Pomolic acid, triterpenoid isolated from Licania pittieri, as competitive antagonist of ADP-induced aggregation of human platelets.

Pomolic acid (PA), triterpenoid isolated from Licania pittieri, has previously shown a potent ability to inhibit adenosine diphosphate (ADP)- and epinephrine-induced human platelet aggregation. To investigate whether PA could be an antagonist of ADP-activated receptors of human platelets (P2Y(1) and P2Y(12)), pharmacological studies were conducted to examining its ability to modulate the platelet shape change induced by a selective P2Y(1) receptor agonist MRS2365 and also the nature of its possible interaction with ADP receptors by analyzing the characteristics of log concentration-response curves of ADP constructed in the absence and in the presence of fixed concentrations of PA, using in vitro platelet aggregation assays. PA did not interfere with the activation of P2Y(1) receptor by MRS2365 to induce platelet shape change and displayed a competitive antagonism of ADP-induced platelet aggregation, which most probably involves competition for a single binding site in platelets. The estimated equilibrium dissociate constant (K(b)) of PA as ADP receptor antagonist was 15.4±0.06nM. Together, these findings give indirect evidence for the idea that PA could be a potent competitive antagonist of P2Y(12) receptor, and open the possibility to consider it as new member of the non-nucleotide generation of antiplatelet drugs.

Inhibitory activity of ginsenosides isolated from processed ginseng on platelet aggregation.

Seven ginsenosides, namely Rg6 (1), F4 (2), Rk3 (3), Rh4 (4), Rs3 (5), Rs4 (6) and Rs5 (7) isolated from processed ginseng were evaluated for their effects on platelet aggregation induced by adenosine diphosphate (ADP), collagen, arachidonic acid (AA) and U46619 (thromboxane A2 mimetic drug). Ginsenosides Rg6, F4 and Rk3 showed inhibitory activity (IC50 = 76 microM, 114 microM and 128 microM, respectively) on AA-induced platelet aggregation. The corresponding IC50 values were comparable to that of acetylsalicylic acid (ASA) (63 microM). Compared to ASA (IC50 = 468 microM) ginsenosides Rg6, F4, Rk3 and Rh4 were found to be more inhibitive (IC50 = 286 microM, 87 microM, 187 microM and 119 microM, respectively) against U46619-induced aggregation. On the other hand, most of the ginsenosides (Rg6, F4, Rh4, Rs3, Rs5) showed negligible effects on ADP and collagen-induced platelet aggregation. The acetylated ginsenosides (Rs3, Rs4 and Rs5) had only mild effects on aggregation induced by four stimulators.

Interaction of salvianolic acids and notoginsengnosides in inhibition of ADP-induced platelet aggregation.

Salvia miltiorrhiza and Panax notoginseng were both considered to be beneficial to cardiovascular diseases in traditional Chinese medicine and often used in combination. To examine the possible interaction between them, the effects of the active fractions of these two herbs, salvianolic acids (SA) and notoginsengnosides (NG), on platelet aggregation were checked respectively or in combination in vitro and in vivo. Both the platelet aggregation of platelet rich plasma (PRP) and washed platelet after ADP induction were checked. In vitro study showed that both SA and NG had an inhibitory effect on platelet aggregation. However, there is no synergistic effect of the combination of SA and NG in vitro. In vivo study showed that i.g. 550 mg/kg/day SA or NG for 5 days could significantly inhibit ADP-induced platelet aggregation of PRP. Moreover, combination of SA and NG at a ratio of 5:1 had a synergistic effect on platelet aggregation of PRP. The mechanism for the synergism of SA and NG in vivo was not clear. High performance liquid chromatography analysis of the plasma of rats received SA, NG or combination of SA and NG showed that co-administration of NG caused change in the plasma distribution profile of SA. The influence of combination on the absorption and/or metabolism of SA may be one of the reasons for the synergism of SA and NG in vivo.

Inhibition of adrenaline and adenosine diphosphate induced platelet aggregation by Lansberg's hognose pit viper (Porthidium lansbergii hutmanni) venom.

The haemostatic components of venom from the genus Porthidium has been poorly studied, although it is known that severe manifestations occur when humans are envenomed, which include invasive oedema and disseminated ecchymosis. The effects of venom on blood platelets are commonly studied and are normally carried out with platelet-rich plasma (PRP). A series of crude venom dilutions was used to determine the effects of adenosine diphosphate (2 microM) and adrenaline (11 microM) induced platelet aggregation. Venom of Porthidium lansbergii hutmanni was fractioned by anionic exchange chromatography, and the fractions were also used to determine the 50% inhibition of adenosine diphosphate (ADP) and adrenaline-induced platelet aggregating dose (AD50). Crude venom has more effect in inhibiting adrenaline-induced aggregation (AD50 = 0.0043 microg) followed by the adenosine diphosphate (AD50 = 17 microg). Peaks I and II obtained by chromatography also inhibited adrenaline-induced platelet aggregation with an AD50 of 3.2 and 0.013 microg, respectively, and both peaks inhibited ADP-induced platelet aggregation with an AD50 of 10 microg. The main purpose of this work was to characterise the in vitro effects caused by P. lansbergii hutmanni crude venom and its fractions on the platelet aggregation mediated by adrenaline and ADP agonists.

Prasugrel.

Clinical trials have demonstrated the superior clinical efficacy of dual antiplatelet therapy with a thienopyridine (a P2Y(12) receptor blocker) and aspirin (COX-1 inhibitor) in patients undergoing stenting as well as patients with acute coronary syndromes. However, clopidogrel treatment is associated with a wide response variability and non-responsiveness in selected patients. The latter phenomenon is linked to the occurrence of recurrent ischaemic events including stent thrombosis in the recent studies. Prasugrel is a new thienopyridine derivative that produces more potent platelet inhibition and a rapid onset of action that is associated with irreversible P2Y(12) receptor blockade. The latter properties of prasugrel may provide a superior alternative to clopidogrel, with less response variability and a decreased prevalence of non-responsiveness.

Antiplatelet and antithrombotic activities of Korean Red Ginseng.

The antiplatelet and antithrombotic activities of Korean Red Ginseng (KRG) were examined on rat carotid artery thrombosis in vivo, and platelet aggregation in vitro and ex vivo. Administration of KRG to rats not only prevented carotid artery thrombosis in vivo in a dose-dependent manner, but also significantly inhibited ADP- and collagen-induced platelet aggregation ex vivo, while failed to prolong coagulation times such as activated partial thromboplastin time (APTT) and prothrombin time (PT), indicating the antithrombotic effect of KRG might be due to its antiplatelet aggregation rather than anticoagulation effect. In line with the above observations, KRG inhibited U46619-, arachidonic acid-, collagen- and thrombin-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC50 values of 620 +/- 12, 823 +/- 22, 722 + 21 and 650 +/- 14 microg/mL, respectively. Accordingly, KRG also inhibited various agonists-induced platelet serotonin secretions as it suppressed platelet aggregation. These results suggest that KRG has a potent antithrombotic effect in vivo, which may be due to antiplatelet rather than anticoagulation activity, and KRG intake may be beneficial to the individuals with high risks of thrombotic and cardiovascular diseases.

The influence of new antithrombotic drugs on regional anesthesia.

PURPOSE OF REVIEW: Antithrombotic drugs are known to increase the risk of spinal epidural hematoma after neuraxial blockade. During the last few years, several new anticoagulants have been introduced, some of them more potent than the drugs currently available. More potency, however, may also indicate a higher risk of bleeding. RECENT FINDINGS: Case series from the last few years indicate that spinal epidural hematoma is more common then previously estimated, with a prevalence from 1: 100,000 in obstetric patients to as high as 1: 3,600 in female orthopedic patients. In order to diminish this risk, most national societies have issued guidelines in which time intervals were established between administration of antithrombotic drugs and performance of neuraxial blockade. SUMMARY: Guidelines are perceived to be capable of reducing the incidence of spinal epidural hematoma with the inherent risk of permanent paraplegia. These guidelines, however, will only be a valuable aid for clinicians if they are constantly updated and newer antithrombotic drugs are included. Although the resurge of peripheral nerve blocks may diminish patient hazards, deep nerve blocks such as lumbar sympathetic blockade are not devoid of serious complications and should probably be handled in the same way as neuraxial blockade.

2-Phenyl-3-(quinolizidin-1-yl)-5-substituted indoles as platelet antiaggregating agents.

A set of ten 2-phenyl-3-(quinolizidin-1-yl)-5-substituted indoles was prepared through the Fischer cyclization of lupinyl- and epi-lupinylphenylketone 4-substituted phenylhydrazones. Compounds were tested for antiaggregating activity on human platelets activated by adenosine diphosphate (ADP), collagen and adrenaline. At 2.5 x 10(-4) M concentration most compounds strongly inhibited the aggregation induced by all the agonists considered and many of them still displayed good activity at 0.625 x 10(-4) M concentration. The least active (1c) and one of the most active (1d) compounds were also tested for antiaggregating activity on rabbit platelets activated by ADP, PAF and sodium arachidonate. Both the compounds were active against ADP and PAF, but only 1d inhibited the arachidonate-induced aggregation (100% at 8 x 10(-6) M concentration) and increased the bleeding time in mice. The same compounds were subjected to a general pharmacological screening and found to display several activities; of particular interest was the dose dependent reduction of serum cholesterol and heparin precipitating betalipoproteins in hypercholesterolemic mice exerted by 1c, which was still significant at the oral dose of 10 mg/kg.

Investigation of platelet aggregation inhibitory activity by phenyl amides and esters of piperidinecarboxylic acids.

A series of anilides and phenyl esters of piperidine-3-carboxylic acid (nipecotic acid) were synthesized and tested for the ability to inhibit aggregation of human platelet rich-plasma triggered by adenosine 5'-diphosphate (ADP) and adrenaline. As a rule, amides were about two times more active than the corresponding esters, and derivatives bearing substituents at the para position of the phenyl ring were significantly more active than the meta-substituted ones. Among the tested compounds, 4-hexyloxyanilide of nipecotic acid (18a) was found to be the most active one, its IC(50) value being close to that of the most active bis-3-carbamoylpiperidines reported in literature (ca. 40 micro M) and aspirin (ca. 60 microM) in ADP- and adrenaline-induced aggregation, respectively. Compared with the isomeric 4-hexyloxyanilides of piperidine-2-carboxylic (pipecolinic) and piperidine-4-carboxylic (isonipecotic) acids, compound 18a showed higher activity, and a Hansch-type quantitative structure-activity relationship (QSAR) study highlighted lipophilicity and increase in electron density of the phenyl ring as the properties which mainly increase the antiplatelet activity (r(2)=0.74, q(2)=0.64). The interaction of nipecotoyl anilides with phosphatidylinositol, a major component of the inner layer of the platelet membranes, was investigated by means of flexible docking calculation methods to give an account of a key event underlying their biological action.

The ability of poloxamers to inhibit platelet aggregation depends on their physicochemical properties.

Poloxamers, block copolymers of polyethylene glycol (PEG) and polypropylene glycol (PPG), are thought to reduce cell-cell adhesion during vascular disorders. We examined how the amphiphilic nature of these polymers may contribute to their ability to inhibit ADP-induced platelet aggregation. Four Poloxamers (184, 188, 335 and 338) with varying PEG and PPG block lengths were examined. Of these, Poloxamer 184 at 2 mM markedly inhibited platelet aggregation. We observed that: i) Typically, less than 10% of the platelet surface is covered by Poloxamers, and greater than 99% of the polymer either remains in solution or binds soluble components in blood plasma. ii) Increasing the PEG side-chain length does not significantly augment the ability of Polox-amers to inhibit platelet aggregation. iii) Poloxamer 184, but not Polox-amers 188 and 335, significantly reduces the ability of stimulated platelets to bind fibrinogen and antibody PAC-1. The study demonstrates that the physical adsorption of some Poloxamers may allow them to inhibit platelet aggregation. The inhibition mechanism involves either binding of Poloxamers to platelet GPIIb-IIIa or inhibition of cellular activation pathways.

Dietary supplementation with aged garlic extract inhibits ADP-induced platelet aggregation in humans.

Garlic has been widely reported to protect against cardiovascular disease by reducing serum cholesterol concentrations and blood pressure and by inhibiting platelet aggregation. However, most of these studies have been performed in hypercholesterolemic subjects or in animal models. We performed a 13-wk study in normolipidemic subjects who ingested 5 mL of aged garlic extract (AGE, Kyolic) per day. Blood was drawn from these subjects at the beginning and end of the study. Aggregation of platelet-rich plasma was induced by ADP; full lipid profiles and liver function tests were determined on serum, and plasma concentrations of eicosanoids were also measured. Dietary supplementation with AGE significantly inhibited both the total percentage and initial rate of platelet aggregation at concentrations of ADP up to 10 micromol/L. The K:(M) for ADP-induced aggregation were approximately doubled after supplementation with AGE, whereas the maximum rate of aggregation was unaffected. No significant changes in plasma thromboxane B(2) and 6-ketoprostaglandin F(1alpha) concentrations or serum lipid profiles were observed. We conclude that AGE, when taken as a dietary supplement by normolipidemic subjects, may be beneficial in protecting against cardiovascular disease as a result of inhibiting platelet aggregation.

Antithrombotic activities of green tea catechins and (-)-epigallocatechin gallate.

The antithrombotic activities and mode of action of green tea catechins (GTC) and (-)-epigallocatechin gallate (EGCG), a major compound of GTC, were investigated. Effects of GTC and EGCG on the murine pulmonary thrombosis in vivo, human platelet aggregation in vitro, and ex vivo, and coagulation parameters were examined. GTC and EGCG prevented death caused by pulmonary thrombosis in mice in vivo in a dose-dependent manner. They significantly prolonged the mouse tail bleeding time of conscious mice. They inhibited adenosine diphosphate- and collagen-induced rat platelet aggregation ex vivo in a dose-dependent manner. GTC and EGCG inhibited ADP-, collagen-, epinephrine-, and calcium ionophore A23187-induced human platelet aggregation in vitro dose dependently. However, they did not change the coagulation parameters such as activated partial thromboplastin time, prothrombin time, and thrombin time using human citrated plasma. These results suggest that GTC and EGCG have the antithrombotic activities and the modes of antithrombotic action may be due to the antiplatelet activities, but not to anticoagulation activities.

Effect of the crude extract of Cestrum parqui on carrageenin-induced rat paw oedema and aggregation of human blood platelets.

An extract of Cestrum parqui aerial parts in methanol:water (1:1) showed inhibition of carrageenin-induced oedema. The aggregation of human blood platelets induced by adenosine diphosphate and platelet activating factor was also inhibited (IC(50)s were 3 and 2 mg/mL, respectively). On the contrary, the extract did not inhibit arachidonic acid-mediated platelet aggregation.

Source of extracellular brain adenosine during hypoxia in fetal sheep.

Microdialysis was performed to determine whether hypoxia increases fetal brain adenosine (ADO) concentration through dephosphorylation of extracellular 5'-adenosine monophosphate (5-AMP). Hypoxia (fetal PaO2 approximately 14 Torr) increased fetal brain ADO levels approximately two-fold when the probes were perfused with synthetic cerebrospinal fluid (CSF) containing inhibitors of the nucleoside transporter but not with this solution plus a blocker of ecto-5'-nucleotidase (AOPCP). The hypoxia-induced rise in fetal brain ADO concentrations depends critically upon the hydrolysis of extracellular 5'-AMP.

Apyrase administration prolongs discordant xenograft survival.

Platelet thrombi and vascular inflammation are prominent features of discordant xenograft rejection. The purinergic nucleotides ATP and ADP, which are secreted from platelets and released by injured endothelial cells (EC), are important mediators of these reactions. Quiescent EC express the ectoenzyme ATP-diphosphohydrolase (ATPDase; an apyrase), which exerts an important thromboregulatory function by hydrolyzing both ATP and ADP. We have shown that ATPDase activity is rapidly lost from the surface of the EC following ischemia-reperfusion injury and during xenograft rejection. The aim of this study was to supplement ATPDase activity within xenografts by infusion of soluble apyrases, and thereby validate the importance of local ATPDase activity in the modulation of xenograft rejection. Lewis rats underwent heterotopic cardiac xenografting from guinea pigs and apyrase was administered intravenously (200 U/kg) as a single dose to evaluate effects on hyperacute rejection (HAR). This initial dose was followed by a continuous apyrase infusion (8.0 U/kg/hr) directly into the graft aorta in combination with systemic cobra venom factor (CVF) administration to deplete complement when delayed xenograft rejection (DXR) was studied. Functional apyrase levels in vivo were assessed by the capacity of blood samples taken at the time of surgery and rejection to inhibit platelet aggregation in vitro. Apyrase administration significantly prolonged graft survival in HAR and DXR. Functional assays showed inhibition of platelet aggregation suggesting effective systemic antiaggregatory effects of the administered apyrases. Histologic studies showed that apyrase administration abrogated local platelet aggregation and activation in HAR and DXR. Our data demonstrate that local administration of apyrase prolonged discordant xenograft survival. These observations emphasize the potential importance of purinergic mediators in platelet activation during xenograft rejection.

Inhibition of platelet aggregation by diterpene acids from Pinus massoniana resin.

The acidic fraction of the resin of Pinus massoniana Lamb. from China was converted to the p-nitrophenyl esters, and the esters separated by chromatography. The separated p-nitrophenyl esters were individually hydrolysed by potassium hydroxide in acetone-water at room temperature to 8 diterpene acids of the pimarane and abietane groups: pimaric acid (8(14),15-pimaradien-18-oic acid) (1), levopimaric acid (8(14),12-abietadien-18-oic acid) (2), palustric acid (8,13-abietadien-18-oic acid) (3), neobietic acid (8(14),13(15)-abietadien-18-oic acid) (4), abietic acid (7,13-abietadien-18-oic acid) (5), dehydroabietic acid (8,11,13-abietatrien-18-oic acid) (6), 7-oxodehydroabietic acid (7-oxo-8,11,13-abietatrien-18-oic acid) (7) and 7 alpha-hydroxydehydroabietic acid (7 alpha-hydroxy-8,11,13-abietatrien-18-oic acid) (8). The structure (and stereochemistry) of the diterpene acids were substantiated by nuclear magnetic resonance spectroscopy (proton and carbon-13, one and two dimensional), by mass spectrometry (electron impact and methane chemical ionization) and by rotation measurements. The 8 diterpene acids were tested for their ability to inhibit the aggregation of washed rabbit platelets induced by platelet activating factor (PAF), adenosine diphosphate (ADP) and by calcium ionophore A23187. With platelet aggregation induced by the latter two agonists, activities comparable with or higher than linolenic acid were given by the first 4 acids. With aggregation induced by PAF, the first 3 acids show activity, but at a level significantly lower than that of linolenic acid. Levopimaric acid has the highest activity among the diterpene acids tested. It is proposed that this activity is related to the folded shape of the molecule.

Effect of the esters of gallic acid on model and human blood platelet membranes studied by Fourier transform infrared spectroscopy.

Gallic acid is one of the components of Chinese herbal drug Radix paeoniae used for promoting blood circulation to remove blood stasis. This paper studied the effects of gallic acid and its esters (e.g. ethyl, propyl, isobutyl and butyl gallate) on model and human blood platelet membranes by FTIR which was used for monitoring the physical state of the acyl chain, interfacial and head group region of the membrane lipid bilayer. From the experimental results it can be seen that the gallic acid and its esters have the modifying function on the pure and cholesterol-containing DPPC model membranes, and have the quantity-effective and structural-effective relationships. In addition, it is discovered that these esters have the modifying effect on the structure of human blood platelet membrane and can reverse the effect of ADP. That the effect of the esters of gallic acid counteracts the effect of cholesterol and ADP on human blood platelet perhaps provides a new explanation of the mechanism of Chinese herbal drugs used for promoting blood circulation to remove blood stasis.

High-dose heparin suppresses platelet alpha granule secretion.

Platelet degranulation has been implicated in the pathophysiology of acute arterial thrombosis, intimal hyperplasia, and atherogenesis. Most previous studies that examined the effect of heparin on platelet function have used platelet aggregometry. These studies have resulted in contradictory data and, by the nature of the assay, reveal no information with regard to platelet degranulation. In contrast, flow cytometry allows accurate quantification of the extent of platelet degranulation by measurement of the platelet surface binding of a GMP-140 specific monoclonal antibody (S12). GMP-140 is only expressed on the platelet surface after platelet alpha granule release. In the present study increasing concentrations of heparin were added to whole blood anticoagulated with sodium citrate. Platelets were activated with a panel of agonists, and the extent of platelet degranulation was quantified by whole blood flow cytometry. Heparin concentrations as high as 100 units/ml were found to suppress platelet alpha granule release induced by either a thromboxane A2 analog (U46619) or a combination of adenosine diphosphate and epinephrine. Heparin suppressed alpha granule release induced by thrombin both in whole blood and in washed platelets. The addition of heparin after platelet activation had no effect on S12 binding. In summary, heparin in high concentrations is a potent inhibitor of platelet degranulation, an action that is unrelated to its effect on the coagulation cascade. Although the heparin concentrations used in this study exceed those used clinically by a factor of 10 or more, future studies of heparin fractions may allow the separation of the anticoagulant and antiplatelet properties of the molecule and allow the administration of an agent that selectively suppresses platelet degranulation without the humoral anticoagulant effect.

[Various aspects of the anti-aggregatory action of diltiazem and cordaphene in patients with ischemic heart disease]. / Nekotorye aspekty antiagregantnogo deistviia dilzema i kordafena u bol'nykh ishemicheskoi bolezn'iu serdtsa.

Some aspects of the antiaggregatory action of calcium antagonists were studied in 50 patients with stable angina pectoris. Dilzem (diltiazem) and cordaphene (nifedipine) were tested for their effects on the erythrocytic component of hemostasis, taking into account their capability of suppressing hemolysis, which made ADP, an important thrombocytic activator, enter the blood flow. The two agents significantly reduce the concentration of plasma ADP, free hemoglobin, diminish mechanical erythrocytic resistance, and block platelet aggregation to a varying degree. A relationship was established between the levels of blood nifedipine and the magnitude of rheological effects. With this, the patients with coronary heart disease showed a good antianginal effect.

Isolation and bioactivity of new tanshinones.

Two new diterpenoids, designated neocryptotanshinone and isotanshinone IIB, have been isolated from "Tan-Shen," the root of Salvia miltiorrhiza, together with a known compound, danshexinkun A. Their structures are established by spectral and physical data. Isotanshinone IIB exhibits significant inhibitory activity in vitro on ADP- and collagen-induced aggregation.