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).     

Effect of Cordycepin-Enriched WIB801C from Cordyceps militaris Suppressing Fibrinogen Binding to Glycoprotein IIb/IIIa

In this study, we investigated the effects of cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha on collagen-stimulated platelet aggregation. CE-WIB801C dose dependently inhibited collagen-induced platelet aggregation, and had a synergistic effect together with cordycepin (W-cordycepin) from CE-WIB801C on the inhibition of collagen-induced platelet aggregation. CE-WIB801C and cordycepin stimulated the phosphorylation of VASP (Ser¹⁵⁷) and the dephosphorylation of PI3K and Akt, and inhibited the binding of fibrinogen to glycoprotein IIb/IIIa (αIIb/β₃) and the release of ATP and serotonin in collagen-induced platelet aggregation. A-kinase inhibitor Rp-8-Br-cAMPS reduced CE-WIB801C-, and cordycepin-increased VASP (Ser¹⁵⁷) phosphorylation, and increased CE-WIB801C-, and cordycepin-inhibited the fibrinogen binding to αIIb/β₃. Therefore, we demonstrate that CE-WIB801C-, and cordycepin-inhibited fibrinogen binding to αIIb/β₃ are due to stimulation of cAMP-dependent phosphorylation of VASP (Ser¹⁵⁷), and inhibition of PI3K/Akt phosphorylation. These results strongly indicate that CE-WIB801C and cordycepin may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.     

Exogenous GTP Increases Cyclic GMP and Inhibits Thrombin-Induced Aggregation of Washed Human Platelets: Comparison with ATP,Adenosine and Guanosine

Abstract: The effects of exogenous guanosine 5′-triphosphate (GTP), guanosine, adenosine 5′-triphosphate (ATP) and adenosine on platelet aggregation, serotonin secretion and cyclic nucleotide accumulation were studied using thrombin-stimulated washed human platelets. GTP (10 μM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion. The inhibition of aggregation was accompanied by an increase in platelet cyclic GMP. GTP did not affect cyclic AMP concentration. Adenosine (1 μM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion, and increased cyclic AMP. ATP at high concentrations (100 μM-1 mM) inhibited aggregation and serotonin secretion, and 1 mM ATP increased cyclic AMP. Guanosine was relatively ineffective in preventing aggregation and serotonin secretion and did not affect cyclic GMP. The rank order of inhibition of thrombin-induced aggregation of washed human platelets was adenosine > GTP > ATP > guanosine. In conclusion, exogenous GTP inhibits thrombin-induced aggregation and serotonin secretion of washed human platelets by increasing cyclic GMP. The results raise the possibility of a cell membrane site of action for GTP in platelets which mediates the activation of soluble guanylate cyclase suggesting that GTP may have a local antithrombotic effect also in vivo.     

Adenosine regulation of canine cardiac adenylate cyclase

Adenosine has a biphasic, [Mg²⁺]-dependent effect on the catalytic activity of dog heart adenylate cyclase. In the presence of 0.5 mM Mg²⁺, adenosine stimulated cyclic AMP formation, but when the cyclase was activated with 4 mM Mg²⁺ plus 0.5 mM Mn²⁺, adenosine inhibited catalytic activity in a dose-dependent fashion. Adenine, 3'-deoxyadenosine and selected purine-modified adenosine analogs stimulated the enzyme, whereas 2'-deoxyadenosine, 5'-deoxyadenosine and adenine-α-l-lyxofuranoside mimicked the inhibitory effect of adenosine on the Mg²⁺ plus Mn²⁺ stimulated enzyme. These results are consistent with the ‘two receptor’ model of Londos and Wolff [C. Londos and J. Wolff, Proc. natn. Acad. Sci. U.S.A.74, 5482 (1978)], but they raise the possibility of subtle organ and species differences in the chemical determinants of adenosine binding. Adenosine in both intracellular and extracellular compartments may modulate adenylate cyclase activity in the beating heart, in addition to its putative role in the regulation of coronary vascular resistance.     

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.     

Exogenous GTP increases cyclic GMP and inhibits thrombin-induced aggregation of washed human platelets: comparison with ATP, adenosine and guanosine.

The effects of exogenous guanosine 5'-triphosphate (GTP), guanosine, adenosine 5'-triphosphate (ATP) and adenosine on platelet aggregation, serotonin secretion and cyclic nucleotide accumulation were studied using thrombin-stimulated washed human platelets. GTP (10 microM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion. The inhibition of aggregation was accompanied by an increase in platelet cyclic GMP. GTP did not affect cyclic AMP concentration. Adenosine (1 microM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion, and increased cyclic AMP. ATP at high concentrations (100 microM-1 mM) inhibited aggregation and serotonin secretion, and 1 mM ATP increased cyclic AMP. Guanosine was relatively ineffective in preventing aggregation and serotonin secretion and did not affect cyclic GMP. The rank order of inhibition of thrombin-induced aggregation of washed human platelets was adenosine > GTP > ATP > guanosine. In conclusion, exogenous GTP inhibits thrombin-induced aggregation and serotonin secretion of washed human platelets by increasing cyclic GMP. The results raise the possibility of a cell membrane site of action for GTP in platelets which mediates the activation of soluble guanylate cyclase suggesting that GTP may have a local antithrombotic effect also in vivo.     

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 of adenosine uptake in human erythrocytes by adenosine-5'-carboxamides,xylosyladenine, dipyridamole,hexobendine, and p-nitrobenzylthioguanosine

Adenosine uptake in human erythrocytes at 0° consists of a saturable and a concentration-proportional component, the latter seems to represent uptake into a pericellulai compartment inaccessible to inulin. Xylosyladenine and derivatives of adenosine-5'-carboxamide were found to be weak inhibitors of the saturable component of adenosine uptake with apparent K_i values at least one order of magnitude higher than the apparent K_m for adenosine (2.4 × 10^?6 M). The affinity of the adenine nucleosides to the saturable uptake process appears to depend not only on the 3'-hydroxy] group and its erythro-configuration but also on the 5'-substituent. Dipyridamole, hexobendine, and p-nitrobenzylthioguanosine, by contrast, had K_i values at least one order of magnitude lower than the K_m for adenosine. The steric requirements for binding of the adenine furanosides to the putative smooth muscle receptors mediating vasodilation. on the the one hand, and to the saturable cellular uptake mechanism, on the other hand, were found to be different.     

Inhibition of cyclooxygenase activity, platelet aggregation and thromboxane B2 production by two environmental toxicants: m- and o-cresol

Cresol is a well-known environmental pollutant, toluene metabolite, uremic toxicant and accidental poisoning product. Formocresol, a preparation of formalin and cresol, is also used as a root canal medicament and for pulpotomy of primary teeth. However, little is known about its effect on cardiovascular system. In this study, m-cresol inhibited the AA-induced platelet aggregation by 43-97% at concentrations ranging from 0.25 to 1 mM. Collagen-induced platelet aggregation was also inhibited by 0.25-1 mM of m-cresol by 47-98%. Accordingly, o-cresol (0.1-0.5 mM) also inhibited the AA-induced platelet aggregation by 46-96% and the collagen-induced platelet aggregation by 35-88% at concentrations of 0.1-1 mM. AA- and collagen-induced platelet thromboxane B(2) (TXB(2)) production was inhibited by even 0.1 mM of m-cresol with 88 and 54% of inhibition, respectively. The o-cresol (0.1 mM) also inhibited the AA- and collagen-induced platelet TXB(2) production with 91 and 97% respectively. Although m- and o-cresol (<1 mM) showed little effect on thrombin-induced platelet aggregation, they effectively inhibited the thrombin-induced platelet TXB(2) production. The m-cresol (2 and 5 mM) inhibited the COX-1 activity by 55-99%, but showed little effect on COX-2 enzyme activity. Moreover, o-cresol (0.5 and 1 mM) inhibited the COX-1 activity by 40-95%. COX-2 enzyme activity was inhibited by 68% at a concentration of 5 mM o-cresol. These results indicate that acute cresol-poisoning, direct root canal medication with formocresol or long-term occupational exposure to cresol and toluene may potentially suppress blood clot formation and lead to tissue hemorrhage via inhibition of platelet aggregation, TXB(2) production and COX enzyme activity.     

Effect of sangivamycin and xylosyladenine on the synthesis and methylation of polysomal ribonucleic acid in Ehrlich ascites cells in vitro

The pyrrolopyrimidine, sangivamycin, and the adenosine analog, xylosyladenine, were examined for their effects on the synthesis and methylation of polysomal RNA in Ehrlich ascites tumor cells in vitro. The synthesis of non-polyriboadenylic acid (non-poly (A) ?) and poly(A)-containing RNA was inhibited 50 per cent at concentrations of 7 × 10^?6 M and 3 × 10^?6 M xylosyladenine, respectively, when adenosine deaminase was inhibited with 2'-deoxycoformycin. Sangivamycin inhibited the synthesis of non-poly(A)- and poly(A)RNA by 50 per cent at concentrations of 5 × 10^?5 M and 2 × 10^?5 M respectively. Electrophoretic separation of non-poly(A)RNA into rRNA and tRNA indicated that the inhibitory effects of both drugs were more pronounced on 28S than on 18S rRNA, and that xylosyladenine but not sangivamycin inhibited the synthesis of tRNA. Assessment of the effects of both analogs on the methylation of polysomal RNA revealed that xylosyladenine inhibited the methylation of nonpoly(A)-and poly(A)RNA, while sangivamycin only weakly affected the latter species of RNA. Base methylation of the affected species of RNA was inhibited slightly more than 2'-O-methylation by both drugs. These results indicate that sangivamycin is a more selective inhibitor of polysomal RNA in comparison to xylosyladenine under conditions where adenosine demainase is not a limiting factor.     

5'-Methylthioadenosine phosphorylase-L. Substrate activity of 5'-deoxyadenosine with the enzyme from Sarcoma 180 cells

5′-Deoxy-5′-methylthioadenosine phosphorylase (MTA phosphorylase), an enzyme involved in the salvage of adenine moieties from 5′-deoxy-5′-methylthioadenosine (MTA) produced primarily during polyamine biosynthesis, is present in Sarcoma 180 cells (0.0026 ± 0.0002 μM units/mg cytosol protein). 5′-Deoxyadenosine (5′-dAdo), an adenosine analog previously thought not to be metabolizable, has been shown [D. Hunting and J.F. Henderson, Biochem. Pharmac. 27, 2163 (1978)] to have a number of biochemical effects on Ehrlich ascites cells. We have now found that 5′-dAdo is a substrate for the MTA phosphorylase from Sarcoma 180 cells, yielding free adenine and 5-deoxyribose-1-phosphate. The reaction was reversible and totally dependent upon phosphate. Evidence that MTA phosphorylase is responsible for 5′-dAdo phosphorylase activity includes the following: (1) Sarcoma 180 MTA phosphorylase preparations did not show additive rates of adenine production in the presence of saturating concentrations of both 5′-dAdo and MTA; (2) double-reciprocal plots of the rates of adenine formation from 5′-dAdo by Sarcoma 180 enzyme preparations in the presence of MTA displayed a pattern characteristic of alternative, competing substrates; (3) the rate of depletion of 5′-dAdo by Sarcoma 180 preparations was inhibited by the presence of MTA; (4) the K_i value of a competitive inhibitor of Sarcoma 180 MTA phosphorylase, 5′-deoxy-5′-chloroformycin, was the same when either MTA or 5′-dAdo was employed as substrate; and (5) the apparent K_m values of phosphate for both MTA and 5′-dAdo phosphorylase activities were identical (3.5mM). The K_m of Sarcoma 180 MTA phosphorylase for MTA is 4 μM; the K_m for 5′-dAdo is 23 μM (V_max relative to MTA = 180 per cent). Incubation of Sarcoma 180 cells with either 5′-dAdo or MTA caused profound elevations of adenine nucleotides, as well as an inhibition of 5-phosphoribosyl-l-pyrophosphate (PRPP) accumulation. The reaction of 5′-dAdo with MTA phosphorylase to yield free adenine, which is then salvaged to adenine nucleotides, can account for many of the previously reported biochemical effects of 5′-dAdo, such as inhibitions of PRPP accumulation, purine de novo synthesis, and glycolysis that have previously been attributed to the unmetabolized nucleoside. The other product of this reaction, 5-deoxyribose-l-phosphate, may also contribute to these effects.     

Inhibitory effects of oligopeptides from hen egg white on both human platelet aggregation and blood coagulation

Egg white proteins have many biological functions and substantial nutritional benefits when used as a food source; however, they also contain allergens such as ovalbumin, ovomucoid, and ovotransferrin. We prepared oligopeptides without allergens from hen egg whites via the use of several proteases, and assessed their effects on platelet aggregation and blood coagulation, known to both of which are known to be major risk factors in thrombogenesis. Egg white oligopeptides (EWOP) inhibited collagen-induced human platelet aggregation in a dose-dependent manner. Additionally, we attempted to determine whether cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), aggregation-inhibiting intracellular molecules, regulate EWOP-inhibited platelet aggregation. EWOP caused an increase in cAMP levels, but did not affect cGMP levels, which suggests that the anti-platelet activity of EWOP operates in a cAMP-dependent manner, rather than via a cGMP-dependent process, in collagen-induced platelet aggregation. In addition, EWOP induced a significantly prolonged prothrombin time (PT) as compared with the controls. These data show that EWOP inhibits the conversion of fibrinogen to fibrin in a plasmatic atmosphere on an extrinsic pathway. Accordingly, these findings suggest that EWOP may be an excellent candidate as a crucial inhibitor of platelet activation, and its anti-platelet effects appear to involve the inhibition of both platelet aggregation and blood coagulation within the cardiovascular system.     

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.

     

Antithrombotic effects of Huanglian Jiedu decoction in a rat model of ischaemia-reperfusion-induced cerebral stroke

ContextHuanglian Jiedu Decoction (HLJJD) has a variety of pharmacological activities, such as anti-inflammatory and neuroprotection against ischaemic brain injury.ObjectivesThis ex vivo study explores its antithrombosis activity and inhibition of platelet aggregation.Material and methodsTo study the antithrombosis activity of HLJJD ex vivo, saline, or HLJDD (100, 200, and 500 mg/kg) was treated prophylactically by gavage for 3 days in Wistar rats (n = 4). Based on the rat model of transient middle cerebral artery infarction (MCAO) or normal rats (n = 4), the antithrombotic activity in the normal group and HLJDD subgroups on prothrombin time, thrombus weight, platelet aggregation, and others was evaluated, followed by the antiplatelet aggregation of its main components (n = 4).ResultsThe weight of the thrombus increased significantly at 24 h after MCAO onset. HLJJD did not influence the change of PT, but significantly inhibited thrombosis by 12.5, 20.0, and 20.5% in reducing the dry weight of thrombus, and blocked collagen-induced platelet aggregation by 25.5, 39.0, and 42.7% and adhesion of blood platelet by 17.3, 26.2, and 27.3%. The IC₅₀ value of HLJJD on collagen-induced platelet aggregation was 670 mg/kg. Geniposide only facilitated antiplatelet aggregation induced by collagen, but not AA or ADP. Both baicalin and berberine showed markedly antiplatelet aggregation induced by all activators. The antithrombotic activity of baicalin was relatively higher than that of berberine (35.0–47.8% vs. 20.6–33.5%).ConclusionOur results indicated that HLJDD regulated blood circulation by inhibiting platelet aggregation and thrombosis, which might also extensively contribute to the clinical prevention and treatment of cerebrovascular diseases.     

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.     

Effect of reserpine on adenosine uptake and metabolism,and subcellular transport of platelet adenosine triphosphate in washed rabbit platelets

There is a slow exchange of adenine nucleotides between the metabolically active (cytoplasmic) pool and the releasable amine storage organelle pool of blood platelets. Reserpine is known to inhibit serotonin uptake into platelet storage organelles. Therefore, we have determined whether reserpine also inhibits the uptake of adenine nucleotides from the cytoplasm into the storage organelles of rabbit platelets. Transport of adenine nucleotides from the metabolically active pool into the releasable amine storage granule pool was followed by labeling the metabolically active pool of adenine nucleotides by incubating the platelets with [¹⁴C]adenosine or [¹⁴C]adenine. Practically complete release of amine storage granule constituents was induced at various times in aliquots of the labeled platelet suspensions by treatment with a high concentration of thrombin (0.45 units/ml. The fraction of the total labeled [¹⁴C]ATP released was taken as a measure of ATP transport from the metabolically active pool into the releasable pool. Reserpine (0.2 and 2 μM) decreased the rate of ATP transport into the storage granules by about 50 per cent. Platelets obtained from rabbits that had received 5 mg/kg of reserpine intraperitoneally 18 hr prior to the collection of blood released less ATP and ADP than control platelets from animals that had not received any drugs. This was not due to inhibition of the release reaction by reserpine. Since reserpine reduces the amount of adenine nucleotides in the storage granules, we conclude that if it affects the rate of efflux of adenine nucleotides from the granules at all, this effect must be slight compared with the inhibition of the uptake into the granules. Reserpine was also found to decrease the incorporation of [8-¹⁴C]adenosine into platelet adenine nucleotides by inhibiting adenosine uptake into the platelets noncompetitively (K_i = 2 μM). Inosine uptake was also inhibited by reserpine. The effect of reserpine on adenosine uptake was reversible. In contrast, the effect of reserpine on ATP transfer from the metabolically active pool into the releasable pool was irreversible. This is in keeping with earlier observations that some reserpine binds to platelets reversibly and some binds irreversibly.     

Neurotoxicity of deoxycoformycin: effect of constant infusion on adenosine deaminase,adenosine, 2'-deoxyadenosine and monoamines in the mouse brain

The tight-binding adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), was continuously infused into mice by intraperitoneal implantation of microosmotic pumps delivering the compound at a rate of 0.16 mg hr^?1 kg^?1 for up to 6 days. The activity of cerebral adenosine deaminase was nearly totally inhibited. The amount of adenosine and 2'-deoxyadenosine was determined in the brain frozen in liquid nitrogen through the intact skull bone. The concentration of adenosine was about 1 $\text{nmol}\text{g}$, and was essentially not altered following treatment with deoxycoformycin. Deoxycoformycin induced a progressive increase in cerebral content of 2'-deoxyadenosine, which after 1 day of treatment equalled the amount of adenosine. The concentrations of serotonin, dopamine and noradrenaline in the brain were not altered.     

Inhibition of platelet aggregation by 2-(p-chlorophenyl)-4-thiazoleacrylic acid (Wy-23,049)--comparison with acetylsalicylic acid

2-(p-Chlorophenyl)-4-thiazoleacrylic acid (Wy-23,049) inhibited the first phase of adenosine diphosphate (ADP) and epinephrine-induced platelet aggregation, while acetylsalicylic acid (ASA) had little effect on the first but effectively inhibited the second phase. ASA was more effective than Wy-23,049 in inhibiting collagen-induced platelet aggregation. An ex vivo guinea-pig experiment demonstrated that Wy-23,049 inhibited the first phase and prevented the appearance of the second phase of ADP-induced platelet aggregation, while ASA inhibited the second phase. Oral administration of Wy-23,049 to rats was much more effective than ASA in protecting the animal against ADP-induced platelet loss. At an oral dose of 40mg/kg, Wy-23,049 prolonged the Lee-White clotting time in rats; ASA did not prolong it.     

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.     

Adenosine analogs and human platelets. Effects on nucleotide pools and the aggregation phenomenon.

The interaction between human blood platelets and adenosine and various adenosine analogs was examined. Effects were found both on the pools of nucleotides, as examined by the technic of high pressure liquid chromatography, and on the phenomenon of ADP-induced platelet aggregation. Although the normal ratio of ATP and ADP in platelets is about 1.5:1, after incubation with adenosine-8-¹⁴C, ATP and ADP were labeled in a ratio of about 7:1. This is consistent with a distribution of the nucleotides among storage and metabolic pools, with the adenosine-8-¹⁴C entering principally the metabolic pool. After 2 hr of incubation with 0–5 mM 2-fluoroadenosine (F-Ado), the concentration of F-ATP was approximately 12 μmoles/10¹¹ platelets. The ratio of F-ATP to F-ADP was approximately 7:1, indicating that it entered primarily the metabolic nucleotide pool. Also, during the first hr of incubation, as the F-ATP concentration increased, the ATP concentration fell. When F-ATP-containing platelets were treated with thrombin, an aggregator and storage granule releaser, the nucleotides released into the medium consisted principally of ATP and ADP in a ratio of about 0.8:1, with very little 2-fluoroadenine-containing nucleotides. After thrombin treatment, the washed platelet pellet contained most of the 2-fluoroadenine nucleotides, but with significant increases in the concentrations of F-AMP and F-ADP. This suggests that F-ATP can replace ATP as the energy donor for the aggregation and release phenomena.As reported elsewhere, adenosine strongly inhibits platelet aggregation induced by ADP. However, this inhibitory effect disappears after preincubation for about 30 min. If the preincubation is carried out in the presence of coformycin, a tight-binding inhibitor of adenosine deaminase (K_i ? 1 × 10^?10M), the inhibition of aggregation by adenosine is markedly prolonged, indicating that the loss of inhibition results from conversion of adenosine to inosine by adenosine deaminase. ADP-induced aggregation is powerfully inhibited by F-Ado, and the inhibition becomes more pronounced on prolonged incubation. This is consistent with the observation that F-Ado has very weak substrate activity with adenosine deaminase. The analog, N⁶-phenyladenosine, an inhibitor of adenosine kinase that does not form analog nucleotides in platelets, inhibits aggregation strongly, and the inhibition is maintained during incubation of 1 hr. Several other adenosine analogs only weakly inhibit ADP-induced aggregation even in the presence of coformycin. These include 2'-deoxyadenosine, 3'-deoxyadenosine (cordycepin), arabinosyladenine, and formycin A, a C-nucleoside. However, significant quantities of nucleotides of formycin A are formed in platelets in the presence of coformycin.