Effects of cryptolepine on collagen-induced aggregation and on the mobilization and metabolism of arachidonic acid by rabbit platelets

1. We examined the effect of cryptolepine on collagen-induced aggregation and on the mobilization, and metabolism of arachidonic acid in rabbit platelets.2. Preincubation of platelets with cryptolepine (50–100 μM) did not affect the primary wave of aggregation but resulted in a dose-dependent, surmountable inhibition of the secondary wave of aggregation induced by collagen (5 μg/ml). The inhibition by cryptolepine was greater when cryptolepine was incubated with the platelets after the peak of the primary wave of aggregation.3. Cryptolepine (50–100 μM) dose-dependently inhibited thrombin (1.5 U/ml) and A23187 (2.5 μM)-induced release of ¹⁴C[AA] from platelet membrane phospholipid pools. The percentage inhibition of A23187-induced ¹⁴C[AA] release was 31.3 ± 4.3% (50 μM) and 79.3 ± 5.4% (100 μM), while thrombin-induced release was inhibited by 39.2 ± 2.4% (50 μM) and 68.2 ± 3.6% (100 μM).4. At near maximal concentration (100 μM) which significantly inhibited secondary aggregatory response and ¹⁴C[AA] release, cryptolepine had no effect on the platelet metabolism of ¹⁴C[AA] to thromboxane B₂, HHT and 12 HETE.5. The present findings suggest that cryptolepine inhibited collagen-induced secondary aggregation through a selective antiphospholipase-like activity. There was not effect on platelet cyclooxygenase and lipoxygenase activities of platelets.     

Acetal phosphatidic acids: novel platelet aggregating agents

1 Palmitaldehyde, olealdehyde and linolealdehyde acetal phosphatidic acids induced rapid shape change and dose-dependent biphasic aggregation of human platelets in platelet-rich plasma; aggregation was reversible at low doses and irreversible at high doses of the acetal phosphatidic acids. The palmitaldehyde congener elicited monophasic dose-dependent aggregation of sheep platelets in platelet-rich plasma.

2 The threshold concentration for palmitaldehyde acetal phosphatidic acid (PGAP)-induced platelet aggregation was 2.5-5 μM for human platelets and 0.25-0.5 μM for sheep platelets. PGAP was 4-5 times as potent versus human platelets as the olealdehyde and linolealdehyde acetal phosphatidic acids, which were equipotent.

3 PGAP-induced irreversible aggregation of [¹⁴C]-5-hydroxytryptamine ([¹⁴C]-5-HT)-labelled human platelets in platelet-rich plasma was accompanied by release of 44.0±2.4% (s.e.) of the platelet [¹⁴C]-5-HT; reversible aggregation was not associated with release. In contrast, PGAP-induced release of [¹⁴C]-5-HT-labelled sheep platelets was dose-dependent.

4 The adenosine diphosphate (ADP) antagonist, 2-methylthio-AMP, and the cyclo-oxygenase inhibitor, aspirin, abolished PGAP-induced second phase aggregation and release in human platelets but did not affect the first, reversible, phase of aggregation. Both the first and second phases of PGAP-induced aggregation were abolished by chlorpromazine, by the phospholipase A₂ inhibitor, mepacrine, and by nmolar concentrations of prostaglandin E₁ (PGE₁); these agents abolished the second, but not the first phase of ADP-induced aggregation.

5 The related phospholipids, lecithin, lysolecithin and phosphatidic acid, at <100 μM, neither induced aggregation of human platelets in platelet-rich plasma, nor modified PGAP-induced aggregation; 1-palmityl lysophosphatidic acid elicited aggregation of human platelets at a threshold concentration of 100 μM.

6 It is concluded that the acetal phosphatidic acids induce platelet aggregation per se by direct action at the platelet membrane, and that the acetal function is of primary importance in their potent platelet-stimulating activity. Moreover, as the acetal phosphatidic acids are the major components of the smooth muscle-contracting acidic phospholipid tissue extract `Darmstoff' (Vogt, 1949), their potent platelet-aggregating properties may be of physiological or pathological significance.

     

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase

Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca²⁺ release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca²⁺ mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.     

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

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) has previously been shown to inhibit cyclic nucleotide phosphodiesterases prepared from various cell homogenates and the function of intact human platelets. We now report that (1) high concentrations of quercetin raise platelet cAMP levels; and (2) quercetin potentiates the inhibitory effect of prostacyclin (PGI₂) on ADP-induced washed human platelet aggregation and the elevation of platelet cAMP levels elicited by PGI₂. These results suggest a role for cAMP in the mechanism of action of quercetin on blood platelets.     

Stereo-dependent inhibition of human platelet function by the optical isomers of trimetoquinol

The stereoisomers of trimetoquinol [1-(3',4',5'-trimethoxybenzyl)-6–7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ] were shown to have potent and selective inhibitory effects on human platelet function in vitro. the R(+)-isomer was 12.1-, 12.3-, 39.2-, 82.9- and 36.0-fold more effective than the S(?)-isomer as an inhibitor of aggregation induced by arachidonic acid (AA), collagen, the epoxymethano-PGH2 analogs U44069 and U46619, and thromboxane A; (TxA₂) respectively. the concentrations of the R(+)-isomer that produced 50 percent inhibition (IC₅₀) of platelet aggregation were 4.2, 4.3, 1.4, 0.14 and 0.64 μM using AA, collagen, U44069, U46619, and TxA₂₀ as respective inducers. the graphical approximation of an inhibitory Constant (K_i = 0.13 μM) for the effect of TMQ on U46619-induced aggregation suggested that a competitive-like inhibition was operative. In other experiments, platelet aggregation and serotonin release induced by U46619 were inhibited differentially by the TMQ stereoisomers with nearly identical concentration-response curves. In addition, racemicTMQ blocked the secondary phase of platelet aggregation and serotonin release induced by ADP. These data, together with the ability of the TMQ stereoisomers to selectively inhibit TxA₂-induced aggregation, suggest that TMQ is an inhibitor of endoperoxide or TxA₂ action, e.g. a thromboxane A₂ receptor antagonist.     

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.     

Effects of taprostene,a stable prostacyclin analogue,on haemodynamics,platelet function and arachidonate metabolism in healthy volunteers

Summary In order to assess the effect of taprostene on haemodynamics, platelet function and arachidonate metabolism in 4 healthy volunteers an intravenous infusion of 25 ng · kg^–1 · min^–1 was given for 6 h.During the infusion period systolic blood pressure dropped from 130 to 111 mm Hg and diastolic blood pressure from 77 to 69 mm Hg. The heart rate rose from 77 to 84 beats/min.During the taprostene infusion the slope and height of the ADP and collagen induced platelet aggregation curves were significantly inhibited and the sensitivity of platelets to PGI₂ and PGE₁ was increased.Plasma and serum thromboxane B₂, conversion of exogenous radiolabelled arachidonic acid, WU-test, circulating endothelial cell count, concentration of platelet factor 4, -thromboglobulin, malondialdehyde and the PGI₂-synthesis stimulating plasma factor did not show any clear drug-related alteration.It is concluded that infusion of taprostene 25 ng · kg^–1 · min^–1 caused measurable inhibition of platelet function ex vivo.     

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.     

Further insights into the anti-aggregating activity of NMDA in human platelets

1. In the present study the effect of N-methyl-D-aspartate (NMDA) on thromboxane B₂ synthesis and on [Ca²⁺]_i was studied in human platelets.
2. NMDA (10⁻⁷ M) completely inhibited the synthesis of thromboxane B₂ from exogenous arachidonic acid (AA), while it did not interfere with the aggregating effect of the thromboxane A₂ receptor agonist U-46619.
3. NMDA (0.1 μM–10 μM) dose-dependently increased intracellular calcium in washed platelets pre-loaded with fura 2 AM, and this effect was not additive with that of AA.
4. NMDA shifted the dose-response curve of AA to the right. At the highest AA concentrations platelet aggregation was not inhibited.
5. The antiaggregating effect of NMDA was not antagonized by N^G-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor.
6. Finally, NMDA (0.01 nM–100 nM) associated with either aspirin or indomethacin significantly potentiated the antiaggregating activity of both cyclo-oxygenase inhibitors.
7. It was concluded that NMDA is a potent inhibitor of platelet aggregation and thromboxane B₂ synthesis in human platelet rich plasma (PRP).

     

The effects of bisantrene on human platelets

Summary Bisantrene, (9,10-anthracenedicarboxaldehyde bis [(4,5-dihydro-lH-imidazol-2yl)hydrazone] dihydrochloride) is one of a series of anthracene dicarboxaldehyde compounds in Phase II trials. Preliminary studies suggested that bisantrene has anti-platelet activity. Therefore, in vitro studies of its effects on platelets were undertaken. Bisantrene in clinically attainable concentrations of 0.625 – 10 M, caused a 95 ± 1% decrease in maximal platelet aggregation to collagen (1 g/ml), and epinephrine (5–40 M) and 90 ± 10% inhibition of arachidonic acid (50 g/ml) induced aggregation. Collagen induced platelet shape change was not affected. Aggregation to calcium ionophore A23187 was inhibited by 10–30%. No effect on ADP induced aggregation was seen at clinically relevant bisantrene concentrations. This inhibition was time dependent, reaching a maximum when platelets were preincubated with bisantrene for 10 minutes before exposure to agonist. Inhibition persisted after bisantrene was removed by washing. To determine the mechanism of platelet inhibition, platelet prostaglandin metabolism, oxygen consumption and release reaction were measured. In the presence of drug: 1. normal cyclooxygenase activity was demonstrated by O₂ consumption studies and normal secondary wave ADP (3.2 M) aggregation; 2. lipoxygenase activity, by O₂ consumption was also normal. 3. There was 30–50% inhibition of thromboxane A2 synthesis induced by arachidonic acid or collagen; 4. ADP, collagen and AA induced release of serotonin was decreased by 30–60% but was never abolished; 5. No effect on basal platelet cAMP levels nor additive effect on PGE₁ induced elevation of platelet cAMP was detectable. These data demonstrate that bisantrene has potent antiplatelet activity probably mediated by several different mechanisms. The inhibition may have important clinical and theoretical consequences.Abbreviations PGl ₂ prostaglandin I₂ - PGEi prostaglandin E₁ - PRP platelet rich plasma - PPP platelet poor plasma - ACD acid citrate dextrose - TBX A₂ thromboxane Az₂ - TBX B₂ thromboxane B₂ - PAF platelet activating factor-acetyl glyceryl ether phosphoryl choline     

Effect of 2(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) on human platelets in vitro

The effect on human platelets of 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) was tested in vitro by measuring cyclic adenosine monophosphate (cAMP) level, cytosolic Ca⁺⁺, [¹²⁵I]fibrinogen binding as well as aggregation induced by several agonists. AP155 dose-dependently inhibited aggregation both in platelet rich plasma (PRP) and in washed platelets (WP), exerting its maximal power in the presence of collagen, ADP and platelet activating factor (PAF). It specifically inhibited the activity of cAMP high affinity phosphodiesterase (PDE), resulting in a sufficient increase in cAMP levels to activate cAMP-dependent protein kinase. AP155 was able to inhibit aggregation, the increase in cytosolic Ca⁺⁺ induced by thrombin, and fibrinogen binding to ADP or thrombin-stimulated platelets. Thus, this new pyridopyrimidine derivative exerts its antiplatelet activity by increasing cAMP intracellular concentration.     

Influence of diethylcarbamazine and mefloquine on PGI2 synthesis by the rat thoracic aorta and myometrial tissues

• 1.1. The influence of the antifilarial drug diethylcarbamazine citrate (D) and dl-erythro mefloquine hydrochloride (Mf) on PGI₂ synthesis by the male rat thoracic aorta and day-20 pregnant rat myometrium was investigated in vitro using a rat platelet antiaggregatory bioassay method.
• 2.2. Pretreatment of the tissues with D (25.5–204 μM) or Mf (24–192 μM) for 30 min at 37°C significantly inhibited PGI₂ synthesis in a concentration-dependent manner.
• 3.3. D exhibited its inhibitory effect even in presence of exogenous arachidonic acid (AA) (16.6 μM) whereas Mf lost its inhibitory effect in presence of AA.
• 4.4. Pretreatment of urethane-anaesthetized rats with D (32 μmol kg⁻¹) but not Mf (7.5 μmol kg⁻¹) for 30 min significantly antagonized AA (4 nmol kg⁻¹)-induced hypotension
• 5.5. Furthermore, D (0.25–0.5 μM) antagonized AA-induced aggregation in rabbit platelet-rich plasma without affecting that of ADP.
• 6.6. D seemed to interfere with the action of the PG endoperoxide synthase (PG cyclooxygenase) whereas Mf seemed to interfere with the action of phospholipase A₂ (PLA₂) enzyme.
• 7.7. D may have exerted its effect via release of toxic O₂ radicals whereas Mf effect may have been due to an interaction with PLA₂ substrate phospholipids.
• 8.8. The demonstrated inherent property of these two drugs to inhibit the synthesis of the potent vasodilator, platelet antiaggregatory, anticonvulsant and antiinflammatory mediator PGI₂ may partly contribute towards better understanding of the biochemical mechanisms that underly some of the previously known but poorly understood actions of these drugs.

     

Modulatory effect of 8-iso-PGE2 on platelets

• 1.1. 8-Iso-PGE₂ induced either reversible or irreversible aggregation of platelets in human platelet-rich plasma (PRP) or in the suspension of washed platelets (WP). The values of EC₅₀ for irreversible aggregation in PRP and WP were 4 and 2 μM, respectively.
• 2.2. In rabbit PRP, 8-iso-PGE₂ (0.1-100 μM) itself did not induce or induced only reversible aggregation.
• 3.3. 8-Iso-PGE₂ (0.1-20μM) potentiated adenosine diphosphate-(ADP) induced platelet aggregation in both human and rabbit. The same effect also was found for adrenaline-induced platelet aggregation in rabbit.
• 4.4. The lower concentrations (0.2-0.5 μM) of 8-iso-PGE₂ decreased, and higher concentrations (1–2 μM) increased platelet aggregating factor- (PAF) induced aggregation in human PRP. In rabbit PRP, 8-iso-PGE₂ (0.02–200 μM) had only a decreasing effect on PAF-induced aggregation.
• 5.5. The results suggest that low concentrations of 8-iso-PGE₂ can amplify or weaken platelet aggregation induced by various aggregatory agents.

     

Effects of dipyridamole in vivo on ATP and cAMP content in platelets and arterial walls and on atherosclerotic plaque formation

Summary In rabbits receiving an atherogenic diet for 2 months, the ATP content of platelet rich plasma (PRP) and arterial tissue was significantly elevated as compared to normal rabbits. This increase in ATP levels of platelets from atherosclerotic rabbits was paralleled by higher basal as well as PGI₂-induced cAMP levels. In arterial tissues, an increase was only obtained in PGI₂-stimulated cAMP content.Treatment with dipyridamole (DPD) for 4 weeks resulted in a reduction of the ATP content in platelets and arterial tissue from atherosclerotic rabbits to values seen in normal animals. Again, the reduction of ATP content was reflected in a decrease of basal as well as PGI₂-induced cAMP levels in platelets, whereas in arterial tissue a decrease was only obtained in PGI₂-induced cAMP content. At the same time, DPD treatment enhanced atherosclerotic plaque formation in the aortic wall.The enhanced atherosclerotic plaque formation seen in DPD treated atherosclerotic rabbits may be linked to the inhibition of adenosine uptake, resulting in a decrease of the adenine nucleotide pools of arterial wall cells. The decrease also caused a reduction in PGI₂-induced cAMP content. This effect may be linked to altered proliferative activity, since in many cell types, stimulation of cAMP levels results in reduced proliferation rates.     

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.     

地塞米松影响血小板聚集、释放机理的探讨

通过对离体兔血小板研究发现:地塞米松(Dex)能抑制胶原诱导的血小板聚集、释放和血栓素A_2(TXA_2)生成等反应,而对花生四烯酸(AA)诱导的上述反应则无明显影响。本研究还发现,Dex能抑制血小板的磷脂酸A_2(PLA_2)活性,细胞外Ca~(2+)可部分拮抗Dex的上述作用,表明Dex的作用可能与其限制细胞外Ca~(2+)进入细胞内有关。Dex对血小板钙调素(CaM)和腺苷酸环化酶(AC)活性,以及对cAMP和cGMP的生成均无明显影响。     

阿魏酸钠和阿斯匹林联用对大鼠血小板聚集的影响及对PGI2-TXA2平衡的调节

单独给大鼠口服阈下剂量的阿魏酸钠(SF)或阿斯匹林(ASA)对胶原诱导的血小板聚集及对颈动脉壁PGI₂样物质的释放均无明显作用。两药并用后大鼠血小板聚集及血小板TXA:样物质的释放均显著地受到抑制,动脉壁PGI₂样物质的释放则无明显变化。说明SF与小剂量ASA联用可增强抗血小板作用。     

Prostaglandin endoperoxides, thromboxane A2 and adenosine diphosphate in collagen-induced aggregation of rabbit platelets

1 A bioassay technique is described for simultaneously monitoring rabbit platelet aggregation with measurement of thromboxane A₂ (TxA₂) and prostaglandins released in response to collagen or arachidonic acid (AA).

2 Five imidazole derivatives were examined as inhibitors of thromboxane synthetase and compared with the effect of the cyclo-oxygenase inhibitor indomethacin; 1-(7-carboxyheptyl) imidazole was identified as the most potent and selective inhibitor of thromboxane synthetase and was used with indomethacin to investigate the relative contribution of the prostaglandin endoperoxides prostaglandin G₂ (PGG₂)/PGH₂ and TxA₂ in mediating platelet aggregation induced by collagen or AA.

3 Platelet aggregation induced by a low concentration of collagen was abolished by indomethacin and carboxyheptylimidazole whilst in response to a high concentration or collagen only partial inhibition of aggregation occurred.

4 The contribution of adenosine diphosphate (ADP) released from platelets during collagen or AA-induced aggregation was examined using the substrate/enzyme complex creatine phosphate/creatine phosphokinase (CP/CPK). The CP/CPK complex abolished aggregation induced by a low dose of collagen whilst aggregation to a high dose of collagen was only partially inhibited.

5 Aggregation induced by a high dose of collagen was abolished by a combination of CP/CPK with indomethacin or carboxyheptylimidazole.

6 AA-induced aggregation was abolished by indomethacin. Carboxyheptylimidazole abolished aggregation induced by a low dose of AA but inhibition was surmounted with increasing concentrations of AA in the absence of TxA₂ formation.

7 PGH₂-induced aggregation was unaffected by indomethacin and only partially inhibited by carboxyheptylimidazole. AA or PGH₂-induced platelet aggregation was unaffected by CP/CPK.

8 In conclusion, aggregation of rabbit platelets induced by a low concentration of collagen was dependent on synergism between TxA₂ and ADP whilst at high concentrations of collagen, sufficient TxA₂ and ADP were released to induce aggregation independently of each other.

9 The small amounts of prostaglandin endoperoxides produced from endogenous arachidonate have apparently no direct pro-aggregatory role. However, the relatively large amount which can be produced by a high concentration of exogenous AA when TxA₂ formation is prevented can cause aggregation of rabbit platelets.

     

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.     

Action of AD6 (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxycarbonylmethoxy coumarin) on human platelets in vitro

Summary The action of AD₆ was tested in vitro on human platelets by measuring beta-thromboglobulin (BTG), platelet factor 4 (PF₄) and thromboxane B₂ (TXB₂) release as well as aggregation. BTG and PF₄ release from blood anticoagulated with sodium citrate was inhibited by AD₆ during a 3 h incubation. Platelet rich plasma (PRP) was stimulated with ADP, collagen, sodium arachidonate, PAF, A23187 and epinephrine, while resuspended washed platelets (WP) were stimulated by thrombin. AD₆ (5–100 M) inhibited dose dependently aggregation, BTG, PF₄ and TXB₂ release induced by threshold concentration of all the tested aggregating agents; however AD₆ action could be overcome by increasing the concentration of the stimulating agents. After cyclo-oxygenase blockade by acetylsalicylic acid (ASA), PRP was stimulated by a supramaximal concentration of PAF. Under these circumstances we could observe a reversible aggregation and a partial release of BTG and PF₄, AD₆ was able to further reduce aggregation and release. Cyclic AMP accumulation induced in WP by prostacyclin was not modified by AD₆ (100 M), while theophylline greatly potentiated prostacyclin action. We conclude that AD₆ is an inhibitor of platelet activation in vitro. Its mode of action is different from cyclo-oxygenase blockade and provides inhibition of platelet activation by a number of different stimuli.