Antiaggregatory Activity Research Articles

Catechin in the Mediterranean diet: vegetable, fruit or wine?

The aim of this study was to determine which type of diet contributes most to plasma concentration of (+)-catechin, a naturally occurring antioxidant flavonoid. Consecutive subjects ( n=180) were screened. A blood sample was collected after a fasting period and (+)-catechin measurement in plasma was performed by high-performance liquid chromatography (HPLC) method using fluorescence detection. Dietary consumption of the last evening meal was assessed by a dietary recall method. Taking fruit, vegetable and wine consumption into account, four types of diet were identified. After adjustment for confounding factors, concentration of (+)-catechin in plasma was three-fold higher in diet with fruit and vegetable but without wine (449.5 μg/l), and four-fold higher in diet with wine but without vegetable and fruit (598.5 μg/l) in comparison to diet without fruit, vegetable and wine (131.6 μg/l). When the consumption of vegetable, fruit and wine was combined, the concentration was the highest (637.1 μg/l) ( P<0.001). Vegetable, fruit and wine were the major determinants of plasma (+)-catechin concentration ( P<0.001). This study demonstrates that the highest plasma concentration of (+)-catechin was observed in subjects consuming fruit, vegetable and wine, and its antioxidant and antiaggregant activity could partly explain the relative protection against coronary heart disease (CHD).

Chaperone-like activity of a synthetic peptide toward oxidized gamma-crystallin.

alphaA-Crystallin can function like a molecular chaperone. We recently reported that the alphaA-crystallin sequence, KFVIFLDVKHFSPEDLTVK (peptide-1, residues 70-88) by itself possesses chaperone-like (anti-aggregating) activity during a thermal denaturation assay. Based on the above data we proposed that the peptide-1 sequence was the functional site in alphaA-crystallin. In this study we investigated the specificity of peptide-1 against gamma-crystallin aggregation in the presence of H2O2 and CuSO4. Peptide-1 was able to completely protect against the oxidation-induced aggregation of gamma-crystallin. Removal of N-terminal Lys or the replacement of Lys with Asp (DFVIFLDVKHFSPEDLTVK, peptide-2) did not alter the anti-aggregation property of peptide-1. However, deletion of KF residues from the N-terminus of peptide-1 resulted in a significant loss of its anti-aggregation property. Bio-gel P-30 size-exclusion chromatography of gamma-crystallin incubated with peptide-2 under oxidative conditions revealed that a major portion of the peptide elutes in the void volume region along with gamma-crystallin, suggesting the binding of the peptide to the protein. Peptide-1 and -2 were also able to prevent the UV-induced aggregation of gamma-crystallin. These data indicate that the same amino acid sequence in alphaA-crystallin is likely to be responsible for suppressing the heat-denatured, oxidatively modified and UV-induced aggregation of proteins.

Synthesis and anti-aggregatory activity of linear retro-inverso RGD peptides.

Six retro-inverso tri- and tetrapeptide analogues of RGD were prepared and their anti-aggregatory activity was determined by platelet aggregation tests in comparison with the corresponding parent peptides. An efficient method for the introduction of a malonyl-aspartic residue into a peptide chain is described for the first time. A 2-3-fold decrease in potency or total loss of bioactivity was observed with the new peptides; structure-activity relationships are discussed.

Effects of endothelial cells and mononuclear leukocytes on platelet aggregation

Although normal vascular endothelium prevents adhesion and aggregation of platelets by the release of nitric oxide (NO) and prostacyclin, the endothelium exposed to oxidized low density lipoprotein (LDL) may be damaged, damage that may include a reduction in its secretory capacity. On the other hand, mononuclear leukocytes (ML) can also release NO to inhibit platelet aggregation. Platelet aggregation was measured with a Chrono-Log aggregometer (USA) in the presence of cultured endothelial cells (EC) or isolated ML, stimulated by collagen. Platelet aggregation was markedly inhibited (28.5 +/- 5.0 versus control 92.2 +/- 1.7%), when EC were added to platelets. Pre-incubation of the EC with 10 micromol/l of indomethacin or 300 micromol/l of L-NG-monomethyl-arginine (L-NMMA) for 6 h substantially reduced their anti-aggregating activity (72.8 +/- 5.1 and 76.6 +/- 7.7%, respectively). However, incubation of the EC in culture with 10 micromol/l of lysophosphatidyl choline (LPC) for 6 h did not affect the anti-aggregating capacity of EC (LPC 28.1 +/- 5.5 vs. EC 28.5 +/- 5.0%). Non-stimulated ML also inhibited (43.2 + 5.9 vs. control 69.2 3.2%) the platelet aggregation induced by collagen. The inhibition was dependent on the number of ML added. In addition, it was enhanced by increasing the concentration of collagen from 2.5 to 10 microg/ml. Platelet aggregation is inhibited mainly by the EC, however, blood ML also inhibit platelet aggregation. Incubation of the EC in culture with LPC did not affect the anti-aggregating capacity of the EC.

A Mass Spectrometry Screening Method for Antiaggregatory Activity of Proteins Covalently Modified by Combinatorial Library Members: Application to Sickle Hemoglobin

A homogeneous assay, based on electrospray mass spectrometry, is described for identifying compounds in a combinatorial library that covalently modify a protein and thereby enhance its solubility. The technique is based on measuring the distribution of modified proteins in the supernatant versus aggregate. Compounds having the greatest anti-aggregatory activity are those with the highest supernatant/aggregate ratio. Mass is used as a marker to identify which covalent modifier in the library is involved. An exploratory study is presented which demonstrates that the antisickling activity of a family of isothiocyanates, as measured by the standard C(sat) assay, correlates well (r(2) = 0.98) with the mass spectrometry analysis of the supernatant/aggregate distribution. The technique has potential for screening libraries capable of covalently modifying other proteins of clinical interest, e.g., Alzheimer's, Huntington's, and various prion related diseases.

4-Dialkylamino-1-(5-substituted or unsubstituted 1-phenyl-1 H-pyrazol-4-yl)butan-1-ols: synthesis and evaluation of analgesic, anti-inflammatory and platelet anti-aggregating activities

A number of 4-dialkylamino-1-(5-substituted or unsubstituted 1-phenyl-1 H-pyrazol-4-yl)butan-1-ols 2a– n were synthesized and tested in vivo for anti-inflammatory and analgesic activities and in vitro for platelet anti-aggregating activity. Dimethylaminoderivatives 2b, e, g showed good analgesic activity; almost all of them had strong platelet anti-aggregating properties at a final concentration of 1×10 −3 M; pyrazoles 2c, d, f– h showed weak anti-inflammatory activity.

Fast narrow-bore HPLC analysis of thiosulfinates in onions and hybrids. Relationship with the platelet anti-aggregant activity.

Fast narrow-bore HPLC analysis of thiosulfinates in onions and hybrids. Relationship with the platelet anti-aggregant activity.

Monoclonal Antibodies against Platelet Membrane Glycoproteins IIb/IIIa and Ibα Inhibit Platelet Dependent Thrombin Generation by Different Mechanisms

The antithrombotic effect of antiplatelet agents is principally due to their anti-aggregatory action, but these agents may also interfere with coagulation. We have investigated the effect of monoclonal antibodies (MAb) to platelet membrane glycoproteins (GP) IIb/IIIa and Ibalpha on thrombin generation. Antibodies to platelet membrane glycoprotein IIb/IIIa (RFGP56 and c7E3) were shown to inhibit platelet-mediated thrombin generation stimulated by both intrinsic and extrinsic methods. An antibody to GP Ibalpha (RFGP37) also inhibited thrombin generation in these systems. FITC-annexin V was used to determine the effect of these antibodies on the exposure of procoagulant phospholipids on the platelet membrane, and it was found that the anti-IIb/IIIa antibodies reduced this, whereas the anti-Ibalpha antibody caused an increase. We conclude that our monoclonal antibodies against platelet membrane glycoproteins IIb/IIIa and Ibalpha inhibit platelet dependent thrombin generation by different mechanisms.

Synthesis, structure and antiaggregatory effects of some N-(4,5-dihydro-1 H-imidazol-2-yl)indoles

A series of 1-(4,5-dihydro-1 H-imidazol-2-yl)indole derivatives was prepared in order to evaluate their antiaggregatory activity in human platelets. The compounds 4a– m were prepared by reacting N-aryl- N-(4,5-dihydro-1 H-imidazol-2-yl)hydroxylamines ( 2a– d) with monosubstituted acetylene derivatives 3a– b. Imidazoline derivatives 4 were further acetylated or sulfonylated to give amides 5a– c and sulfonamides 6a– c and 7a– c, respectively. Eight compounds were taken as representative aryliminoimidazoline analogs. Among them only one, 4m, showed a good concentration-dependent action against the primary or α 2-adrenoreceptor mediated phase of noradrenaline-induced aggregation in platelets.

Endothelin-B-receptors-dependent-inhibition of platelet aggregation in the CD-1 mouse.

An experimental protocol of adenosine diphosphate-(ADP) induced platelet aggregation in the mouse was designed in order to study the roles played by endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors in the ET-1-induced inhibition of ex vivo platelet aggregation. The pressor effects of ET-1 or IRL-1620 were firstly determined in vivo in anesthetized (ketamine/xylazine) CD-1 mice (males and females; 25-30 g). All agents were administered intravenously (via the jugular vein) and blood samples were collected from the carotid artery into heparinized Eppendorfs (15 U/ml). To obtain platelet-rich plasma (PRP) the blood was immediately centrifuged for 12 min at low speed (1100 g). Platelet-poor plasma (PPP) was then prepared by centrifugation of the whole blood sample at high speed (3700 g) for 30 min. PPP was used to calibrate the aggregometer at 100% transmission. Platelet aggregation was monitored ex vivo as a change in light transmission through PRP following the injection of ADP (5 microM). ET-1 (0.01-2.0 nmol/kg) induced a significant and dose-dependent inhibition of platelet aggregation ex vivo (12-84%). The selective ET(B) agonist, IRL-1620 (0.05-2.0 nmol/kg), also triggered a marked inhibition of platelet aggregation. Indomethacin (10 mg/kg), a nonselective cyclooxygenase inhibitor, abolished the inhibitory effect of ET-1. The selective ET(A) antagonist, BQ-123 (1 nmol/kg), abolished the in vivo pressor effect of exogenous ET-1, without affecting its anti-aggregatory activity. The selective ET(B) antagonist, BQ-788 (0.5 nmol/kg), did not modify the elevation of blood pressure produced by the ET-1; however, it did abrogate dose-dependently the inhibitory effect of ET-1 on platelet aggregation. These results suggest that the anti-aggregatory effect of ET-1, in anesthetized CD-1 mice, relies mainly upon the activation of ET(B) receptors. The mechanism whereby ET-1 exerts this effect, is partially indirect and requires at least the production and the release of prostanoids (possibly PGI2) into the blood stream.

Endothelin-B-Receptors-Dependent-Inhibition of Platelet Aggregation in the CD-1 Mouse

Summary: An experimental protocol of adenosine diphosphate-(ADP) induced platelet aggregation in the mouse was designed in order to study the roles played by endothelin-A (ETA) and endothelin-B (ETB) receptors in the ET-1-induced inhibition of ex vivo platelet aggregation. The pressor effects of ET-1 or IRL-1620 were firstly determined in vivo in anesthetized (ketamine/xylazine) CD-1 mice (males and females; 25-30 g). All agents were administered intravenously (via the jugular vein) and blood samples were collected from the carotid artery into heparinized Eppendorfs (15 U/ml). To obtain platelet-rich plasma (PRP) the blood was immediately centrifuged for 12 min at low speed (1100g). Plateletpoor plasma (PPP) was then prepared by centrifugation of the whole blood sample at high speed (3700 g) for 30 min. PPP was used to calibrate the aggregometer at 100% transmission. Platelet aggregation was monitored ex vivo as a change in light transmission through PRP following the injection of ADP (5 μM). ET-1 (0.01-2.0 nmol/kg) induced a significant and dose-dependent inhibition of platelet aggregation ex vivo (12-84%). The selective ETB agonist, IRL-1620 (0.05-2.0 nmol/kg), also triggered a marked inhibition of platelet aggregation. Indomethacin (10 mg/kg), a nonselective cyclooxygenase inhibitor, abolished the inhibitory effect of ET-1. The selective ETA antagonist, BQ-123 (1 nmol/kg), abolished the in vivo pressor effect of exogenous ET-1, without affecting its anti-aggregatory activity. The selective ETB antagonist, BQ-788 (0.5 nmol/kg), did not modify the elevation of blood pressure produced by the ET-1; however, it did abrogate dose-dependently the inhibitory effect of ET-1 on platelet aggregation. These results suggest that the anti-aggregatory effect of ET-1, in anesthetized CD-1 mice, relies mainly upon the activation of ETB receptors. The mechanism whereby ET-1 exerts this effect, is partially indirect and requires at least the production and the release of prostanoids (possibly PGI2) into the blood stream.

Effect of Exercise Training on Oxidized LDL-Mediated Platelet Function in Rats

This study investigates how exercise training affects Oxidized LDL (Ox-LDL) mediated-platelet activation. Five-week-old male Wistar rats were assigned to either control or trained groups. Trained rats were treadmill-trained for 10 weeks after familiarization. The following measurements were taken in both control and trained groups: Ox-LDL-mediated platelet aggregability and [Ca2+]i, plasma and platelet-derived nitric oxide (NO) metabolite (nitrite plus nitrate) levels, and antiaggregating activity of NO derived from endothelial cells. Based on those measurements, major findings in this study can be summarized as follows: 1) the trained group had a higher plasma -NO metabolite level than the control group; 2) the trained group had a lower platelet aggregability and [Ca2+]i elevation and a higher platelet derived-NO metabolite level than the control group; 3) the trained group had lower Ox-LDL-potentiated platelet aggregability and [Ca2+]i elevation and Ox-LDL-attenuated NO metabolite in platelet than the control group; 4) treating the platelet with L-arginine inhibited Ox-LDL-potentiated platelet activation in both control and trained groups; 5) Ox-LDL enhances platelet aggregation directly although impairing NO bioactivity but not guanylate cyclase activity in both control and trained groups. Results in this study demonstrate that exercise training decreases Ox-LDL-potentiated platelet activation most likely by enhancing platelet-derived NO release.

Interaction of 12-lipoxygenase with cellular proteins

Eicosapentaenoic acid (EPA) has been reported to have a potent anti-aggregatory activity and to be efficiently metabolized by 12-lipoxygenase, not by cyclooxygenase in platelets. In vitro effect of 12-lipoxygenase metabolites of EPA on platelet function was studied and compared with those of arachidonic acid (AA). The 12-lipoxygenase metabolites of AA and EPA; 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-hydroperoxyeicosapentaenoic acid (12-HPEPE), and their hydroxy derivatives, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE) were prepared enzymatically using human platelet lysate. These compounds were purified by high performance liquid chromatography and identified by gas chromatography-mass spectrometry. 12-HPETE and 12-HPEPE inhibited dose-dependently washed human platelet aggregation and serotonin (5-HT) release induced by AA and collagen. The potency of 12-HPEPE was almost equal to that of 12-HPETE. Their hydroxy derivatives, 12-HETE and 12-HEPE were less potent. 12-hydroperoxy derivatives of AA and EPA were the most potent in inhibiting platelet aggregation and 5-HT release among 5-, 12- and 15-hydroperoxy isomers of AA and EPA. The inhibitory effects of 12-HPETE and 12-HPEPE on platelet aggregation were additive.

New antithrombotic aryl-sulfonylthiosemicarbazide derivatives synthesized from natural safrole

As part of a research program aiming at the synthesis and pharmacological evaluation of novel lead-compounds exploring Brazilian abundant natural products, we describe herein the synthesis and the antithrombotic profile of new aryl-sulfonylsemicarbazides and aryl-sulfonylthiosemicarbazides (10a-d). The new derivatives, designed with basis on the molecular hybridization concept, were prepared in good yields from natural safrole (9), isolated from sassafras oil. The anti-aggregating activity of these new derivatives (10a-d) on platelet aggregation induced by ADP, collagen, arachidonic acid and U-46619, indicates an important antithrombotic profile for the 6-methyl-3,4-methylenedioxyphenyl-sulfonyl-N-phenylthiosemicarbazide derivative (10d), acting at the arachidonic acid cascade and representing a new lead-compound with antithrombotic activity.

ChemInform Abstract: Synthesis, Antiaggregating and Hypotensive Activity of Azomethines of the 3,3‐Dialkylisoquinoline Series and Their Derivatives.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Antithrombotic Effect of SM-20302, a Nonpeptide GPIIb/IIIa Antagonist, in a Photochemically Induced Thrombosis Model in Guinea Pigs

SM-20302, a synthetic inhibitor of the fibrinogen receptor of platelets, has been shown to inhibit the platelet aggregation induced by various stimuli. In the present study, we performed ex vivo platelet aggregation studies by using heparinized platelet-rich plasma (PRP) as well as citrated PRP and compared the antiaggregatory activity with the in vivo antithrombotic efficacy of SM-20302. The oral administration of SM-20302 (0.3–10 mg/kg) to guinea pigs completely inhibited the ADP-induced ex vivo platelet aggregation in citrated PRP. In heparinized PRP, SM-20302 (1–10 mg/kg) showed a dose-dependent inhibition of ex vivo platelet aggregation, and it exhibited complete inhibition at a dose of 3 and 10 mg/kg, respectively. The concentration of ionized calcium in the citrated samples was approximately 35 times lower than that in heparinized samples. Chelation of ionized calcium caused an enhancement of the antiaggregatory activity of SM-20302 in guinea pig heparinized PRP in vitro. And addition of CaCl 2 to citrated PRP reversed the enhancement. Citrate therefore appeared to enhance the inhibitory activity of SM-20302 by lowering the ionized calcium levels. We also examined the in vivo efficacy of SM-20302 in a photochemically induced femoral artery thrombosis model in guinea pigs. The photochemical injury of the endothelium of femoral artery resulted in a progressive decline in the blood flow. The oral administration of SM-20302 (0.1–3 mg/kg) produced a dose-dependent maintenance of the femoral artery patency and significantly prolonged the time to occlusive thrombus formation at a dose of 1 and 3 mg/kg, respectively. These results suggest that SM-20302 may be an orally active antithrombotic agent, and its in vivo antithrombotic efficacy appeared to correlate well with the ex vivo platelet inhibition in PRP prepared from heparinized blood but not in PRP anticoagulated with citrate.

The antithrombotic efficacy of the GP IIb/IIIa antagonist SR121787 is potentiated by antithrombin-dependent factor Xa inhibition without an increase in the bleeding risk in the rabbit.

Current antithrombotic therapy in acute coronary symptoms is only partially effective and exhibits bleeding complications. These experiments were designed to address the antithrombotic and hemorrhagic interactions of the novel glycoprotein (GP) IIb/IIIa antagonist SR121787 in combination with the indirect inhibitor of factor Xa, SR90107/ORG31540. Thrombogenesis was initiated by electrolytic injury of the intimal surface of the carotid artery, and thrombus formation was assessed by recording carotid blood flow and by measuring thrombus weight 45 min after electrical stimulation. SR121787 was an efficacious antithrombotic agent in this model (ED50 = 16.3+/-0.3 mg/kg, p.o.), whereas heparin (4.5 mg/kg, i.v.) and SR90107/ORG31540 [1 mg/kg (850 aXa anti-units/kg), i.v.] were only marginally effective (17 and 27% inhibition of carotid blood flow reduction, respectively). Coadministrations of heparin (4.5 mg/kg, i.v.) or SR90107/ORG31540 (0.5 mg/kg, i.v.) were found to potentiate the antithrombotic efficacy of threshold doses of SR121787 (5 or 10 mg/kg, p.o.). The enhancement of the antithrombotic efficacy of SR121787 by SR90107/ORG31540 was--contrary to the association of SR121787 with heparin--not accompanied by an increased blood loss from the incised rabbit ear. Coadministrations of heparin or SR90107/ORG31540 did not influence the ex vivo antiaggregatory activity of SR121787. SR121787 coadministration did not alter the systemic anticoagulant activities in heparin or SR90107/ORG31540-treated animals. These data suggest the potential for optimized antithrombotic treatment in acute coronary syndromes when a GP IIb/IIIa antagonist (SR121787) is combined with an antithrombin-dependent factor Xa inhibitor (SR90107/ORG31540).

Synthesis and study of antiaggregative and hypotensive activity of 3,3-dialkyl- isoquinoline azomethines and their derivatives

Synthesis and study of antiaggregative and hypotensive activity of 3,3-dialkyl- isoquinoline azomethines and their derivatives

Tissue distribution of phosphodiesterase families and the effects of sildenafil on tissue cyclic nucleotides, platelet function, and the contractile responses of trabeculae carneae and aortic rings in vitro

Sildenafil is a selective inhibitor of phosphodiesterase type 5 (PDE5), which has been shown to be a clinically effective treatment for erectile dysfunction. Its action results from increased levels of cyclic guanosine monophosphate (cGMP), which is normally degraded by PDE5. This cyclic nucleotide is a second messenger for nitric oxide, which is involved in the regulation of numerous functions, including vascular smooth muscle tone. In an attempt to better predict the effects of sildenafil on cardiovascular function, the distribution of PDE activity was determined with anti-PDE1 and anti-PDE5 antibodies in the human cardiac ventricle and saphenous vein, and in vitro studies were performed on the isolated human cardiac ventricle, corpus cavernosum, saphenous vein, and mesenteric artery as well as on rabbit aorta, dog coronary artery, dog trabecular tissue, and rabbit and human platelets. The major PDE activity in the human cardiac ventricle was shown to be calcium/calmodulin-dependent PDE1, but there was no detectable level of PDE5. In contrast, the human saphenous vein contained PDEs 1, 4, and 5, and the human mesenteric artery contained PDEs 1, 2, 3, 4, and 5. The distribution of PDE5 in the cardiovascular system is consistent with the observed pharmacodynamic and clinical effects of sildenafil. Sildenafil, unlike milrinone, a selective PDE3 inhibitor, had no effect on the isolated trabeculae carneae; this is consistent with the lack of PDE5 expression in cardiac myocytes. Sildenafil selectively increased cGMP levels in coronary vascular smooth muscle tissue but produced no change in cyclic adenosine monophosphate (cAMP) levels, which is consistent with the drug’s selectivity for PDE5. In phenylephrine-contracted isolated rabbit aortic rings, sildenafil enhanced the relaxation induced by the nitric oxide donor glyceryl trinitrate, suggesting that sildenafil may potentiate the hypotensive effects of nitric oxide donor agents on the vasculature, an effect that has been observed clinically. Human platelets were found to contain PDE5, which was inhibited by 50% (IC 50) by sildenafil at a concentration of 6.3 nM, consistent with the IC 50 value in the corpus cavernosum. Sildenafil alone had no direct effect on platelet function, but it potentiated the in vitro antiaggregatory activity of sodium nitroprusside on rabbit and human platelets. The pharmacodynamic and adverse event profiles observed in clinical trials with sildenafil are consistent with the in vitro profile of the tissue distribution of PDE5 and its known mechanism of action as a selective inhibitor of PDE5.

Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A 2 synthesis in human blood platelets

S-Nitroso-cysteine (SNC), a putative endothelium-derived relaxing factor, potently inhibited collagen- and arachidonic acid-induced platelet aggregation (IC 50=100 nM) and thromboxane A 2 (TxA 2) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC-induced formation of cGMP but did not reverse inhibition by SNC of collagen- and arachidonic acid-induced platelet aggregation. Combination of ODQ with SQ-29548, a specific platelet TxA 2 receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP-independent mechanisms that may involve inhibition of TxA 2 synthesis in human platelets.