Mechanism of Action of p-Chlorobiphenyl on the Inhibition of Platelet Aggregation

p-Chlorobiphenyl (1–50 μm ) concentration-dependently inhibited the aggregation and release reaction of rabbit washed platelets induced by arachidonic acid and collagen, but not those induced by platelet-activating factor (PAF), U46619 and thrombin. The IC50 values of p-chlorobiphenyl on the arachidonic acid and collagen-induced platelet aggregation were 2.9 ± 0.5 and 12.8 ± 2.3 μm , respectively. The formation of both platelet thromboxane B₂ and prostaglandin D₂ caused by arachidonic acid was inhibited by p-chlorobiphenyl concentration-dependently. In myo-[³H]inositol-labeled and fura-2-loaded platelets, [³H]inositol monophosphate generation and the rise in intracellular Ca²⁺ stimulated by arachidonic acid were inhibited by p-chlorobiphenyl. In human platelet-rich plasma, p-chlorobiphenyl and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by adenosine 5′-diphosphate and adrenaline without affecting the primary aggregation. It is concluded that p-chlorobiphenyl may be a cyclo-oxygenase inhibitor and its antiplatelet action is mainly due to the inhibition of thromboxane formation.     

Adenosine-3′,5′-diphosphate and coenzyme a—Effects on platelet function

The effects in vitro of adenosine-3′5′-diphosphate and coenzyme A on human platelet aggregation and [¹⁴C]hydroxytryptamine release were studied. Whereas coenzyme A, at concentrations between 0.06 and 0.24 mM, inhibited the aggregation of platelets induced by ADP, adenosine-3 ′, 5 ′-disphosphate, which is part of the coenzyme A molecule, blocked both ADP- and thrombin-induced platelet aggregation. The ADP-induced platelet aggregation was inhibited at a lower adenosine-3′, 5′-diphosphate (10–20μM) concentration than was thrombin-induced aggregation (60–200 μM). Adenosine-3′,5′-diphosphate also inhibited [¹⁴C]adenosine uptake by platelets in a concentration-dependent manner (20–200 μM), but only to a maximum of 40 per cent of total [¹⁴C]adenosine radioactivity incorporated into the platelets. The inhibitory effect of adenosine-3′,5′-diphosphate and coenzyme A on the release reaction was further documented by the decrease in aggregation-induced release of [¹⁴C]5-hydroxytryptamine from prelabeled platelets into the medium. The extent of inhibition caused by coenzyme A and adenosine-3′,5′-diphosphate was found to depend upon the concentration of inhibitor and incubation time. If these agents are indeed inhibitors of platelet aggregation, then they may serve as valuable tools to study platelet function.     

Inhibition in vitro of platelet aggregation and arachidonic acid metabolism by flavone

The effects of flavone on platelet aggregation and arachidonic acid (AA) metabolism were tested in vitro. When incubated at a concentration of 50 μM, flavone completely suppressed platelet aggregation induced by 150 μM AA in thirty-six out of forty-three subjects tested. A lower concentration (10 μM) was effective in about 50% of the donors. Flavone also inhibited the second wave of aggregation induced by epinephrine and ADP. Platelet thromboxane formation, estimated both by radioimmuno-assay measurements and by studies of ¹⁴C-labeled AA metabolism, was depressed by flavone. Flavone-treated platelets preferentially utilized [¹⁴C]AA for the lipoxygenase pathway while cyclo-oxygenase activity was depressed. Adenosine 3':5'-cyclic monophosphate (cAMP) was measured in flavone-treated and control platelets. While their baseline levels were similar, flavone-treated platelets showed a lower stimulation of cAMP induced by prostacyclin (PGI₂) than did controls. Phosphodiesterase activity was not affected by flavone as judged from the decay rates of PGI₂-stimulated cAMP levels. From these findings we conclude that the antiaggregating activity of flavone is not a consequence of changes in platelet cAMP but is due to inhibition of cyclo-oxygenase.     

Inhibitory effect of 8-bromo cyclic GMP on an extracellular Ca2+-dependent arachidonic acid liberation in collagen-stimulated rabbit platelets

The inhibitory effect of cyclic GMP on collagen-induced platelet activation was studied using 8-bromo cyclic GMP (8brcGMP) in washed rabbit platelets. Addition of collagen (1 micrograms/ml) to platelet suspension caused shape change and aggregation associated with thromboxane (TX) A2 formation. 8brcGMP (10-1000 microM) inhibited collagen-induced platelet aggregation and TXA2 formation in a concentration-dependent manner. 8brcGMP did not affect platelet cyclooxygenase pathways, but markedly inhibited collagen-induced arachidonic acid (AA) liberation from membrane phospholipids in [3H]AA-prelabeled platelets, indicating that the inhibitory effect of 8brcGMP on collagen-induced aggregation is due to an inhibition of AA liberation. In [32P]orthophosphate-labeled platelets, collagen stimulated phosphorylation of a 20,000 dalton (20-kD) and 40-kD proteins. 8BrcGMP stimulated phosphorylation of a specific protein having molecular weight of 46-kD and inhibited collagen-induced both 20- and 40-kD protein phosphorylation. Collagen could stimulate the AA liberation without activation of phospholipase C or Na+-H+ exchange, but could not in the absence of extracellular Ca2+. These findings suggest that cyclic GMP inhibits collagen-induced AA liberation which is mediated by an extracellular Ca2+-dependent phospholipase A2. However, cyclic GMP seems to inhibit the Ca2+-activated phospholipase A2 indirectly, since 8brcGMP had no effect on Ca2+ ionophore A23187-induced platelet aggregation or AA liberation. It is therefore suggested that cyclic GMP may regulate collagen-induced increase in an availability of extracellular Ca2+ which is responsible for phospholipase A2 activation in rabbit platelets.     

Effect of anethole dithiolthione on human platelet aggregation.

Anethole dithiolthione (ADT) (10 mumol/l) inhibited platelet aggregation and the formation of thromboxane (Tx)B2 in plasma in response to adenosine diphosphate (ADP), epinephrine and arachidonic acid (AA). ADT partially inhibited platelet aggregation and TxB2 formation in plasma induced by thrombin, phorbol myristate acetate and calcium ionophore A23187 and increased the lag time of collagen-induced aggregation at concentrations in the range 10-40 mumol/l. ADT (100 mumol/l) completely inhibited the aggregation of washed platelets challenged with thrombin. ADT had no additive effect on the inhibition of thrombin-induced platelet aggregation by acetylsalicylic acid. ADT was a more effective inhibitor of AA-induced platelet aggregation than butylated hydroxytoluene. ADT inhibited the release of 3H-AA from platelet phospholipids in response to ADP and collagen. It is suggested that ADT inhibits platelet aggregation by inhibiting thromboxane synthesis and preventing AA release.     

Bakkenolide G,a Natural PAF-receptor Antagonist

Because platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) participates in many physiopathological responses, including inflammatory reaction, endotoxic shock, allergic diseases and platelet aggregation, PAF-receptor antagonists are important in the treatment of these diseases. A biologically active compound, bakkenolide G, extracted from the plant Petasites formosanus selectively and concentration-dependently inhibited PAF-induced platelet aggregation and ATP release. The IC50 of bakkenolide G for PAF (2 ng mL^?1)-induced platelet aggregation was 5.6 ± 0.9 μm . Bakkenolide G also concentration-dependently inhibited PAF-induced intracellular signal transductions, including thromboxane B₂ formation, and increased intra-cellular calcium concentration and phosphoinositide breakdown without affecting those caused by thrombin (01 units mL^?1), collagen (10 μg mL^?1), arachidonic acid (100 μm ) and U46619 (1 μm ). Bakkenolide G shifted the concentration-response curves of PAF-induced platelet aggregation parallel to the right; the Schild plot slope and the pA₂ value were 1.31 ± 0.31 and 6.21 ± 0.75, respectively. Moreover, bakkenolide G concentration-dependently competed with [³H]PAF binding to platelets, with an IC50 value of 2.5 ± 0.4 μm . These data strongly indicate that bakkenolide G is a specific PAF-receptor antagonist as an antiplatelet aggregatory agent.     

15,16-Dihydrotanshinone I, a major component from Salvia miltiorrhiza Bunge (Dansham), inhibits rabbit platelet aggregation by suppressing intracellular calcium mobilization

Radix Salviae miltiorrhiza (RSM, ‘Dansham’ in Korea, ‘Danshen’ in Chinese), the root of Salviae miltiorrhiza Bunge (Labiate) has been used as Chinese fork medicine for the treatment of cardiovascular diseases such as angina pectoris, coronary heart disease, myocardial infarction, and hypertension. In the present study, we evaluated the inhibitory effects of 15, 16-Dihydrotanshinone I, one of the major ingredients of Salvia miltiorrhiza Bunge, on platelet aggregation, with elucidation of its mechanisms of action. 15,16-Dihydrotanshinone I concentration-dependently inhibited collagen-induced aggregation of rabbit washed platelets with IC₅₀ of 8.7±5.6 μM, the potency being about seven-fold greater than EGCG, an active Green tea catechin component (IC₅₀: 56.6±48.7 μM). 15,16-Dihydrotanshinone I significantly inhibited the intracellular calcium ([Ca²⁺]_i) mobilization in a concentration-dependent manner. 15,16-dihdydrotanshinone I also significantly suppressed collagen (50 μg/mL)-induced liberation of [³H]Arachidonic acid from [³H]Arachidonic acid-incorporated rabbit platelet. In addition, 15,16-Dihydrotanshinone I at 50 μM slightly but significantly inhibited collagen-induced production of thromboxane B₂. These results indicate that 15,16-Dihydrotanshinone I exert potent anti-platelet activity via suppression of [Ca²⁺]_i mobilization and arachidonic acid liberation.     

Anti-platelet activity of KR-32560, a novel sodium/hydrogen exchanger-1 inhibitor.

We investigated the anti-platelet effect of a newly synthesized guanidine derivative KR-32560, a sodium/hydrogen exchanger-1 (NHE-1) inhibitor, together with the elucidation of the possible mode of action. KR-32560 concentration dependently inhibited the aggregation of washed rabbit platelets induced by collagen (10 microg mL(-1)) and arachidonic acid (AA; 100 microM), with IC50 values of 25 and 46 microM, respectively. Whereas, KR-32560 showed weaker potency against aggregation induced by thrombin (0.05 UmL(-1)) and U46619 (1 microM), and had no effect on thapsigargin (0.5 microM)- or A23187 (5 microM)-induced platelet aggregation up to 50 microM. KR-32560 inhibited the collagen-induced [3H]AA liberation in a concentration-dependent manner. In addition, KR-32560 significantly suppressed TXB2 formation in AA-exposed platelets, but had no effect on production of PGD2, indicating an inhibitory effect on TXA2 synthase. This finding was supported by a TXA2 synthase assay that KR-32560 inhibited the conversion of PGH2 into TXB2 with a similar magnitude to suppression of TXB2 formation. Furthermore, KR-32560 significantly inhibited the collagen-induced [Ca2+]i mobilization and serotonin secretion. Taken together, these observations suggest that the anti-platelet activity of KR-32560 may be mediated by the inhibition of cytoplasmic Ca2+ mobilization and AA liberation.     

Effects of clofibrate and 6-substituted chroman analogs on human platelet function:: Mechanism of inhibitory action

The effects of cloflbrate (CPIB) and two related cyclic analogs, 6-chlorochroman-2-carboxylic acid (CCCA) and 6-phenylchroman-2-carboxylic acid (PCCA), on human platelet function were evaluated. CPIB, CCCA and PCCA all inhibited platelet activation, i.e. aggregation and secretion of [¹⁴C]serotonin induced by ADP, epinephrine, collagen and thrombin, in a concentration-dependent manner. PCCA was at least fifty-two times more effective as an inhibitor of ADP-, epinephrine- and collagen-induced platelet activation and only 2-fold more effective as an inhibitor of thrombin-induced platelet activation when compared with CPIB or CCCA. Only PCCA inhibited platelet aggregation and [¹⁴C]serotonin secretion induced by arachidonic acid (AA) in a concentration-dependent manner. CPIB and CCCA did not inhibit AA-induced platelet activation. In fact, both of these agents had a potentiating effect on the onset of platelet aggregation by AA. All three compounds inhibited thrombin-induced release of [³H]arachidonic acid ([³H]AA) from platelet phospholipids and thrombin-mediated malondialdehyde (MDA) production. Only PCCA, however, inhibited AA-induced MDA production. These results indicate that CPIB, CCCA and PCCA all inhibit platelet activation by inhibiting prostaglandin biosynthesis. PCCA blocked AA-induced platelet activation, and this additional inhibitory action of PCCA appears to be responsible for its comparatively higher inhibitory potency. A comparison of the structure-activity relationship of the inhibitors indicated that replacement of the chloro group by a phenyl group produced a compound (PCCA) that was a potent inhibitor of prostaglandin biosynthesis and was thereby a more effective antiaggregatory agent than either CPIB or CCCA.     

Effects of dilazep on cytoplasmic calcium ion concentrations and arachidonic acid metabolism in activated human platelets

The effects of dilazep (tetrahydro-1H-1,4-diazepine-1,4(5H)-dipropanol bis(3,4,5-trimethoxybenzoate)-di-hydrochloride monohydrate, Comelian), a coronary and cerebral vasodilator and an antiplatelet drug, on the cytoplasmic Ca2+ concentration ([Ca2+]i) and arachidonic acid (AA) metabolism in activated human platelets were investigated. [Ca2+]i (free calcium ion concentration) of aequorin-loaded platelets was estimated by using the platelet ionized calcium aggregometer. AA metabolism was studied by the determination of AA metabolites, hydroxyheptadecatrienoic acid and 12-hydroxyeicosatetraenoic acid, using reversed-phase high performance liquid chromatography. When platelets were preincubated with dilazep (0-0.5 mmol/l), the drug inhibited both platelet aggregation and [Ca2+]i elevation induced by thrombin, AA and collagen in a concentration dependent manner, while only aggregation was inhibited after stimulation with the Ca ionophore A23187 (calcimycin). Both influx and release of Ca2+ into platelet cytoplasm induced by thrombin or AA were inhibited by dilazep, while neither of them was affected when induced by A23187. Oral ingestion of dilazep as a 100-mg capsule significantly depressed the [Ca2+]i elevation induced by thrombin, AA and collagen after 3 h. Dilazep inhibited endogenous AA metabolism by platelets induced by thrombin, although it enhanced exogenous one. Thus, dilazep inhibited platelet aggregation induced by any agonists including A23187, while [Ca2+]i elevation was inhibited by the drug only when the receptor-mediated agonist was used. Furthermore, it is suggested that dilazep inhibited AA liberation from platelet membrane phospholipids, leading to reduced production of all endogenous AA metabolites after platelet activation although metabolites of exogenous AA could be increased.     

Inhibition of platelet aggregation by 5′-nucleotidase purified from Trimeresurus gramineus snake venom

By means of DEAE-Sephadex A-50 column chromatography and gel filtrations on Sephadex G-75, Sephacryl S-300 and Sephadex G-100, successively, a potent 5′-nucleotidase was purified from Trimeresurus gramineus venom. The venom 5′-nucleotidase is a single polypeptide chain and homogeneous as judged by SDS-polyacrylamide gel electrophoresis. It is a thermostable glycoprotein consisting of 589 amino acid residues. Its molecular weight was estimated to be 74,000 by SDS-polyacrylamide gel electrophoresis. It possessed nucleotidase activities toward adenosine monophosphate and adenosine diphosphate. The specific activities toward AMP and ADP were 504 ± 28 and 101 ± 8 μg P_i/min per mg, respectively. Pre-incubation of this venom's 5′-nucleotidase with ADP resulted in the cleavage of ADP and formation of adenosine. The 5′-nucleotidase activity was inhibited by EDTA. Both Zn²⁺ and Co²⁺ reversed the inhibitory effect of EDTA.In rabbit platelet-rich plasma, it inhibited completely the ADP (2 × 10^-5 g/ml)-induced platelet aggregation. It also inhibited the platelet aggregations induced by sodium arachidonate (100 μM), collagen (20 μg/ml) and ionophore A-23187 (5 μM). In rabbit platelet suspensions, it inhibited the platelet aggregation induced by ADP (2 × 10^-5 g/ml), sodium arachidonate (100 μM) and low concentration of thrombin (0.03 U/ml). The collagen (20 μg/ml)- and ionophore A-23187 (5 μM)-induced platelet aggregations were not affected significantly by this venom 5′-nucleotidase. In ADP-refractory platelet-rich plasma, the venom 5′-nucleotidase inhibited the platelet aggregations induced by collagen (20 μg/ml) or sodium arachidonate (100 μM). The venom 5′-nucleotidase showed a more pronounced inhibitory effect on sodium arachidonate-induced platelet aggregation than creatine phosphate/creatine phosphokinase and apyrase did. No lactate dehydrogenase was released by this venom 5′-nucleotidase, indicating that no platelet lysis occurred. It is concluded that removal of ADP, which is released by these platelet aggregation inducers, and the subsequent accumulation of adenosine are responsible for the inhibitory effect of the venom 5′-nucleotidase on platelet aggregations.     

Triphenyltin fluoride in vitro inhibition of rabbit platelet collagen-induced aggregation and ATP secretion and blockade of arachidonic acid mobilization from membrane phospholipids

Recent studies have demonstrated that triphenyltin fluoride (TPTF) inhibits collagen-induced aggregation and ATP secretion of rabbit platelets in vivo [S. Manabe and O. Wada, J. Toxic. Sci. 6, 236 (1981)]. The aim of the present investigation was to test the effects in vitro of TPTF on platelet aggregation and to elucidate the mechanism of the inhibitory action by studying the release and metabolism of arachidonic acid and the cyclic AMP contents of rabbit platelets treated in vitro with TPTF. Although no inhibitory effect of TPTF was found on sodium arachidonate-induced platelet aggregation and ATP secretion, TPTF inhibited both reactions induced by collagen. Triphenylarsine and triphenylantimony did not inhibit, even at a concentration of 10(-3) M. The anti-aggregating concentration (IC50) of TPTF was 6.0 x 10(-6) M against collagen. TPTF had no inhibitory effect on the conversion of exogenous arachidonic acid to malondialdehyde (MDA) by platelets, while the collagen-induced production of arachidonate metabolites [MDA, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2] was remarkably inhibited by TPTF. Furthermore, TPTF apparently inhibited the collagen-induced release of arachidonic acid from platelets, although the formation of phosphatidic acid was not inhibited. Total cyclic AMP content after TPTF exposure was not changed significantly. These results indicate that TPTF inhibited the collagen-induced arachidonic acid release from platelet phospholipids, presumably by acting on phospholipase A2. Furthermore, it seems unlikely that the inhibition of arachidonic acid release by TPTF can be explained by the level of cyclic AMP in platelets.     

Effects of reserpine on rabbit platelet aggregation and adherence to collagen or injured rabbit aorta

Effects of reserpine in vivo and in vitro on rabbit platelets in citrated platelet-rich plasma and in suspensions of washed platelets have been studied. Administration of reserpine ($\text{5}\text{mg}\text{kg}$) intraperitoneally 18 hr before platelets were isolated caused inhibition of collagen-induced aggregation but not of aggregation induced by ADP or thrombin. Thrombin-induced aggregation was slightly enhanced. Platelets from reserpine-treated rabbits were less adherent than control platelets to collagen-coated glass surfaces or to the subendothelium of the rabbit thoracic aorta. Similar effects on aggregation were obtained when reserpine (0.2 to 10 μM) was added to suspensions of washed rabbit platelets as little as 2 sec before the addition of collagen. Collagen-induced release of nucleotides and [¹⁴C]serotonin from prelabeled washed rabbit platelets was not affected by the presence of reserpine, whereas thrombin-induced release was slightly enhanced. Inhibition by reserpine (2–10 μM) of platelet adherence to a collagen-coated surface or to the subendothelium was also observed within a time interval too short for the reserpine to have caused depletion of platelet granule contents. Thus, reserpine has an immediate effect on the plasma membrane of the platelets which is responsible for inhibition of platelet adherence to collagen and hence of collagen-induced aggregation. This inhibitory effect differs from a much slower effect of reserpine at the granule membrane which results in the depletion of the granule contents of serotonin and adenine nucleotides. The effect of reserpine is not abolished by washing and resuspending platelets that have been exposed to reserpine in vivo. By inhibiting the interaction of platelets with collagen, reserpine may interfere with one of the components of hemostatic plug and thrombus formation.     

Biopharmaceutics: Biochemical Pharmacology: Inhibition of Human Platelet Aggregation by Diazeniumdiolates: Extent of Inhibition Correlates with Nitric Oxide Load Delivered

The profile of nitric oxide (NO) release from the diazeniumdiolate class of NO donors was evaluated using inhibition of platelet aggregation as a model. At 37°C, the NO complexes (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}-diazen-1-ium-1, 2-diolate (dimethylhexanediamine complex), sodium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (diethylamine complex), (Z)-1-{N-3-aminopropyl-N-[N-(3-aminopropylammonio)butyl]amino}diazen-1-ium-1,2-diolate (spermine complex), and (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,22-diolate (diethylene-triamine complex) have half-lives of 1, 2 and 39min, and 20 h, respectively. All the diazeniumdiolates caused concentration-dependent inhibition of platelet aggregation; IC50 values (values for which the effect was half the maximum) were 26.0 ± 24.1, 34.9 ± 24.0 and 14.9 ± 6.4 nM for dimethylhexanediamine complex, diethylamine complex and spermine complex, respectively, when pre-incubated with platelets for one half-life. Inhibition by all compounds was time-dependent. Pretreatment of platelets with spermine complex for 5 and 39 min resulted in IC50 values of 1.7 ± 0.85 μM and 19.7 ± 0.12 nM, whereas IC50 values for sodium nitroprusside were 27.3 ± 1.25 nM and 25 nM (average, n = 2), at 5 and 39 min, respectively. Pre-incubation of each diazeniumdiolate at a concentration of 100 nM for 5 min at 37°C, which resulted in the theoretical delivery of NO loads from 96.9% down to 0.3%, resulted in decreasingly efficacious inhibition of platelet aggregation. Linear regression analysis of the theoretical NO load delivered against the actual maximum inhibition (%) showed a strong correlation (r² = 0.975). All four diazeniumdiolates caused concentration- and time-dependent inhibition of agonist-stimulated elevation of intra-platelet Ca²⁺ levels; IC50 values were, respectively, 8.7 ± 1.49nM and 11.5 ± 1.36nM for dimethylhexanediamine complex and diethylamine complex at their half-lives, and 176 ± 16.9 nM and > 100 μM, for spermine complex and diethylenetriamine complex at 2 min pre-incubation time. The respective nucleophiles not complexed with NO did not show anti-aggregatory properties or inhibition of agonist-induced elevation of intra-platelet Ca²⁺ levels. The inhibitory effects of all diazeniumdiolates tested were attenuated by 10 μm haemoglobin. These studies indicate that these compounds induce controlled, predictable release of NO at biological pH and temperature.     

Antiplatelet activity of [5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl]guanidine (KR-32570), a novel sodium/hydrogen exchanger-1 and its mechanism of action

The antiplatelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen (10 microg/mL), thrombin (0.05 U/mL), arachidonic acid (100 microM), a thromboxane (TX) A2 mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2, 1 microM) and a Ca2+ ATPase inhibitor thapsigargin (0.5 microM) (IC50 values: 13.8 +/- 1.8, 26.3 +/- 1.2, 8.5 +/- 0.9, 4.3 +/- 1.7 and 49.8 +/- 1.4 microM, respectively). KR-32570 inhibited the collagen-induced liberation of [3H]arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at 50 microM. The TXA2 synthase assay showed that KR-32570 also inhibited the conversion of the substrate PGH2 to TXB2 at all concentrations. Furthermore, KR-32570 significantly inhibited the [Ca2+]i mobilization induced by collagen at 50 microM, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen (10 microg/mL)-induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, TXA2 synthase, the mobilization of cytosolic Ca2+ and NHE-1.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

Arachidonate metabolism, 5-hydroxytryptamine release and aggregation in human platelets activated by palmitaldehyde acetal phosphatidic acid

Palmitaldehyde acetal phosphatidic acid ( PGAP ) caused dose-dependent aggregation of human platelets resuspended in modified Tyrode medium, with a threshold concentration of 0.5-1 microM and an EC50 of 4 microM. Concentrations of PGAP which elicited biphasic irreversible aggregation concomitantly induced formation of 1.02 +/- 0.029 nmol (mean +/- s.e. mean) of malondialdehyde (MDA) per 10(9) platelets and caused release of 58 +/- 2.8% of platelet [14C]-5-hydroxytryptamine ([14C]-5-HT) from prelabelled platelets; no MDA formation or [14C]-5-HT release occurred at lower doses of PGAP which elicited only monophasic reversible aggregation. Adenosine 5'-pyrophosphate (ADP)-induced platelet activation resulted in formation of 0.344 +/- 0.004 nmol of MDA per 10(9) platelets in association with irreversible aggregation and 49.1 +/- 1% release of [14C]-5-HT. Mepacrine, a phospholipase A2 inhibitor, at 2.5 microM reduced PGAP -induced MDA formation and [14C]-5-HT release by the resuspended platelets without affecting irreversible aggregation; higher concentrations of mepacrine abolished all three responses. Chlorpromazine, a calmodulin antagonist, similarly inhibited PGAP -induced MDA formation and irreversible aggregation, and at 100 microM abolished monophasic aggregation. The cyclo-oxygenase inhibitor indomethacin caused a concentration-dependent reduction of PGAP -induced MDA formation by resuspended human platelets without significantly inhibiting [14C]-5-HT release or irreversible aggregation; concentrations (greater than or equal to 1.75 microM) which inhibited MDA formation by more than 94% abolished [14C]-5-HT release, and converted second phase irreversible aggregation to an extensive reversible response. 2-Methylthioadenosine 5'-phosphate (2 methylthio-AMP), an ADP antagonist, inhibited PGAP -induced MDA formation, [14C]-5-HT release and second phase aggregation in the human platelet suspensions in a parallel, concentration-dependent manner; at 9.4 microM 2-methylthio-AMP, both MDA formation and [14C]-5-HT release were abolished and monophasic, reversible aggregation remained. Albumin was required for aggregation of washed human platelets to PGAP . Irreversible PGAP -induced aggregation of washed [14C]-arachidonate-labelled platelets was accompanied by a low net loss of 14C from platelet phospholipids, an equivalent increase in 14C in free fatty acids, and the appearance of 14C in thromboxane (Tx)B2; mepacrine reduced the loss in 14C from phospholipids and inhibited aggregation and formation of [14C]-TxA2.(ABSTRACT TRUNCATED AT 400 WORDS)     

The inhibitory effects of cannabinoids, the active constituents of Cannabis sativa L. on human and rabbit platelet aggregation

Olivetol, cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN) and tetrahydrocannabinol (delta 1-THC) were assessed for their ability to inhibit agonist-induced platelet aggregation and [14C]5-HT release. With the exception of olivetol, (40% maximal effectiveness), none of the compounds inhibited tetradecanoylphorbolacetate (TPA)-induced aggregation of human or rabbit platelets. All of these cannabinoids partially inhibited primary aggregation and totally inhibited secondary aggregation of human platelets when adrenaline was used as the agonist. Inhibition was dose-dependent over the range 10(-3)-10(-5) M. Both rabbit and human platelet aggregation induced by adenosine diphosphate was inhibited in a dose-dependent manner and the order of potency was CBG greater than CBD greater than olivetol greater than THC greater than CBN, the IC50 of CBG being 2.7 x 10(-4) M. PAF-induced aggregation of rabbit platelets was also inhibited by these compounds in a dose-dependent manner over the concentration range 10(-3) to 10(-4) M, however [14C]5-HT release was only partially prevented by the cannabinoids in a manner which did not correlate with inhibition of aggregation.     

Antiplatelet Mechanism of 2‐Chloro‐3‐(4‐hexylphenyl)‐amino‐1,4‐naphthoquinone (NQ304), an Antithrombotic Agent

Abstract: The effects of 2‐chloro‐3‐(4‐hexylphenyl)‐amino‐1,4‐naphthoquinone (NQ304), an antithrombotic agent, on aggregation, binding of fibrinogen to glycoprotein IIb/IIIa and intracellular signals were investigated using human platelets. NQ304 inhibited thrombin‐, arachidonic acid‐ and thapsigargin‐induced aggregation of washed human platelets with the IC₅₀ values of 22.2±0.7, 6.5±0.2, and 7.6±0.1 μM, respectively. NQ304 significantly inhibited fluorescein isothiocyanate‐conjugated fibrinogen binding to human platelet surface glycoprotein IIb/IIIa receptor by 75%, but failed to inhibit the fibrinogen binding to purified glycoprotein IIb/IIIa receptor. This result suggests that NQ304 inhibit platelet aggregation by suppression of an intracellular pathway that involves exposure of the glycoprotein IIb/IIIa receptor, rather than by direct inhibition of fibrinogen‐glycoprotein IIb/IIIa binding. NQ304 significantly inhibited thrombin‐induced increase in intracellular Ca²⁺ mobilization at the dose of 30 μM and ATP secretion in a dose‐dependent manner. It also inhibited thrombin‐ and arachidonic acid‐induced thromboxane A₂ formation in human platelet dose‐dependently. In conclusion, the antiplatelet mechanism of NQ304 may be due to the reduction of the thromboxane A₂ formation, inhibition of adenosine triphosphate release and intracellular calcium mobilization.     

Inhibition of platelet aggregation by papaverine-like drugs: evidence for a novel mechanism of action.

Trimethoquinol [6,7-dihydroxy-1-(3′,4′,5′-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline] (TMQ) was chosen as a model compound for studying inhibition of platelet aggregation in vitro, because of its β-adrenoceptor agonist properties and structural resemblance to the anti-aggregatory agent, papaverine. TMQ inhibited collagen-induced aggregation of human platelet-rich plasma (I₅₀, 2 μM), the second wave of aggregation induced by 2.5 μM ADP (I₅₀, 0.9 μM), and the second wave of aggregation induced by 45 μM epinephrine (I₅₀, 2.5 μM). Collagen-induced aggregation of human washed platelets was inhibited by TMQ (I₅₀, 1 μM). TMQ was a better inhibitor than aspirin and papaverine and had an inhibitory activity similar to indomethacin in all of the systems studied. TMQ retained inhibitory activity in the presence of both β-adrenoceptor antagonist: propranolol (50 μM), and α-adrenoceptor antagonist: phentolamine (2.5 μM). Platelet adenylate cyclase was not activated and neither cAMP nor cGMP-phosphodiesterase activities were inhibited by TMQ, PGF_2α, biosynthesis by aggregating platelets during the coagulation of blood obtained from rats pretreated with aspirin (10 mg/kg, p.o.) or indomethacin (1 mg/kg, p.o.) was inhibited. However, similar pretreatment with TMQ (100 mg.kg, p.o.) and papaverine hydrochloride (100 mg/kg, p.o.) had no effect. TMQ acted synergistically with the aggregation inhibitors: papaverine, aspirin, and PGE₁. The in vitro inhibitory action of papaverine, aspirin, and TMQ was enhanced by increasing calcium concentration. These data indicate that the platelet anti-aggregation activity of TMQ, in contrast to its myocardial stimulating and bronchodilating mechanism, is independent of adrenergic activation. Cyclic AMP accumulation or prostaglandin biosynthesis also seem not to be involved in TMQ action. Therefore, it appears that TMQ may have a novel anti-aggregatory mechanism of action.