L8027 and 1-nonyl-imidazole as non-selective inhibitors of thromboxane synthesis

The inhibition of prostaglandin-related actions on the platelets, respiration and hemodynamics of guinea pigs by 1-(isopropyl-2-indolyl)-3-pyridyl-3-ketone (compound L8027) and by 1-nonyl-imidazole (NI) was studied. L8027 (1–10 μg/kg, i.v.) inhibited arachidonic acid (AA)-initiated bronchoconstriction, thrombocytopenia and hypotension in a dose-dependent and reversible manner. NI, however, in doses as high as 8 mg/kg did not inhibit these actions. AA-induced platelet aggregation was antagonized by L8027 (50–500 nM) and NI (25–100 μM), and this action was dose-dependent and competitive. Thromboxane A2 (TxA2) synthesis in blood and platelets, as measured by the rabbit aorta bioassay, was suppressed in a concentration-related manner by L8027 at anti-aggregatory doses. NI did not inhibit TxA2 synthesis in guinea-pig platelet-rich plasma (PRP) at concentrations which blocked platelet aggregation. However, it did block TxA2 synthesis in rabbit and human PRP and in washed guinea-pig platelets, indicating a species or plasma specificity. Both compounds inhibited ATP release from platelets as they inhibited aggregation. The radioimmunoassay revealed that L8027 decreased formation of both TxB2 and PGE2, unlike imidazole which only blocked formation of TxB2 and resulted in an increase of PGE2. These results show that although L8027 does block TxA2 formation, it is not a selective antagonist and it inhibits cyclo-oxygenase as well. NI appears to have the ability to act via mechanisms other than prostaglandin metabolism.     

Some properties of rat platelet aggregation and effects of butylated hydroxytoluene, warfarin and aspirin

The platelet aggregation characteristics of male Sprague-Dawley (Jcl:SD) rats were investigated. Epinephrine, ristocetin, serotonin and platelet-activating factor were ineffective in rat platelets. Heparinized platelet-rich plasma (PRP) was more sensitive than citrated PRP to three aggregating agents, ADP, collagen and arachidonic acid. Butylated hydroxytoluene (BHT) and BHT quinone methide (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone) inhibited ADP- and collagen-induced aggregation at concentrations over 10(-3) M in vitro. The ADP-, collagen- and arachidonic acid (0.5-2.0 mM)-induced aggregations of PRP obtained from rats given 1.20% BHT in the diet for 7 days were normal, while arachidonic acid (3.9 mM)-induced aggregation of PRP from BHT-fed rats was significantly lower than control. PRP from rats given aspirin and warfarin also aggregated normally with ADP or collagen addition. These results suggest that heparinized PRP may be preferable in platelet aggregation analyses in rats and reaffirmed that effects on platelet aggregation may not play a key role in BHT-induced bleeding. Platelet aggregation capacity also does not necessarily reduce in haemorrhages induced by aspirin or warfarin.     

牛心内膜腺苷三磷酸双磷酸酶对血小板聚集的抑制作用

AIM: To study the anti-aggregatory effect of bovine endocardial endothelial cell (EEC)-associated apyrase. METHODS: Cultured bovine EEC was used. Adenosine diphosphate (ADP) was analyzed by reversed phase HPLC, and rabbit platelet aggregation was measured turbimetrically. RESULTS: Incubation of EEC with ADP 500 mumol.L-1 resulted in a progressive decrease in ADP concentration, which was paralleled by the decrease in platelet aggregating potential of the unmetabolized ADP. In the presence of aspirin (Asp 1 mmol.L-1)-treated EEC 1 x 10(9) cells.L-1, the aggregation of Asp (1 mmol.L-1) and methylene blue (10 mumol.L-1)-treated platelets in response to thrombin 500 U.L-1 and platelet activating factor (PAF 1 nmol.L-1) was markedly inhibited and was reversible, which was very similar to that in apyrase-treated platelets. The supernatants of EEC had no effect on platelet aggregation. EEC inhibited ADP (5 mumol.L-1)-induced platelet aggregation, but failed to inhibit adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S, an unmetabolizable structural analog of ADP, 15 mumol.L-1)-induced platelet aggregation. CONCLUSION: ADP hydrolysis by EEC-associated apyrase is a major anti-thrombotic mechanism of bovine EEC.     

Effects of PAF-acether and structural analogues on platelet activation and bronchoconstriction in guinea-pigs

PAF-acether (platelet-activating factor) (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine) induces platelet-dependent bronchoconstriction in the guinea-pig which correlates with its in vivo thrombocytopenic effect. We investigated the influence of modifications of the polar head group in position 3 of the glycerol skeleton of PAF-acether on guinea-pig platelet activation and bronchoconstriction. PAF-acether itself induced concentration-dependent platelet activation (EC50 for aggregation = 0.41 nM and EC20 for secretion of ATP = 0.56 nM). The 3-phosphoryl-N-methyl-morpholino ethanol analogue was slightly more active than PAF-acether and the 3-phosphoryl-N-methyl-piperidinium ethanol, 3-phopshoryl-(N-methyl-piperidino-3') methanol and 3-phosphoryl-(N-methyl-hydroxy-4') piperidine analogues were equieffective to PAF-acether in activating platelets. The 3-phosphoryl-piperidino ethanol analogue was 8 times less active than PAF-acether; the 3-phosphoryl-morpholino ethanol analogue and the 1-O-octadecyl-2-O-acetyl-3-O-[trimethyl-ammonio)-propyl) glycerol were inactive up to 1 microM. Our data show that the choline head group is not a compulsory requirement for activity. When injected i.v. to the propranolol-treated guinea-pigs, the platelet-activating analogues also induced bronchoconstriction. Two PAF-acether antagonists, compounds 48740 RP and BN 52021, inhibited PAF-acether-induced platelet activation when added to PRP at the final concentration of 0.1 mM (aggregation inhibited by 91 +/- 4 and by 94 +/- 3% respect.; secretion inhibited by 80 +/- 12 and 79 +/- 10% respectively, mean +/- S.E.M., n = 4). Both antagonists also suppressed platelet activation and in vivo bronchoconstriction, thrombocytopenia, leukopenia and hypotension induced by PAF-acether and the various analogues. Our results indicate that PAF-acether and the analogues studied trigger platelet activation and the consequent bronchoconstriction through mechanisms which share sensitivity to same antagonists.     

Shape change and aggregation of blood platelets: interaction between the effects of adenosine and diphosphate, 5-hydroxytryptamine and adrenaline.

1 The interaction of effects between 5-hydroxytryptamine (5-HT) and adenosine diphosphate (ADP) on human or rabbit platelets was investigated in vitro. The initial platelet shape change and their aggregation were measured in stirred, citrated platelet-rich plasma (PRP) at 37 degrees C by recording the rate and extent of changes in light scattering and light transmission. 2 Both the velocity and extent of aggregation and the velocity and extent of the rapid morphological change caused by ADP were enhanced by simultaneous addition of 5-HT. Methysergide but not imipramine inhibited the 5-HT effects. 3 Platelets were made refractory to the aggregating and shape changing effect of either ADP or 5-HT by repeated aggregation with the particular agent; platelets made refractory to ADP retained their responsiveness to 5-HT and platelets made refractory to 5-HT responded to ADP. Platelets pre-incubated for 3-10 min with 5-HT without aggregation showed greatly reduced aggregation on subsequent addition of ADP. Methysergide inhibited all the effects of 5-HT whilst imipramine was inactive. 4 When the shape change or aggregation of platelets induced by ADP was submaximal, addition of 5-HT increased it further. Pre-incubation of PRP with 5-HT before the addition of ADP resulted in failure of the secondary induction of aggregation or shape change by 5-HT. The secondary induction by 5-HT also did not occur in the presence of methysergide; imipramine had no inhibitory effect. Similar secondary induction of aggregation was shown by adrenaline injected during aggregation by ADP; the adrenaline effect was removed by phentolamine but not by propranolol. 5 Our results show that the initial change in shape of platelets and their aggregation can be induced by ADP or 5-HT in specific manner. The interaction of the effect of these substances on platelets can result in either increase in platelet sensitivity or, under certain conditions, decrease in platelet responsiveness. The increase or depression of platelet reactivity appears to be a highly specific effect and is probably mediated at specific receptors involved with platelet activation.     

Pharmacological modulation of the respiratory and haematological changes accompanying active anaphylaxis in the guinea-pig

The intrathoracic accumulation of radiolabelled platelets was recorded in anaesthesized, sensitized guinea-pigs concomitantly with changes in airways resistance during active anaphylaxis. Antigen challenge induced an increased airway resistance associated with intrathoracic platelet accumulation, thrombocytopenia and leukopenia. Bronchoconstriction, but not the haematological changes, was modified by a combination of mepyramine, methysergide and aspirin. The addition of FPL 55712 and PGI2 to this cocktail of drugs additionally inhibited the thrombocytopenia and leukopenia but not the intrathoracic accumulation of platelets induced by antigen challenge. PGI2 was alone able to significantly inhibit the intrathoracic platelet accumulation, but not the bronchoconstriction induced by antigen challenge. These results suggest that active anaphylaxis is associated with activation of blood elements and subsequent pulmonary entrapment of platelets but that the platelet release reaction rather than the embolisation of platelet aggregates is the critical step in the induction of allergic bronchoconstriction in the guinea-pig.     

Dicentrine, a novel antiplatelet agent inhibiting thromboxane formation and increasing the cyclic AMP level of rabbit platelets.

Dicentrine is an antiplatelet agent isolated from the Chinese herb Lindera megaphylla. We examined the in vitro effects of dicentrine on various aspects of platelet reactivity. Dicentrine inhibited the aggregation and ATP release of washed rabbit platelets induced by arachidonic acid (AA), collagen, ADP, platelet-activating factor (PAF), thrombin and U46619. Dicentrine also inhibited the thromboxane B2 formation caused by AA, collagen and thrombin in washed intact platelets or that induced by AA in lysed platelet homogenate, while prostaglandin D2 formation caused by AA was not increased. The generation of inositol monophosphates (in the presence of indomethacin) caused by thrombin, collagen and PAF was not suppressed significantly, nor did dicentrine suppress fibrinogen-induced aggregation of elastase-treated platelets. Dicentrine inhibited the intracellular Ca2+ increase in quin-2/AM-loaded platelets caused by thrombin, PAF, collagen and AA. The cyclic AMP level was elevated by dicentrine in a concentration-dependent manner. These data indicate that the inhibitory effect of dicentrine on platelet aggregation and ATP release was due to the inhibition of thromboxane formation and the elevation of the level of cyclic AMP.     

Dual effects of aspirin in guinea-pig lungs.

Dual effects of aspirin were demonstrated in guinea-pig lungs: (a) aspirin (3.3 mg/kg i.v.) antagonized bronchoconstriction induced by slow reacting substance of anaphylaxis (SRS-A); (b) aspirin produced bronchoconstriction when injected in the presence of propranolol into guinea-pigs in vivo at 330 mg/kg, or into guinea-pig isolated lungs in vitro as a 4% solution (40 mg/ml). 2 The severity of bronchoconstriction following administration of aspirin was directly related to the degree of beta-adrenoceptor blockade and to the age of the guinea-pigs. Aspirin-induced bronchoconstriction was prevented in vivo and in vitro by atropine and it could be reversed in vivo by atropine. Aspirin-induced bronchoconstriction was not inhibited by vagotomy or phenoxybenzamine. 3 These data suggest that the mechanism involved in aspirin-induced bronchoconstriction may be local cholinergic stimulation and that reduced beta-adrenergic drive may be a predisposing factor.     

Effects of plumbagin on platelet aggregation and platelet-neutrophil interactions

The effects of plumbagin were investigated on platelet aggregation in vitro and ex vivo, on the binding of thrombin-stimulated platelets to neutrophils, and platelet aggregation induced by intact neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet activating factor (PAF) activated neutrophils, by use of the methods of Hamburger, McEver and Born, respectively. The results showed that plumbagin in vitro significantly inhibited adenosine diphosphate (ADP)-, arachidonic acid (AA)-, or platelet activating factor (PAF)-induced platelet aggregation, in a concentration-dependent manner. The medium inhibitory concentrations (IC 50 ) were 39.4, 82.7 and 38.1 microM, respectively. Intragastric plumbagin at 10 mg/kg markedly suppressed platelet aggregation induced by ADP, AA, or PAF. Plumbagin decreased the binding between thrombin-stimulated platelets and neutrophils with an IC 50 of 62.9 microM. Plumbagin significantly inhibited washed platelet aggregation stimulated by fMLP- or PAF-activated neutrophils. The IC 50 values were 54.3 and 47.6 microM, respectively. On the other hand, plumbagin and aspirin increased the inhibition of intact neutrophils on AA-induced platelet aggregation. It is suggested that plumbagin inhibited platelet aggregation in vitro and ex vivo, suppressed the binding of activated platelets to neutrophils, inhibited platelet aggregation induced by activated neutrophils, and increased inhibition of intact neutrophils on platelet reactivity. Abbreviations. DMSO:dimethyl sulphoxide fMLP: N-formyl-methionyl-leucyl-phenylalanine ADP:adenosine diphosphate AA:arachidonic acid PAF:platelet activating factor     

Pharmacological properties of the novel anti-platelet aggregating agent 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid

Various pharmacological properties of a new antiplatelet aggregating agent, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid (E-5510), were examined in order to elucidate its mode of action, Firstly, the inhibitory effect on in vitro aggregation of platelets from humans and various experimental animals was studied. E-5510 inhibited human platelet aggregation induced by collagen, arachidonic acid, adenosine diphosphate (ADP), platelet activating factor (PAF) and epinephrine. Thrombin-induced platelet aggregation, which was not inhibited by acetylsalicylic acid (ASA) or the thiazole drug, 4,5-bis(4-methoxyphenyl)-2-(trifluoromethyl) thiazole, was inhibited by E-5510. E-5510 inhibited collagen-induced platelet aggregation in platelet-rich plasma (PRP) from guinea pigs, beagle dogs and monkey to the same degree as in human PRP, but its effect was weaker in rat PRP. Human platelet adhesion to a collagen-coated plastic disk and thrombin-induced adenosine triphosphate (ATP) release from human platelets were also inhibited by this compound. Next, the ex vivo anti-platelet effect of E-5510 was examined in guinea pigs and beagle dogs. E-5510 was the most potent among the tested drugs (ticlopidine, ASA, cilostazol and the thiazole drug. The anti-platelet effect of this compound appeared within 1 h and lasted more than 8 h after oral administration. The above results suggest that E-5510 may antagonize platelet activation by inhibiting phospholipase C and/or A2, which results in suppression of both phosphatidylinositol breakdown and arachidonic acid release from phospholipids, as well as by inhibiting cyclooxygenase. E-5510 exerted its anti-platelet action without affecting prostaglandin I2 production in the blood vessels. It is considered that E-5510 has a highly potent anti-platelet aggregating effect and a unique multi-site mode of action. This compound is a promising candidate as an antithrombotic drug for clinical use.     

Platelet release reaction and aggregation induced by canatoxin, a convulsant protein: evidence for the involvement of the platelet lipoxygenase pathway.

Canatoxin is a toxic protein isolated from Canavalia ensiformis seeds. It induces death preceded by convulsions of spinal cord origin and also produces in vitro aggregation of platelets in rabbit, human and guinea-pig plasma. The aggregating effect is dose-dependent at nanomolar concentrations. Rabbit platelets pretreated with canatoxin became refractory to a second exposure to this protein or to collagen, but were still responsive to ADP, Paf-acether or arachidonic acid. [14C]-5-hydroxytryptamine was released from pre-labelled platelets on stimulation with canatoxin. Washed rabbit platelets, but not thrombin-degranulated ones, aggregated on stimulation with canatoxin provided that fibrinogen was added before the toxin. Canatoxin's pro-aggregating activity was inhibited by mepacrine, EDTA, caffeine, prostacyclin, adenosine monophosphate and also by the ADP scavenger system, creatine phosphokinase/creatine phosphate. Furthermore, 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW 755C), eicosatetraynoic acid (ETYA) and nordihydroguaiaretic acid (NDGA) were potent inhibitors of canatoxin-induced aggregation. In contrast, no inhibition was seen with indomethacin. The data indicate that canatoxin is mainly a release-reaction-promoting agent, being devoid of any direct aggregating activity. Thus the aggregation is totally dependent on the release of ADP. Furthermore, canatoxin-induced platelet activation is probably dependent on platelet phospholipase A2 and lipoxygenase activity but is not dependent on cyclo-oxygenase products or the release of Paf-acether.     

Pharmacological modulation of the effects of N-formyl-L-methionyl-L-leucyl-L-phenylalanine in guinea-pigs: involvement of the arachidonic acid cascade.

The intravenous administration of the chemotactic and secretagogue peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 0.3-30 micrograms kg-1) to the guinea-pig induces bronchoconstriction and dose-dependent leukopenia accompanied by mild thrombocytopenia. No electron microscopic evidence of platelet aggregation in lungs or significant accumulation of 111In-labelled platelets in the thoracic region at the height of bronchoconstriction was noted. Bronchoconstriction and leukopenia induced by FMLP were not affected by prostacyclin, by platelet depletion, by the platelet-activating factor (Paf-acether) antagonist BN 52021 or by the histamine H1-antagonist mepyramine. Bronchoconstriction, but not leukopenia, was inhibited by aspirin, whereas the peptido-leukotriene antagonist compound FPL 55712 and the cyclo-oxygenase lipoxygenase inhibitor indomethacin reduced bronchoconstriction to a limited extent only. The mixed cyclo-oxygenase/lipoxygenase inhibitor compound BW 755C was very effective in blocking bronchoconstriction by the highest dose of FMLP used, but failed to interfere with leukopenia. FMLP-induced dose-dependent contraction of parenchymal lung strips was accompanied by the formation of immuno-reactive thromboxane B2 in amounts markedly less than those formed from exogenous arachidonic acid at concentrations equieffective in inducing contractions. FMLP-induced contractions of the guinea-pig lung strip were not modified by mepyramine nor by FPL 55712. They were reduced by indomethacin and aspirin and an even greater reduction was obtained with aspirin used in combination with FPL 55712. BW 755C suppressed the effects of all the concentrations of FMLP tested, whereas tert-butyloxy-carbonyl-L-methionyl-L-leucyl-L-phenylalanine, a chemical analogue of FMLP, displaced the concentration-response curve to the right, without reducing the maximal contraction obtained. The present results indicate that: (a) bronchoconstriction by FMLP is not due to platelet activation, to cyclo-oxygenase-dependent mechanisms or to peptido-leukotriene formation. The inhibitory effect of aspirin and BW 755C involves a property other than cyclo-oxygenase inhibition, which is not shared by indomethacin. (b) The contractile effects of FMLP on parenchymal lung strips follow an interaction with specific receptor sites, as shown by the effectiveness of tert-butyloxy-carbonyl-L-methionyl-L-leucyl-L-phenylalanine, and involves the combined effects of cyclo-oxygenase and lipoxygenase metabolites.     

In-vitro and Ex-vivo Inhibition of Blood Platelet Aggregation by Naftazone

Because of the considerable interest in the role of platelets and antiplatelet therapy in cardiovascular disease, including the aggregation of platelets to each other during arterial thrombosis and atherogenesis, we have studied the effect of naftazone (Etioven), an original vasculotropic drug on platelet aggregation. Rat and human platelets were prepared and incubated in-vitro with different concentrations of naftazone. We found that naftazone inhibited both platelet secretion and aggregation in platelet-rich plasma (PRP) and washed platelets after stimulation with thrombin or ADP. Rats were also treated intraperitoneally for five days with various naftazone doses (0.125-10 mg kg^?1) and ex-vivo platelet aggregation compared, at various times after the last injection, with that of control animals. Inhibition by naftazone was dose-dependent in both PRP and isolated platelets. The inhibition was transient, a maximum value (～ 50%) being obtained about 3–6 h after the last injection, with a return to near-control values after 24 h. Naftazone also facilitated platelet deaggregation after in-vitro stimulation with thrombin or ADP. In another series of experiments, rats were treated intraperitoneally for five days with 10 mg kg^?1 of aspirin, ticlopidine, dipyridamole or naftazone. Platelets were prepared and tested for aggregation 90 min after the last injection. Thrombin-induced aggregation in PRP and washed platelets was significantly reduced after in-vivo treatment with ticlopidine and naftazone. Except for dipyridamole, all the drugs inhibited ex-vivo ADP-induced aggregation in PRP. In isolated platelet preparation, only naftazone induced a significant inhibition of ADP-or thrombin-stimulated aggregation. We conclude that naftazone inhibits platelet aggregation in-vitro and ex-vivo.     

Relationship of arachidonic acid concentration to cyclooxygenase-dependent human platelet aggregation

Inhibition of ex vivo arachidonic acid (AA)-induced aggregation is a biomarker for the isotype selectivity of cyclooxygenase (COX) inhibitors since platelets express COX-1 but not COX-2. At low concentrations, there is broad inter- and intrasubject variability in AA-induced aggregation of platelets ex vivo. This study defined a concentration that reliably induces aggregation without overcoming inhibition by therapeutic aspirin therapy (ASA, 81-mg) treatment. Logistic regression analysis of ex vivo aggregation, induced with increasing concentrations of AA in platelet-rich plasma (PRP), estimated that platelets from > or = 90% of subjects would aggregate at > or = 1.5 mM AA (95% confidence interval [CI], 1.1, 2.1). A concentration of 1.6 mM AA failed to aggregate platelets from 26 healthy volunteers, who had previously aggregated at this concentration, following six daily oral doses of 81 mg of ASA. These data demonstrate that 1.6 mM AA reproducibly induces platelet aggregation in PRP from healthy volunteers without overcoming the antiplatelet effect of daily low-dose aspirin therapy.     

Estimation of anti-platelet drugs on human platelet aggregation with a novel whole blood aggregometer by a screen filtration pressure method

1. The effects of anti-platelet drugs on human whole blood aggregation were evaluated using a novel whole blood aggregometer by a screen filtration pressure (SFP) method. 2. The SFP whole blood aggregometer was found to successfully detect whole blood aggregation induced by ADP, collagen and TRAP by measuring the SFP of blood samples. The platelet aggregation threshold index (PATI), the concentration of agonist required with an inducing pressure rate of 50%, varied time-dependently after collection of blood. High values for ADP and collagen were noted immediately after blood collection, suggesting low aggregation activity of platelets, and gradually increase thereafter. 3. Cilostazol (phosphodiesterase 3 inhibitor), dipyridamole, aspirin and tirofiban all inhibited whole blood aggregation in vitro. Inhibitory effects of cilostazol and dipyridamole, but not tirofiban, were markedly enhanced 6 or 7 fold by long pre-incubation (60 min), compared with short pre-incubation (2 min). Such enhancement was only observed with ADP- and not collagen-induced whole blood aggregation. A similar phenomenon was also observed for aggregation with platelet rich plasma (PRP). Cilostazol inhibition of ADP-induced platelet aggregation was more potent with PRP than whole blood (PATI(200)=3.80+/-0.95 microM for whole blood; 2.04+/-0.61 microM for PRP). Inhibitory effects of dipyridamole were attenuated in PRP without erythrocytes. 4. These results demonstrate that the SFP aggregometer can sensitively detect anti-platelet aggregatory effects of various kinds of drugs. So that it is a useful tool for evaluation of anti-platelet drugs.     

Captopril augments both basal and frusemide-induced natriuresis in normal man by suppression of circulating angiotensin II.

1. The effect of increasing doses of orally administered aspirin (30-900 mg) on platelet aggregation and ATP release induced by arachidonic acid (AA), collagen and platelet activating factor (PAF) was assessed in 12 normal volunteers. 2. Aspirin ingestion was associated with a significant increase in EC50 for AA (P less than 0.0001) and collagen (P less than 0.0001) but not for PAF (P greater than 0.495) although the normal biphasic aggregation response for the latter was abolished. Maximum ATP release was reduced by aspirin for all three agonists. 3. The mean maximum degrees of inhibition of platelet aggregation induced by aspirin for AA, collagen and PAF were 100%, 48% and 21% of baseline, respectively. The corresponding mean maximum inhibition of ATP release was 100%, 63% and 57%. The minimum cumulative doses of aspirin producing these effects were 240, 240 and 90 mg for AA, collagen and PAF respectively. For collagen alone, there was a significant decrease in the degree of inhibition of aggregation between the last dose on day 1 (150 mg) and the baseline measurement on day 2. 4. Platelets from female subjects were more sensitive to collagen (P less than 0.05) and AA (P less than 0.01) stimulation compared with males. However, prior to aspirin ingestion, PAF produced a greater maximum response in platelets from females (P less than 0.02) while following aspirin ingestion PAF-induced activation was inhibited to a greater degree in females (P less than 0.02). 5. These results indicate that collagen- and PAF-induced platelet activation are only partially dependent on cyclo-oxygenase and for PAF this seems related only to the second phase of aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function

1. The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2. Siguazodan selectively inhibited the major cyclic AMP-hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP-inhibited PDE or PDE-III. 3. In platelet-rich plasma (PRP), siguazodan inhibited U46619-induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4. In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium [( Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5. Siguazodan has anti-platelet actions over the same concentration range that it is an inotrope and vasodilator.     

Mechanisms of aggregation inhibition by aspirin and nitrate-aspirin prodrugs in human platelets

Objectives Aspirin is the mainstay of anti‐platelet therapy in the secondary prevention of cardiovascular disease. However, problems with aspirin safety and resistance demand clinical strategies based on multiple pharmacological approaches. Prodrugs of aspirin may offer beneficial effects in terms of gastro‐intestinal safety and multiple pharmacological approaches. However, the pharmacological profile of aspirin prodrugs in human platelets has not been completed yet. We aimed to compare the effects of aspirin and prodrugs of aspirin ( 1 – 5 ) on human platelet aggregation stimulated by ADP and collagen and associated receptor expression (GPIIb/IIIa and P‐selectin) in platelet‐rich plasma (PRP) and washed platelets (WP). Methods As aspirin is released from prodrugs following esterase hydrolysis we studied the expression and activity of butyrylcholineterase (BuChE) and carboxyesterase (CE) in plasma and platelets. The mechanism of prodrug‐induced platelet aggregation inhibition was explored by studying the effects of plasma and purified human BuChE on aggregation. Finally, the relative contribution of nitric oxide (NO) bioactivity to nitrate‐containing prodrugs of aspirin‐induced inhibition of aggregation was determined using 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ,) a selective inhibitor of the soluble guanylyl cyclase. Key findings ST0702, 2 , a nicotinic acid‐aspirin codrug was equipotent with aspirin with respect to inhibition of collagen‐induced platelet aggregation. Compound 4 , a NO releasing aspirin was the most potent inhibitor of ADP‐induced platelet aggregation, an effect partially reversed by ODQ. The platelet inhibitory effects of aspirin prodrugs were time‐dependent as the maximal inhibitory effects against collagen‐induced aggregation were achieved by aspirin at 2 min, 1 at 5 min and ST0702 at 15 min. The aspirin prodrugs were significantly less potent in WP than in PRP and the reverse was true of aspirin. In the presence of complete BuChE inhibition in PRP, there was almost complete loss of aspirin prodrug, but not aspirin anti‐aggregatory activity. Interestingly, CE activity was observed in WP and platelet lysate with pNPA substrate. Accordingly, 1 and ST0702 retained 50% and 100% anti‐aggregatory activity at maximal concentrations in WP, which was attenuated in the presence of esterase inhibitor phenylmethylsulphonyl fluoride. Conclusions The inhibitory effect of aspirin prodrugs in PRP is due to prodrug activation by BuChE. In contrast, the platelet‐inhibitory effects of aspirin prodrugs in WP may be mediated through the activity of platelet CE. Compound 4 , a NO‐containing aspirin prodrug, may exert dual inhibitory effects in platelets. Thus, aspirin prodrugs effectively inhibit human platelet aggregation and as such may be an alternative to conventional aspirin.     

Effect of cobaltous chloride on aggregation of platelets from normal and afibrinogenaemic human blood

The aggregation of normal human platelets in vitro induced by ADP was severely inhibited after preincubation of platelet-rich plasma (PRP) with CoCl2. Platelets from a patient with congenital afibrinogenaemia did not aggregate until fibrinogen was added. This recovered response was also inhibited by CoCl2. The impairment of aggregation seemed to be due to the action of cobalt on surrounding fibrinogen and not to a direct action on the platelets themselves. These results illustrate another aspect of the potential toxicity resulting from the use of cobaltous salts in treating the anaemia of renal failure, in which bleeding disorders have been reported.     

木瓜蛋白酶体外对血小板聚集的抑制作用

目的：以洗涤血小板为模型,观察木瓜蛋白酶在体外对血小板聚集的抑制作用,以探讨其抗血栓作用的可能机制。方法：将不同剂量木瓜蛋白酶与洗涤血小板作用,以血小板聚集分析仪检测ADP、花生四烯酸（AA）、胶原和凝血酶诱导的血小板最大聚集率,以流式细胞仪检测活化血小板膜纤维蛋白原受体（FIB-R）和P-选择素表达水平,以SDS-PAGE分析血小板肌动蛋白聚合体的变化。检测ADP诱导的原发性高血压（PH）及急性心肌梗死（AMI）患者血小板最大聚集率和FIB-R表达水平。结果：木瓜蛋白酶剂量依赖性地抑制血小板聚集,降低血小板最大聚集水平,血小板聚集水平与木瓜蛋白酶剂量呈负相关（P〈0.01）。木瓜蛋白酶剂量依赖性地抑制ADP诱导的血小板FIB-R表达,降低FIB-R表达水平（P〈0.01）。木瓜蛋白酶降低ADP诱导的血小板膜P-选择素表达水平和抑制肌动蛋白聚合体增加（P〈0.01）。木瓜蛋白酶抑制PH及AMI患者血小板聚集和FIB-R表达（P〈0.01）。结论：木瓜蛋白酶通过抑制活化血小板膜纤维蛋白原受体的表达,并抑制肌动蛋白聚合以及释放反应从而剂量依赖性地抑制血小板的聚集反应,有抗血栓形成作用。