Inhibition Of Human Platelet Aggregation Research Articles

Inhibitory effects of tryptophan pyrolysis products on human platelet aggregation through inhibition of prostaglandin endoperoxide synthetase

To determine the effects of the carcinogenic heterocyclic amines on the stimulus-reaction system in cells, the effects of several such amines, including 3-amino-1,4-dimethyl-5 H-pyridol[4,3- b]indole (Trp-P-1) and 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2), on human platelet aggregation and of Trp-P-1 and Trp-P-2 on human polymorphonuclear leucocyte aggregation were investigated. Of the carcinogens studied, only Trp-P-1 and Trp-P-2 had potent inhibitory effects on human platelet aggregation induced by sodium arachidonate. The concentrations of Trp-P-1 and Trp-P-2 causing 50% inhibition of human platelet aggregation induced by sodium arachidonate were 15 and 25 μM, respectively. The heterocyclic amines examined had no significant effects on human polymorphonuclear leucocyte aggregation. Moreover, radiochemical studies of arachidonate metabolism showed that Trp-P-1 and Trp-P-2 inhibited, in a dose-dependent manner, the formation of cyclooxygenase products in platelets induced by sodium arachidonate. These results indicate that Trp-P-1 and Trp-P-2 have potent inhibitory effects on prostaglandin endoperoxide synthetase in the stimulus-reaction system of human platelets.

The effect of chloramphenicol and cycloheximide on platelet aggregation and protein synthesis

This study investigated the role of platelet protein synthesis in platelet aggregation. Cycloheximide (Cx) and chloroamphenicol (Cm) were used as inhibitors of cytoplasmic (80S ribosome directed) and mitochondrial protein synthesis respectively. The effect of these agents on human platelet aggregation and l-[U- 14C]leucine incorporation into platelet protein was investigated. Cm exhibited dose-dependent inhibition of collagen, thrombin and secondary ADP aggregatory responses, but had no effect on arachidonate or primary ADP responses over a similar concentration range (3.1 × 10 −3, 3.1 × 10 −4 and 3.1 × 10 −5 M). Cm also inhibited platelet secretion associated with collagen and secondary ADP responses. Furthermore, Cm exhibited a similar dose-dependent inhibition of l-[U- 4C]leucine incorporation into platelet protein reaching 80% inhibition of incorporation at 3 × 10 −3M. At similar concentrations (3.5 × 10 −3, 3.5 × 10 −4 and 3.5 × 10 −5M) Cx failed to show inhibition of human platelet aggregation by all agonists used with the exception of collagen where some inhibition was seen at high Cx concentration (3.5 × 10 −3 M). Cx was also found to be ineffective at inhibiting l-[U-10 14C] leucine incorporation into platelet protein at all concentrations tested. These results suggest that the majority of platelet protein synthesis is mitochondrial and that this protein synthesis may have a role in human platelet aggregation.

Inhibition of human platelet aggregation and cytoplasmic calcium response by calcium antagonists: studies with aequorin and quin2.

Calcium antagonists inhibit platelet aggregation, but whether this action is due to inhibition of the effect of agonists on cytoplasmic ionized calcium concentration is unknown. We studied this problem by loading gel-filtered platelets with either quin2 or aequorin and stimulating them with epinephrine, arachidonate, thrombin, the calcium ionophore A23187, 1-oleoyl-2-acetyl glycerol, or adenosine diphosphate in media with or without extracellular calcium. In response to all of these agonists, aequorin indicated an increase in cytoplasmic calcium that accompanied or preceded platelet aggregation. In calcium-containing media, verapamil, nifedipine, and diltiazem inhibited these effects in a concentration-dependent fashion, except for those produced by thrombin and A23187. Removal of extracellular calcium with EGTA reduced the calcium response to arachidonate, adenosine diphosphate, and 1-oleoyl-2-acetyl glycerol, and the calcium response and aggregation were further inhibited by the calcium antagonists. In general, strong inhibition of the aequorin cytoplasmic calcium signal by approximately 100 microM concentrations of nifedipine, verapamil, and diltiazem was correlated with inhibition of platelet aggregation, but high concentrations of the inhibitors were required. Since inhibition by the calcium antagonists of the cytoplasmic calcium response and aggregation exceeded the effect of simple removal of extracellular calcium, these drugs may affect internal redistribution of calcium in human platelets.

Inhibition of human platelet aggregation by dihydropyrano- and dihydrofuranocoumarins, a new class of cAMP-phosphodiesterase inhibitors

Certain esters of dihydropyranocoumarin and dihydrofuranocoumarin alcohols have previously been shown to inhibit the cAMP-phosphodiesterase from bovine heart. We now report that these naturally occurring coumarins inhibit the high affinity ( K m = 1.1 μM) cAMP-phosphodiesterase from human platelets with activities that closely correlate with those obtained using phosphodiesterase from bovine heart tissue. Additionally the coumarins inhibit the aggregation of human platelets induced with ADP, adrenaline and collagen with activities comparable to those of dipyridamole. A lack of significant correlation between these metabolic and functional activities indicates that there exist, besides cAMP-phosphodiesterase inhibition, additional mechanisms of action for the platelet aggregation inhibitory effect of dihydropyrano- and dihydrofuranocoumarins.

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F 2α

The inhibition of human platelet aggregation produced by PGF 2α is not specific for thromboxane A 2 mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF 2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH 2). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC 50 = 0.5 μM), displaces the specific binding of [ 3H] 9,11-epoxymethano PGH 2 to washed human platelets. PGF 2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF 2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF 2α. We suggest that the very weak effect of PGF 2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.

Inhibition of human platelet aggregation by vitamin K

The effect of several vitamin K (Vit K) analogues on the aggregation of human platelets was examined. The analogues were potent inhibitors of aggregation induced by ADP, thrombin, collagen and arachidonate but were less active against aggregation induced by the calcium ionophore A23187. Vit K 3 also prevented platelet membrane phosphatide breakdown induced by collagen. These effects were not due to a direct inhibition of enzymes involved in the liberation of arachidonate or its subsequent transformation. The analogues exerted no effects on enzymes regulating intraplatelet cAMP. However, these effects could be overcome by increasing extracellular Ca ++ levels, indicating a possible interaction with Ca ++ regulation in platelets.

Inhibition of human platelet aggregation by parathyroid hormone. Is cyclic AMP implicated?

Parathyroid hormone (PTH) is a polypeptide which in different in vitro systems raises intracellular cyclic AMP (cAMP) levels via adenyl cyclase activation and stimulates Ca2+ transport across cell membranes. We tested whether, on the basis of this mechanism, PTH would inhibit human platelet aggregation. The latter was tested in vitro by a photometric technique. Platelet aggregation induced by the calcium ionophore A 23187 was inhibited by PTH at concentrations (0.5-3 USP U/ml) similar to those effective in other in vitro systems. Higher concentrations of PTH were required to prevent aggregation initiated by adenosine-5'-diphosphate, arachidonic acid, or platelet-aggregating factor. The terminal synthetic fragment 1-34 b PTH was ineffective against all aggregation stimuli. The antiaggregating effect of PTH was potentiated by verapamil and theophylline and was additive to that of PGI2. However, PTH did not appear to increase platelet cAMP levels and was not counteracted by an inhibitor of platelet adenyl cyclase. It is therefore unlikely that PTH inhibits platelet aggregation through an adenyl cyclase stimulated increase of cAMP. Since PTH levels are markedly increased in uremic plasma, it might contribute to the defective platelet function and the bleeding tendency frequently occurring in uremic patients.

A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom

Y.-S. Li, K.-F. Liu, Q.-C. Wang, Y.-L. Rpan and G.-C. Tu. A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom. Toxicon 23, 895–903, 1985. — By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G — 75, a potent platelet function inhibitor was purified from Vipera russelli siamensis venom. It appeared as a single protein band on polyacrylamide gel electrophoresis in the presence or absence of SDS, and consists of 123 amino acid residues. Its NH 3-terminal residue is serine. It showed the following characteristics: molecular weight, 13,800; isoelectric point, 10.4; ld(In50), 0.5 ± 0.12 mg/kg (i.v.). The platelet inhibitor exhibited phospholipase A 2 activity with a specific activity of 35 μmoles/min/mg. From 2 g of the venom, 70 mg of the purified inhibitor was obtained. Inhibition of human platelet aggregation induced by ADP or adrenaline was dose-dependent, with id(In50) of 1.14μg/ml or 0.37 μg/ml, respectively. The platelet aggregation induced by thrombin or collagen was also inhibited and the inhibitory activity on platelet aggregation was heat stable (at 100°C, 20 min) in an acidic medium (pH 5.8), while its phospholipase A 2 activity was relatively heat labile under the same condition. The release of 3H-serotonin in platelets stimulated by ADP was also inhibited and this was positively correlated with inhibition of platelet aegregation induced by ADP ( r = 0.998, P < 0.002).

Compound [formula omitted] is a potent inhibitor of human platelet aggregation antagonizing the calmodulin-dependent platelet reaction in vitro

Compound [formula omitted] is a potent inhibitor of human platelet aggregation antagonizing the calmodulin-dependent platelet reaction in vitro

Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor sepharose chromatography

Cilostamide derivatives are potent inhibitors of human platelet aggregation and selectively inhibit human platelet cyclic adenosine monophosphate (cyclic AMP) phosphodiesterase. N-Cyclohexyl- N-(2-hydroxybutyl)-5-[6-1,2,3,4-tetrahydro-2oxoquinolyloxy)]-butyramide (OPC-13135) is one of these derivatives, and the concentration of OPC-13135 producing 50% inhibition of human platelet aggregation induced by 2 μg/ml collagen was 5 μM. On the other hand, the concentrations of OPC-13135 producing 50% inhibition of human platelet cyclic AMP phosphodiesterase and cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase were 0.073 and 21.8 μM, respectively. We purified over 480-fold the soluble low K m form of cyclic AMP phosphodiesterase from human platelets, using OPC-13135 Sehparose column as a final step in the purification procedure. The purified protein has a molecular weight of 175,000, determined by gel filtration and is an acidic protein, as determined by isoelectric focussing (pI = 4.9). Kinetic measurements indicated that the enzyme protein had a K m , value for the substrate cyclic AMP and cyclic GMP of 0.34 and 0.11 μM respectively, and a V max value of 85.3 and 19.8 nmole/min/mg protein, respectively. K i value of the OPC-13135 for the enzyme was 0.015 μM and was of competitive fashion against cyclic AMP.

Mepacrine-induced inhibition of human platelet cyclic-GMP phosphodiesterase

The effect of mepacrine ( DL-quinacrine-HCl), a specific inhibitor of phospholipase C, on cyclic-GMP levels in human platelets was investigated. The concentrations of mepacrine producing 50% inhibition of human platelet aggregation induced by 5 μM ADP and 3 μg/ml of collagen were 50±8 and 70 ± 15 μM, respectively. Addition of mepacrine to human platelet suspension resulted in increases in cyclic GMP. In contrast to cyclic-GMP levels, cyclic-AMP content was not affected by mepacrine. Mepacrine did not stimulate guanylate cyclase, but did specifically inhibit human platelet cyclic-GMP phosphodiesterase, separated from cyclic-AMP phosphodiesterase or other forms of phosphodiesterase on DEAE-cellulose columns. Stimulation by cyclicGMP of human platelet cyclic-GMP-stimulated cyclic-AMP-phosphodiesterase activity was not inhibited by mepacrine. The IC 50 value of the drug for cyclic-GMP phosphodiesterase was 40μM, and IC 50 for cyclic-AMP phosphodiesterase was 1.2 mM. Mepacrine was 30-times more potent as an inhibitor of human platelet cyclic GMP than of cyclic-AMP phosphodiesterase. Mepacrine blocks arachidonate release from human platelets by inhibiting phosphatidylinositol-specific phospholipase C. The increase in cyclic-GMP levels produced by addition of mepacrine will explain part of the pharmacological action of this drug.

Effects of SQ 27,427, a thromboxane A2 receptor antagonist, in the human platelet and isolated smooth muscle

The TxA2 receptor antagonist properties of SQ 27,427 [a cyclohexylcarbinol-7-oxabicyclo(2.2.2)heptenoic acid analog] were studied in vitro both in the human platelet and various isolated smooth muscle preparations. SQ 27,427 was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid, ADP, epinephrine, collagen and the stable TxA2 agonists 9,11-azoPGH2 and SQ 26,655. Inhibition of platelet aggregation was achieved at concentrations of SQ 27,427 which did not alter TxB2 levels. SQ 27,427 was found to weakly inhibit the formation of TxB2 from arachidonic acid and had no effect on the synthesis of PGE2 or PGI2 from arachidonic acid. SQ 27,427 was also found to be a weak stimulator of platelet adenylate cyclase, being 1000 times less potent than PGI2. In isolated smooth muscle experiments, SQ 27,427 was shown to be a potent and specific TxA2 receptor antagonist. It caused competitive antagonism of 9,11-azoPGH2-induced contractions of vascular, respiratory and gastrointestinal smooth muscles. This antagonism was specific, as responses to norepinephrine, serotonin, PGE2, PGI2, PGF2α, histamine, carbachol and KCl were not altered by SQ 27,427.

Endotoxin-mediated inhibition of human platelet aggregation

A variety of endotoxins, when added to human platelet-rich plasma (PRP) or to suspensions of washed platelets (WP), demonstrated an inhibitory effect on platelet aggregation induced by various aggregating agents. Endotoxin blocked the release of 14C serotonin from platelets but had no influence on cyclic AMP production. Endotoxin did not interfere with thromboxane generation by platelets. However, endotoxin-treated platelets failed to respond to added thromboxane. The inhibitory effect of endotoxin on platelet aggregation was more pronounced in the presence of ionophore A23187 as compared to other aggregating agents and was effectively reversed by calcium but not by magnesium, another divalent cation. Furthermore, endotoxin failed to inhibit the ristocetin-induced agglutination of formaldehyde-fixed platelets; a non-calcium dependent phenomenon. These findings appear to suggest that endotoxin-mediated inhibitory activity of platelet aggregation is related to the interference in the role of calcium. The antiaggregatory activity of endotoxin appears to be due to a direct and rapid action on platelets and not due to a non-specific binding, as the effect was not abolished by washing the endotoxin-incubated platelets. Endotoxin-mediated alteration of platelet function may contribute to bleeding diathesis in septecemic and endotoxemic patients.

9-Deoxy-delta 9, delta 12-13,14-dihydroprostaglandin D2, a metabolite of prostaglandin D2 formed in human plasma.

Incubation of prostaglandin D2 (PGD2) with human plasma yielded a product that has been identified as 9-deoxy-9,10-didehydro-12,13-didehydro-13,14-dihydro-PGD2 (9-deoxy-delta 9, delta 12-13,14-dihydro-PGD2). The identification was based on mass spectrometry, UV spectrometry, mobilities and retention time on TLC and HPLC, and NMR. The conversion of PGD2 to this product was dependent on the incubation time and the amount of plasma added to a reaction mixture and was abolished by prior boiling. The conversion rate of PGD2 to this metabolite was 0.03 nmol/min per mg of protein of whole plasma at pH 8.0 at 37 degrees C. Similar conversion was also found by incubating PGD2 with human serum albumin added at the concentration found in plasma. These results suggest that the conversion of PGD2 to this product is catalyzed by the enzymatic action of a plasma protein, probably serum albumin. The biological activities of this compound were examined in several systems. It showed negligible activity in inhibition of human platelet aggregation and relaxation of rabbit stomach strip. On the other hand, it exhibited a three times stronger inhibitory activity (IC50, 1.8 microM) than PGD2 (IC50, 5 microM) on the growth of L-1210 cultured cells.

Lack of interaction of heparin with ADP and prostacyclin on rat platelets

It has recently been reported that heparin is able to counteract the ir hibitory effect of prostacyclin (PGI2) on human platelet aggregation (l-3). The mechanism of this interaction, however, still awaits clarification.There is growing evidence that heparin neutralizes the effect of prostanoid and non prostanoid inhibitors of human platelet aggregation by potentiating pletelet response to aggregating stimuli (3,4). If this is so, in a system where heparin does not potentiate platelet aggregation it should also fail to blunt the inhibitory effect of PG12. We report here evidence that in rat platelets the positive interacticn between ADP and heparin does not occur and heparin does not modify the inhibitory potency of PGI2.

Forskolin (a powerful inhibitor of human platelet aggregation)

Forskolin (a powerful inhibitor of human platelet aggregation)

Role of cyclic AMP in the inhibition of human platelet aggregation by quercetin, a flavonoid that potentiates the effect of prostacyclin

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) has previously been shown to inhibit cyclic nucleotide phosphodiesterases prepared from various cell homogenates and the function of intact human platelets. We now report that (1) high concentrations of quercetin raise platelet cAMP levels; and (2) quercetin potentiates the inhibitory effect of prostacyclin (PGI 2) on ADP-induced washed human platelet aggregation and the elevation of platelet cAMP levels elicited by PGI 2. These results suggest a role for cAMP in the mechanism of action of quercetin on blood platelets.

A comparative study of collagen induced thromboxane release from platelets of different species: Implications for human athero sclerosis models

The role of thromboxane in human, rabbit, dog, pig and sheep platelet aggregation has been studied. Each of these species showed a concentration-dependent relationship between collagen concentrations and the thromboxane B 2 (TXB 2) released from aggregating platelets, however sheep platelets produced only 17.5% of the amount of TXB 2 released from human platelets under the same collagen stimulus. Indomethacin did not inhibit sheep platelet aggregation in the concentration range 0.114 mM – 0.114 μM, yet proved to be a potent inhibitor of human platelet aggregation. This prostaglandin endoperoxide analogue, U.46619, induced human platelet aggregation at a dose of 100 ng, however the analouge, at doses as high as 500 μg was much less active on sheep platelets when compared to human platelets. Thus we have demonstrated that there are differences in the extent of thromboxane involvement in platelet aggregation between different species, which must be considered with other criteria, before choosing an animal model for studying human atherosclerosis.

Specific but noncompetitive inhibition by 2-alkylthio analogues of adenosine 5'-monophosphate and adenosine 5'-triphosphate of human platelet aggregation induced by adenosine 5'-diphosphate.

Some 2-alkylthio derivatives of adenosine 5'-monophosphate (AMP), adenosine 5'-monophosphorothioate (AMPS) and adenosine 5'-triphosphate (ATP) were examined as inhibitors of human platelet aggregation. 2-Methylthio-AMP, 2-ethylthio-AMP, 2-(pentan-l-yl)thio-AMP, 2-ethylthio-AMPS, 2-methylthio-ATP and 2-ethylthio-ATP (100 microM) each inhibited aggregation induced by adenosine 5'-diphosphate (ADP) but not by 11 alpha, 9 alpha-epoxymethano prostaglandin H2, a stable endoperoxide analogue. Log dose-response curves to ADP in the absence and presence of each inhibitor were not parallel and the inhibition could not be overcome by high concentrations of ADP. The ATP analogues achieved greater inhibition of aggregation induced by ADP (5 microM) than did the AMP analogues. The order of potency of the AMP analogues was 2-ethylthio-AMPS greater than 2-ethylthio-AMP greater than 2-(pentan-l-yl)thio-AMP greater than 2-methylthio-AMP, and 2-methylthio-ATP was more potent than 2-ethylthio-ATP. These 2-alkylthio substituted analogues of AMP and ATP are specific but noncompetitive inhibitors of ADP-induced human platelet aggregation.

Cardiovascular effects of prostaglandin D 3 and D 2 in anesthetized dogs

Prostaglandin (PG) D 3 has been identified as an inhibitor of human platelet aggregation, but little is known of the hemodynamic activity of this material. In morphine pretreated, chloralose-urethan anesthetized dogs, bolus intravenous injections (1, 3.2 and 10 μg/kg) of PGD 3 and also PGD 2 were associated with marked, dose-related increases in pulmonary arterial pressure. Cardiac index and rate increased, while peripheral vascular resistance decreased in response to injections of PGD 3. A biphasic (depressor followed by a pressor phase) effect on systemic arterial pressure was observed after PGD 2, while PGD 3 was associated with dose-related depressor responses. Graded intravenous infusions (0.25, 0.50 and 1.0 μg/kg/min) of PGD 3 and PGD 2 were associated with qualitatively similar cardiovascular responses. Quantitatively, PGD 3 infusions were associated with greater decreases in peripheral vascular resistance and greater increases in cardiac output, heart rate, and peak left ventricular dp/dt than were infusions of PGD 2. In contrast, PGD 3 was less potent than PGD 2 as a pulmonary pressor material. Systemic arterial pressure responses to infusions of the prostaglandins were variable. In these experiments, PGD 3 and PGD 2 were associated with qualitatively similar cardiovascular responses characterized by peripheral vasodilatation.