ADP-induced Aggregation Of Rabbit Platelets Research Articles

In silico consensus activity prediction, rational synthesis, and evaluation of antiglycation and antiplatelet activities of 3,6-disubstituted 1,2,4,5-tetrazines

According to the in silico consensus activity prediction, a series of 3,6-disubstituted 1,2,4,5-tetrazines were synthesized as promising inhibitors of the Maillard reaction. In addition to the studies of antiglycation activity, a comparative in vitro evaluation of the effect of the synthesized compounds on the ADP-induced rabbit platelet aggregation model was carried out. Compounds with antiglycation and antiplatelet activities significantly higher than that of the reference drugs, aminoguanidine and acetylsalicylic acid, were revealed.

Screening of some vegetables that inhibit platelet aggregation and their mechanism:a preliminary study

Objective To study the mechanism of some vegetables inhibiting human platelet aggregation function.Methods Some vegetable juice was mixed(tomato juice as positive control)with platelet-rich plasma(PRP),and maximum ratio of platelet aggregation was measured after induction by agonists on the aggregometer.The expression levels of platelet activation marker were detected,including fibrinogen receptor(Fib-R),P-selectin(CD62P),and the combination amount of fibrinogen on the surface of platelet by flow cytometry to validate the inhibitory effect of platelet aggregation by animal tests.Ordinary white rabbits were randomly divided into five groups,each group was fed with saline,cooked tomato juice,blanched garlic leaves juice,Chinese cabbage juice or spinach juice,respectively.And the maximum ratio of rabbit platelet aggregation in different time were observed.Results Cooked vegetable juice couldn't inhibit human platelet aggregation induced by AA,but blanched garlic leaves juice,Chinese cabbage juice and spinach juice could inhibit human platelet aggregation obviously induced by ADP.collagen or epinephrine.The inhibition intensity of platelet aggregation increased markedly along with the increase of the concentration of the cooked vegetables juice.Cooked juice of blanched garlic leaves,Chinese cabbage,spinach could not inhibit the expression of Fib-R and CD62P,Whereas they were able to significantly decrease the amount of Fib-R and fibrinogen binding. ADP-induced rabbit platelet aggregation ratios in the group fed with cooked juice of blanched garlic leaves,Chinese cabbage or spinach were significantly lower than control group after 3,5,8 weeks,respectively.The inhibition ratios of the platelet aggregation in cooked Chinese cabbage juice group were 45.7%,53.6%,48.5% after 3,5,8 weeks,respectively.Cooked juice of blanched garlic leaves group were 10.7%,66.7%,46.3%,respectively. Cooked spinach juice group were 8.7%,21.0%,42.6%,respectively.The collagen-induced rabbit platelet aggregation ratios in the groups fed with cooked juice of blanched garlic leaves or Chinese cabbage were significantly lower than control group after 5 and 8 weeks respectively. The inhibition ratios of the platelet aggregation in cooked Chinese cabbage juice group were 54.9%,56.3%after 5 and 8 weeks,respectively. Cooked juice of blanched garlic leaves group were 28.4%and 86.7%,respectively. Cooked tomato juice could not inhibit rabbit platelet aggregation in 8 weeks. Conclusions After induced by ADP or collagen,cooked juice of blanched garlic leaves,Chinese cabbage and spinach could significantly decrease the amount of Fib-R and fibrinogen binding,and inhibit platelet aggregation function significantly.It may have potential application of therapy or prlevention of thrombotic diseases. Key words: Platelet aggregation; Vegetables; In vitro; Animal experimentation

Distribution of low molecular weight platelet aggregation inhibitors from snake venoms

An assay of platelet aggregation inhibitors measured by the turbidimeter using Aggregometer PAM 8C (Mebanix) was performed after each crude snake venom (57 species) was subjected to ultrafiltration using MILLIPORE UFP 1 LGC. The snake venoms of Viperidae (three species), Elapidae (11 species), and Hydrophiidae (two species) inhibited ADP-induced rabbit platelet aggregation. In particular, six venoms of Bitis gabonica, Pseudocerastes persicus, Dendroaspis angusticeps, D. polylepis, Ophiophagus hannah, and N. nigricollis crawshawii strongly inhibited platelet aggregation. Furthermore, adenosine was identified from Bitis gabonica venom using HPLC and FAB/MS analysis.

Characterization and cloning of a novel phospholipase A 2 from the venom of Trimeresurus jerdonii snake

A phospholipase A 2 (PLA 2), called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS–polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 μmol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 μg/ml enzyme was incubated for 90 min in the presence of PC and Ca 2+. No detectable hemolysis was noticed when PC was not added. Ca 2+ was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca 2+ was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC 50 of 1.0 μM. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA 2s, especially the D49 PLA 2s. Also, the residues concerned to Ca 2+ binding were conserved. This is the first report of cDNA sequence of T. jerdonii venom PLA 2.

γ-Mangostin, a novel type of 5-hydroxytryptamine 2A receptor antagonist

Gamma-mangostin, purified from the fruit hull of the medicinal plant Garcinia mangostana caused a parallel rightwards shift of the concentration/response curve for the contraction elicited by 5-hydroxytryptamine (5-HT) in the rabbit aorta (pA2 = 8.2) without affecting the contractile responses to KCl, phenylephrine (alpha1) or histamine (H1). The perfusion pressure response of rat coronary artery to 5-HT (5-HT2A) was reduced concentration dependently by gamma-mangostin (IC50 = 0.32 microM). 5-HT amplified, ADP-induced aggregation of rabbit platelets (5-HT2A) was inhibited by gamma-mangostin (IC50 = 0.29 microM), whereas that induced by thrombin was not affected, nor did gamma-mangostin affect 5-HT-induced contraction of the guinea-pig ileum (5-HT3)in the presence of 5-HT1, 5-HT2 and 5-HT4 receptor antagonists. Furthermore, 5-HT-induced contraction of the rat fundus (5-HT2B) and 5-HT-induced relaxation of the rabbit aorta in the presence of ketanserin (5-HT1) and carbachol-induced contraction of the guinea-pig ileum (muscarinic M3) were not affected by gamma-mangostin (5 microM). Gamma-mangostin inhibited [3H]spiperone binding to cultured rat aortic myocytes (IC50 = 3.5 nM). The Kd for [3H]spiperone binding was increased by gamma-mangostin (3 nM) from 11.7 to 27.4 nM without affecting Bmax. These results suggest that gamma-mangostin is a novel competitive antagonist, free from a nitrogen atom, for the 5-HT2A receptors in vascular smooth muscles and platelets.

Bassiatin, a new platelet aggregation inhibitor produced by Beauveria bassiana K-717.

A new platelet aggregation inhibitor, bassiatin, was isolated from the cultured broth of Beauveria bassiana which had been isolated from a soil sample collected in Yunnan province, China. The structure of bassiatin was determined to be (3S, 6R)-4-methyl-6-(1-methylethyl)-3-phenylmethyl-1, 4-perhydrooxazine-2,5-dione by NMR analysis, X-ray crystallographic analysis and chemical synthesis. Bassiatin inhibited ADP-induced aggregation of rabbit platelets with the IC50 being 1.9 x 10(-4) M.

Isolation of a Phospholipid Inhibitor of Platelet Activating Factor-Induced Activity from Perfused Rat Liver: Identification as Phosphatidylglycerol

An endogenous inhibitor of platelet activating factor action was isolated from perfused rat liver. It was purified by thin-layer chromatography and high-performance liquid chromatography and subjected to chemical modifications in order to identify its structure. On the basis of its fast atom bombardment-mass spectrum it was characterized as phosphatidylglycerol composed mainly of 16:0/18:1 and 16:0/20:2 fatty acyl chains ([M + H] + at m/ z 749 and 775, respectively) and very minor levels of 18:0/18:1 and 18:0/20:2. The purified compound exhibited inhibition on rabbit platelet aggregation induced by 5 × 10 −10 M platelet activating factor (PAF) at an EC 50 value near 2.5 × 10 −6 M and on the serotonin secretion at an EC 50 7 × 10 −6 M. Other phospholipids isolated from the liver preparations, such as phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin (diphosphatidylglycerol), and phosphatidic acid, exhibited no inhibitory activity in the concentration range from 1 × 10 −4 to 1 × 10 −7 M nor did they induce any aggregation, or lysis, of the platelets. Of importance, phosphatidylglycerol could inhibit thrombin- and ADP-induced aggregation of rabbit platelets. These results suggested a possible site of inhibition common to the signal transduction pathway of these agonists. Preliminary binding experiments showed a noncompetitive type of inhibition on PAF binding to intact rabbit platelets.

Inhibition of smooth muscle contraction and platelet aggregation by peptide 204–212 of lipocortin 5: an attempt to define some structure requirements

Peptide 204–212 of lipocortin (LC) 5 inhibited porcine pancreatic phospholipase A2 (PLA2) induced rat stomach strip contractions and ADP induced rabbit platelet aggregation in a concentration dependent manner (IC30 of 10 μM and 400 μM, respectively). The first two amino acids are not necessary since the eptapeptide 206–212 was equipotent in both assays (IC30 of 12.5 μM and 420 μM). Of the two pentapeptides 204–208 and 208–212 only the latter showed inhibitory activity in both models although the potency was much reduced (IC30 of 170 μM and 630 μM) compared with that of the parent nonapeptide. Comparison of peptide 204–212 effects with those of its analogues on LC1 and LC2 indicate that lysine 208 and aspartic acid 211 are essential in order to maintain a fully active nonapeptide.

PI-200 and PI-201, new platelet aggregation inhibitors produced by Streptomyces sp. A7498. Taxonomy, fermentation, isolation, physico-chemical properties, structure determination and biological properties.

Two new platelet aggregation inhibitors, PI-200 and PI-201 were isolated from the fermentation broth of Streptomyces sp. A7498. PI-200 and PI-201 inhibited ADP-induced aggregation of rabbit platelets with an IC50 of 3.8 x 10(-4) M and 7.1 x 10(-4) M, respectively.

Proaggregatory effect of epinephrine on rabbit platelets inhibited by ticlopidine

Ticlopidine is a potent inhibitor of ADP-induced aggregation of rabbit platelets ex vivo . In vivo , however, multiple agonists play a role in platelet activation. In this study, we examined the effect of epinephrine on the antiplatelet action of ticlopidine in rabbit platelets. Epinephrine reversed the inhibitory effect of drug on ADP-induced platelet aggregation. The potentiating effect of epinephrine was mediated through α 2-adrenergic receptors, was reversed by pretreatment with the Na +/H + exchange inhibitor dimethylamiloride, and was mimicked by agents that increased intracellular sodium or pH. Ticlopidine had no effect on resting intracellular pH, an indication that the effect of epinephrine was not compensating for a drug-induced intracellular acidification. While this potentiation was also found to be inhibited by aspirin, it did not involve enhanced release of thromboxane A 2. Our results demonstrate that epinephrine can overcome the inhibitory effect of ticlopidine on ADP-induced aggregation through a mechanism involving activation of Na +/H + exchange and through an as yet unidentified mechanism sensitive to aspirin.

Effects of Ethanol on Pathways of Platelet Aggregation In Vitro

Ethanol, at physiologically tolerable concentrations, did not affect the primary phase of ADP-induced aggregation of human or rabbit platelets, which is not associated with the secretion of granule contents. Potentiation by epinephrine of the primary phase of ADP-induced aggregation of rabbit platelets was also not inhibited by ethanol. However, ethanol did inhibit the secondary phase of ADP-induced aggregation which occurs with human platelets in citrated platelet-rich plasma and is dependent on the formation of thromboxane A2. Inhibition by ethanol of thromboxane production by stimulated platelets is likely due to inhibition of the mobilization of arachidonic acid from membrane phospholipids, as ethanol had little or no effect on aggregation and secretion induced by arachidonic acid or the thromboxane mimetic U46619. Rabbit platelet aggregation and secretion in response to low concentrations of collagen, thrombin, or PAF were inhibited by ethanol. Inhibition of the effects of thrombin and PAF was also observed with aspirin-treated platelets. Thus, in addition to inhibiting the mobilization of arachidonate for thromboxane formation that occurs with most agonists, ethanol can also inhibit aggregation and secretion through other effects on platelet responses.

Involvement of phosphoinositide metabolism in potentiation by adrenaline of ADP-induced aggregation of rabbit platelets.

Changes in phosphoinositide metabolism were examined in washed rabbit platelets stimulated with 0.5 microM-ADP, 50 microM-adrenaline, or ADP and adrenaline in combination. Adrenaline does not stimulate platelet aggregation when used alone, but does potentiate aggregation stimulated by ADP. In platelets prelabelled with [32P]Pi and [3H]glycerol, adrenaline was found to potentiate the ADP-induced changes in platelet phospholipids, causing larger increases in the amount and labelling of phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid than was observed with ADP alone. The combination of ADP and adrenaline did not produce a greater decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) than was produced by ADP alone. In platelets prelabelled with [3H]inositol, adrenaline potentiated the increases in labelling of inositol phosphate and inositol bisphosphate stimulated by ADP; no increase in inositol trisphosphate labelling was detected with ADP alone or with the combination of ADP and adrenaline. Phentolamine, an alpha-adrenergic-receptor antagonist, blocked potentiation by adrenaline of ADP-induced changes in phosphoinositide metabolism. Propranolol and sotalol, beta-adrenergic-receptor antagonists, augmented the potentiation; this is consistent with the concept that the effect of adrenaline is mediated by beta-adrenergic receptors. The effect of adrenaline on phosphoinositide metabolism appears to be to potentiate the mechanisms by which ADP causes turnover of PIP and possibly degradation of PI, rather than the mechanism by which PIP2 is decreased.

An effect on ADP-induced aggregation of rabbit platelets by dietary supplementation with polyunsaturated fatty acids and pyridoxal 5-phosphate

An effect on ADP-induced aggregation of rabbit platelets by dietary supplementation with polyunsaturated fatty acids and pyridoxal 5-phosphate

Plasma Fibronectin Is Not Involved in ADP-Induced Aggregation of Rabbit Platelets

Plasma Fibronectin Is Not Involved in ADP-Induced Aggregation of Rabbit Platelets

Inhibition of Platelet Aggregation by Sulfinpyrazone

The action mechanism of sulfinpyrazone (SUL) was examined with rabbit platelets by testing its influence on the aggregations induced by ADP and collagen, on platelet content of cyclic AMP, on the generation of labile aggregation-stimulating substance (LASS) by platelets, and on platelet aggregation induced by LASS. The generation of LASS was examined by incubating 20μg/ml of arachidonic acid (AA) with washed platelets in the presence or absence of SUL, and measuring platelet aggregation when the incubated mixture was added to platelet-rich plasma (PRP) containing 20μM indomethacin. SUL was administered in a dose of 200mg or 400mg to healthy adults, and plasma concentrations and aggregations induced by ADP, collagen, and epinephrine were measured before and 1, 2, 4, and 6 hours after administration.At 3mM concentration, SUL exhibited little inhibitory effect on ADP-induced aggregation of rabbit platelets; but at concentrations of 1mM and above SUL inhibited collagen-induced aggregation, showing suppression of the release reaction. At 10mM, SUL elicited no change in platelet levels of cyclic AMP, and did not promote the PGE1-induced increase in cyclic AMP. LASS-induced aggregation was slightly inhibited by 3mM SUL. LASS formation was markedly inhibited by 0.3mM SUL, and at higher concentrations the inhibition increased dose-dependently. After administration of SUL in a dose of 200mg, the plasma level of the drug reached a maximum (average 10.5μg/ml) after 1 hour and after 6 hours had fallen to one-third of that level (3.0μg). With a does of 400mg, the plasma levels reached a maximum (35.5μg/ml) after 2 hours and fell to approximately one-third of that level (12.7μg/ml) after 6 hours. The maximum inhibition of platelet aggregation was seen between 4 and 6 hours after administration, showing a discrepancy with the peak plasma level.SUL inhibited the secondary but not the primary aggregation induced by ADP, only slightly inhibited collagen-induced aggregation, but strongly suppressed epinephrine-induced aggregation. SUL also inhibited the biosyntheses of PG endoperoxides and thromboxane A2, and at high concetration showed a direct inhibitory effect against these mediators. The results can be interpreted in terms of the mechanism of inhibition of LASS generation, in view of the report that SUL, unlike aspirin, does not inhibit the activity of cyclo-oxygenase at concentrations that inhibit platelet aggregation.

Fibrinogen and ADP-induced platelet aggregation

Fibrinogen is required for ADP-induced aggregation of washed human platelets and enhances ADP-induced aggregation of rabbit platelets. To achieve these effects, fibrinogen had to be either present before ADP was added or added with the ADP. When fibrinogen was added after platelets had changed in shape in response to ADP, fibrinogen did not support ADP-induced aggregation. The association of 125l-fibrinogen with platelets during ADP-induced aggregation was investigated. A mixture of 125l-fibrinogen and trivalent 51Cr(as a measure of trapped fluid) was added to suspensions of Washed human or rabbit platelets. Samples were taken for rapid centrifugation before and at several times after the addition of ADP. The percentage of labeled fibrinogen associated with the platelets was calculated. Fibrinogen associated transiently with platelets during their response to ADP. The largest amount of fibrinogen became associated with the platelets almost immediately after the addition of ADP; less fibrinogen was associated with the platelets at the peak of aggregation, and the associated fibrinogen was lost during platelet deaggregation. Association occurred only if conditions permitted platelet aggregation in response to ADP; inhibitory conditions included lack of divalent cations, inhibition of platelet metabolism, or the presence of inhibitors of platelet aggregation such as prostaglandin E1 or adenosine. Thus the association of fibrinogen with platelets appeared to take place during the initial changes induced by ADP. These results indicate that fibrinogen may be involved in transient linkages between platelets in ADP-induced aggregates.

Inhibitory effect of tiaramide on ADP-induced aggregation in rabbit platelets.

Tiaramide in 10(-4) or 10(-5) M depressed the ADP-induced aggregation of rabbit platelets using the turbidimetric method. In modified Chandler's loop method, tiaramide in the same concentration accelerated the restoration of the time course of disaggregation.

The Role of Prostaglandins in the ADP-Induced Aggregation of Rabbit Platelets Shown by the Use of 15-Hydroxyprostaglandin Dehydrogenase

The initial shape change and subsequent aggregation of platelets in citrated rabbit platelet-rich plasma caused by ADP in vitro was inhibited by 15-hydroxyprostaglandin dehydrogenase. This inhibition was NAD-dependent and was also seen when shape change and aggregation were initiated by sodium arachidonate or by collagen. The aggregation of gel-filtered rabbit platelets by thrombin was not, however, affected by removal of 15-hydroxyprostaglandins. Indomethacin was found to inhibit ADP-induced aggregation but at a concentration (250 micron) much higher than that required to inhibit collagen-induced aggregation. Moreover the platelet release reaction had not taken place 3 min after ADP stimulation. The direct role 15-hydroxyprostaglandin production in ADP-induced aggregation of rabbit platelets is proposed. The involvement of 15-hydroxyprostaglandins in platelet aggregation caused by other inducers is also discussed.

ChemInform Abstract: PLATELET AGGREGATION INHIBITORS. II ‐ N‐HETEROCYCLIC ALDOXIME METHIODIDES

On the basis of an hypothesis according to which suitable nucleophilic agents may convert adenosine diphosphate (ADP) into adenosine monophosphate (AMP) and adenosine, well known inhibitors of ADP-induced platelet aggregation, some N-heterocyclic aldoxime methiodides were tested as inhibitors of ADP-induced rabbit platelet aggregation. Several 1-aryl-2-hydroxyiminomethly-3-methylimidazolium iodides significantly inhibit in vitro and in vivo-in vitro ADP-induced rabbit platelet aggregation.

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

The effects of bupranolol, a new beta-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known beta-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 beta-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.