The effect of mitomycin C on platelet aggregation and adenosine 3',5'-monophosphate metabolism.

The effect of Mitomycin C on aggregation, adenosine 3',5'-monophosphate (cyclic AMP) metabolism and reactions induced by thrombin was studied in rabbit platelets. Mitomycin C inhibited the platelet aggregation induced by adenosine diphosphate or thrombin. The level of radioactive cyclic AMP derived from 8-14C adenine or 8-14C adenosine increased after incubating intact platelets with Mitomycin C. Formation of radioactive adenosine triphosphate also increased though mitochondrial oxidation was not stimulated. Similar effect was observed also in rabbit liver. Mitomycin C failed to stimulate platelet adenyl cyclase but inhibited cyclic AMP phosphodiesterase in the absence of theophylline. In the platelets preincubated with Mitomycin C, thrombin-induced inhibition of adenyl cyclase, stimulation of membrane-bound cyclic AMP phosphodiesterase, and release of 250,000 dalton protein from platelet membranes were prevented. These results suggest that Mitomycin C will affect cellular membrane structure and function, and this extranuclear effect of Mitomycin C will lead to inhibition of aggregation in blood platelets.     

Synergistic inhibition of platelet aggregation by endothelium-derived relaxing factor and prostacyclin

The anti-aggregatory effect of endothelium-derived relaxing factor (EDRF) on aggregation of washed, aspirin-treated platelets was compared with that of nitric oxide. Nitric oxide produced a dose-dependent inhibitory effect on PAF-induced aggregation: the antiaggregatory activity was unstable and was completely preventable by pretreating the platelets with haemoglobin (10 mumol/l). Bovine aortic endothelial cells (EC) were grown to confluence on microcarrier beads, pretreated with aspirin (1 mmol/l), and their addition to the platelet cuvette also caused a dose-dependent inhibition of aggregation induced by PAF, thrombin and A23187. The inhibitory effect of the EC on platelet aggregation was partly prevented in the presence of haemoglobin (10 mumol/l). Both nitric oxide and EC showed synergy with prostacyclin, in that the latter potentiated the anti-aggregatory action of both these factors against PAF-induced platelet aggregation. Thus cultured endothelial cells release a non-prostanoid anti-aggregatory factor, which, like nitric oxide, shows a synergistic interaction with prostacyclin and is blocked by haemoglobin. This anti-aggregatory factor has the characteristics of EDRF.     

Alteration of adenine nucleotide metabolism in coronary smooth muscle cells by activated platelets.

Cultured porcine coronary smooth muscle cells were preloaded with [3H]adenine and the inside and outside radioactive metabolites of the cells were analyzed following exposure to activated platelets. Incubation of the cells with human platelets activated by collagen enhanced intracellular conversion of ATP to ADP and caused dose- and time-dependent increase in radioisotopic release, mainly adenosine. Isolation of cyclic AMP revealed decreased cyclic AMP levels in the treated cells, both intra- and extracellularly. Of the substances released by the activated platelets, thromboxane A2 and serotonin enhanced radioisotopic release. The modulation of adenine metabolism by the activated platelets was preceded by increase in accumulation of inositol phosphates in the cells and was prevented by Iloprost (1 microM), a prostacyclin analog, cilostamide (10 microM), a cyclic AMP-specific phosphodiesterase inhibitor, or dibutyryl cyclic AMP (1 mM). Nifedipine showed only minor preventive effect. The agents which elevate cyclic AMP accumulation also attenuated phosphoinositide hydrolysis, whereas nifedipine had no effect. These results suggest that activated platelets may stimulate adenine metabolism in coronary smooth muscle cells, presumably due to activation of phosphoinositide turnover resulting in increased intracellular calcium. Enhanced adenosine release from the cells exposed to activated platelets may be a compensatory mechanism to prevent further platelet aggregation and contraction of coronary smooth muscle.     

Investigation of possible mechanisms of pyridoxal 5′-phosphate inhibition of platelet reactions

Pyridoxal 5′-phosphate (PLP) inhibits ADP-induced platelet shape change and aggregation. PLP also causes rapid deaggregation of platelets aggregated by ADP. Effects on platelet aggregation and coagulation are demonstrable following administration to man. Several suggestions concerning the possible mechanism of inhibition have been investigated. PLP did not inhibit the nucleoside diphosphokinase activity of intact rabbit platelets nor of isolated platelet membranes. PLP had no measurable effect on rabbit platelet cyclic AMP either alone or in the presence of prostaglandin E₁ and caffeine. ADP-induced aggregation and ¹²⁵I-fibrinogen binding of rabbit platelets were inhibited concomitantly; however, fibrinogen-induced aggregation of chymotrypsin-treated platelets was only slightly inhibited by PLP. ¹²⁵I-fibrinogen binding to chymotrypsintreated platelets was partially inhibited by PLP; when PLP was added to these platelets, bound fibrinogen was displaced. With chymotrypsin-treated platelets, PLP inhibited the ADP component of the synergistic aggregating effect of the combination of fibrinogen and ADP. It seems unlikely that the inhibitory effects of PLP are due to interference with nucleoside diphosphokinase, increasing platelet cyclic AMP or inhibition of fibrinogen binding. The mechanism of inhibition remains to be established.     

In vitro effects of ethanol on rabbit platelet aggregation, secretion of granule contents, and cyclic AMP levels in the presence of prostacyclin

Ethanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin-treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.     

Different effects of aspirin, dipyridamole and UD-CG 115 on platelet activation in a model of vascular injury: studies with extracellular matrix covered with endothelial cells

Cultured endothelial cells produce an extracellular matrix (ECM) which activates platelets, similarly to deendothelialized vascular segments. Platelet-rich plasma (PRP) was incubated with endothelial cells cultures seeded in various densities on ECM. The interaction of the platelets with this artificial intima was evaluated by phase microscopy and by thromboxane A2 (TXA2) and prostacyclin (PGI2) measurement. Large platelet aggregates were formed on exposed ECM. Platelets aggregation but not adhesion on the ECM was markedly inhibited by the presence of endothelial cells. Pretreatment of the endothelial cells with 0.1 mM aspirin reduced their PGI2 synthesis and was associated with platelet aggregation on the ECM. 10 microM dipyridamole markedly inhibited platelet activation by ECM when the drug was added to citrated whole blood before PRP preparation. UD-CG 115 which elevates cyclic AMP in cardiac muscle, inhibited platelet aggregation and TXA2 production induced by ECM, in the presence as well as in the absence of endothelial cells, without any effect on endothelial PGI2 production.     

Lithium inhibits adenylate cyclase of human platelets

The ADP-induced aggregation of human platelets is markedly increased after preincubation with lithium chloride, whereas lithium has the opposite effect on rabbit platelets. Since this phenomenon might be related to cAMP metabolism, the influence of lithium on total cAMP content and adenylate cyclase activity was investigated. Lithium does not significantly change the total cAMP content of human platelets neither during incubation nor during ADP-induced aggregation. Basal adenylate cyclase activity, however, was slightly inhibited by lithium in human platelets. The inhibition of adenylate cyclase by ADP appeared to be enhanced by lithium. Prostacyclin induced stimulation of adenylate cyclase is counteracted by this ion. In rabbit platelets the prostacyclin stimulated adenylate cyclase activity is not affected by lithium. These data suggest that a correlation exists between the influence of lithium on the aggregation of human and rabbit platelets and the sensitivity of their adenylate cyclase for this ion.     

Ethanol stimulates prostacyclin biosynthesis by human neutrophils and potentiates anti-platelet aggregatory effects of prostacyclin

Previous reports on the direct effects of ethanol on human platelet aggregation function have been inconsistent. Ethanol ingestion produces vasodilation and raises intracellular cyclic AMP concentrations, effects similar to those of prostacyclin. We, therefore, hypothesized that ethanol may influence biosynthesis and/or bioactivity of prostacyclin. In our experiments, ethanol in concentrations up to 400 mg% had no consistent inhibitory effect on platelet aggregation in response to epinephrine, ADP, or combination of subthreshold concentrations of epinephrine plus ADP. However, ethanol in concentrations as low as 10 mg% potentiated the platelet aggregation inhibitory effects of prostacyclin. In addition, ethanol (20 mg%) decreased formation of thromboxane A₂ in whole blood by 41% and stimulated formation of prostacyclin by 160% (both P c 0.01). Additional studies using isolated human cells demonstrated synthesis of prostacyclin by neutrophils in the presence of platelets, and this neutrophil prostacyclin formation was enhanced in the presence of ethanol. These effects of alcohol in concentrations achieved after moderate intake may relate to the hemodynamic, biochemical, and cardioprotective effects of ethanol.     

Angiotensin delays platelet aggregation after injury of cerebral arterioles

Endothelium of cerebral surface vessels (pial arterioles and venules) was injured with a light/dye technique in anesthetized mice. This induced platelet aggregation at the site of injury. The onset of aggregation was monitored through a microscope in mice given angiotensin II acetate, 4 micrograms i.v. 30 minutes earlier. Aggregation latency was compared with that in vehicle treated (saline) mice. Angiotensin II caused a highly significant delay in aggregation within the arterioles which was not related to a change in shear rate of blood. Angiotensin II added to platelet rich plasma, failed to influence the aggregation produced by subsequent addition of 0.5 microM ADP or 0.5 mM sodium arachidonate. Angiotensin is a well known stimulator of prostacyclin synthesis or release, and angiotensin has been reported to inhibit platelet aggregation ex vivo by increasing prostacyclin in the effluent superfusing the mass of aggregating platelets. Our data represent the first report of an antiaggregating effect of angiotensin II in vivo in an intact microvascular bed. The data is consonant with the literature describing increased prostacyclin levels following angiotensin II infusion. The antiaggregating effect of angiotensin in cerebral microvessels may help explain a recent observation describing increased survival of gerbils treated with angiotensin following carotid ligation.     

Sodium arachidonate induced platelet aggregation is independent of secreted adenosine diphosphate

Human gel-filtered platelets or platelet-rich plasma were stimulated by sodium arachidonate or by ADP in the presence of two compounds known to inhibit ADP mediated aggregation and secretion - ATP and N-ethylmaleimide. Using gel-filtered platelets and the lowest concentration of agonist necessary to elicit maximum aggregation, fifty percent inhibition of ADP-mediated aggregation required 9 μM N-ethylmaleimide or 23 μM ATP. Sodium arachidonate-mediated aggregation was significantly less sensitive; equivalent inhibition required 30 uM N-ethylmaleimide or >500 μM ATP. Concentrations of both inhibitors were determined that would completely inhibit ADP-induced aggregation yet would not completely prevent sodium arachidonate-induced aggregation. Furthermore, this concentration of N-ethylmaleimide could not be overcome by up to 500 uM ADP, demonstrating that the observed arachidonate-induced aggregation was not due to the effects of a small amount of secreted ADP acting at the platelet surface. Therefore, aggregation of human platelets induced by arachidonic acid can occur by a mechanism that is independent of secreted ADP.     

Inhibitory mechanism of dipyridamole on platelet aggregation ex vivo

The effects of dipyridamole on platelet aggregation ex vivo and in vitro and on platelet cyclic AMP phosphodiesterase (PDE) were studied, and the mechanism of the ex vivo effects was assessed. Both the ADP- and collagen-induced aggregations ex vivo were inhibited dose-responsively by oral administration of dipyridamole. Maximum dipyridamole levels in the plasma were reached at 30 min after the administration. The inhibitory effects of dipyridamole on platelet aggregation ex vivo reached a maximum at between 1 and 2 hrs. On the other hand, the ADP-induced aggregation in vitro and cyclic AMP PDE activity were not inhibited until after 10 min of incubation at a low concentration of dipyridamole. This mode of inhibition of platelet aggregation in vitro and of cyclic AMP PDE activity agreed with the mode of inhibition in the case of platelet aggregation ex vivo. It is suggested therefore that the ex vivo effects, observed with only a low dipyridamole concentration in the plasma, may be due primarily to inhibition by dipyridamole of the cyclic AMP PDE in platelets.     

Antithrombogenic effects of calcium channel blockers: synergism with prostacyclin and thromboxane synthase inhibitors

Four calcium channel blockers (nimodipine, nifedipine, verapamil and diltiazem) of three chemical classes were tested in vitro for inhibition of platelet aggregation using heparinized human platelet rich plasma. Both ADP- and thrombin-induced aggregation were inhibited as was the biosynthesis of thromboxane A2 in response to ADP or thrombin. However, the IC50's for the calcium channel blockers were greater than or equal to 110 microM. Nimodipine was also tested in combination with prostacyclin, the potent platelet antiaggregatory agent, or with a thromboxane synthase inhibitor, U63557A. At concentrations at which neither nimodipine or prostacyclin inhibited platelet aggregation greater than or equal to 10%, the two compounds is combination synergistically inhibited both ADP- and thrombin-induced platelet aggregation. U63557A inhibited biosynthesis of thromboxane A2 by platelets in response to ADP or thrombin, but did not inhibit either ADP- or thrombin-induced platelet aggregation. However, U63557A in combination with a threshold inhibitory concentration of nimodipine resulted in a synergistic inhibition of platelet aggregation induced by ADP or thrombin. These results suggest that calcium channel blockers may be of therapeutic value as a new class of antithrombogenic agents when used in combination with agents that inhibit either platelet aggregation or synthesis of platelet thromboxane A2.     

Enhanced platelet sensitivity to prostacyclin in patients in an active stage of Takayasu arteritis.

Patients in an active stage of Takayasu arteritis are often complicated with thrombosis in the affected vessels. We investigated whether alteration of platelet sensitivity to prostacyclin is involved in platelet function in these patients. Twelve female patients in an active stage (48.3+/-11.8 years, mean+/-S.D.), diagnosed clinically by a persistently elevated erythrocyte sedimentation rate (>40 mm/h) with typical symptoms, along with 10 gender- and age-matched patients in an inactive stage and 12 control subjects were enrolled. Half-maximal concentration (EC(50)) for platelet aggregation to collagen was determined in the presence and absence of 1 nM iloprost, a stable prostacyclin analog. Sensitivity of platelets to prostacyclin was quantified by the ratio of EC(50) (R) in the presence of iloprost to that in its absence. Patients in an active stage exhibited enhanced platelet aggregation, as demonstrated by significantly lower EC(50) to collagen and increased plasma thromboxane B(2) concentration. However, R values in these patients were significantly higher (4.00+/-1.05; P<.001) than those in the inactive patients or controls (2.58+/-0.62 and 2.43+/-0.68, respectively), suggesting enhanced sensitivity to prostacyclin in patients with active disease. Plasma 6-keto-PGF1 alpha levels were lower in the active patients than those in other groups of subjects. We conclude that platelets in an active stage of TA may be sensitive not only to collagen but also to prostacyclin. The increase in sensitivity of the platelets to prostacyclin could be a compensatory mechanism against a decrease in the prostanoid production, presumably associated with endothelial dysfunction.     

Functional fractionation of platelets: aggregation kinetics and glycoprotein labeling of differing platelet population

Platelet heterogeneity has been studied with a technique called functional fractionation which employs gentle centrifugation to yield subpopulations ("reactive" and "less-reactive" platelets) after exposure to small doses of aggregating agent. Aggregation kinetics of the different platelet populations were investigated by quenched-flow aggregometry. The larger, "reactive" platelets were more sensitive to ADP (Ka = 1.74 microM) than the smaller "less-reactive" platelets (Ka = 4.08 microM). However, their maximal rate of aggregation (Vmax, % of the platelets aggregating per sec) of 23.3 was significantly lower than the "less-reactive" platelets (Vmax = 34.7). The "reactive" platelets had a 2.2 fold higher level of cyclic AMP. Platelet glycoproteins were labeled using the neuraminidase-galactose oxidase--[H3]-NaBH4 technique. When platelets were labeled after reversible aggregation, the "reactive" platelets showed a two-fold decrease in labeling efficiency (versus control platelets). However, examination of whole cells or membrane preparations from reversibly aggregated platelets revealed no significant difference in Coomassie or PAS (Schiff) staining. These results suggest that the large, "reactive" platelets are more sensitive to ADP but are not hyperaggregable in a kinetic sense. Reversible aggregation may cause a re-orientation of membrane glycoproteins that is apparently not characterized by a major loss of glycoprotein material.     

A fibrin(ogen) derived pentapeptide induces vasodilation, prostacyclin release and an increase in cyclic AMP

A pentapeptide derived from fibrin(ogen), Ala-Arg-Pro-Ala-Lys, is known to increase microvascular permeability. This peptide induced dilation of bovine mesenteric arteries and caused a release of prostacyclin and an increase in cyclic AMP in these vessels. These changes were abolished by pretreatment with indomethacin, indicating that the vasodilation and the increase in cyclic AMP are due to the prostacyclin release. One mechanism underlying the effect of this peptide on microvascular permeability might thus be a triggering of the arachidonic acid cascade, with release of prostacyclin and an increased blood flow, which may potentiate the effect of other substances with a direct effect on vascular endothelium, e.g. histamine. The pentapeptide is known to release histamine from mast cells.     

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.     

The effect of a medicinal Chinese herb on platelet function

We investigated the effect of the Chinese herb Injectio Salvia Miltiorrhizae (ISM) on human platelet function in vitro. ISM inhibited platelet aggregation and serotonin release induced by either ADP or epinephrine in a dose dependent manner. This effect of ISM was observed with both gel-filtered platelets (ID50 = 8-30 micrograms ISM/ml gel-filtered platelets) and platelets in plasma (ID50 = 400-900 micrograms ISM/ml of platelet-rich plasma). The active molecule(s) in ISM was heat stable, resistant to acid, base and proteolysis and fractionated on Sephadex 6-25 at MW approximately 280. ISM did not interact with the platelet alpha-adrenergic receptor, but increased cAMP in intact platelets. The results are consistent with the concept that ISM inhibition of platelet aggregation and release is mediated by an increase in platelet cAMP. The exact mechanism whereby ISM increases platelet cAMP appears to be that of inhibition of cyclic AMP phosphodiesterase. The effect of ISM on platelet function is one mechanism which might explain the therapeutic effect of ISM in experimental and clinical coronary artery disease.     

Selective antagonism of prostaglandin (PG) E1, PGD2 and prostacyclin (PGI2) on human and rabbit platelets by DI-4-phloretin phosphate (DPP)

The inhibition of human and rabbit platelet aggregation by prostaglandin (PG) E₁, PGD₂ and prostacyclin (PGI₂) was examined to determine if the three PGs inhibit platelets via a common receptor. Di-4-phloretin phosphate (DPP) was found to antagonise PGD₂, but not PGE₁ or PGI₂ on human platelets. In contrast, on rabbit platelets, DPP antagonised PGI₂ and PGE₁ but not PGD₂. The results suggest that there are two distinct types of PG receptors on human and rabbit platelets activated either by prostacyclin or PGD₂.     

Reduced prostacyclin survival after fasting-induced elevation of plasma free fatty acids

The anti-thrombotic effect of prostacyclin (PGI2) may be determined not only by its synthetic rate but also by its subsequent survival in blood. After its release from the vascular wall, prostacyclin binds to plasma albumin which stabilizes the molecule and prolongs its inhibitory effects on platelets. In vitro studies have shown that free fatty acids compete for the same albumin binding sites and may therefore displace PGI2 and substantially shorten its survival. To see if this competition could also occur in vivo, we produced a three-fold rise of plasma free fatty acid concentrations in ten normal volunteers by four days of fasting, which led to a significant reduction in prostacyclin survival as measured by a functional assay based on inhibition of ADP-induced platelet aggregation. The shortening of prostacyclin survival was associated with evidence of increased platelet reactivity as measured by the circulating platelet aggregate ratio test. Diseases that produce marked elevations of free fatty acids such as acute myocardial infarction may also lead to shortened PGI2 survival with potentiation of platelet mediated thrombosis.     

Effects of dipyrone on prostaglandin production by human platelets and cultured bovine aortic endothelial cells

Dipyrone and its metabolites 4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine inhibited the formation of thromboxane A2 (TXA2) during in vitro platelet aggregation induced by ADP, epinephrine, collagen, ionophore A23187 and arachidonic acid. Inhibition occurred after a short incubation (30--40 sec) and depended on the concentration of the drug or its metabolites and the aggregating agents. The minimal inhibitory concentration of dipyrone needed to completely block aggregation varied between individual donors, and related directly to the inherent capacity of their platelets to synthesize TXA2. Incubation of dipyrone with cultured bovine aortic endothelial cells resulted in a time and dose dependent inhibition of the release of prostacyclin (PGI2) into the culture medium. However, inhibition was abolished when the drug was removed from the culture, or when the cells were stimulated to produce PGI2 with either arachidonic acid or ionophore A23187. These results indicate that dipyrone exerts its inhibitory effect on prostaglandins synthesis by platelets or endothelial cells through a competitive inhibition of the cyclooxygenase system.