Effects of garlic extract and of three pure components isolated from it on human platelet aggregation, arachidonate metabolism, release reaction and platelet ultrastructure

We studied the effect of the methanol extract of garlic bulbs (EOG) and of three pure components isolated from it (F1, F2, F3), on human platelet aggregation induced by ADP, epinephrine, collagen, thrombin, arachidonate, PAF, and the ionophore A-23187. Incubation of PRP with EOG, either in methanol or in homologous PPP, inhibits platelet aggregation induced by all of the above mentioned agonists. F1, F2, and F3 also inhibit platelet aggregation, however, F3 was about four times more potent. Addition of EOG or F3 to platelets that have already been irreversibly aggregated by 10 microM ADP, induces rapid deaggregation. Inhibition of aggregation was still present after three hours. The inhibitory effect persisted even after the treated platelets were Gel-Filtered (GFP) or separated from plasma through a metrizamide gradient and resuspended in new homologous PPP. Thrombin-induced release of ATP from GFP was inhibited by 75-80% after EOG or F3 treatment. Incorporation of [3-H]-arachidonate by intact platelets was decreased by 50-60% in treated platelets. However, platelets incubated with the inhibitors after incorporation of radiolabeled arachidonate, although did not aggregate, produced, after thrombin activation similar amounts of radiolabeled TXB2 and lipoxygenase products as the controls. Electron microscopy of inhibited platelets, in the presence of thrombin, showed no degranulation but an increase of spherical forms. Our results suggest that the effects described might be mediate by a perturbation of the physicochemical properties of the plasma membrane rather than by affecting arachidonate or calcium metabolism in the cells. Chemical structures of F1, F2 and F3 have been provisionally assigned: F1 is diallytrisulfide, F2 is 2-vinyl-1,3-dithiene, and F3 is most probably allyl 1,5-hexadienyltrisulfide.     

Flurbiprofen,a new potent inhibitor of platelet aggregation

Flurbiprofen [dl-2-(2-fluoro-4-biphenylyl)propionic acid] is a non-steroidal anti-inflammatory compound whose inhibitory activity on collagen-induced platelet aggregation approaches the activity seen with prostaglandin E₁ (PGE₁). However, unlike PGE₁, it does not cause an increase in platelet cAMP levels, is orally active when given to animals or man, and is without side effects at platelet anti-aggregating doses. An oral dose of 1 mg/kg in rats inhibited collagen-induced platelet aggregation for 48 hours. At this dose, the mesenteric bleeding time in rats was prolonged from 197.2±24.4 sec. to 470.4±48.4 sec. The compound prevented death in mice due to pulmonary thromboembolism following collagen injection and inhibited platelet aggregation in man following oral administration. The drug appears to function by blocking the interaction of platelets with surfaces such as collagen but does not bind to the platelet membrane. It has no effect on the uptake of glucose or serotonin. The platelet anti-aggregating effect of flurbiprofen resides in the d-isomer while the l-isomer is without platelet anti-aggregating effect and does not counteract or enhance the effect of the d-isomer. These studies suggest that flurbiprofen may be a potentially useful antithrombotic drug.     

Verapamil is a potent inhibitor of 5-HT-induced platelet aggregation

We have studied the effects of verapamil, diltiazem and amlodipine on 5-HT-induced platelet aggregation and compared the results with those obtained for other platelet aggregating agents. Experiments were carried out using both human whole blood and platelet-rich plasma (PRP). Verapamil (but not diltiazem or amlodipine) inhibited 5-HT-induced platelet aggregation at much lower concentrations (IC50 = about 1 microM) than were required for inhibition of aggregation induced by other aggregating agents. Like some other selective inhibitors of 5-HT-induced platelet aggregation, it was not possible to completely overcome the inhibition by increasing the concentration of 5-HT. The antiaggregatory effects of verapamil were similar, but not identical, in whole blood and PRP. These results show that the Ca2+ channel blocker verapamil has some selectivity as an inhibitor of 5-HT-induced platelet aggregation and that this behaviour as a 5-HT antagonist should be taken into account when interpreting any therapeutic benefit ascribed to this drug.     

The effect of lithium on platelet aggregation and platelet release reaction

Lithium increases the aggregation of human platelets in vivo as well as in vitro. One of the mechanisms presumed to underlie this aggregation-promoting effect, potentiation of ADP release, was investigated in relation to lithium uptake. It appeared that lithium is not taken up by human platelets against a concentration gradient, and equilibrium is achieved in about 120 min. When platelets are incubated for this fixed time with different lithium concentrations, the aggregation response is related to the intraplatelet lithium concentration. When platelets are incubated during increasing periods with a fixed lithium concentration, no direct correlation exists between intraplatelet lithium concentration and extent of aggregation. After blocking the release with aspirin, the stimulatory effect of lithium on platelet aggregation was still observed. Low ADP concentrations, causing no measurable release, still caused more marked aggregation of platelets preincubated with lithium chloride than of platelets incubated with sodium chloride. It is concluded that the increase in platelet aggregation which occurs after incubation of platelets with lithium, is not caused merely by potentiation of the release reaction.     

Increased platelet aggregation and release reaction in myotonic dystrophy

Platelet aggregation (PA) induced by (-)-epinephrine and adenosine diphosphate (ADP) was studied in 16 patients with myotonic dystrophy (MyD) and 14 healthy subjects. Plasma beta-thromboglobulin level (beta-TG), a useful marker of in vivo platelet release reaction, as well as in vitro 5-[14C]hydroxytryptamine (5-HT) release, were also studied. The extent of PA induced by (-)-epinephrine at 1 and 3 min and by ADP at 3 min was significantly higher in the patients than in controls. Plasma beta-TG and ADP- or epinephrine-induced platelet 5-HT release were also increased in MyD patients. These results suggest that an abnormality in release as well as in alpha 2-receptor functioning occurs in the platelets of MyD patients. The relation of this abnormality to changes in Ca2+ fluxes through the platelet membrane is discussed.     

Human platelet aggregation and release reaction induced by platelet activating factor (PAF-acether)--effects of acetylsalicylic acid and external ionized calcium

The effects of the cyclo-oxygenase inhibition on PAF-acether-induced human platelet aggregation and secretion are controversial. We studied the above parameters on citrated platelet-rich plasma of 12 normal subjects before and after the in vivo administration of acetylsalicylic acid (ASA). Individual sensitivities to PAF-acether were highly variable. ASA completely inhibited the platelet secretion induced by low concentrations of PAF-acether, but caused only partial inhibition when platelets were exposed to high concentrations of PAF-acether. The concentration of PAF-acether which overcame the cyclo-oxygenase inhibition varied substantially, depending on the individual sensitivity of the platelets to it. The addition of CaCl2 2 mM to the samples did not affect the extent of the platelet secretion, but increased irreversible aggregation in samples taken both before and after the ASA administration. These data suggest that low concentrations of PAF-acether stimulate the human platelet secretion by activating the cyclo-oxygenase pathway, whereas higher concentrations also trigger other mechanism(s) that suffice to induce human platelet secretion and full aggregation.     

Purification and characterisation of a snake venom phospholipase A2: A potent inhibitor of platelet aggregation

An inhibitor of human platelet aggregation was identified from the venom of an Australian Copperhead snake, Austrelaps superba, as a novel phospholipase A₂. The inhibitor was purified to homogeneity by chromatography on Q-Sepharose, S-Sepharose and C8 reverse phase HPLC. The purified phospholipase A₂ has a molecular weight of 15 kDa as assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence analysis of the platelet inhibitor revealed 70–80% sequence identity to other previously described secretory phospholipase A₂. Phospholipase activity of the purified protein was confirmed by the ability of the enzyme to hydrolyse lecithin. Pretreatment of the purified protein with the specific phospholipase A₂ inhibitor p-bromophenacyl bromide, resulted in abrogation of both its enzyme and platelet inhibitory activity. The phospholipase A₂ inhibited platelet aggregation and serotonin release, induced by a variety of platelet agonists, in a time and dose dependent manner.     

Studies on the mechanism of platelet aggregation and release reaction induced by collagen and adrenaline

The effect of hirudin on collagen- and adrenaline-induced platelet aggregation was studied in order to evaluate the possible role of thrombin formation in platelet aggregation. The results were compared with those obtained with heparin, dextran-sulphate and diethylaminoethyl-dextran (DEAE-dextran). Hirudin, heparin, dextran-sulphate and DEAE-dextran potentiated adrenaline-induced first and second wave platelet aggregation. Collagen-induced platelet aggregation was to various degrees inhibited by heparin, hirudin and DEAE-dextran. The results indicate that thrombin formation is not an essential step in adrenaline-induced platelet aggregation and release reaction but may to some extent be involved in collagen-induced platelet release.     

Anti-thrombotic action of ticlopidine, a new platelet aggregation inhibitor

Anti-thrombotic action of Ticlopidine, 5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-C]pyridine hydrochloride, was studied by using a lactic acidosis-induced pulmonary thrombosis model in rats. Ticlopidine was highly effective in preventing thrombus formation at an oral dose as low as 50 mg/kg, and the action was well retained even after repeated daily administration of the compound at 100 mg/kg for 4 weeks. No difference was observed between control and the treated rats in morphology of thrombi formed, which were composed of platelet aggregates, fibrin and coagulated protein-like substances. Ticlopidine was also effective in preventing ADP- and collagen-induced acute pulmonary thromboembolic death in mice, collagen-induced transient thrombocytopenia in rats and platelet aggregation in β-aminopropionitrile-treated rats.

Ticlopidine was found to be superior to aspirin and dipyridamole in the treatment of these thrombosis models.     

Smooth muscle proliferation in chronically injured canine pulmonary arteries is reduced by a potent platelet aggregation inhibitor U-53,059

Dirofilaria immitis (DI) infection chronically injures canine pulmonary arteries. This injury produces endothelial cell loss, platelet/leukocyte adhesion, and smooth muscle proliferation. In the present study we assessed the effect of the cyclooxygenase inhibitor, U-53,059, on platelet function, platelet kinetics, coagulation, and smooth muscle proliferation in DI infected dogs. Platelet aggregation to the combination of arachidonic acid/ADP was significantly inhibited by U-53,059. Coagulation and hematologic parameters were not effected by either DI infection or U-53,059 treatment. Platelet survival and the number of platelet dense granules were reduced in DI infection. Quantification of the lesions demonstrated that U-53,059 reduced both severity and density compared to non-treated dogs. U-53,059 is a potent and effective inhibitor of platelet aggregation which modifies smooth muscle proliferation produced by chronic vascular injury.     

Prevention of canine coronary artery thrombosis with echistatin, a potent inhibitor of platelet aggregation from the venom of the viper, Echis carinatus

A model of acute, platelet-dependent canine coronary artery thrombosis was utilized to assess the antithrombotic effect of a synthetic, RGD-containing 49-residue protein termed echistatin. This protein is derived from the venom of the viper, Echis carinatus. In vitro, echistatin inhibited ADP (10 microM)-induced platelet aggregation with IC50 values in human and canine platelet-rich plasma of 101 +/- 4 and 127 +/- 32 nM, respectively. In vivo, in the dog, infusion of echistatin for 30 min at 20 micrograms kg-1 min-1 or 2.6 nM kg-1 min-1 resulted in total abolition of acute platelet-dependent coronary thrombus formation in all dogs tested (n = 5). Infusion of a lower dose (10 micrograms kg-1 min-1) was not effective in prevention of thrombus formation. Blood samples were taken before and after infusion of echistatin in order to determine ex vivo platelet aggregatory responses. Echistatin (20 micrograms kg-1 min-1, i.v.) attenuated ex vivo platelet aggregation elicited by ADP, U-46619 and collagen and increased bleeding time by 2.9 +/- 0.5-fold over control. Thus, in the dog, echistatin is an effective antithrombotic agent inhibiting both platelet aggregation in vivo in the coronary artery as well as ex vivo with a concomitant increase in bleeding time. Furthermore, the effects of echistatin on platelet aggregation and bleeding time are reversible with restoration to control levels occurring 30-60 min after termination of the infusion.     

Inhibition of platelet aggregation and the release of P-selectin from platelets by cilostazol

To evaluate the in vitro effects of cilostazol, a phosphodiesterase III inhibitor, on platelet responses, we measured platelet aggregation and the levels of soluble P-selectin, a glycoprotein present on the -granule membrane in resting platelets, and cAMP. Platelet-rich plasma and washed platelets from healthy human volunteers were treated with cilostazol (5, 25 and 50 μM). Platelet-rich plasma was stimulated by ADP (1 and 5 μM) or collagen (5 μg/ml). Washed platelets were stimulated by thrombin (4 U/ml) in the presence or absence of 1 μM forskolin. In vehicle-treated samples, soluble P-selectin levels in response to 1 μM ADP-induced primary aggregation were similar to those of circulating levels of healthy volunteers but the levels in response to 5 μM ADP-induced secondary aggregation and collagen-induced aggregation increased markedly compared to those in response to primary aggregation. This result suggests that P-selectin is released from platelets according to the extent of platelet aggregation. Cilostazol inhibited platelet aggregation as well as P-selectin release in a concentration-dependent manner. Cilostazol inhibited completely thrombin-induced aggregation in the presence of 1 μM forskolin, when cAMP levels were two-fold higher than those in the absence of forskolin. Cilostazol, which increases intracellular cAMP in platelets, may be useful in the treatment of arterial occlusive diseases.     

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3′,4′-tetrasulphate (QTS) and quercetin 3-acetyl-7,3′,4′-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 μM, final concentration), and the platelet aggregation was induced by using adenosine 5′-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 μM) and Qc (250 μM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 μM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA₂) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B₂ (TxB₂) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA₂, as judged by TxB₂ formation, in stimulated WP and a marked interference on the TxA₂ receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.     

Factors influencing shear-induced platelet alterations: platelet lysis is independent of platelet aggregation and release

To gain further insight into the mechanisms involved in fluid shear-induced platelet alterations, we examined conditions and factors that might affect shear-induced platelet reactions in human citrated platelet-rich plasma (C-PRP) under well-defined laminar flow conditions at shear stresses between 0 and 160 dyn/cm² using a Couette rotational viscometer. Prevention of excessive alkalinization of C-PRP during shear due to CO₂ loss did not appreciably affect shear-induced platelet aggregation (PAG), adenine nucleotide (AN) release or platelet lysis. Shear-induced PAG and AN release were significantly greater in C-PRP stored and sheared at 24°C as compared to C-PRP stored at 24°C or 37°C and sheared at 37°C whereas platelet lysis was not affected by temperature. When C-PRP was sheared in the presence of EDTA or EGTA, shear-induced PAG up to shear stresses of 80 dyn/cm² was almost completely suppressed whereas AN release and lysis were unaffected. Exposure of C-PRP to PGE₁ and theophylline before shear virtually abolished shear-induced PAG and AN release at shear stresses up to 80 dyn/cm² but had no demonstrable effect on shear-induced platelet lysis. These findings seem to indicate that ADP released from platelets by shear and extracellular Ca⁺⁺ or in the presence of PGE₁ and theophylline. These findings seem to indicate that the structural and biochemical changes associated with shear-induced PAG and release in our system do not predispose platelets to shear-induced lysis.     

Pharmacological profile of G619, a new platelet aggregation inhibitor.

G619, a 4-OH-isophthalic acid derivative, was studied for its capacity to inhibit platelet aggregation. G619 dose-dependently inhibited U46619, collagen, ADP, PAF, thrombin and epinephrine-induced platelet aggregation in vitro. The IC50 values for inhibition of U46619-induced human and rabbit platelet aggregation were 39 and 43 microM, respectively. G619, at 100 microM, inhibited high concentration collagen (10 micrograms/ml)-induced aggregation of rabbit platelets pretreated with indomethacin and increased the level of cAMP in washed rabbit platelets by 30% (p less than 0.01 vs basal). However, G619, did not inhibit fibrinogen binding to GPIIb/IIIa receptor, phosphodiesterase, U46619-induced contractile responses on canine saphenous vein or rabbit aorta, calcium-induced vasoconstriction and thrombin or PAF-induced elevation of [Ca++]i in platelets in vitro. In vivo, the U46619-induced maximal thrombocytopenia in rats was reduced from 40% (vehicle) to 22% and 18% by 10 and 30 mg/kg of G619 i.v., respectively. G619 (30 mg/kg) had no effect on the U46619-induced vasopressor response or sudden death in rats, and had no effect on TxB2 formation. Our results indicate that G619 is a broad-spectrum platelet aggregation inhibitor and may have its effect on a common mechanism for platelet aggregation besides an effect on the thromboxane A2 receptor.