Inhibition of rabbit platelet aggregation by 1,4-naphthoquinones

The effects of four 1,4-naphthoquinone derivatives on the aggregation of rabbit platelets were examined. All the four 1,4-naphthoquinone derivatives inhibited the platelet aggregation of washed rabbit platelets induced by thrombin (0.1 U/ml) and the IC50 is: 2-chloro-3-methyl-1,4-naphthoquinone (CMN), 5 micrograms/ml; 3-methyl-5,8-dihydroxy-1,4-naphthoquinone, 13 micrograms/ml; 5,8-dihydroxy-1,4-naphthoquinone, 18 micrograms/ml; 3-methyl-1,4-naphthoquinone (vitamin K3), 53 micrograms/ml. CMN was the most potent in inhibiting the aggregation and release reaction induced by ADP, arachidonic acid, PAF, ionophore A23187, collagen and thrombin in a dose-dependent manner in washed platelets, platelet-rich-plasma and whole blood. The thromboxane B2 formation caused by collagen and ionophore A23187 was inhibited by CMN. However, the thromboxane B2 formation by arachidonic acid was markedly increased. The platelet inhibitory effect of CMN could not be antagonized either by raising the concentrations of extracellular Ca++ or by wash out. The phosphoinositides breakdown induced by thrombin was inhibited by CMN. Phospholipids (PE, PC, PI) could slightly antagonize the antiplatelet effect of CMN. It is concluded that the inhibitory effect of CMN on rabbit platelet aggregation may be due to the inhibition of phosphoinositides breakdown caused by the inducers.     

Effect of amino sugars on platelet aggregation and on fibrinogen binding

The amino sugars glucosamine, galactosamine and mannosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction. The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectin-like activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.     

Malondialdehyde formation by blood platelets: a diagnostic test to assess acetylsalicylic acid induced thrombocytopathy?

We investigated whether the measurement of N-ethylmaleimide stimulated malondialdehyde (MDA) formation by blood platelets from normal subjects is equally sensitive to acetylsalicylic acid intake as are platelet aggregation studies. MDA production and platelet aggregation by collagen and arachidonate were assayed in ten healthy volunteers before and up to ten days after a single oral dose of 500 mg aspirin. Discordant results of the two tests were seen in several subjects 4 to 6 days after aspirin intake. In three cases with still suppressed MDA values on day 4, collagen or arachidonate induced aggregation was normalized. However, on day 6, when MDA was normalized in all subjects, the aggregation response to arachidonate was still pathologic in 5 of the ten volunteers. In case of a patient with abnormal aggregation response to arachidonate and/or collagen, therefore, a normal MDA value does not permit to exclude aspirin as the cause of the platelet dysfunction.     

Collagen-induced calcium influx is enhanced in platelets from hypercholesterolemic rabbits

We investigated the mechanism of hyperreactivity to collagen of the platelets from hypercholesterolemic rabbits, as compared with cholesterol-enriched platelets prepared in vitro by incubation of normal platelets with hypercholesterolemic plasma or cholesterol-lecithin dispersion. The platelets from rabbits fed high cholesterol diet for only 1 to 2 weeks, had an enhanced reactivity in aggregation and secretion, as well as an increase in cholesterol content and a decrease in membrane lipid fluidity. The cholesterol-enriched platelets prepared in vitro also had the same properties. In all these cholesterol-enriched platelets, two membrane functions, Ca2+-influx and arachidonate liberation from membrane phospholipids, were markedly enhanced in response to collagen, probably due to the influence of cholesterol upon physical properties of the membranes. These data suggest that in platelets from hypercholesterolemic rabbits, the decrease in membrane fluidity, accompanying the increase in membrane cholesterol content, brings about the enhancement of Ca2+-influx and liberation of arachidonate, thus resulting in hypersensitivity to collagen.     

Elucidation of a role of plasma albumin during collagen-induced aggregation of bovine platelets

Bovine platelets of which phospholipids were labeled with [14C] arachidonate were stimulated by collagen. Omitting albumin in suspending medium suppressed aggregation and arachidonate liberation from phospholipids. Analysis of [14C] arachidonate metabolites released into medium revealed that release of 12S-hydroxyeicosatetraenoic acid (12-HETE) from stimulated platelets was dependent on the existence of albumin, while thromboxane B2 release was not. Moreover, exogenously added 12-HETE inhibited collagen-induced aggregation. These observations suggest that albumin potentiates aggregation by preventing intracellular accumulation of 12-HETE, and strengthen our previous finding that albumin in plasma is very essential for collagen-induced aggregation of platelets (Sekiya, F., Takagi, J., Kasahara, K., Inada, Y. and Saito, Y. (1988) Thrombos. Res. 50, 837-846.).     

XC 386: A new antiplatelet agent

XC386 was a new antiplatelet compound, which inhibited the aggregation and release reaction of rat platelet-rich plasma induced by collagen. This inhibition was dose-dependent and the IC₅₀was calculated to be 1 mM on collagen-induced aggregation. In washed platelets, the aggregations induced by ADP and collagen were much more markedly inhibited by XC386 than those induced by thrombin, A23187 and arachidonate. High calcium (4 to 8 mM) could not antagonize the inhibition. XC386 did not alter the malondialdehyde (MDA) and thromboxane B (TXB₂) levels of resting platelets. But MDA formation induce by collagen, thrombin and A23187, and TXB₂ formation induced by collagen and thrombin were significantly inhibited, while both formations induced by arachidonate were not changed. Combination of indomethacin or CP/CPK and XC386 enhanced markedly the inhibitory effect of XC386 on collagen- or A23187-induced aggregation. It was concluded that XC386 might inhibit platelet aggregation before the step of arachidonic acid release by phospholipase A₂.     

Effects of tris on responses of human and rabbit platelets to aggregating agents

Despite reports that Tris [tris (hydroxymethyl)aminomethane] affects platelets, it is often used to buffer suspending media. Human or rabbit platelets were washed and resuspended in Tyrode solution containing apyrase and 0.35% albumin. Addition of 15 mM Tris partially inhibited primary aggregation induced by 10 microM ADP and inhibited aggregation and release of 14C-serotonin from prelabelled platelets stimulated with low concentrations of thrombin (0.05-0.2 U/mL), or collagen. Platelets resuspended in 15 mM Tris, 0.15 M NaCl, 0.35% albumin, pH 7.5, did not aggregate in response to 10 microM ADP whereas platelets in Tyrode-albumin aggregated extensively. Ca2+ (5 mM) did not overcome the inhibition of thrombin-induced aggregation. Tris (15 or 1.5 mM) potentiated aggregation and release induced by sodium arachidonate (20-50 microM) or the ionophore A23187 (0.6-1 microM). Pretreatment of platelets with aspirin did not prevent potentiation by A23187, indicating that it is not mediated through activation of the arachidonate pathway. The inhibitory and potentiating effects of Tris are similar to those of amino sugars, lysine, arginine and primary amines such as methylamine and cadaverine, and may represent general effects of amines on platelets. Potentiation of the effects of some aggregating agents and inhibition of others re-emphasizes the concept that there are several different mechanisms through which aggregation can occur. Tris-based buffers are unsuitable for platelet suspending media and their use as solvents for aggregating agents or inhibitors should be limited.     

Some properties of mouse platelets

Mouse platelets were aggregated by arachidonate, thrombin, collagen and ADP. In general they were, like rat platelets, more aggregable in heparinized PRP than in citrated (3.8%) PRP. Mouse platelets underwent the release reaction when aggregated by arachidonate, collagen and thrombin, but not when stimulated by ADP. The aggregation of the platelets to arachidonate was inhibited by cyclooxygenase inhibitors and by prostacyclin. Studies with tritiated arachidonate showed that mouse platelets possess the lipoxygenase and cyclooxygenase pathways found in other mammalian platelets and produce thromboxane and 12-HETE. The mouse provides a convenient model for the study of many conditions known to affect platelet aggregation. The similarity of mouse platelets to the platelets of other mammals together with the ability to study large numbers of animals at low cost, should encourage further use of mouse platelets.     

Ouabain enhances basal and stimulus-induced cytoplasmic calcium concentrations in platelets

Incubation of platelet-rich plasma (PRP) with ouabain, an inhibitor of sodium/potassium ATPase (Na+/K+ ATPase), induced a significant rise in basal platelet intracellular calcium concentration [( Ca2+]i) when measured using fura 2. Ouabain induced an enhanced aggregation response to low doses of collagen in both PRP and washed platelets loaded with aequorin. In aequorin loaded platelets this enhanced aggregation response was associated with an enhanced rise in [Ca2+]i such that the relationship between [Ca2+]i and aggregation was unchanged. As inhibition of plasma membrane Na+/K+ ATPase would lead to a raised intracellular sodium ion concentration [( Na+]i) the results suggest that in the platelet, [Na+]i can modulate [Ca2+]i and hence influence the response of platelets to stimuli such as collagen.     

Factors influencing the deaggregation of human and rabbit platelets

The mechanisms involved in platelet deaggregation are unclear. Washed platelets from rabbits or humans aggregated by ADP can be deaggregated by EDTA or PGI2 if the release reaction has not occurred; during deaggregation 125I-fibrinogen dissociates from the platelets. Human platelets suspended in a medium without calcium undergo the release reaction during ADP-induced aggregation; EDTA, PGE1 or PGI2 do not deaggregate these platelets although EDTA displaces much of the 125I-fibrinogen that associates with them during aggregation. Rabbit platelets aggregated by low concentrations of release-inducing stimuli (sodium arachidonate, collagen or thrombin) can be deaggregated by EDTA, PGI2 or PGE1 and 125I-fibrinogen dissociates from them; with high concentrations of collagen or thrombin, deaggregation and dissociation of 125I-fibrinogen is slower. Human platelets that have undergone the release reaction in response to thrombin, collagen or a combination of sodium arachidonate and ADP are not readily deaggregated by EDTA or PGE1. Since aggregation and fibrinogen binding involving the glycoprotein IIb/IIIa complex are readily reversed by EDTA, and since Ca2+ is required for thrombospondin binding to activated platelets, there may be a third type of platelet-platelet adherence that is not disrupted by EDTA; this type of binding plays a greater role with human than with rabbit platelets.     

Congenital deficiency of alpha-2-adrenoceptors on human platelets: description of two cases

The biochemistry and functionality of platelets from two related subjects (mother and son) with alpha-2-adrenoceptor-deficient platelets has been evaluated. Radioligand binding experiments with the specific alpha-2-adrenergic-receptor antagonist, 3H-yohimbine, showed a drastic reduction of alpha-2-adrenoceptors in platelets from both subjects in comparison with the control values. Electron microscopy studies revealed a normal morphology and a normal number of alpha granules and dense bodies. Levels of adenine nucleotides; 5-hydroxytryptamine; B-thromboglobulin; platelet-factor-4 and thromboxane A2 production were within normal limits. Platelet aggregation and 5-hydroxytryptamine production in response to adrenalin (at concentrations up to 50 microM) were absent, whereas ADP, AA, PAF, collagen and thrombin-induced aggregation, secretion, Ca++ flux and thromboxane A2 production were normal. The inhibitory effect caused by different concentrations of prostacyclin on Ca++ flux, aggregation, secretion and thromboxane A2 production of platelet functionally lacking of alpha-2-adrenoceptor was not distinguishable from control platelets and platelets preincubated with yohimbine.     

Effect of the calcium-entry blocking agent nifedipine on activation of human platelets and comparison with verapamil

The effects of the calcium-entry blocking agent nifedipine on the activation of human platelets by various agonists has been studied and compared with verapamil. Like verapamil, nifedipine inhibited platelet aggregation and secretion caused by collagen, the second phase of ADP-induced aggregation, and aggregation caused by the ionophore A23187. Both agents inhibited the formation of TXB2 from endogenous arachidonate, whereas only nifedipine inhibited platelet aggregation and decreased TXB2 formation caused by exogenous arachidonate without inhibiting uptake. These results indicate that both calcium-blocking agents may be inhibiting the release of arachidonate in platelets by phospholipases, and that nifedipine also inhibits the formation and action of thromboxane A2 in platelets. Epinephrine-induced aggregation was inhibited by low concentrations of verapamil while nifedipine only inhibited aggregation by epinephrine at much higher concentrations. It is suggested that low concentrations of verapamil inhibit epinephrine-induced aggregation by interacting with platelet alpha-adrenergic receptors, and that higher concentrations of both calcium-blocking agents inhibit platelet responses to other aggregating agents by preventing intracellular calcium mobilization.     

The inhibitory effect of 9-amino-1,2,3,4-tetrahydroacridine (THA) on platelet function.

Tetrahydroacridine (THA), or Cognex, is currently awaiting FDA approval for the treatment of Alzheimer's disease. In addition to reports indicating that THA improves the symptoms of patients with Alzheimer's disease, we have found that THA possesses potent antiplatelet activity. THA produced dose-dependent inhibition of human platelet aggregation induced by collagen, ADP, A23187, and phorbol ester. THA, when added to activated platelets, dispersed the platelet aggregates. We have also examined the effects of THA on intracellular Ca++ mobilization, ATP release, and production of cyclic AMP.     

Identification of receptors for fibrinogen and von Willebrand factor mediating aggregation in guinea pig platelets

Monoclonal antibodies were prepared to guinea pig platelets and selected for their ability to inhibit ADP-induced platelet aggregation and ristocetin induced, Ca++-independent platelet agglutination. One antibody, PG-2, produced strong inhibition of aggregation induced by ADP, thrombin, collagen and arachidonic acid, while not inhibiting ristocetin-induced agglutination. A second antibody, PG-1, blocked ristocetin-induced agglutination, but did not inhibit aggregation induced by the previous agents. PG-2 blocked ADP-induced 125I-fibrinogen binding to washed guinea pig platelets by approximately 50%, but did not inhibit ristocetin-induced binding of 125I-vWF. Conversely, PG-1 selectively inhibited ristocetin-induced 125I-vWF binding, with the degree of inhibition inversely related to the ristocetin concentration. These studies suggest that in guinea pig platelets, fibrinogen and von Willebrand factor binding to different membrane sites are responsible for the aggregation response of stimulated platelets and the ristocetin-induced agglutination response respectively. These antibodies offer significant promise for the further development of a guinea pig animal model for studying platelet and megakaryocyte function.     

Unexpected effects of aurin tricarboxylic acid on human platelets.

Aurin tricarboxylic acid (ATA) is a potent inhibitor of ristocetin-mediated platelet agglutination and of shear-induced, von Willebrand factor (vWf)-mediated platelet aggregation, probably via inhibition of vWf interaction with glycoprotein Ib (GPIb). We examined the effects of ATA (both the sodium salt and a solution of ATA in ethanol) on platelet functions in citrated plasma (PRP) and in suspensions of washed platelets in Tyrode-albumin solution (contains 2 mM Ca2+). ATA (42-211 micrograms/ml) blocked aggregation and release of granule contents induced by thrombin (0.15 U/ml in PRP; 0.03 U/ml in platelet suspension). Responses to higher concentrations of thrombin were not inhibited. ATA also prolonged thrombin-induced clotting of fibrinogen. Since ATA had no effect on fibrinogen-induced responses of chymotrypsin-treated platelets, ATA probably acts on thrombin rather than on fibrinogen. In PRP and platelet suspensions, ATA (acid form 106 micrograms/ml; sodium salt 122 micrograms/ml) had little effect on ADP-induced platelet aggregation. The sodium salt of ATA (61-122 micrograms/ml) enhanced collagen-induced aggregation and release by platelets in citrated plasma and by washed platelets; the enhancement was extensively inhibited by aspirin. With platelet suspensions, ATA significantly enhanced aggregation and release caused by low concentrations of sodium arachidonate (15-50 microM); aggregation and release caused by higher concentrations of arachidonate were somewhat inhibited by ATA. Arachidonate-induced aggregation and release were also enhanced by ATA in PRP. ATA enhanced aggregation and release induced by the calcium ionophore A23187; aspirin had little effect on the enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ethanol on pathways of platelet aggregation in vitro

Ethanol, at physiologically tolerable concentrations, did not affect the primary phase of ADP-induced aggregation of human or rabbit platelets, which is not associated with the secretion of granule contents. Potentiation by epinephrine of the primary phase of ADP-induced aggregation of rabbit platelets was also not inhibited by ethanol. However, ethanol did inhibit the secondary phase of ADP-induced aggregation which occurs with human platelets in citrated platelet-rich plasma and is dependent on the formation of thromboxane A2. Inhibition by ethanol of thromboxane production by stimulated platelets is likely due to inhibition of the mobilization of arachidonic acid from membrane phospholipids, as ethanol had little or no effect on aggregation and secretion induced by arachidonic acid or the thromboxane mimetic U46619. Rabbit platelet aggregation and secretion in response to low concentrations of collagen, thrombin, or PAF were inhibited by ethanol. Inhibition of the effects of thrombin and PAF was also observed with aspirin-treated platelets. Thus, in addition to inhibiting the mobilization of arachidonate for thromboxane formation that occurs with most agonists, ethanol can also inhibit aggregation and secretion through other effects on platelet responses.     

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.     

Synergism between platelet aggregating agents: the role of the arachidonate pathway.

Aggregation of platelets by low concentrations of ADP is augmented by non-aggregating concentrations of collagen, thrombin, arachidonate or the divalent cation ionophore A23,187. Release-inducing agents act synergistically with ADP and with each other. Both collagen and thrombin cause aggregation by releasing ADP and by freeing platelet arachidonate to form prostaglandin endoperoxides which give rise to thromboxane A₂. In these experiments the role of the arachidonate pathway in the synergism between pairs of aggregating and release-inducing agents was examined. Indomethacin was used to inhibit conversion of arachidonate to prostaglandin endoperoxides and thromboxane A₂ and creatine phosphate/creatine phosphokinase (CP/CPK) was used in some experiments to convert released ADP to ATP. Synergism of collagen with ADP, arachidonate or thrombin was inhibited by indomethacin indicating that the arachidonate pathway plays a major role in the synergistic effects to which collagen contributes. Synergism of thrombin with collagen or arachidonate was inhibited by indomethacin but synergism of thrombin with ADP was only slightly affected. Indomethacin had little influence on the combined effects of these two agents on platelet aggregation. Thus it appears that the conversion of platelet arachidonate to prostaglandin endoperoxides and thromboxane A₂ plays a minor part in the synergistic effects in which thrombin or A23,187 are involved. Thus, the non-steroidal anti-inflammatory drugs may have only limited use in inhibiting the contribution of thrombin and ADP to the formation of platelet thrombi at sites of vessel injury.     

Interaction of beta 2-glycoprotein-I with human blood platelets: influence upon the ADP-induced aggregation

The interaction of beta 2-glycoprotein-I (beta 2-G-I), a plasma constituent of unknown function, with blood platelets was studied. The following results were obtained: 1) beta 2-G-I binds to washed human platelets isolated by centrifugation (WP) at one kind of specific, saturable binding sites. The dissociation constant was found to be approx. 1 X 10(-6) M. In the presence of physiological concentrations of Ca++ (2.5 mM), this specific binding is markedly reduced. Unspecific binding of beta 2-G-I to platelets, however, is not influenced by Ca++. Platelets prepared by gel filtration (GFP), differing in their in vitro aggregability from WP, exhibit no specific binding of beta 2-G-I. Binding to GFP is also not induced by activation with thrombin, collagen or ADP. beta 2-G-I causes significant alteration of the ADP-induced aggregation of GFP. Aggregation induced by thrombin, collagen, arachidonic acid or PAF-acether, however is not altered by beta 2-G-I. It is suggested, that pelleting during centrifugation causes irreversible rearrangements in the membrane of platelets.     

Effects of etamsylate on platelet functions and arachidonic acid metabolism

The effects of etamsylate on human platelet aggregation and ATP release as well as on the arachidonate metabolism by the platelet have been studied. Etamsylate enhanced these platelet functions induced by arachidonic acid (AA), thromboxane A2, collagen and calcium ionophore A23187 but not those induced by ADP and epinephrine. In experiments with cyclooxygenase-inhibited platelets, AA-induced platelet aggregation was completely inhibited and it was not enhanced by etamsylate, while A23187-induced aggregation was partially inhibited and this aggregation was enhanced by etamsylate. Platelet AA metabolism including thrombin-induced AA liberation from phospholipids as well as the lipoxygenase and cyclo-oxygenase pathways was not significantly affected by etamsylate. These results suggested that etamsylate enhanced platelet response to thromboxane A2 and calcium ionophore and that this could be included as a mechanism for its potentiating effect on platelet functions.