Differences among betaadrenoceptor blocking drugs in modifying platelet aggregation and arachidonic acid liberation under thrombin stimulation

The dose-dependent inhibition of thrombin stimulated platelet aggregation due to betaadrenoceptor blocking drugs followed the rank order of potency:propranolol >alprenolol > metipranolol and correlated with arachidonic acid (³H-AA) liberation. Atenolol which slightly potentiated stimulated aggregation increased also the liberation of ³H-AA from membrane phospholipids of isolated platelets. Stimulation of platelets resulted in decreased ³H-AA incorporation into phosphatidylcholine, phosphatidylinositol and phosphatidic acid and increased incorporation into phosphatidylethanolamine and phosphatidylserine. A₁-prenolol, metipranolol and propranolol enhanced the incorporation of ³H-AA into phosphatidylcholine of stimulated platelets.     

Effect of cobra venom phospholipase A2 on platelet aggregation in comparison with those produced by arachidonic acid and lysophophatidylcholine

Cobra venom phospholipase A₂ induced a biphasic effect on washed rabbit platelets. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The aggregation and thromboxane B₂ formation were inhibited by indomethacin, mepacrine, tetracaine and imipramine, while PGE₁ and sodium nitroprusside inhibited only the aggregation, but not the thromboxane B₂ formation. The second phase was an inhibitory effect on platelet aggregation induced by arachidonic acid, PAF, ADP or collagen but not that by thrombin or ionophore A23187. The longer the incubation time of cobra venom phospholipase A₂ with platelets, the more the inhibitory effect. The aggregating and anti-aggregating effects could be overcome by bovine serum albumin. Lysophosphatidylcholine (Lyso-PC) and arachidonic acid showed synergistic inhibition in platelet aggregation. Lyso-PC decreased thromboxane B₂ formation in platelets formed by collagen. The inhibitory effect of Lyso-PC on platelet aggregation was more marked at lower calcium concentrations. It is concluded that the aggregating effect of exogenous addition of venom phospholipase A₂ is due to thromboxane formation and the antiplatelet effect is similar to those produced by arachidonic acid and lysophosphatidylcholine.     

The loss of sialic acid and its prevention in stored human platelets

The loss of sialic acid was determined in human platelets stored during a seven day period in their homologous plasma. Approximately 30% of the sialic acid was lost during the first three days of incubation at room temperature and a total of 73% was lost after seven days. The rapid $\text{in vitro}$ loss of sialic acid may mimic a slower $\text{in vivo}$ loss. It was found that platelets with a greater density had a higher sialic acid content than the less dense platelets. The loss of sialic acid from stored platelets could be completely inhibited by the addition of sialyl compounds to the incubation plasma. The trisaccharide, N-acetylneuramin-lactose, gave a greater degree of protection than fetuin at comparable concentrations.     

Inhibition of cyclooxygenase-independent platelet aggregation by sodium salicylate

The effect of acetylsalicylic acid (ASA) on platelet aggregation (PA) and thromboxane A₂ (TxA₂) formation was investigated in vitro and ex vivo after 1 g or 300 mg ASA administration to healthy subjects. 50–100 μM ASA inhibited PA by single aggregating agent such as platelet aggregating factor (PAF) or epinephrine and reduced to 5% of control platelet TxB₂ formation, but did not influence PA by epinephrine plus PAF. The latter was inhibited by increasing ASA concentration. In samples incubated with 100 μM ASA and stimulated with epinephrine plus PAF, PA could be inhibited by the addition of 100–300 μM sodium salicylate. After 300 mg-1 g ASA administration to healthy subjects, the inhibition of PA by epinephrine plus PAF was more marked by highest doses of ASA. This study suggests that aspirin inhibits PA with a cy clooxygenase-independent mechanism; this effect is mediated, at least in vitro, by salicylic acid.     

Properties of PAF-acether-induced platelet aggregation and secretion. Studies in gel-filtered human platelets

Human platelets rapidly lose their responsiveness to PAF-acether after blood collection. We collected blood from fasting donors and prepared gel-filtered platelets that remained responsive to PAF-acether for about 6 hours. Log-dose response studies showed bi-phasic aggregation between 20 and 100 nM PAF-acether with secretion of dense-, - and lysosomal granule contents during the second wave of aggregation. Between 0.2 and 10 nM PAF-acether aggregation was weak and no secretion occurred whereas 300 nM PAF-acether or more induced maximal aggregation and secretion. Secretion, however, was never more than 70, 55, and 30% of maximal secretable amount of 5HT, βTG and βN, respectively. Aggregation and secretion were enhanced by fibrinogen (optimal concentration 0.3–0.7 g.l⁻¹), required Ca²⁺ or Mg²⁺ but were inhibited when Mg²⁺ or Ca²⁺ were present at a concentration of 2 mM or more. These date show that human platelets are almost equally sensitive to PAF-acether as rabbit platelets, and respond with incomplete secretion of dense-, - and lysosomal granule contents.     

Effect of fibrinogen on ADP-induced platelet aggregation

The effect of fibrinogen on ADP-induced platelet aggregation was studied. In absence of ADP no binding of fibrinogen to platelets was observed. On addition of ADP to platelets, fibrinogen induced a dose-related aggregation and release reaction. Binding of approximately 1,000 fibrinogen molecules per platelet was observed. Since fragments of fibrinogen could inhibit this reaction it is suggested that ADP exposes structures on the platelet membrane which bind to fibrinogen. It is suggested that this binding of fibrinogen is of importance for the aggregation of platelets in presence of ADP.

Addition of ADP at high concentrations to platelets induces release of intracellullar fibrinogen which may explain the aggregation occurring at high concentration of ADP in the absence of added fibrinogen.     

Effect of beta-pyridyl-carbinol on platelet aggregation

We studied the anti-platelet aggregation activity of beta-pyridyl-carbinol (b-PC) (Ronicol, Roche). This compound has a chemical structure similar to nicotinic acid and is therapeutically indicated in functional and organic circulatory processes.

We determined the in vitro antiaggregation activity induced by ADP (4 μM) and collagen (20 μg/ml) using Cardinal and Flower's technique.

Antithrombotic activity was assessed in rats by measuring the duration of ADP-induced (12.40 mg/kg i.v.) respiratory dysfunction in 2 groups: one given 17.14 mg/kg beta-pyridyl-carbinol for 8 consecutive days before testing and controls.

In vitro and in vivo results were highly significant. Circulating platelet count increased in b-PC treated animals.

Systolic and diastolic pressures remained unmodified during administration of b-PC.     

Effect of phorbol 12-myristate 13-acetate on collagen-induced signal transduction in rabbit platelet

Investigations were made on the inhibitory effect of phorbol 12-myristate 13-acetate (PMA), a powerful activator on protein kinase C, on collagen-induced signal transduction in washed rabbit platelets. Upon activation of the platelets with a low-dose of collagen (5 μg/ml), which was suppressed by 10 μM indomethacin, pretreatment of the platelets with 2 nM PMA caused prolongation of lag phase (2 min) before the onsets of the aggregation and ATP secretion as compared with PMA-untreated platelets (30 sec). Under this condition, appearance of the cell responses including the phosphatidic acid formation, thromboxane (Tx) generation and Ca²⁺-influx was similarly retarded for 2–3 min, whereas arachidonic acid liberation from the membrane phospholipids was not significantly affected by the PMA pretreatment. After such lag phase, every response appeared rapidly and reached almost the control value (without PMA). Upon activation of the same platelets with a high-dose of collagen (50 μg/ml), which was only half suppressible by indomethacin, PMA in the presence of indomethacin almost completely suppressed the phosphatidic acid formation as well as the aggregation and ATP secretion. Thus, our results suggest that collagen-platelet interaction may elicit direct activation of phospholipase A2 and C, and that the latter enzyme activation may be regulated by a negative effect of protein kinase C. However, the phospholipase A2 activation may be regulated by a mechanism independent of such effect. In PMA-pretreated platelets in response to a low-dose of collagen, the prolonged lag phase for aggregation appears to be due to impaired conversion of liberated arachidonic acid to TxA₂.     

Effects of eicosapentaenoic acid on arachidonic acid metabolism in cultured vascular cells and platelets: Species difference

The metabolism of eicosapentaenoic acid (EPA) in cultured vascular smooth muscle cells isolated from human, rat, rabbit and miniture pig and bovine endothelial cells were studied. EPA was not able to be converted to any prostaglandins (PGs) in murine and porcine smooth muscle cells. However, in human and rabbit smooth muscle cells and bovine endothelial cells EPA was easily converted to Δ 176-ketoPGF_1α, which is a stable metabolite of PGI₃. Cyclooxygenase and 12-lipoxygenase activities in platelets isolated from human citrated blood were almost completely inhibited by EPA at the dose over 4 μg. But in platelets isolated from rat the inhibitory effects of EPA on arachidonic acid (AA) metabolism were much smaller than that in human platelets. In rat, EPA was not only being converted to no PGI₃, but also being a blocker to PGI₂ synthesis in vascular cells. Moreover, in rat EPA has much less activity in inhibiting thromboxane A₂ (TXA₂) synthesis in platelets. On the contrary, in human EPA was not only easily converted to PGI₃ in vascular cells, but also blocking TXA₂ synthesis in platelets.Thus, anti-aggregatory effects of EPA was positive in human and negative in rat perhaps due to species difference in sensitivity to EPA.     

Low platelet apachidonic acid in young patients with brain infarction

Fatty acid patterns of plasma and platelet lipids, platelet aggregation and thromboxane A₂(TxA₂) production were studied in young patients (n = 12) with brain infarction and in healthy controls (n = 13). Platelet arachidonic acid content was significantly reduced in the stroke patients, but in vitro platelet aggregation was similar in the two groups. A low dose of acetosalicylic acid (ASA) (100 mg) suppressed thromboxane production and normalized the platelet arachidonic acid values. The low arachidonic acid in platelets is probably due to its increased consumption, indicating platelet activation in vivo.     

The effect of arachidonic acid metabolism on intravascular platelet aggregation in rats

The effects of BW755C, benoxaprofen, indomethacin and piroxicam were studied on intravascular platelet aggregation using continuous platelet counting. Plasma levels of thromboxane B₂(TxB₂) and 6-keto prostaglandin F₁(6-keto PGF_1α) were measured by radioimmunoassay. BW755C, a dual inhibitor of arachidonic acid metabolism, potentiated or inhibited aggregation depending on dose. BW755C increased TxB₂and 6-keto PGF_1α in plasma levels at low doses. At higher doses BW755C inhibited aggregation and reduced TxB₂plasma levels. At 16mg/kg BW755C, 6-keto PGF_1α was detected? Penoxaprofen also potentiated collagen-induced aggregation. 8 mg/kg indomethacin was shown to have a short lasting increased inhibitory action on collagen-induced aggregation when compared with the more specific cyclooxygenase inhibitor, piroxicam. 6-keto PGF_1α was detected in plasma fro rats treated with indomethacin and piroxicam. The results obtained using low doses of BW755C suggest that the lipoxygenase pathway is involved in platelet aggregation. Interpretation of the results obtained using higher doses of drugs also suggests this involvement though some nonspecific actions of the drugs must be taken into consideration. Further work is required to detail the role of lipoxygenase products in collagen-induced intravascular platelet aggregation.     

Transient aggregation resistance of human blood platelets in fresh plasma. I. Methodology

A novel analytical method, using turbidcmetry, for reporting the time-dependent decay in the threshold concentration of adenosine diphospahte (ADP), required to elicit a secondary aggregation response in fresh human citrated platelet-rich plasma (PRP),is described. The phenomenon, called “transient aggregation resistance” (TAR) ends, usually within one hour after venepuncture, in a steady state or “baseline aggregation resistance” (BAR) . Back extrapolation of the mathematically transformed data to T = 0, yields a maximal threshold concentration of ADP, representing the initial aggregation resistance (TARmax) at the time of blood withdrawal, which threshold is usually many orders of magnitude higher than the BAR-value. The exponential decay of TAR can be characterized by its half-life ( ) . Mixing fresh, rapidly prepared, plasma with PRP, kept long enough to show only the stable low BAR-value, could restore the initial high (transient) aggregation resistance found in fresh PRP, suggesting that it concerns a natural, labile plasmatic factor. One hour old PRP, deliberately made refractory to ADP, did not show the TAR phenomenon again, but had a higher BAR-value. It is suggested that the level of clinical relevance of these early in vitro aggregation measurements is higher than that of classical, delayed aggregometry (e.g.BAR-values).     

An analysis of inhibitory effect of cyclic GMP on arachidonic acid- or collagen-induced aggregation in rabbit platelets

Aggregation of washed rabbit platelets induced by arachidonic acid (AA) or collagen was inhibited by nitroprusside (NP) and 8-bromo cyclic GMP in a concentration-dependent manner. Although NP and 8-bromo cyclic GMP inhibited the AA-induced aggregation, these agents did not affect the conversion of exogenous AA to PG endoperoxides and TXA2 (which were observed as TXB2). On the other hand, collagen caused release of AA from phospholipids and sequential formation of TXB2 in [14C]AA prelabeled platelets. In contrast with the case in which exogenous AA was used, NP and 8-bromo cyclic GMP inhibited the collagen-induced formation of TXB2 by preventing the liberation of endogenous AA. These results indicate that cyclic GMP has at least two different inhibitory actions in platelets; one is the inhibition of AA release from phospholipids and the other is the inhibition of the action of TXA2 in platelets.     

Interactions between sodium salicylate and acetyl salicylic acid evaluated using ADP induced platelet aggregation and bleeding time

1 g acetyl salicylic acid orally significantly enhanced the initial rate of platelet aggregation induced by 1 pmol/1 and 2.5 μ mol/1 ADP.

Sodium salicylate was without effects on the platelet aggregation and specifically it did not prevent acetylsalicylic acid from inhibiting the secondary aggregation. Sodium salicylate was without effect on the bleeding time and did not inhibit the prolongation induced by acetyl salicylic acid. Our study do not lend support to the concept of an important interaction in vivo between acetyl salicylic acid and its first metabolite salicylate in man.     

Effects of arachidonic acid on the metabolism of eicosapentaenoic acid in washed human platelets

We examined effects of arachidonic acid (AA) on eicosapentaenoic acid (EPA) metabolism in washed human platelets. Although human platelets had been considered to metabolize scarcely EPA, a simultaneous addition of EPA and AA to washed platelet suspensions stimulated markedly EPA metabolism. In addition, the stimulatory effect was more potent over the formation of thromboxane (TX) B3 than that of 12-hydroxy-5,8,10,14,17-eicosapentaenoic acid (HEPE). The stimulation by AA can be due to AA itself and/or AA metabolites. Indomethacin decreased the stimulatory effect of AA on the HEPE formation, suggesting that cyclooxygenase product(s) of AA stimulated the HEPE formation. Among the metabolites of AA investigated, prostaglandin (PG)G2 and 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid had the stimulatory effect on both TXB2 and HEPE formations, whereas PGH2, PGD2, TXB2 and 12-hydroxy-5, 8,10,14-eicosatetraenoic acid were ineffective.     

Inhibitory effect of activated protein C on platelet aggregation induced by the prothrombin-converting reaction

The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaction mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation; maximum aggregation rates, 31.3–92.5%, and times to reach to maximum aggregation, 11.6 to 20.1 min. This aggregation was inhibited by the addition of APC with 50% inhibition concentration (IC₅₀) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dose-dependent manner with IC50 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.