Concentration-dependent Inhibition Of Platelet Aggregation Research Articles

Saccharides from Arctium lappa L. root reduce platelet activation and thrombus formation in a laser injury thrombosis mouse model.

Arctium lappa L., also known as burdock, is a popular medicinal plant in traditional Chinese medicine due to its potential therapeutic properties. Saccharides from Arctium lappa L. root (ALR-S) have been extensively studied for their anti-inflammatory and anti-diabetes effects. Platelets play a pivotal role in thrombosis. The present study describes the effects of ALR-S on platelet activation and thrombosis using a laser injury thrombosis in vivo model. The study also measured the effects of ALR-S on platelet activation by analysing aggregation, ATP release, platelet spreading, adhesion and clot retraction in vitro. Specifically, the effects were ALR-S concentration-dependent inhibition of platelet aggregation and ATP release. Activated platelets pretreated with ALR-S showed diminished CD62P expression levels and fibrinogen binding, as measured by flow cytometry. ALR-S inhibited platelet spreading on fibrinogen and adhesion on collagen under shear. ALR-S attenuated platelet activation by decreasing oxidative stress and thrombus formation. These results demonstrated the antiplatelet effects of ALR-S, suggesting the antithrombotic and cardiovascular protective activities of ALR-S as a functional food.

Application of a Simple Microfluidic Chip Analysis Technology to Evaluate the Inhibitory Role of Protocatechuic Acid on Shear-Induced Platelet Aggregation.

This study aimed to develop a simple microfluidic chip analysis technology to study the inhibitory effect of protocatechuic acid on shear-induced platelet aggregation. The microfluidic chip designed in this study simulates 80% fixed narrow microchannels. This microchannel narrow model uses the finite element analysis module of the three-dimensional modeling software solidwork to analyze fluid dynamic behavior. Blood treated with protocatechuic acid at 1, 2, 4, 8, or 16 µg/mL was passed through the microchannel stenosis model at a shear rate of 10,000 s−1. The platelet adhesion and aggregation behaviors were then measured using fluorescence microscopy and observed in real time. Simultaneously, the antiplatelet aggregation effect of protocatechuic acid was analyzed using thromboelastography and photoelectric turbidimetry. The designed stenosis model of the microfluidic chip can produce a gradient of fluid shear rate, and the gradient of fluid shear rate can induce platelet aggregation. Under this model, the degree of platelet adhesion and aggregation increased as the shear rate increased. In the experimental concentration range of 0–8 µmol/mL, protocatechuic acid exerted a concentration-dependent inhibition of platelet aggregation. In contrast, thromboelastography and photoelectric turbidimetry failed to demonstrate an inhibitory effect. The microfluidic chip analysis technology developed in this study can be used to study the effect of protocatechin in inhibiting platelet aggregation induced by shear rate in vitro. This technology is simple to operate and can be used as a new type of antiplatelet aggregation analysis technology for screening studies of novel potential antiplatelet aggregation drugs.

Characterization of UBO-QIC as a Gαq inhibitor in platelets

Gαq plays an important role in platelet activation by agonists such as thrombin, adenosine diphosphate (ADP) and thromboxane. The significance of Gαq signaling in platelets was established using YM254890, a Gαq/11-specific inhibitor and Gαq knockout murine platelets. However, YM-254890 is no longer available for investigators and there is a need to characterize other Gαq inhibitors. The aim of this study is to characterize the specificity of a compound, {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4-methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)} (UBO-QIC), as a Gαq inhibitor in platelets. Human platelets treated with UBO-QIC showed a concentration-dependent inhibition of platelet aggregation and secretion by protease-activated receptors (PAR) agonists, U46619 and ADP. UBO-QIC also abolished Gαq pathway signaling events such as calcium mobilization and pleckstrin phosphorylation. UBO-QIC had no nonspecific effects on the Gα12/13 pathway since platelet shape change was intact in Gαq knockout murine platelets stimulated with PAR agonists in the presence of the inhibitor. In addition, UBO-QIC-treated platelets did not affect collagen-related peptide-induced platelet activation suggesting that this inhibitor had no non-specific effects on the GPVI pathway. Furthermore, Akt phosphorylation downstream of the Gαi and Gαz pathways, and vasodilator-stimulated phosphoprotein phosphorylation downstream of the Gαs pathway were not inhibited in UBO-QIC-treated platelets. UBO-QIC is a specific inhibitor for Gαq, which can be a useful tool for investigating Gαq-coupled receptor signaling pathways in platelets.

The active metabolite of prasugrel effectively blocks the platelet P2Y12 receptor and inhibits procoagulant and pro-inflammatory platelet responses

The aim of these studies was to investigate the extent of platelet P2Y12 receptor inhibition by the thienopyridine active metabolite of prasugrel, R-138727. Blood was taken from healthy volunteers and pre-incubated with R-138727 or cangrelor (AR-C66931MX). Platelet aggregation was assessed in platelet rich plasma (PRP) and whole blood (WB). Vasodilator stimulated phosphoprotein (VASP) phosphorylation, platelet procoagulant activity (annexin V binding and microparticle formation) and calcium mobilisation were measured by flow cytometry. Platelet-leukocyte co-aggregate formation and sCD40L release, both pro-inflammatory responses of platelets, were measured by flow cytometry and ELISA, respectively. P2Y12 receptor antagonism was determined using a radioligand binding assay (33P 2-MeSADP) in resting and stimulated platelets and the effects of clopidogrel administration were also assessed. R-138727 yielded concentration-dependent inhibition of platelet aggregation, VASP phosphorylation inhibition, procoagulant activity and pro-inflammatory responses. In the presence of R-138727 or cangrelor there was increased calcium reuptake following agonist stimulation. R-138727 30 µmol/L and cangrelor 1 µmol/L completely inhibited 33P 2-MeSADP binding, compared to partial inhibition following clopidogrel administration. Platelet activation and granule secretion did not expose an additional pool of P2Y12 receptors. Prasugrel's active metabolite effectively blocks the P2Y12 receptor with the highest concentrations tested yielding complete inhibition of P2Y12-mediated amplification of several important platelet responses.

Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation

Nitric oxide (NO) inhibits platelet aggregation primarily via a cyclic 3′5′-guanosine monophosphate (cGMP)-dependent process. Sildenafil is a phosphodiesterase type 5 (PDE5) inhibitor that potentiates NO action by reducing cGMP breakdown. We hypothesised that sildenafil would augment the inhibitory effects of NO on in vitro platelet aggregation. After incubation with sildenafil or the soluble guanylate cyclase inhibitor H-(1,2,4)oxadiazolo(4,3- a)quinoxallin-1-one (ODQ), collagen-mediated human platelet aggregation was assessed in the presence of two NO donors, the cGMP-dependent sodium nitroprusside (SNP) and the cGMP-independent diethylamine diazeniumdiolate (DEA/NO). SNP and DEA/NO caused a concentration-dependent inhibition of platelet aggregation. ODQ inhibited and sildenafil augmented the effect of SNP, and to a lesser extent the effect of DEA/NO. We conclude that sildenafil potentiates NO-mediated inhibition of platelet aggregation through blockade of cGMP metabolism and that PDE5 inhibitors may have important antiplatelet actions relevant to the prevention of cardiovascular disease.

Pharmacological profiles of R-96544, the active form of a novel 5-HT 2A receptor antagonist R-102444

We examined the pharmacology of (2 R,4 R)-4-hydroxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride (R-96544), the active form of a novel 5-HT 2A receptor antagonist, (2 R,4 R)-4-lauroyloxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride (R-102444). R-96544 produced a concentration-dependent inhibition of platelet aggregation induced by serotonin (5-hydroxytryptamine, 5-HT) alone or in combination with ADP in platelets from humans, monkeys, cats, rabbits, rats and mice. An intravenous administration of R-96544 to rabbits significantly inhibited ex vivo platelet aggregation induced by 5-hydroxytryptamine (5-HT) combined with epinephrine. An oral administration of R-102444 to rats also resulted in significant inhibition of ex vivo platelet aggregation, whereas R-102444 was ineffective in an in vitro platelet aggregation assay. These antiplatelet effects of R-96544 and R-102444 were more potent than those of two other 5-HT 2A receptor antagonists, sarpogrelate and its active metabolite (±)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-propanol hydrochloride (M-1). A binding study using cat platelet membranes showed that R-96544 has high affinity for 5-HT 2A receptors but no effect on non-serotonergic [ 3H]ketanserin-binding sites. R-96544 caused a parallel shift to the right of concentration–response curves for 5-HT in rat caudal artery contraction mediated by 5-HT 2A receptors. Schild plot analysis gave a pA 2 value of 10.4 with a slope near unity (1.04). R-96544 also inhibited 5-HT 2A receptor-mediated contraction of guinea pig trachea but not 5-HT 3 receptor-mediated contraction of guinea pig ileum and 5-HT 2B receptor-mediated contraction of rat fundus preparation. R-96544 (i.v.) attenuated the pressor responses evoked by 5-HT (15 μg/kg, i.v.) but not by phenylephrine (5 μg/kg, i.v.) and angiotensin II (0.1 μg/kg, i.v.), after ganglionic blockade in anesthetized spontaneously hypertensive rats. These results show that R-96544, the active form of R-102444, is a novel 5-HT receptor antagonist with potent, competitive, and 5-HT 2A-selective activity.

In vitro adrenaline and collagen-induced mobilization of platelet calcium and its inhibition by naftopidil, doxazosin and nifedipine.

The aim of the study was to obtain further information regarding the modes of action of doxazosin, naftopidil and nifedipine on platelet function. We conducted an in vitro study of drug influences on adrenaline and collagen-induced mobilization of platelet calcium. In the presence of fibrinogen (300 micrograms ml-1) both collagen (5 micrograms ml-1) and adrenaline (16 microM) stimulated the aggregation of washed platelets. Collagen induced a transient rise (+4.97 +/- 0.63 microM) in platelet Ca2+ concentration, [Ca2+]i, as measured using the photoprotein aequorin, which coincided with the onset of aggregation. Adrenaline induced a smaller rise (+3.6 +/- 0.96 microM) which, however, occurred after the onset of aggregation. Naftopidil, an alpha 1-adrenoreceptor antagonist produced a concentration-dependent inhibition of collagen-induced Ca2+ mobilization, maximum inhibition (22.9 +/- 4%, P < 0.05) occurring with 40 microM naftopidil. The inhibition of Ca2+ mobilization was not reflected by a concentration-dependent inhibition of platelet aggregation, although 40 microM naftopidil produced statistically significant inhibition (23.3 +/- 11.7%, P < 0.05). The adrenaline-induced rise in [Ca2+]i was inhibited dose dependently by naftopidil (e.g. 40 microM naftopidil, 100 +/- 0%, P < 0.05), as was aggregation (40 microM naftopidil, 100 +/- 0%, P < 0.05). Doxazosin, another alpha 1-adrenoreceptor blocker, inhibited Ca2+ mobilization induced by collagen to similar extents as for naftopidil (30 microM doxazosin, 17.4 +/- 2.5%, P < 0.05), but did not inhibit platelet aggregation. It also inhibited the adrenaline-induced rise in [Ca2+]i in a concentration-dependent manner (30 microM doxazosin, 37.6 +/- 13.7%, P < 0.05), significant inhibitions of platelet aggregation also being produced (30 microM, 49.6 +/- 17.2%, P < 0.05). As expected, the calcium channel blocker nifedipine produced concentration-dependent inhibitions of both collagen-induced Ca2+ mobilization (e.g. 28 microM nifedipine, 47.8 +/- 2.7%, P < 0.05) and aggregation (28 microM, 55.1 +/- 9.2%, P < 0.05). These data indicate that the alpha 1-adrenoreceptor blockers, naftopidil and doxazosin, inhibit Ca2+ mobilization, this mechanism being possibly the means whereby these drugs inhibit platelet aggregation.

Effects of Acetylsalicylic Acid on Inhibition of Ex Vivo Platelet Aggregation and Secretion by SKF 107260, a Novel GPIIb/IIIa Receptor Antagonist

SKF 107260 is a potent pentapeptide antagonist of the platelet membrane glycoprotein receptor GP IIb/IIIa. The in vitro platelet inhibitory effects of SKF 107260, acetylsalicylic acid (ASA), and their combination, on collagen-induced platelet aggregation and secretion (ATP release) were assessed in human whole blood. Additionally, the concentration-response relationships for these inhibitors were compared for males and females in order to explore gender differences in platelet responsiveness. SKF 107260 caused a concentration-dependent inhibition of platelet aggregation which was significant at concentrations > or = 30 nM. ASA also caused a concentration-dependent inhibition of platelet aggregation which was significant at concentrations > or = 1 mg/dl. The addition of ASA 1 mg/dl to increasing concentrations of SKF 107260 resulted in a more pronounced inhibition of platelet aggregation than when either agent was used alone. These data suggest a pharmacologic interaction, especially at SKF 107260 concentrations < or = 30 nM. Since ATP release was significantly inhibited at concentrations > or = 1 nM, platelet secretion appears to be more sensitive than aggregation to inhibition by SKF 107260. These data suggest that platelet secretion in response to collagen is dependent on the aggregation response mediated by GP IIb/IIIa. In conclusion, SKF 107260 is a potent inhibitor of both whole blood platelet aggregation and secretion and these anti-aggregatory effects may be augmented by concomitant ASA administration.

Vascular and anti‐platelet actions of 1,2‐ and 1,3‐glyceryl dinitrate

1. The aim of this study was to investigate whether two metabolites of glyceryl trinitrate (GTN), 1,2 and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN) could account for the pharmacological effects of GTN. To this end the formation of nitric oxide (NO) from 1,2- and 1,3-GDN in the presence of bovine aortic smooth muscle cells (SMC) or endothelial cells (EC) was studied. The effects of various thiols on NO formation from these dinitrates was also evaluated. 2. 1,2-GDN or 1,3-GDN (10(-10)-10(-5) M) caused a dose-dependent relaxation of rabbit aortic strips denuded of endothelium and precontracted with phenylephrine. The dinitrates were less than one tenth as potent as GTN. 3. Incubation of 1,2-GDN or 1,3-GDN (75-2400 microM) with SMC for 30 min led to a concentration-dependent increase in nitrite (NO2-) formation but this increase was less than that produced from GTN. Likewise incubation of 1,2-GDN or 1,3-GDN with N-acetylcysteine (NAC), glutathione (GSH) or thiosalicylic acid (TSA) (all at 1 mM) for 30 min at 37 degrees C produced a concentration-dependent increase in NO2- formation. 4. Platelet aggregation induced by thrombin (40 mu ml-1) was not modified by high concentrations of 1,2-GDN or 1,3-GDN (175-700 microM). However, aggregation was inhibited when platelets were exposed to 1,2-GDN or 1,3-GDN (700 microM) in the presence of SMC (0.24-1.92 x 10(5) cells) or EC (0.8-3.2 x 10(5) cells). These effects were abrogated by co-incubation with oxyhaemoglobin (OxyHb, 10 microM) indicating that they were due to NO release. The concentrations of the dinitrates required to inhibit platelet aggregation by 50% were about 15 times higher than for GTN in the presence of the same numbers of SMC or EC.5. When NAC or TSA (both at 0.5 mM) were co-incubated with platelets for 3 min in the presence of1,2-GDN or 1,3-GDN, a concentration-dependent inhibition of platelet aggregation was observed. These anti-platelet effects were abolished by co-incubation with OxyHb (10 microM). Glutathione had no potentiating effects.6. Thus the dinitrate metabolites of GTN are metabolized to NO by SMC or EC and are acted upon by thiols to form NO at concentrations about 10 times higher than those of GTN. In vivo, after oral or intravenous GTN, GDN levels are reached which are more than 10 times higher than those of GTN.These data support the notion that part of the effects of GTN are due to the generation of NO from 1,2-GDN and 1,3-GDN by the cells of the vascular wall.

The effect of doxazosin on platelet aggregation in normotensive subjects and patients with hypertension: An in vitro study

An in vitro assay was used to investigate the effects of doxazosin on the platelet aggregation induced by epinephrine, collagen, and adenosine diphosphate. Platelet-rich plasma from normotensive subjects and patients with hypertension was compared. Doxazosin produced a concentration-dependent inhibition of platelet aggregation in both groups, but significantly lower concentrations were required to inhibit platelet aggregation in plasma taken from patients with hypertension. The concentrations of doxazosin that inhibited platelet aggregation in vitro were similar to those that are used clinically to control blood pressure in patients with hypertension.

Potentiation of Antiaggregating Activity of Adenosine by a Phosphodiesterase Inhibitor, EG626 (Oxagrelate), in Human Platelets in Vitro

EG626 (oxagrelate), a specific inhibitor of cyclic AMP phosphodiesterase, produced in vitro a concentration-dependent inhibition of platelet aggregation induced by collagen and ADP in human platelets. When adenosine was added to the platelet rich plasma (PRP) in the presence of a threshold concentration of EG626, the potency of adenosine in inhibiting platelet aggregation was markedly potentiated. This potentiating effect of EG626 proved to be synergistic, but not additive and was accompanied by a marked accumulation of cyclic AMP in the platelets. The antiaggregating and cyclic AMP increasing activities of adenosine were little affected by S-(p-nitrobenzyl)-6-thioguanosine (6TG), an uptake inhibitor of adenosine, or 2′-deoxycoformycin, an inhibitor of adenosine deaminase. The incorporation of adenosine into platelets was abolished by 6TG. These observations indicate that incorporation of adenosine into platelets is not required for inhibition of aggregation or an increase in cyclic AMP and that the site of action of adenosine is probably extracellular. It also appears that the synergistic action by EG626 is not the result of an inhibition of adenosine uptake and/or adenosine deaminase. This speculation is supported in part by the finding that EG626 also potentiates the antiaggregating activity of 2-chloroadenosine. Antiaggregating activity of prostaglandin E1, an activator of adenylate cyclase, was markedly potentiated in combination with EG626. Dibutyryl cyclic AMP showed a time-dependent inhibition of the platelet aggregation, and the inhibitory action was markedly potentiated by EG626. Qualitatively similar results were obtained with another phosphodiesterase inhibitor, 3-isobutyl-1 -methylxanthine (IBMX). All these data suggest that the synergistic potentiation of the antiaggregating activity of adenosine by EG626 might be due to the synergistic accumulation of cyclic AMP in the platelets. This action is mediated through activation of adenylate cyclase by adenosine in combination with the inhibition of cyclic AMP phosphodiesterase by EG626.

Potentiation of antiaggregating activity of adenosine by a phosphodiesterase inhibitor, EG626 (oxagrelate), in human platelets in vitro.

EG626 (oxagrelate), a specific inhibitor of cyclic AMP phosphodiesterase, produced in vitro a concentration-dependent inhibition of platelet aggregation induced by collagen and ADP in human platelets. When adenosine was added to the platelet rich plasma (PRP) in the presence of a threshold concentration of EG626, the potency of adenosine in inhibiting platelet aggregation was markedly potentiated. This potentiating effect of EG626 proved to be synergistic, but not additive and was accompanied by a marked accumulation of cyclic AMP in the platelets. The antiaggregating and cyclic AMP increasing activities of adenosine were little affected by S-(p-nitrobenzyl)-6-thioguanosine (6TG), an uptake inhibitor of adenosine, or 2'-deoxycoformycin, an inhibitor of adenosine deaminase. The incorporation of adenosine into platelets was abolished by 6TG. These observations indicate that incorporation of adenosine into platelets is not required for inhibition of aggregation or an increase in cyclic AMP and that the site of action of adenosine is probably extracellular. It also appears that the synergistic action by EG626 is not the result of an inhibition of adenosine uptake and/or adenosine deaminase. This speculation is supported in part by the finding that EG626 also potentiates the antiaggregating activity of 2-chloroadenosine. Antiaggregating activity of prostaglandin E1, an activator of adenylate cyclase, was markedly potentiated in combination with EG626. Dibutyryl cyclic AMP showed a time-dependent inhibition of the platelet aggregation, and the inhibitory action was markedly potentiated by EG626. Qualitatively similar results were obtained with another phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). All these data suggest that the synergistic potentiation of the antiaggregating activity of adenosine by EG626 might be due to the synergistic accumulation of cyclic AMP in the platelets. This action is mediated through activation of adenylate cyclase by adenosine in combination with the inhibition of cyclic AMP phosphodiesterase by EG626.