Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.     

Characterization of the normal bovine platelet aggregation response

1. Bovine platelets are more sensitive to stimulation by platelet activating factor (PAF) than adenosine-di-phosphate (ADP) or thrombin. 2. While epinephrine, arachidonic acid and serotonin are ineffective by themselves as aggregatory stimulants of bovine platelets they enhance the aggregation response of other platelet agonists. 3. There is no correlation between thromboxane A2 production and release and the extent of platelet aggregation in bovine platelets. 4. The dependence of bovine platelet aggregation on a phospholipid pathway and calcium mobilization is indicated.     

Antiplatelet effects of clausine-D isolated from Clausena excavata

Clausine-D inhibited concentration-dependently the aggregation and release of washed rabbit platelets induced by arachidonic acid and collagen, without affecting those induced by U46619, PAF and thrombin. The IC₅₀ values of clausine-D on arachidonic acid-and collagen-induced platelet aggregation were calculated to be 9.0±1.1 and 58.9±0.9 μM, respectively. Thromboxane B₂ and prostaglandin D₂ formation in platelets caused by arachidonic acid were also suppressed. Clausine-D inhibited increased intracellular concentration of calcium in platelets caused by arachidonic acid and collagen, and also abolished the generation of inositol monophosphate caused by arachidonic acid, but not that by collagen U46619, PAF and thrombin. In human citrated platelet-rich plasma, clausine-D inhibited the secondary phase, but not the primary phase, of aggregation induced by epinephrine and ADP. These results indicate that the antiplatelet effect of clausine-D is due to inhibition of the formation of thromboxane A₂.     

Inhibitory effect of sulfur-containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets

The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5'-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 microM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10(-4) M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane.     

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.     

Beclobrinic acid--a new hypolipidemic agent--inhibits in vitro human platelet activation by blocking prostaglandin synthesis

Effects and the mechanism of the antiplatelet actions of beclobrinic acid, free acid form of a new hypolipidemic agent beclobrate [(+)-2-[d-(P-chlorophenyl)p-tolyl)oxy)-2-methyl-butyrate), were examined using human platelets. Platelet-rich plasma (PRP) which has been prelabeled with (14C)-serotonin was incubated with beclobrinic acid (BBA) for one minute before the addition of various agonists. BBA (0.1-1.5 mM) inhibited platelet aggregation and serotonin secretion induced by ADP, epinephrine, arachidonic acid and collagen in a concentration dependent manner. BBA also inhibited arachidonic acid-induced production of malondialdehyde (MDA), a byproduct of prostaglandins, in a concentration dependent manner. However, up to 1.0 mM BBA did not inhibit platelet aggregation induced by U46619, a stable analog of prostaglandin H2. In other experiments BBA also blocked thrombin-induced release of (3H)-arachidonic acid from platelet phospholipids. These findings suggest that: (a) BBA inhibits platelet aggregation and serotonin secretion by inhibiting prostaglandin synthesis at two steps. First by interfering in the release of arachidonic acid from platelet phospholipids and second by inhibiting its conversion into prostaglandins; and (b) BBA does not inhibit the action of prostaglandins on human platelets.     

Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion.

It was previously demonstrated that C-reactive protein (CRP) inhibits platelet aggregation and release reactions, activation of platelet factor 3, and platelet-dependent clot retraction. Multiple considerations including selective inhibition of secondary wave aggregation suggested that CRP exerted its inhibitory effects by interfering with the release of endogenous ADP. In the present investigation, CRP was found by direct assay to inhibit the release of endogenous ADP and/or serotonin concomitant with inhibition of platelet aggregation stimulated by ADP, epinephrine, thrombin, and AHGG. CRP did not induce an increase in the basal level of platelet cAMP, suggesting independence of a direct effect upon this mediator system. Furthermore, CRP did not inhibit the aggregation and secretion induced by the antibiotic ionophore A23187, suggesting the absence of a direct effect upon the activation of platelet contractile elements. By contrast, CRP did inhibit both thrombin-induced release of malondialdehyde, a prostaglandin endoperoxide nonprostanoate endproduct, and platelet aggregation induced by the prostaglandin endoperoxide precursor arachidonic acid. These data, therefore, raise the possibility that CRP inhibits platelet reactivities by interfering with an aspect of porstaglandin metabolism, and that this occurs subsequent to the hydrolytic accumulation of arachidonic acid and prior to the movement of calcium from the platelet dense tubules. These studies support the concept that CRP serves to modulate platelet reactivities during acute inflammatory reactions.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2 alpha

The inhibition of human platelet aggregation produced by PGF2 alpha is not specific for thromboxane A2 mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF2 alpha (8 microM); 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 PGH2). In contrast the thromboxane receptor antagonist EP 045 (up to 20 microM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC50 = 0.5 microM), but not PGF2 alpha (28 microM), displaces the specific binding of [3H] 9,11-epoxymethano PGH2 to washed human platelets. PGF2 alpha 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 PGF2 alpha 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 PGF2 alpha. We suggest that the very weak effect of PGF2 alpha 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.     

Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans

Chelerythrine chloride is an antiplatelet agent isolated from Zanthoxylum simulans. Aggregation and ATP release of washed rabbit platelets caused by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin were inhibited by chelerythrine chloride. Less inhibition was observed in platelet-rich plasma. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was decreased by chelerythrine chloride. Phosphoinositides breakdown caused by collagen and PAF was completely inhibited by chelerythrine chloride, while that of thrombin was only partially suppressed. Chelerythrine chloride inhibited the intracellular calcium increase caused by arachidonic acid, PAF, collagen and thrombin in quin-2/AM-loaded platelets. The cyclic AMP level of washed platelets did not elevated by chelerythrine chloride. The antiplatelet effect of chelerythrine chloride was not dependent on the incubation time and the aggregability of platelets inhibited by chelerythrine chloride was easily recovered after sedimenting the platelets by centrifugation and then the platelet pellets were resuspended. Chelerythrine chloride did not cause any platelet lysis, since lactate dehydrogenase activity was not found in the supernatant. These data indicate that the inhibitory effect of chelerythrine chloride on rabbit platelet aggregation and release reaction is due to the inhibition on thromboxane formation and phosphoinositides breakdown.     

Bioactive amide of prostaglandin E1 and ethanolamine plasmalogen analog of platelet-activating factor inhibits several pathways of human platelet aggregation

The influence of an amide of prostaglandin E1 and ethanolamine plasmalogen platelet-activating factor analog 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-11alpha, 15alpha-dioxy-9-keto-13-prostenoyl)ethanolamine (PGE1-PPAF) on platelet-activating factor (PAF)-, ADP-, and thrombin-induced human platelet aggregation has been studied. It was found that PGE1-PPAF inhibits the PAF-, ADP-, and thrombin-induced platelet aggregation in platelet-rich plasma. 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine inhibited PAF-induced aggregation up to 50% but had no influence on platelet aggregation induced by ADP or thrombin. The ethanolamine plasmalogen analog of PAF 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolami ne, having a palmitoyl residue instead of PGE1, did not inhibit platelet aggregation induced by PAF, ADP, or thrombin. We propose that inhibition of human platelet aggregation by PGE1-PPAF is mediated by its action on platelet PAF-receptors and the adenylate cyclase system.     

Effect of docosahexaenoic acid (DHA) on arachidonic acid metabolism and platelet function

Studies from our laboratory have suggested a role for ferrous iron in the metabolism of arachidonic acid and demonstrated that inhibitors of prostaglandin synthesis exert their effect by complexing with the heme group of cyclooxygenase. Docosahexaenoic acid (DHA) is a potent competitive inhibitor of arachidonic acid metabolism by sheep vesicular gland prostaglandin synthetase. In this study we have evaluated the effect of exogenously added DHA on platelet function and arachidonic acid metabolism. DHA at 150 microM concentration inhibited aggregation of platelets to 450 microM arachidonic acid. At this concentration DHA also inhibited the second wave of the platelet response to the action of agonists such as epinephrine, adenosine diphosphate and thrombin. Inhibition induced by this fatty acid could be overcome by the agonists at higher concentrations. DHA inhibited the conversion of labeled arachidonic acid to thromboxane by intact, washed platelet suspensions. However, platelets in plasma incubated first with DHA then washed and stirred with labeled arachidonate generated as much thromboxane as control platelets. These results suggest that the polyenoic acids, if released in sufficient quantities in the vicinity of cyclooxygenase, could effectively compete for the heme site and inhibit the conversion of arachidonic acid.     

Effects of pituitary adenylate cyclase-activating polypeptide on the cyclooxygenase pathway of rat platelets and on platelet aggregation

Several data suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of local circulation. One possible role of PACAP in the regulation of circulation is that, it may modify the cyclooxygenase pathway of the arachidonate cascade in platelets. Our study was designed to study the effect of PACAP on the cyclooxygenase pathway of rat platelets and on platelet aggregation. PACAP (10(-7) and 10(-6) M) significantly inhibited the cyclooxygenase pathway of platelets, mostly the thromboxane synthesis. Pretreatment with a PACAP receptor antagonist, PACAP(6-38), or with an inhibitor of protein kinase A, H-89, shows that the effects of PACAP on the cyclooxygenase pathway were diminished. In the aggregation studies, PACAP inhibited both the arachidonic acid-induced and the thrombin-induced platelet aggregation. It can be concluded that PACAP inhibits the cyclooxygenase pathway of rat platelets via a specific PACAP receptor-activated, cAMP-dependent pathway, and these effects of PACAP are involved in the inhibition of platelet aggregation.     

trans-2,5-Bis-(3,4,5-trimethoxyphenyl)tetrahydrofuran. An orally active specific and competitive receptor antagonist of platelet activating factor

trans-2,5-Bis(3,4,5-trimethoxyphenyl)tetrahydrofuran (L-652,731) is found to be a potent and orally active platelet activating factor (PAF)-specific and competitive receptor antagonist. It potently inhibits [3H]PAF (1 nM) binding to receptor sites on rabbit platelet membranes with an ED50 of 2 X 10(-8) M under the assay condition without the addition of mono- or divalent cations. In a comparative study, it is more potent than CV-3988, kadsurenone, and ginkgolide B as a receptor antagonist. The equilibrium dissociation constants (KB) of L-652,731 obtained either from the inhibition of receptor binding or from the inhibition of PAF-induced aggregation of gel-filtered rabbit platelet are 2.7 X 10(-8) and 2.1 X 10(-8) M, respectively. The agreement of these KB determinations based on receptor and cellular function suggests that L-652,731 does not inhibit other steps following PAF-receptor binding. L-652,731 does not antagonize the binding of several radioligands to their respective receptor. It shows no inhibitory effect on platelet aggregation induced by other aggregating agents including thrombin, collagen, A-23187, arachidonic acid, epinephrine, and ADP. L-652,731 is orally active; it inhibits PAF-induced rat cutaneous vascular permeability with an ED50 of 30 mg/kg orally. Significant inhibitory results of L-652,731 suggest that PAF may be partially involved in cutaneous vascular permeability induced by histamine and bradykinin.     

Effect of heparin on platelet aggregation in vitro

Heparin added to citrated platelet rich plasma influences shape change and aggregation of platelets in different ways. In the presence of heparin neither ADP nor collagen induces shape change, while shape change after thrombin or arachidonic acid remains unaltered. Heparin potentiates the first aggregation step induced by ADP and epinephrine but inhibits aggregation induced by thrombin and ristocetin. The second phase of aggregation and the release reaction are not directly influenced by heparin no matter which aggregation agent is used.     

CV-3988 - a specific antagonist of platelet activating factor (PAF)

CV-3988, rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate was shown to be a specific inhibitor of platelet activating factor (PAF). This compound in concentrations of 3 x 10(-6) to 3 x 10(-5)M inhibited aggregation of rabbit platelets induced by PAF (3 x 10(-8)M), while it had no effect on the aggregation induced by arachidonic acid, ADP, collagen or A-23187. CV-3988 alone even at a concentration of 10(-3)M had no effect on platelet aggregation. The inhibitory action of CV-3988 on the PAF-induced aggregation was independent of the formation of micelles. The PAF (0.1 to 1.0 micrograms/kg, i.v.)-induced hypotension in anesthetized rats was also inhibited dose-dependently by the i.v. administration of CV-3988 (1 and 10 mg/kg), while the hypotensive actions induced by the i.v. administration of acetylcholine (1 micrograms/kg), arachidonic acid (1 mg/kg), bradykinin (10 micrograms/kg), isoproterenol (1 microgram/kg) and histamine (100 micrograms/kg) were not altered by CV-3988 (10 mg/kg, i.v.). All these findings indicate that CV-3988 specifically inhibits the action of PAF in vitro and in vivo. This is the first report of a PAF antagonist which can specifically inhibit the PAF-induced hypotension as well as the PAF-induced platelet aggregation.     

Thiopental enhances human platelet aggregation by increasing arachidonic acid release.

Conflicting results have been reported regarding the effect of thiopental on aggregation and cytosolic calcium levels in platelets. The present study attempted to clarify these phenomena. Using platelet-rich plasma or washed suspensions, platelet aggregation, thromboxane (TX) B2 formation, arachidonic acid (AA) release, and cytosolic free calcium concentrations ([Ca2+]i) were measured in the presence or absence of thiopental (30-300 microM). Platelet activation was induced by adenosine diphosphate (ADP, 0.5-15 microM), epinephrine (0.1-20 microM) arachidonic acid (0.5-1.5 mM), or (+)-9,11-epithia-11,12-methano-TXA2 (STA2, 30-500 nM). Measurements of primary aggregation were performed in the presence of indomethacin (10 microM). Low concentrations of ADP and epinephrine, which did not induce secondary aggregation in a control study, induced strong secondary aggregation in the presence of thiopental (> or = 100 microM). Thiopental (> or = 100 microM) also increased the TXB2 formation induced by ADP and epinephrine. Thiopental (300 microM) increased ADP- and epinephrine-induced 3H-AA release. Thiopental (300 microM) also augmented the ADP- and epinephrine-induced increases in [Ca2+]i in the presence of indomethacin. Thiopental appears to enhance ADP- and epinephrine-induced secondary platelet aggregation by increasing AA release during primary aggregation, possibly by the activation of phospholipase A2.     

Inhibition of blood platelet aggregation by dioxo-ene compounds

Compounds with a conjugated oxo-ene-oxo system were tested for inhibition of blood platelet aggregation. All compounds with this structure in trans configuration were effective inhibitors of aggregation induced by thrombin and by arachidonic acid. While the oxo-trans-ene-oxo system is prerequisite for such activity, other structural features of the compounds may be varied without loss of activity. Inhibition is exemplified by 9,12-dioxo-trans-10- and 10,13-dioxo-trans-11-octadecenoic acids and their methyl esters, by 11,14-dioxo-trans-12- eicosenoic acid, by 4,7-dioxo-trans-5- decene and by trans- dibenzoylethylene . The half-inhibition concentrations are in the order of 2-6 microM, with complete inhibition at 8-20 microM. According to experiments with the inhibiting 9,12-dioxo-trans-10-octadecenoic acid, the normal oxygenation of exogenous arachidonic acid by platelets is not affected but the thrombin-induced internal release of this acid seems to be abolished by the inhibitor. The inhibition of aggregation in the presence of exogenous arachidonic acid and its products suggests that the inhibitor also interferes with other events leading to aggregation. By implication from other properties of the oxo-trans-ene-oxo system, reaction with SH groups may be a mechanism for inhibition.     

Triazolobenzodiazepines competitively inhibit the binding of platelet activating factor (PAF) to human platelets

PAF causes dose dependent platelet aggregation of human platelet rich plasma or gel filtered platelets (GFP). The benzodiazepines alprazolam and triazolam, but not diazepam (1-10 microM), inhibit PAF induced aggregation but have no effect on aggregation induced by other platelet agonists such as ADP, epinephrine and collagen. The IC50 for aggregation by PAF (4 nM) in GFP is 1 microM for both alprazolam and triazolam. The mechanism for this inhibition was explored by studying the binding of 3H-PAF(0.08 nM) to GFP in Tyrodes buffer containing albumin (0.35%), Mg++ (1mM) and Ca++ (0.5mM). GFP was incubated with different doses of the drug for 5 min prior to addition of 3H-PAF. Incubation was then carried out for 60 min at 25 degrees C to achieve binding equilibrium, as previously established. Alprazolam and triazolam, but not diazepam, caused competitive displacement of 3H-PAF from specific binding sites of GFP. The IC50 of alprazolam was 3.8 microM while that of triazolam was 0.82 microM. Lineweaver-Burk plots of 3H-PAF binding in the presence of inhibitor were also consistent with competitive inhibition. These results are consistent with the interpretation that the specific inhibition of PAF induced platelet aggregation by alprazolam and triazolam, respectively, is due to competitive inhibition of binding of PAF to its receptor.     

Effects of acrolein on human platelet aggregation

Acrolein, a component of tobacco smoke, potentiated platelet aggregation and increased thromboxane A2 (TXA2) formation caused by thrombin and arachidonic acid (AA). Acrolein produced these effects at concentrations in the range 50-5000 microM. Acrolein had no effect on platelet responses to ADP, epinephrine, collagen or the ionophore A23187. Acrolein increased the mobilization of [3H]arachidonic acid from prelabelled platelets in response to thrombin and arachidonic acid. The increased availability of substrate could partly explain the enhanced production of TXA2 and increased aggregation observed in the presence of acrolein. These findings could provide an explanation for the increased incidence of vascular disease in cigarette smokers.     

血小板聚集的药理性解聚

在进行中的不可逆聚集的富血小板血浆(PRP)中,加入不同浓度的解聚剂,测定其解聚程度。以一系列作用机制不同的血小板解聚剂对ADP、胶原、花生四烯酸、U_(46619)(血栓素A_2类似物)、PAF所诱发的血小板聚集的拮抗作用的结果显示,血小板聚集作用得以维持是一个复杂的过程,涉及多种机制的参与,并和促聚剂种类有关。维持ADP诱发的聚集,需要外源性Ca~(2 )及细胞内Ca~(2 )的动员。PAF U_(46619)和花生四烯酸诱发的聚集作用的维持也需要细胞内钙的动员。但是胶原诱发的聚集作用的维持,有除Ca~(2 )、ADP以外的其他途径。维持持续的聚集并不依赖于血小板TXA_2(血栓素A_2)的持续合成,钙调节蛋白在血小板的持续聚集中起重要作用。钙调蛋白抑制剂都是有效的血小板解聚剂。各种血小板解聚剂的拮抗效果取决于(1) 采用促聚剂的种类、(2) 加入解聚剂时血小板聚集的时相、(3) 解聚剂的种类。