Potentiating Effect Of 5, 8, 11-Eicosatrienoic Acid (20:3ω9) On Platelet Aggregation

1981 ◽  
Author(s):  
M Lagarde ◽  
M Burtin ◽  
H Sprecher ◽  
M Dechavanne ◽  
S Renaud
Keyword(s):  
Saturated Fat ◽  
Eicosatrienoic Acid ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Lipoxygenase Pathway ◽  
Fatty Acid Deficiency ◽  
Vitro Effect ◽  
Induced Aggregation ◽  
Saturated Fat Intake

The in vitro effect of 20:3ω9 (5×10-6M) was tested on human platelet suspensions, with aggregation induced by thrombin, ionophore A23187, ADP, collagen and arachidonic acid. When 20:3ω9 was added simultaneously with the aggregating agent, the response was increased by 100 to 300 % with all agents. It was solely to thrombin and ionophore that the aggregation was increased (by 200 to 300 %) when 20:3ω9 was preesterified in the platelet phospholipids by a 2 hour incubation period and subsequent platelet washing. Aspirin at a concentration inhibiting cyclooxygenase activity, did not block the potentiating effect of 20:3ω9. However, that effect was inhibited by a higher aspirin concentration, also inhibiting the peroxydase from the lipoxygenase pathway. Monohydroperoxy (HOO) - and monohydroxy (HO) - 20:3ω9 have been prepared from human platelets and purified by TLC. When added to platelets in vitro, solely the HO-20:3ω9 (0.2×10-6M) increased by several folds the response to thrombin and ionophore. Finally, both thrombin and ionophore induced the formation of HO-20:3ω9 (detected by a radiochemical technique) from platelets enriched with 20:3ω9. Thus, an increase level of 20:3ω9 in the platelet phospholipids seems to markedly enhance the susceptibility of platelets to aggregation by thrombin (and ionophore), probably through the lipoxygenase end product. Consequently the increase in the susceptibility of platelets to thrombin induced aggregation reported in essential fatty acid deficiency as well as with high saturated fat intake, both in man and in animals, might be due to the higher level of 20:3ω9 in the platelet phospholipids, which has been observed under those conditions.

The In Vitro Effect of Ticlopidine on Fibrinogen and Factor VIII Binding to Human Platelets

1981 ◽  
Vol 46 (03) ◽  
pp. 590-592 ◽  
Author(s):  
Helen Lee ◽  
R C Paton ◽  
C Ruan ◽  
J P Caen
Keyword(s):  
Factor Viii ◽  
Antiplatelet Agent ◽  
Human Platelets ◽  
Dependent Manner ◽  
Fibrinogen Binding ◽  
The Mean ◽  
In Vitro Effects ◽  
Vitro Effect ◽  
Induced Aggregation

SummaryThe mode of action of the antiplatelet agent ticlopidine is not yet fully understood. Its multiple effects on platelet function include prolongation of the bleeding time, reduction in primary and secondary Waves of ADP-induced aggregation and inhibition of collagen and thrombin-induced aggregation. We have studied the in vitro effects of ticlopidine on fibrinogen binding induced by ADP and adrenaline as well as factor VIII/vWF binding induced by ristocetin. 125I-fibrinogen binding was measured in suspensions of freshly washed normal platelets stimulated by 10 μM ADP or 10 μM adrenaline. The binding of 125I-factor VIII/vWF in the presence of 1 mg/ml ristocetin was measured in both washed and paraformaldehyde-fixed platelets. Ticlopidine at final concentrations of 200, 100, 50 and 25 μM inhibited both ADP and adrenaline-induced fibrinogen binding in a dose-dependent manner. The mean % inhibition of ADP-induced fibrinogen binding was 82, 73, 42 and 32 respectively. The mean % inhibition of adrenaline induced fibrinogen binding was 86, 82, 60 and 35 respectively. In contrast, the factor VIII/vWF binding was unaffected by ticlopidine at all concentrations except at 200 μM using fresh platelets where a slight inhibition (19%) was observed.These results suggest that ticlopidine either inhibits platelet activation and consequently fibrinogen binding, or inhibits the binding directly, presumably by having an effect on the specific configuration of the platelet membrane required for normal fibrinogen binding.

Monosubstituted Coumarins Inhibit Epinephrine-Induced Platelet Aggregation Antiplatelet Effect of Monosubstituted Coumarins

2021 ◽  
Vol 19 ◽  
Author(s):  
Fausto Alejandro Jiménez-Orozco ◽  
Sergio Galicia-Zapatero ◽  
Edgar López-López ◽  
José L. Medina-Franco ◽  
Fernando León Cedeño ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
In Silico ◽  
Drug Targets ◽  
Platelet Reactivity ◽  
Antiplatelet Agents ◽  
Human Platelets ◽  
In Silico Studies ◽  
Vitro Effect ◽  
Induced Aggregation

Aim: Evaluate the in vitro effect of coumarin and 15 monosubstituted derivatives on the inhibition of human platelet aggregation induced by various pro-aggregatory agonists, particularly by epinephrine. Background: The emergence of residual platelet reactivity during the use of conventional antiplatelet agents (acetylsalicylic acid and clopidogrel) is one of the main causes of double therapy´s therapeutic failure. Platelet adrenoceptors participate in residual platelet reactivity. Therefore, it is necessary to develop new antiplatelet agents that inhibit epinephrine-induced platelet aggregation as a new therapeutic strategy. Information on the antiplatelet activity of coumarins in inhibiting epinephrine-induced aggregation is limited. Objective: Establish the structure-activity relationship (SAR) of coumarin derivatives with hydroxy, methoxy, and acetoxy groups in different positions of the coumarin nucleus to identify the most active molecules. Using in silico studies, suggest potential drug targets to which the molecules bind to produce antiplatelet effects. Methods: The platelet aggregation was performed using a Lumi-aggregometer; the inhibitory activity of 16 compounds were evaluated by inducing the aggregation of human platelets (250 × 103/μl) with epinephrine (10 µM), collagen (2 µg / ml) or ADP (10 µM). The aggregation of controls platelets was considered 100% of the response for each pro-aggregatory agonists. Results: Eleven molecules inhibited epinephrine-induced aggregation, with 3-acetoxycoumarin and 7-methoxycoumarin being the most active. Only coumarin inhibited collagen-induced platelet aggregation, but no molecule showed activity when using ADP as an inducer. Conclusions : In silico studies suggest that most active molecules might have antagonistic interactions in the adrenoceptors α2 and β2. The antiplatelet actions of these coumarins have the potential to reduce residual platelet reactivity and thus contribute to the development of future treatments for patients who do not respond adequately to conventional agents.

The In Vitro Effect Of Ticlopidine On Fibrinogen And Factor VIII Binding To Human Platelets

1981 ◽  
Author(s):  
H Lee ◽  
R C Paton ◽  
C Ruan
Keyword(s):  
Factor Viii ◽  
Human Platelets ◽  
Dependent Manner ◽  
Prolonged Bleeding Time ◽  
Fibrinogen Binding ◽  
The Mean ◽  
In Vitro Effects ◽  
Vitro Effect ◽  
Induced Aggregation

Ticlopidine is an anti-aggregating drug whose mode of action is not yet fully understood. Its multiple effects on platelet function include prolonged bleeding time, reduction in primary and secondary waves of ADP-induced aggregation and inhibition of collagen and thrombin- induced aggregation. We have studied the in vitro effects of ticlopidine on fibrinogen binding induced by ADP and adrenaline as well as Factor VIII/vWF binding induced by ristocetin. 125I fibrinogen binding was measured in suspensions of freshly-washed normal platelets stimulated by 10 μM ADP or 10 μM adrenaline. The binding of 125I-Factor VIII/vWF in the presence of 1 mg/ml ristocetin was measured in both washed and paraformaldehyde-fixed platelets. Ticlopidine at final concentrations of 200, 100, 50 and 25 μM inhibited both ADP and adrenaline-induced fibrinogen binding in a dose-dependent manner. The mean % inhibition of ADP-induced fibrinogen binding was 82, 73, 42 and 32 respectively. The mean % inhibition of adrenaline-induced fibrinogen binding was 86, 82, 60 and 35 respectively. In contrast, the Factor VIII/vWF binding was unaffected by ticlopidine at all concentrations except at 200 μM using fresh platelets where a slight inhibition (19 %) was observed.These results suggest that ticlopidine either inhibits platelet activation and consequently fibrinogen binding, or inhibits the binding directly, presumably by having an effect on the specific configuration of the platelet membrane required for normal fibrinogen binding.

AUSTRALIAN SNAKE VENOMS AND THEIR EFFECT UPON HUMAN PLATELETS

1987 ◽  
Author(s):  
L R Marshall ◽  
R P Herrmann
Keyword(s):  
Human Platelets ◽  
Lag Phase ◽  
Maximal Effect ◽  
Snake Venoms ◽  
Dual Channel ◽  
Common Clinical Presentation ◽  
Vitro Effect ◽  
Induced Aggregation ◽  
Phosphate Buffered Saline

The in vitro effect of Australian snake venoms on human citrated plasma has been documented and the majority induce coagulation, in keeping with the common clinical presentation of D.I.C. following envenomation. The effect of these venoms upon platelets in vitro has hitherto not been studied extensively and clinical evidence is conflicting, some cases with thrombocyto-paenia have been reported. Twenty Australian venoms were tested, 19 elapids and one hydrophiid (Enhydrina schistosa). Four crotalid snake venoms from the Americas and S.E. Asia were also tested. All of the venoms (1 mg/ml) were investigated for t^eir ability to aggregate both fresh washed platelets (200 × 109/l) resuspended in modified Ardlie’s buffer pH 7.35 and formaldehyde fixed platelets (200 × 109/l) in phosphate buffered saline pH 7.35 using a dual channel Chronolog aggregometer. Samples were taken for electron microscopy (EM).All elapid venoms induced aggregation in fresh platelets, some only minimally and often after a long lag phase. EM studies revealed only clumping without degranulation of the platelets. This was in marked contrast to the crotalid venoms where rapid aggregation and gross degranulation occurred. The hydrophiid venom failed to induce aggregation of the fresh platelets, however upon addition of normal plasma gross aggregation and degranulation was demonstrated. Aggregation of fixed platelets was negligible in the presence of the majority of elapid and the hydrophiid venoms. The crotalid venoms however did induce aggregation, although to a lesser extent than with the fresh platelets.The elapid venoms, along with the others studied, required metabolically active platelets to exert their maximal effect. Crotalid and hydrophiid venoms were more active against platelets than the elapid venoms. The hydrophiid venom’s action on platelets was unique in that a plasma co-factor appeared to be required and this is the subject of further investigations.

Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro

1976 ◽  
Vol 36 (02) ◽  
pp. 376-387 ◽  
Author(s):  
Teruhiko Umetsu ◽  
Kazuko Sanai ◽  
Tadakatsu Kato
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Rabbit Platelets ◽  
Β Blocker ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Platelet Functions

SummaryThe effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.

Effects of Dipyrone on Prostaglandin Production by Human Platelets and Cultured Bovine Aortic Endothelial Cells

1983 ◽  
Vol 49 (02) ◽  
pp. 132-137 ◽  
Author(s):  
A Eldor ◽  
G Polliack ◽  
I Vlodavsky ◽  
M Levy
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Competitive Inhibition ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Aortic Endothelial

SummaryDipyrone and its metabolites 4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoan- tipyrine inhibited the formation of thromboxane A2 (TXA2) during in vitro platelet aggregation induced by ADP, epinephrine, collagen, ionophore A23187 and arachidonic acid. Inhibition occurred after a short incubation (30–40 sec) and depended on the concentration of the drug or its metabolites and the aggregating agents. The minimal inhibitory concentration of dipyrone needed to completely block aggregation varied between individual donors, and related directly to the inherent capacity of their platelets to synthesize TXA2.Incubation of dipyrone with cultured bovine aortic endothelial cells resulted in a time and dose dependent inhibition of the release of prostacyclin (PGI2) into the culture medium. However, inhibition was abolished when the drug was removed from the culture, or when the cells were stimulated to produce PGI2 with either arachidonic acid or ionophore A23187.These results indicate that dipyrone exerts its inhibitory effect on prostaglandins synthesis by platelets or endothelial cells through a competitive inhibition of the cyclooxygenase system.

Effect Of Solvents On Indium-111 Oxine Transport In Human Platelets And On Platelet Function In Vitro

1981 ◽  
Author(s):  
T Tsukada
Keyword(s):  
Platelet Function ◽  
Kinetic Constant ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Polysorbate 80 ◽  
Autologous Plasma ◽  
Indium 111 ◽  
Induced Aggregation ◽  
Water Space

Mechanism of Indium-111 oxine(In) transport in human platelets in buffered saline and the effect of In-labeling on platelet function were studied using In dissolved in 25% of ethanol in saline (In-ES) or 0.01% of polysorbate 80 in HEPES buffer(In-PH). Increase in temperature up to 37° C progressively enhanced the transport of In-ES, while transport of In-PH reached to plateau at 15°C. A states of equilibrium was not reached during 2 hr incubation at 22°C in In-ES. Uptake of In-PH reached to plateau after only 15 min of incubation. Distribution of In taken up by platelets in InES was 57% in cytosol and 27% in stroma, while in In-PH 69% in stroma and 22% in cytosol. 88% of In in cytosol was bound to lipids(46% in cholesterol and 27% in PS+PI). 82% of In in stroma was found in PS+PI fraction.The fact that the ratio of free In between the platelet water space and the outside medium after 30 min of incubation at up to 0.1 uM of In exceeded unity, suggests satura- , ble component of In transport prevails at this concentration in In-ES and In-PH. Kinetic constant could be calculated, Kt= 2nM, Vmax= 2.5 pmol/min/ml in In-ES, and Kt= InM, Vmax=0.7 pmol/min/ml in In-PH.Elution of In from radiolableled platelets in autologous plasma incubated at 37°C for 5 hr was less than 10% in the case of In-ES and 56% in the case of In-PH. Less than 3% of labeled-In was eluated from platelets in collagen-induced aggregation and 4-7% of In was eluated in thrombin-induced aggregation.Although 0.3% of ethanol and/or 6nM of oxine have no inhibitory effect of platelet aggregation, collagen-induced aggregation and release reaction of In-labeled platelet was impaired. 0.003% of polysorbate 80 itself abolished completely the aggregability of platelets by collagen or thrombin.It is concluded In-PH is unsuitable for platelet labeling. In-111 oxine also seems to have problems which Cr-51 has, i.e. inhomogenous distribution of In in a platelet population, elution of In from labeled platelets in circulation.

Effects of gas bubbling and other forms of convection on platelets in vitro

1989 ◽  
Vol 67 (3) ◽  
pp. 1250-1255 ◽  
Author(s):  
D. M. Pickles ◽  
D. Ogston ◽  
A. G. MacDonald
Keyword(s):  
Shear Stress ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Air Plasma ◽  
Physiological Level ◽  
Gentle Agitation ◽  
Rocking Motion ◽  
Experimental Treatments ◽  
Induced Aggregation

Citrated platelet-rich human plasma was subjected to one of three experimental treatments at 37 degrees C for 15 min: stirring, bubbling (with stirring), and gentle agitation achieved by a rocking motion. The last two were “equiconvective” as judged by equilibration rates with CO2 and O2 but presumably differed in the shear stress they imposed on the cells. Stirring platelets in normal air or 5% CO2-air caused no significant aggregation. Bubbling air through platelet-rich plasma increased its pH and marked aggregation occurred. Bubbling CO2-air caused the platelet-rich plasma pH to attain its physiological level of 7.4 with less aggregation. In both cases, subsequent ADP-induced aggregation was diminished. Rocking (without stirring) in the presence of CO2-air caused negligible aggregation in platelets and an enhanced response to ADP. Because of the marked difference between the two equiconvective treatments, bubbling and rocking, the main factor in activating the human platelets is suggested to be shear stress (potentiated by high pH), with perhaps a lesser contribution from the air-plasma interface.

Inhibition Of Platelet Aggregation, Malonyldialdehyde And Thromboxane Formation By Hydroperoxides Of Arachidonic Acid

1981 ◽  
Author(s):  
D Aharonv ◽  
J B Smith ◽  
M J Silver
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Lipoxygenase Pathway ◽  
Dose Dependent Fashion ◽  
Induced Aggregation ◽  
Layer Chromatography ◽  
Dose Dependent ◽  
Thromboxane Formation

The arachidonate hydroperoxides 12-HPETE and 15-HPETE were biosynthesized from arachidonic acid using partially purified human platelet lipoxygenase or soybean lipoxidase respectively, and isolated by thin layer chromatography. Both compounds inhibited the arachidonic acid- induced aggregation of washed human platelets, suspended in calcium-free Krebs Henseleit solution, in a dose dependent fashion at concentrations between 1 and 50 uM. No inhibition was seen with up to 100 uM of these hydroperoxides when platelet -rich plasma was used. 12-HPETE (in micromolar concentrations) inhibited the formation of both thromboxane B2 (radioimmunoassay) and malonyldialdehyde (spectrophotometrie assay) when washed platelets were incubated with arachidonic acid. The 12-hydroxide, 12-HETE also inhibited platelet aggregation and thromboxane formation, but was less potent than 12-HPETE. We suggest that arachidonate hydroperoxide generated in platelets via the lipoxygenase pathway modulates platelet aggregation induced by arachidonic acid by inhibiting thromboxane formation.

scholarly journals PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses

2014 ◽  
Vol 111 (03) ◽  
pp. 508-517 ◽  
Author(s):  
Carol Dangelmaier ◽  
Bhanu Kanth Manne ◽  
Elizabetta Liverani ◽  
Jianguo Jin ◽  
Paul Bray ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein A ◽  
Human Platelets ◽  
Clot Retraction ◽  
Functional Responses ◽  
Atp Secretion ◽  
Dependent Protein ◽  
Induced Aggregation

Summary3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

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