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.     

Superoxide dismutase cooperates with prostacyclin to inhibit platelet aggregation: a comparative study in washed platelets and platelet rich plasma.

The role of superoxide anions (O2-) in human platelet aggregation in Krebs' buffer or plasma was investigated. In indomethacin (10 microM)-treated washed platelets superoxide dismutase (SOD; 60 U/ml) or ferricytochrome c (FCC; 70 microM) inhibited platelet aggregation by thrombin but not that by collagen or ADP. In addition, in indomethacin (10 microM)-treated washed platelets, SOD significantly potentiated the anti-aggregatory activity of prostacyclin (PGI2) or iloprost when thrombin but not collagen was used as the aggregating agent. In platelet rich plasma, SOD (60 U/ml) did not inhibit platelet aggregation nor did it potentiate the anti-aggregatory activity of iloprost when ADP, collagen or thrombin were used as aggregating agents. Thus, O2- participate in the aggregatory activity of thrombin but not collagen or ADP and PGI2 or iloprost, by reducing the sensitivity of platelets to thrombin, co-operate with SOD to inhibit thrombin-induced platelet aggregation. The interpretation of the use of SOD in experiments involving endothelium-derived relaxing factor (NO) is discussed.     

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.     

Inhibitory mechanisms of dantrolene on platelet aggregation

The antiplatelet effect of dantrolene and possible inhibitory mechanisms were studied in rabbit platelets. Preincubation of rabbit washed platelets with dantrolene (50-300 microM) inhibited the platelet aggregation and adenosine triphosphate release induced by arachidonic acid (100 microM), collagen (10 microg/mL), or thrombin (0.1 U/mL) in a dose-dependent manner. The thromboxane B2 formation caused by collagen or thrombin was inhibited by dantrolene, while formation of thromboxane B2 and prostaglandin D2 induced by arachidonic acid were not inhibited. In addition, the formation of phosphoinositide breakdown and the rise of intracellular calcium level induced by collagen or thrombin were also inhibited in a dose-dependent manner by dantrolene in the presence of indomethacin (2 microM). However, the platelets cyclic AMP level was not affected by dantrolene. In conclusion, the present study demonstrates that dantrolene inhibits platelet activation mainly due to suppression of phosphoinositide breakdown.     

Low molecular weight heparin as an anticoagulant for in vitro platelet function studies

We evaluated the suitability of low molecular weight (LMW) heparin as an anticoagulant for in vitro platelet function tests in 11 normal volunteers. Results with citrated platelets were considered as the standard. Spontaneous platelet aggregation and the aggregation responses to ADP, epinephrine, collagen, ristocetin and thrombin were measured turbidimetrically in an aggregometer. Dose-response and dose-rate curves were constructed for ADP- and epinephrine-induced aggregation. The maximum aggregation response (EDmax) and rate (EDRmax), and the estimated dose of agonist to induce 50% of the maximum response (ED50) and rate (EDR50) were calculated from these curves. The inhibition of ADP-induced aggregation with PGI2 was expressed as per cent inhibition. The release of ATP and TxA2, from platelets aggregated with collagen was measured. No spontaneous aggregation occurred with either anticoagulant. The ED50 and the EDR50 for heparinized platelets were significantly lower for ADP induced aggregation (0.8 +/- 0.3 microM vs 2.1 +/- 1.0 microM [p = 0.001] and 0.4 +/- 0.1 microM vs 0.8 +/- 0.3 microM [p = 0.003]). The EDRmax with ADP was significantly higher (p = 0.004) for heparinized platelets (64.6 +/- 17.0 units/ml vs 50.4 +/- 7.6 units/ml). The heparinized platelets aggregated slightly, but significantly, less in response to ristocetin than the citrated platelets. The response of washed heparinized and citrated platelets to thrombin was not significantly different. The per cent inhibition of ADP aggregation with PGI2, was significantly lower with heparinized platelets. The release of TxA2 and ATP was similar for both anticoagulants. These results indicate that LMW heparin is a satisfactory anticoagulant for platelet aggregation tests.     

Effect of 4-hydroxy-2,3-trans-nonenal on platelet function

4-Hydroxy-2,3-trans-nonenal (HNE), an aldehyde end-product of lipid peroxidation, potentiated aggregation and increased thromboxane A2 formation in platelets challenged with ADP, thrombin or the ionophore A23187. These effects were observed at HNE concentrations in the range 10-100 microM. Platelet responses to collagen, epinephrine and arachidonic acid were not affected by HNE. Concentrations of HNE in excess of 100 microM inhibited platelet activation. HNE increased the release of 3H-arachidonic acid from prelabelled platelet phospholipids in response to thrombin or ADP. It is proposed that HNE may play an important role in controlling platelet function by regulating the activity of phospholipase A2.     

The anti-aggregation effects of ondansetron on platelets involve IP3 signaling and MAP kinase pathway, but not 5-HT3-dependent pathway

Ondansetron is a 5-HT3 receptor antagonist with potent antiemetic, analgesic, and antiphlogistic effects. Literature concerning 5-HT3 antagonists on platelets is limited. In this report we examined the pharmacological effects of ondansetron on human washed platelets. Platelet aggregation induced by thrombin (0.1 U/mL), collagen (2 μg/mL), arachidonic acid (0.5 mM), ADP (10 μM), or U46619 (2 μM) was observed. The effects of ondansetron on platelet aggregation and ATP release were investigated at different concentrations. Cytosolic Ca²⁺ influx concentration, TXB2, IP3, and the levels of cAMP and cGMP were monitored, and flow cytometric analysis and immunoblotting were performed to investigate downstream signaling components. Our results showed that ondansetron, in a concentration-dependent manner, inhibited agonist-induced platelet aggregation. At 75 μM, ondansetron significantly attenuated intracellular Ca²⁺ mobilization, thromboxane B2 formation, and ATP release by human washed platelets activated by thrombin, collagen, or U46619, whereas it only partially attenuated arachidonic acid-driven platelet activation. Administration of ondansetron resulted in attenuated IP3 production in the washed platelets stimulated by thrombin, as determined by reduced IP1 levels, as well as diminished p38 and ERK2 phosphorylation in response to thrombin. No effect of ondansetron on the levels of either cAMP or cGMP in washed platelets was observed. Furthermore, ondansetron-mediated inhibition of platelet aggregation was not impacted by SR 57227A, the 5-HT3 agonist. Thus, rather than involving the 5-HT3-dependent pathway, the negative effect of ondansetron on platelet aggregation is instead manifested through the attenuation of agonist-induced IP3 production and MAPK (p38 and ERK2) phosphorylation that results in suppressed intracellular Ca²⁺ mobilization, TXB2 formation, and ATP release.     

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.     

Antiplatelet effects of protopine isolated from Corydalis tubers

Protopine inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, PAF, collagen and ionophore A23187. Although the platelet aggregation caused by thrombin was not inhibited by protopine (100 micrograms/ml), the release reaction was partially suppressed. In rabbit platelet-rich plasma, protopine also inhibited the platelet aggregation caused by ADP, arachidonic acid, PAF and collagen. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was suppressed by protopine. Protopine inhibited the intracellular calcium increase caused by arachidonic acid in quin-2/AM loaded rabbit platelets. In the presence of indomethacin, the intracellular calcium increase caused by collagen and PAF was completely suppressed by protopine, and the intracellular calcium increase caused by thrombin was partially inhibited. The phosphoinositides breakdown caused by collagen and PAF was inhibited by protopine, but that by thrombin was not affected significantly. Protopine did not cause the elevation of cyclic AMP level of platelets. It is concluded that the antiplatelet effects of protopine is due to inhibition on thromboxane formation and phosphoinositides breakdown and then lead to the decrease of intracellular calcium concentration.     

Potentiation with epinephrine of macaque platelet aggregation by other agonists: implications for studies on human atherosclerosis

The effects of low concentrations of epinephrine on the aggregation of macaque and human platelets by arachidonic acid (AA), collagen, and thrombin were studied. When epinephrine (0.05 to 1 microM) was added to macaque or human citrated or macaque heparinized platelets, either before or after the addition of near-threshold concentrations of AA, significant increases in aggregability were always seen. Epinephrine alone did not aggregate macaque platelets from citrated blood. When near-threshold concentrations of collagen or thrombin were present in the medium, low concentrations of epinephrine (0.05 to 0.50 microM) potentiated the aggregation of macaque and human citrated platelets and macaque heparinized platelets. The P values for the addition of epinephrine were less than 0.01 in all series. The ability of low epinephrine concentrations to potentiate aggregation of macaque platelets by other agonists is of particular significance because in humans the most important effect of epinephrine on platelets in vivo is probably the potentiation, by low concentrations, of aggregation induced by other aggregatory agents normally present in the blood in low concentrations.     

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.     

Increased aggregation and secretion responses of human platelets when loaded with the calcium fluorescent probes quin2 and fura-2

Incorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAF and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cyclooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethyl-ammonium-diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.     

In vitro studies of a new synthetic thrombin inhibitor

(2R, 4R)4-methyl-1-[N alpha-(3-methyl-1,2,3,4-tetrahydro-8-quinoline- sulfonyl)-L-arginyl]-2-piperidine carboxylic acid monohydrate (MCI-9038) was found to be a potent synthetic inhibitor of thrombin. In concentrations as low as 1 microM, the thrombin time, prothrombin time, and partial thromboplastin time were more than doubled. The venom (Bothrops atrox) time was similarly prolonged. The drug also inhibited the thrombin-induced activation of factors VIII and XIII. While MCI-9038 in concentrations of 10(-4) M had no effect on platelet aggregation induced by collagen, ADP, epinephrine, and arachidonate, nanomolar concentrations inhibited thrombin-induced platelet aggregation and the release of platelet ADP. The drug also significantly inhibited the adhesion of thrombin-treated platelets to cultured bovine aortic endothelial cells. We conclude that MCI-9038 is an extremely potent inhibitor of the effects of thrombin on platelets and clotting factors.     

Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents

The effect of the concentration of Ca2+ in the suspending medium of human and rabbit platelets on aggregation, release of 14C-serotonin, and TXB2 formation in response to ADP, thrombin, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF), collagen and arachidonic acid was studied in either platelet-rich plasma anticoagulated with D-phenylalanyl-prolyl-arginyl chloromethylketone (PPACK) or citrate, or suspensions of washed platelets in modified Tyrode-albumin solutions containing 1 mM Mg2+ and concentrations of added Ca2+ ranging from 0 to 5 mM. In response to ADP, thrombin, or PAF, human platelets were stimulated to form TXA2 by close platelet contact in a low-Ca2+ medium; at physiological concentrations of Ca2+, TXB2 formation was much less and declined progressively as the concentration of Ca2+ was raised. When the formation of TXA2 was blocked with aspirin or indomethacin, aggregation and release by human platelets were strongest at physiological concentrations of Ca2+. Rabbit platelet responses differed markedly from those of human platelets because close contact of rabbit platelets in a low-Ca2+ medium did not promote TXA2 formation. Rabbit platelet responses were more strongly inhibited by the lack of added Ca2+ in the medium than the responses of human platelets, possibly because rabbit platelets do not contain releasable Ca2+. In all studies of human platelets in media with low concentrations of Ca2+, the additional contribution to platelet responses of TXA2 formed because of close platelet contact should be considered because TXA2 formation is not usually stimulated in this way at physiological concentrations of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aggregation of washed platelets from non-anticoagulated human blood is not reversible

Most of the knowledge acquired on platelet function and biochemistry has been obtained from platelets prepared from blood anticoagulated with sodium citrate. Using washed platelets from human blood (PNB) to which no anticoagulants were added, we report on responses not observed with platelets prepared from citrate-anticoagulated blood. Native blood was passed rapidly (within 5 min of venepuncture) through a Sephadex G-25/G-50 column to remove divalent ions and thus prevent coagulation. Platelets were separated from the gel-filtered blood by differential centrifugation. Responses of PNB to thrombin, collagen, calcium ionophore, ristocetin, release of 14C-5hydroxytryptamine and beta-thromboglobulin, and generation of thromboxane A2 were similar to those observed for citrated platelets. Comparison of PNB with thrombin-treated platelets, which demonstrate an increase of platelet factor 3 activity, a reduction of the adenylate energy charge and an impairment of clot retraction, indicated the absence of platelet activation. Unlike citrated platelets, however, aggregation of PNB in response to ADP was irreversible in the presence of Ca2+ and fibrinogen, even at concentrations as low as 0.2 microM ADP, with aggregation taking up to 25 times longer to reach the same extent of aggregation as for citrated platelets. PNB did not aggregate to epinephrine even in the presence of Ca2+ and fibrinogen. Sodium citrate impaired ADP-induced aggregation and clot retraction of PNB. Thus citrate affects platelet function and may cause changes resulting in the unphysiological behaviour and responses of platelets.     

Antiplatelet activity of Staphylococcus aureus lipoteichoic acid is mediated through a cyclic AMP pathway

In this study, Gram-positive Staphylococcus aureus lipoteichoic acid (LTA) dose dependently (0.1-1.0 microg/mL) and time dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists (i.e., thrombin and collagen). LTA also dose dependently inhibited intracellular Ca(2+) mobilization in human platelets stimulated by collagen. In addition, LTA (0.5 and 1.0 microg/mL) dose dependently increased the formation of cyclic AMP but not cyclic GMP in platelets. LTA (0.5 and 1.0 microg/mL) did not significantly increase the production of nitrate within a 10-min incubation period. Rapid phosphorylation of a platelet protein of M(r) 47,000, a marker of protein kinase C activation, was triggered by PDBu (0.03 microM). This phosphorylation was dose dependently inhibited by LTA (0.5 and 1.0 microg/mL) within a 10-min incubation period. Furthermore, LTA (0.5 and 1.0 microg/mL) also inhibited platelet aggregation induced by PDBu (0.03 microM) in human platelets.These results indicate that the antiplatelet activity of LTA may be involved in the increase of cyclic AMP, leading to inhibition of intracellular Ca(2+) mobilization and protein kinase C activity. Therefore, LTA-mediated alteration of platelet function may contribute to bleeding diathesis in septicemic and endotoxemic patients.     

Aggregation to thrombin and collagen of platelets from a Glanzmann thrombasthenic patient lacking glycoproteins IIb and IIIa

The aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 micrograms/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 microM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 micrograms/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained two-dimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on IIIa), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.     

Evidence that the rat is not an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation

Abnormal platelet aggregation seen in experimentally induced diabetic, hypercholesterolemic and spontaneously hypertensive rats (SHR) has been linked with increased prostaglandin synthesis. The present study was conducted to examine the role of prostaglandins in rat platelet activation using normal Wistar Kyoto (WKY) and SHR rats. Up to 30 microM ADP did not induce secondary phase of platelet aggregation in rat PRP and up to 30 microM epinephrine did not produce any response in rat PRP. In other experiments ADP (1.0 microM) and epinephrine (2.0 microM) induced typical biphasic aggregation responses in human PRP. Up to 20 microM U46619, a stable analog of prostaglandin H2, did not induce platelet aggregation in rat PRP or washed rat platelets. In contrast 2.0 microM U46619 caused maximal aggregation in human PRP and washed human platelets. Arachidonic acid (1.5-2.0 mM) induced aggregation in washed rat platelets. However, this was associated with excessive (67% and 94%) loss of cytoplasmic LDH. The low concentrations of thrombin (0.04 and 0.05 U/ml), induced two to three-fold increase in aggregation response in SHR platelets as compared to WKY platelets. Higher concentrations of thrombin (0.1 and 0.3 U/ml) induced similar aggregation responses in SHR and WKY platelets. Thrombin (0.04-0.3 U/ml) induced serotonin secretion in a concentration dependent manner. The extent of secretion was the same in SHR and WKY platelets at all concentrations. Thrombin-induced synthesis of thromboxane A2 (TXA2) in WKY and SHR platelets was quantified using a radioimmunoassay for TXB2. Thrombin (0.04-0.3 U/ml) produced TXB2 in WKY and SHR platelets in a concentration dependent manner. The SHR platelets produced significantly larger amounts of TXB2 as compared to WKY platelets. In other experiments aspirin (500 microM) inhibited thrombin (0.05 U/ml) induced TXB2 synthesis by 75% in both WKY and SHR platelets but failed to inhibit aggregation or secretion in either WKY or SHR platelets. Based on these data it is suggested that: (a) rat platelets inspite of their ability to synthesize TXA2 do not require TXA2 for aggregation; and (b) the rat may not be an appropriate model to study the role of prostaglandins in normal or abnormal platelet aggregation.     

Endothelin and platelet function

The effects of newly discovered vasoconstrictor peptide endothelin was studied on human, rabbit and canine platelet function. Endothelin (0.01 nM - lμM) did not promote platelet aggregation. In human platelets, endothelin (0.1 μM) did not significantly affect aggregation responses to ADP, collagen, epinephrine, arachidonic acid, PGH₂ or thrombin. Endothelin did not promote the mobilization of intracellular calcium in Fura2 loaded human platelets. In rabbit and canine platelets endothelin produced signficant potentiation of platelet aggregation mediated by low concentrations of ADP. Aggregation responses to higher concentration of ADP (5 μM) were unaffected by endothelin. These data reveal that under certain circumstances endothelin may potentiate rabbit and canine platelet aggregation responses to ADP, however endothelin does not produce direct effects on human platelet function.     

The novel effect of a new prostacyclin analogue ZK36374 on the aggregation of human platelets in whole blood

Platelet aggregation was studied at 37 degrees C in citrated whole human blood, using the Ultra Flo 100 Whole Blood Platelet Counter. Aggregation was measured as a fall in the number of single platelets following addition of an aggregating agent. At peak aggregation, the fall in the number of platelets induced by ADP (10 microM), collagen (1 microgram/ml) or thrombin (0.2 U/ml) was about 90%. When blood was incubated with the prostacyclin-analogue ZK36374, the aggregation responses to ADP, collagen and thrombin were reduced with IC50's = 0.5, 1.5 and 3 nM respectively and the corresponding IC100's were: 1, 3 and 12 nM. When ZK36374 was added at peak aggregation, the number of single platelets increased significantly due to disaggregation of preformed platelet aggregates. It is concluded that the present technique represents a rapid, sensitive and more physiological approach for investigating the effects of pharmacological agents on platelet aggregation.