03) ◽  
pp. 455-466 ◽  
Author(s):  
R. B Davis ◽  
G. C Holtz
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Lead Acetate ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Blood Platelet ◽  
Human Platelets ◽  
Serotonin Release ◽  
Aggregation Of Platelets

SummaryThe effects of lead on blood platelet function and ultrastructure have been investigated. Lead acetate was injected intravenously in 27 rats and was added to rat and human platelet rich plasma in vitro. In vitro studies showed that concentrations of 2.5 × 10-3 M lead acetate reduced or blocked aggregation of rat and human platelets by adenosine diphosphate, collagen, and thrombin. Radioactive serotonin release from human platelets was inhibited by 10-4 M lead acetate. One hour after the injection of lead, platelet aggregation by thrombin was reduced, but platelet aggregation by adenosine diphosphate and collagen showed little change. Three days after lead, aggregation of platelets by collagen and thrombin was blocked and aggregation by adenosine diphosphate reduced. Thrombocytopenia was present 4 days after intravenous lead acetate. Electron micrographs of platelets showed that the mean number of mitochondria per platelet was increased, whereas alpha granules were reduced. Dense bodies were not significantly changed. Lead acetate affects platelet function in concentrations reported in human bone marrow in lead poisoning, and may relate to the binding of free sulfhydryl groups by lead.

Effects of an Epoxymethano Stable Analogue of Prostaglandin Endoperoxides (U-46619) on Human Platelets

1981 ◽  
Vol 45 (02) ◽  
pp. 103-106 ◽  
Author(s):  
G di Minno ◽  
V Bertelé ◽  
L Bianchi ◽  
B Barbieri ◽  
C Cerletti ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Healthy Volunteers ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Plasma Samples ◽  
Similar Extent ◽  
Reversible Aggregation ◽  
Thromboxane Production ◽  
Cyclic Endoperoxide ◽  
Aspirin Ingestion

SummaryU-46619 is a stable epoxymethano analogue of cyclic endoperoxide PGH2. We studied platelet aggregation, 14C-5HT release, LDH extrusion and prostaglandin and thromboxane production induced by this compound in platelet-rich plasma samples from 15 healthy volunteers. Each subject was tested both before and 90 min after aspirin (500 mg) ingestion. The threshold aggregating concentration (TAC) of U-46619 ranged between 0.18 and 0.90 µM. Aggregation was maximal between 40 and 60 min after venipuncture and was concentration-dependent. At concentrations below the TAC, U-46619 induced primary reversible aggregation with minimal 14C-5HT release. At TAC or higher concentrations aggregation and release proceded as parallel events. Neither prostaglandin or thromboxane production nor LDH loss could be detected in any of the situations tested. Aspirin ingestion did not modify the pattern of platelet responses. In unstirred, not aggregated platelet samples 14C-5HT release by U-46619 occurred to a similar extent as in stirred, aggregated platelet samples. Addition to citrated PRP of 0.3 mM Na2 EDTA blocked both aggregation and release induced by U-46619. This compound, however, aggregated washed platelets resuspended in Ca++-free-tyrode-albumin containing fibrinogen. The mechanism by which U-46619 activates platelets differs from that of all other common aggregating agents.

In Vitro Clumping of Human Platelets by Dextran

1968 ◽  
Vol 19 (01/02) ◽  
pp. 070-076 ◽  
Author(s):  
D. P Dhall ◽  
N. A Matheson
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Optical Density ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Time Dependent ◽  
Molecular Weights ◽  
Biphasic Effect ◽  
Delayed Time

SummaryPlatelet aggregation in vitro by dextrans of various molecular weights has been examined in platelet rich plasma and in suspensions of human platelets with a turbidi-metric method and microscopy. Addition of dextran to platelet rich plasma and platelet suspensions causes a biphasic effect on the optical density, consisting of an immediate decrease which seems independent of the molecular weight of the dextran and a delayed, time-dependent decrease influenced by it. Microscopically, platelet aggregation by dextran corresponds more closely to the delayed decrease. Aggregation of human platelets by dextran, for which stirring is necessary, is related to the final dextran concentration, is affected by the platelet population and is not spontaneously reversible. Platelet clumping by dextran is of particular interest since dextran has been shown to possess antithrombotic properties.

High glucose rapidly activates the nitric oxide/cyclic nucleotide pathway in human platelets via an osmotic mechanism

2005 ◽  
Vol 93 (03) ◽  
pp. 517-526 ◽  
Author(s):  
Paola Massucco ◽  
Luigi Mattiello ◽  
Isabella Russo ◽  
Monica Traversa ◽  
Gabriella Doronzo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Platelet Aggregation ◽  
Guanylate Cyclase ◽  
High Glucose ◽  
Cyclic Nucleotide ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
No Synthase ◽  
No Production ◽  
Cyclic Monophosphate

SummaryThe aim was to evaluate whether high glucose influences the nitric oxide (NO)/cyclic nucleotide pathway in human platelets via osmotic stress and to clarify the role of protein kinase C (PKC) in this phenomenon. The study was carried out on 33 healthy lean male volunteers, aged 28.3±1.3 years. NO synthesis was detected as L-citrulline production after L-arginine incubation in platelets incubated for 6 min with 22.0 mM D-glucose and isoosmolar concentrations of mannitol, L-glucose and fructose. To evaluate the influence of PKC, experiments with D-glucose and mannitol were repeated in the presence of the PKC-beta selective inhibitor LY379196, and NO synthesis was detected after a 6-min incubation with phorbol 12-mirystate 13-acetate (PMA), a non-selective PKC activator. Platelet content of guano-sine-3’, 5’-cyclic monophosphate (cGMP) and adenosine-3’, 5’-cyclic monophosphate (cAMP) was measured by radio-immunoassay in platelets incubated with iso-osmolar concentrations of D-glucose, mannitol, L-glucose and fructose. NO-dependence of cyclic nucleotide enhancements was evaluated by inhibiting NO synthase and guanylate cyclase. Platelet aggregation to ADP and collagen was evaluated in Platelet-Rich Plasma (PRP) in the presence of a 6-min incubation with D-glucose and mannitol, both without and with LY379196 and the guany-late cyclase inhibitor (H-[1, 2, 4]Oxadiazolo [4, 3-a]quinoxaline-1-one)(ODQ). Iso-osmolar concentrations of D-glucose, mannitol, L-glucose and fructose, and PMA increased NO production (p=0.0001). Effects of D-glucose and mannitol were blunted by LY379196. D-glucose and mannitol enhanced platelet cGMP and cAMP (p=0.0001) with a mechanism blunted by NO synthase and guanylate-cyclase inhibition, but did not modify platelet aggregation. In conclusion, glucose activates the NO/cyclic nucleotide pathway in human platelets with an osmotic mechanism mediated by PKC-beta.

PLATELET-ACTIVATING FACTOR (PAF)-INDUCED INTRACELLULAR Ca MOBILIZATION IN HUMAN PLATELETS.

1987 ◽  
Author(s):  
K Matsuno ◽  
F Katabami ◽  
M Koyama ◽  
K Abe ◽  
K Sakurada ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Dependent Manner ◽  
Platelet Suspension ◽  
Fura 2 ◽  
Intracellular Ca ◽  
Specific Antagonist

PAF-induced intracellular Ca2+ mobilization and platelet aggregation were investigated in human platelets. Cytosolic free Ca2+ concentration ([Ca2+]cyt) was measured by using fluorescent 45Ca2+ probe quin2 and fura-2, and photoprotein aequorin. Ca2+ uptake was measured after stimulation by PAF. Platelet aggregation was studied by recording the change in light transmission with platelet rich plasma (PRP) or washed platelet suspension (WPS).These three Ca2+ -indicators could determine the elevation of [Ca2+ ]cyt that was stimulated by PAF in the presence of extra- cellular Ca2+ (quin2 method: 98.2nM to 289.7nM; fura-2 method: 102.OnM to 351.4nM; aequorin method: 4.1μM to 8.2μM). In the absence of extracellular Ca2+ , however, little elevation of [Ca2+ ]cyt was detected after stimulation by PAF. PAF could evoke the transient Ca2+ uptake.New PAF specific antagonist, ONO-6240 inhibited PAF-induced platelet aggregation at a concentration from lμM dose-dependently, whereas it didn’t inhibit collagen- and thrombin-induced platelet aggregation at a concentration of lOOyM. ONO-6240 inhibited PAF- induced increase in [Ca2+ ]cyt in a dose-dependent manner as deter mined by these Ca2+ -indicators, as well as platelet aggregation.These results suggest the increase in [Ca2+ ]cyt is responsible for platelet aggregation induced by PAF, and the increased Ca2+ is derived from external Ca2+ influx chiefly.

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