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.

EFFECTIVENESS AND TOLERABILITY OF CV-3988, A SELECTIVE PAF ANTAGONIST, AFTER INTRAVENOUS ADMINISTRATION TO MAN

1987 ◽  
Author(s):  
J Arnout ◽  
A Van Hecken ◽  
I Delepeleire ◽  
Y Miyamoto ◽  
I Holmes ◽  
...  
Keyword(s):  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Biological Activities ◽  
Wide Spectrum ◽  
Platelet Activating Factor ◽  
Controlled Study ◽  
Dependent Manner ◽  
Double Blind ◽  
Health And Disease

Platelet activating factor (PAF) is a naturally occurring phospholipid with a wide spectrum of biological activities. Although PAF has been ascribed a potential role in various conditions including inflammation, asthma, glomerulonephritis and thrombosis, its precise function in physiologic/pathophysiologic processes remains unclear. The introduction of selective PAF receptor antagonists could represent a useful tool to extend our knowledge of the role of this mediator in health and disease.We have investigated the efficacy and tolerability of (RS)-2-methoxy-3-(octadecylcarbomoyloxy)propy1 2-(3-thiazolio)-ethyIphosphate (CV-3988, Takeda Chem. Ind), a selective PAF antagonist with structural analogies with PAF, after intravenous infusion in man in a double-blind, placebo-controlled study. The compound, in doses from 750 to 2,000 pg/kg, significantly reduced platelet sensitivity to PAF. The threshold aggregating concentration (TAC) of PAF was defined as the minimal concentration causing an irreversible aggregation with a maximal amplitude of at least 50% of the difference in light transmission between platelet rich plasma and platelet poor plasma. It increased in a dose-dependent manner reaching 3.6 times the basal TAC (p<0.0005) at the end and 2.60 times the basal TAC (p<0.0005) 4 hours after infusion of the highest dose. The TAC of PAF returned to the basal value within 24 hours after the end of the infusion.CV-3988 did not cause major side effects nor changes in blood pressure, pulse or respiratory rate. However, small but clinically insignificant changes in plasma haemoglobin and serum haptoglobin were seen at the end and four hours after the end of the infusion, indicating a slight haemolysis probably by high local concentrations at the infusion site.Our results indicate that, when adequate infusion volumes and infusion rates are used, CV-3988 can be safely administered to man and should be useful in elucidating the role of PAF in health and disease.

scholarly journals Fibrinogen-independent aggregation and deaggregation of human platelets: studies in two afibrinogenemic patients

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 221-226 ◽  
Author(s):  
M Cattaneo ◽  
RL Kinlough-Rathbone ◽  
A Lecchi ◽  
C Bevilacqua ◽  
MA Packham ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Plasma Proteins ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Human Fibrinogen ◽  
Control Subjects ◽  
Platelet Aggregates ◽  
And Control

Abstract Platelets from two afibrinogenemic patients were used to determine whether fibrinogen is essential for platelet aggregation and to examine whether released fibrinogen contributes to the stabilization of platelet aggregates when platelets have been induced to aggregate and release their granule contents by stimulation with thrombin. The addition of adenosine diphosphate (ADP) to platelet-rich plasma (PRP) or to suspensions of washed platelets from the afibrinogenemic patients caused the formation of small aggregates, which was either not inhibited or only slightly inhibited by the F(ab')2 fragments of an antibody to fibrinogen but was inhibited by an antibody (10E5) to glycoprotein IIb/IIIa. Thus there is a component of ADP-induced platelet aggregation that is not dependent on fibrinogen or other plasma proteins but is dependent on glycoprotein IIb/IIIa. There was little difference in the extent of aggregation and the release of granule contents of normal and afibrinogenemic platelets in response to the release-inducing agents collagen, platelet-activating factor (PAF), sodium arachidonate, or thrombin. With normal or afibrinogenemic platelets, aggregation by thrombin (0.2 U/mL or higher) was not inhibited by the F(ab')2 fragments of an antibody to human fibrinogen. Deaggregation by combinations of inhibitors of platelets aggregated by 1 U/mL thrombin showed no difference between platelets from afibrinogenemic and control subjects, indicating that released fibrinogen does not make a major contribution to the stabilization of platelet aggregates formed by thrombin stimulation.

scholarly journals Studies on the Biological Effects of Ozone: 10. Release of Factors from Ozonated Human Platelets

1999 ◽  
Vol 8 (4-5) ◽  
pp. 205-209 ◽  
Author(s):  
G. Valacchi ◽  
Velio Bocci
Keyword(s):  
Platelet Aggregation ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Platelet Rich Plasma ◽  
Biological Effects ◽  
Transforming Growth Factor Β1 ◽  
Human Platelets ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Tgf Β1

In a previous work we have shown that heparin, in the presence of ozone (O3), promotes a dose-dependent platelet aggregation, while after Ca2+chelation with citrate, platelet aggregation is almost negligible. These results led us to think that aggregation may enhance the release of platelet components. We have here shown that indeed significantly higher amount of platelet-derived growth factor (PDGF), transforming growth factor β1 (TGF-β1) and interleukin-8(IL-8) are released in a dose-dependent manner after ozonation of heparinised platelet-rich plasma samples. These findings may explain the enhanced healing of torpid ulcers in patients with chronic limbischemia treated with O3autohaemoteraphy (O3-AHT).

scholarly journals Use of increased concentrations of adenosinediphosphate for high residual platelet reactivity in patients with coronary heart disease

2021 ◽  
Vol 70 (1) ◽  
pp. 129-132
Author(s):  
O.A. Trubacheva ◽  
S.N. Belyaeva ◽  
T.E. Suslova ◽  
I.V. Petrova
Keyword(s):  
Coronary Heart Disease ◽  
Heart Disease ◽  
Platelet Aggregation ◽  
Antiplatelet Therapy ◽  
Light Transmission ◽  
Platelet Reactivity ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Transmission Curve ◽  
Platelet Suspension

Detection of a tendency to increased thrombosis in patients with coronary heart disease (CHD) is of important prognostic value in the selection of drugs aimed at achieving a persistent antithrombotic effect. The aim of the study was to evaluate the use of elevated ADP inducer concentrations to improve the accuracy of ADP-induced platelet aggregation in patients with coronary heart disease. Material and method. Material and method. We studied 48 patients with CHD who were on continuous double antiplatelet therapy for 6 months (aspirin 75mg and clopidogrel 75mg per day). The aggregation activity of the platelet suspension was studied using the Born method G. in the modification of Gabbasov Z. A. Platelet activity was evaluated by the degree of aggregation of platelet-rich plasma along the light transmission curve under the influence of the inducer adenosine diphosphate (ADP) at a concentration of 2 mmol/l and by its own patented method against the background of additional ADP application. Results. In patients, platelet aggregation decreased to 5-35% (p<0.005) compared to the standard values, which are 50-60%. The values of platelet aggregation with the additional introduction of the inducer of aggregation ADP in a ratio of 2:1 to 2 µmol/l for 1, 2, 3, and 4-minute registration of platelet aggregation, resulted in increased aggregation from 55% to 75% (p<0.001), indicating high residual platelet reactivity on the background of double antiplatelet therapy. Correlations of the degree of aggregation for elevated ADP concentrations with multivessel arterial lesion and dyslipidemia were also found, r=0.86 and r=0.92, respectively. Conclusion. The use of elevated concentrations of adenosine diphosphate in platelet aggregation in patients with ischemic heart disease increases the accuracy of assessing ADP-induced platelet aggregation against the background of dual antiplatelet therapy and contributes to the detection of high residual platelet reactivity.

scholarly journals Intracellular Ca2+ rise in human platelets induced by polymorphonuclear-leucocyte-derived cathepsin G

1992 ◽  
Vol 288 (3) ◽  
pp. 741-745 ◽  
Author(s):  
M Molino ◽  
M Di Lallo ◽  
G de Gaetano ◽  
C Cerletti
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Platelets ◽  
Polymorphonuclear Leucocyte ◽  
Cation Channel ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Bivalent Cation ◽  
Platelet Suspension

Cathepsin G, a serine protease released by polymorphonuclear-leucocyte azurophilic granules upon stimulation, activates human platelets, inducing an increase in intra-platelet Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (50-200 nM). The [Ca2+]i rises elicited by low (50-80 nM) cathepsin G concentrations in fura-2-loaded platelets showed a biphasic mode, with a first small peak followed by a greater and more prolonged Ca2+ transient. Higher (100-200 nM) cathepsin G concentrations induced a monophasic increase in intracellular Ca2+. Acetylsalicylic acid, nordihydroguaiaretic acid and ketanserin did not affect platelet activation by cathepsin G, whereas the ADP-scavenger system phosphocreatine/creatine kinase significantly decreased Ca2+ mobilization, platelet aggregation and 5-hydroxytryptamine secretion by cathepsin G. Preventing cathepsin G-induced platelet aggregation with the synthetic peptide RGDSP (Arg-Gly-Asp-Ser-Pro) did not significantly affect cathepsin G-induced Ca2+ transients. Ni2+ (4 mM), a bivalent-cation-channel inhibitor, decreased the cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either decreasing Ni2+ or increasing cathepsin G concentration. Preventing Ca2+ influx across the plasma membrane with 4 mM-EGTA totally abolished Ca2+ transients. However, EGTA also strongly decreased catalytic activity of cathepsin G, which is essential for platelet activation. Evidence of a rapid and sustained bivalent-cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 s or 3 min after cathepsin G. No accumulation of InsP3 could be detected when platelets were stimulated with cathepsin G. All these data indicate that cathepsin G induces a [Ca2+]i increase mainly through an influx across the plasma membrane. This massive Ca2+ entry is probably due to opening of receptor-operated channels and is amplified by endogenous ADP release.

scholarly journals Effect of platelet-activating factor (PAF) on human platelets

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 582-585 ◽  
Author(s):  
CM Chesney ◽  
DD Pifer ◽  
LW Byers ◽  
EE Muirhead
Keyword(s):  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Platelet Activating Factor ◽  
Creatine Phosphate ◽  
Major Effect ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Platelet Factor ◽  
Platelet Suspension ◽  
Second Wave

Abstract The effect of pure synthetic PAF (1–0-alkyl-2-acetyl-sn-glycero-3- phosphorylcholine) was studied in human platelets. PAF (0.2--2.0 micrograms/ml) produced a dose-dependent aggregation in human platelet- rich plasma (PRP) or platelet suspension obtained by gel-filtration (GFP). In addition, PAF (0.8 microgram/ml) induced secretion of 14C- serotonin (45% +/- 10%; mean +/- SD, n = 9) and platelet factor 4 (PF4) (12.89 +/- 3.81 micrograms/10(9) platelets; n = 9) in PRP. Similar results were obtained in GFP. Aggregation and release of 14C-serotonin and PF4 were inhibited by the metabolic inhibitors 2-deoxyglucose (16.7 mM) and antimycin-A (8.3 micrograms/ml), by the membrane-active drugs mepacrine (10 microM) and chlorpromazine (0.025 mM), by PGI2 (5.34 nM), which elevates intracellular c-AMP, by indomethacin (10 microM) or aspirin (100 microM). The ADP scavengers, creatine phosphate and creatine phosphokinase (CP/CPK), inhibited the second wave of aggregation but not secretion. These data suggest that the major effect of PAF on human platelets is mediated through the cyclo-oxygenase pathway and not through a third pathway.

scholarly journals Consumption of plant extract supplement reduces platelet activating factor-induced platelet aggregation and increases platelet activating factor catabolism: a randomised, double-blind and placebo-controlled trial

2019 ◽  
Vol 121 (09) ◽  
pp. 982-991 ◽  
Author(s):  
Lamprini Gavriil ◽  
Maria Detopoulou ◽  
Filio Petsini ◽  
Smaragdi Antonopoulou ◽  
Elizabeth Fragopoulou
Keyword(s):  
Platelet Aggregation ◽  
Healthy Volunteers ◽  
Plant Extracts ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Controlled Trial ◽  
Platelet Activating Factor ◽  
Double Blind ◽  
Metabolism Enzymes ◽  
Baseline Levels

AbstractPlatelet-activating factor (PAF) is a potent mediator of inflammation that plays a crucial role in atherosclerosis. The purpose of this study was to evaluate the effect of a dietary supplement containing mainly plant extracts on PAF actions and metabolism in healthy volunteers. A double-blind, placebo-controlled, 8 weeks’ duration study was performed. Healthy volunteers were randomly allocated into the supplement or the placebo group and fifty-eight of them completed the study. The supplement contained plant extracts (Aloe gel, grape juice, Polygonum cuspidatum) and vitamins. The activities of PAF metabolic enzymes: the two isoforms of acetyl-CoA:lyso-PAF acetyltransferase, cytidine 5’-diphospho-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-cholinephosphotransferase) and platelet-activating factor–acetylhydrolase (PAF-AH) in leucocytes and lipoprotein associated phospholipase-A2 in plasma were measured along with several markers of endothelial function. Platelet aggregation against PAF, ADP and thrombin receptor activating peptide was measured in human platelet-rich plasma by light transmission aggregometry. No difference was observed on soluble vascular cell adhesion molecule-1, sP-selectin and IL-6 levels at the beginning or during the study period between the two groups. Concerning PAF metabolism enzymes’ activity, no difference was observed at baseline between the groups. PAF-AH activity was only increased in the supplement group at 4 and 8 weeks compared with baseline levels. In addition, supplement consumption led to lower platelet sensitivity against PAF and ADP compared with baseline levels. However, a trial effect was only observed when platelets were stimulated by PAF. In conclusion, supplementation with plant extracts and vitamins ameliorates platelet aggregation primarily against PAF and secondarily against ADP and affects PAF catabolism by enhancing PAF-acetylhydrolase activity in healthy subjects.

scholarly journals Effect of platelet-activating factor (PAF) on human platelets

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 582-585
Author(s):  
CM Chesney ◽  
DD Pifer ◽  
LW Byers ◽  
EE Muirhead
Keyword(s):  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Platelet Activating Factor ◽  
Creatine Phosphate ◽  
Major Effect ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Platelet Factor ◽  
Platelet Suspension ◽  
Second Wave

The effect of pure synthetic PAF (1–0-alkyl-2-acetyl-sn-glycero-3- phosphorylcholine) was studied in human platelets. PAF (0.2--2.0 micrograms/ml) produced a dose-dependent aggregation in human platelet- rich plasma (PRP) or platelet suspension obtained by gel-filtration (GFP). In addition, PAF (0.8 microgram/ml) induced secretion of 14C- serotonin (45% +/- 10%; mean +/- SD, n = 9) and platelet factor 4 (PF4) (12.89 +/- 3.81 micrograms/10(9) platelets; n = 9) in PRP. Similar results were obtained in GFP. Aggregation and release of 14C-serotonin and PF4 were inhibited by the metabolic inhibitors 2-deoxyglucose (16.7 mM) and antimycin-A (8.3 micrograms/ml), by the membrane-active drugs mepacrine (10 microM) and chlorpromazine (0.025 mM), by PGI2 (5.34 nM), which elevates intracellular c-AMP, by indomethacin (10 microM) or aspirin (100 microM). The ADP scavengers, creatine phosphate and creatine phosphokinase (CP/CPK), inhibited the second wave of aggregation but not secretion. These data suggest that the major effect of PAF on human platelets is mediated through the cyclo-oxygenase pathway and not through a third pathway.

scholarly journals Fibrinogen-independent aggregation and deaggregation of human platelets: studies in two afibrinogenemic patients

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 221-226
Author(s):  
M Cattaneo ◽  
RL Kinlough-Rathbone ◽  
A Lecchi ◽  
C Bevilacqua ◽  
MA Packham ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Plasma Proteins ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Human Fibrinogen ◽  
Control Subjects ◽  
Platelet Aggregates ◽  
And Control

Platelets from two afibrinogenemic patients were used to determine whether fibrinogen is essential for platelet aggregation and to examine whether released fibrinogen contributes to the stabilization of platelet aggregates when platelets have been induced to aggregate and release their granule contents by stimulation with thrombin. The addition of adenosine diphosphate (ADP) to platelet-rich plasma (PRP) or to suspensions of washed platelets from the afibrinogenemic patients caused the formation of small aggregates, which was either not inhibited or only slightly inhibited by the F(ab')2 fragments of an antibody to fibrinogen but was inhibited by an antibody (10E5) to glycoprotein IIb/IIIa. Thus there is a component of ADP-induced platelet aggregation that is not dependent on fibrinogen or other plasma proteins but is dependent on glycoprotein IIb/IIIa. There was little difference in the extent of aggregation and the release of granule contents of normal and afibrinogenemic platelets in response to the release-inducing agents collagen, platelet-activating factor (PAF), sodium arachidonate, or thrombin. With normal or afibrinogenemic platelets, aggregation by thrombin (0.2 U/mL or higher) was not inhibited by the F(ab')2 fragments of an antibody to human fibrinogen. Deaggregation by combinations of inhibitors of platelets aggregated by 1 U/mL thrombin showed no difference between platelets from afibrinogenemic and control subjects, indicating that released fibrinogen does not make a major contribution to the stabilization of platelet aggregates formed by thrombin stimulation.

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.

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