Rat platelet aggregation by ATP. Aggregometrical and ultrastructural comparison with aggregations induced by ADP and collagen

This paper describes the aggregation of rat platelets by adenosine triphosphate (ATP). The aggregometry of ATP-induced aggregation and the ultrastructure of ATP-aggregated platelets were compared and contrasted with those of adenosine diphosphate (ADP)-treated and collagen-treated samples. Human platelets were also studied alongside with rat specimens. Several lines of evidence indicate that the ATP-induced aggregation of rat platelet-rich plasma (PRP) is not a result of contaminating ADP in the ATP preparation. ATP did not cause aggregation of human platelets; it inhibited ADP- and collagen-induced human platelet aggregation. ATP pretreated with a creatine phosphate/creatine phosphokinase system caused similar rat platelet aggregation as did ATP not treated with this system. The aggregometry of ATP-induced aggregation of rat PRP was similar to that of collagen-induced aggregation but markedly different from that of ADP-induced aggregation. However, the nature of ATP-induced aggregation was similar to that induced by ADP. Both ATP- and ADP-induced rat platelet aggregations were not affected by adenosine, adenosine monophosphate, or acetylsalicylic acid. The ultrastructure of ATP-aggregated platelets was similar to that of ADP-aggregated ones. It appears that either platelets of rats possess specific ATP receptors or the rat plasma contains a material, lacking or insufficiently present in human plasma, that converts ATP to ADP in a fashion similar to the release of ADP from platelet storage granules.     

ADP-induced platelet activation

Platelet activation is central to the pathogenesis of hemostasis and arterial thrombosis. Platelet aggregation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is the first known and an important agonist for platelet aggregation. ADP not only causes primary aggregation of platelets but is also responsible for the secondary aggregation induced by ADP and other agonists. ADP also induces platelet shape change, secretion from storage granules, influx and intracellular mobilization of Ca2+, and inhibition of stimulated adenylyl cyclase activity. The ADP-receptor protein mediating ADP-induced platelet responses has neither been purified nor cloned. Therefore, signal transduction mechanisms underlying ADP-induced platelet responses either remain uncertain or less well understood. Recent contributions from chemists, biochemists, cell biologists, pharmacologists, molecular biologists, and clinical investigators have added considerably to and enhanced our knowledge of ADP-induced platelet responses. Although considerable efforts have been directed toward identifying and cloning the ADP-receptor, these have not been completely successful or without controversy. Considerable progress has been made toward understanding the mechanisms of ADP-induced platelet responses but disagreements persist. New drugs that do not mimic ADP have been found to inhibit fairly selectively ADP-induced platelet activation ex vivo. Drugs that mimic ADP and selectively act at the platelet ADP-receptor have been designed, synthesized, and evaluated for their therapeutic efficacy to block selectively ADP-induced platelet responses. This review examines in detail the developments that have taken place to identify the ADP-receptor protein and to better understand mechanisms underlying ADP-induced platelet responses to develop strategies for designing innovative drugs that block ADP-induced platelet responses by acting selectively at the ADP-receptor and/or by selectively interfering with components of ADP-induced platelet activation mechanisms.     

Inhibition of rat platelet aggregation by mycalolide-B, a novel inhibitor of actin polymerization with a different mechanism of action from cytochalasin-D

In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.     

Characterization of platelet hypofunctions in rats under SART stress (repeated cold stress)

Platelet hypoaggregability has been reported in rats exposed to a chronic form of environmental stress induced by long-lasting fluctuation in air temperature, known as SART (specific alternation of rhythm in temperature) stress. This study examines functional characteristics of platelets from stressed rats in more detail. Exposure to stress reduced aggregation and ATP release in platelets stimulated with collagen, as determined using platelet-rich plasma (PRP). The resting levels of ATP but not ADP in platelets from stressed rats were lower than those from unstressed ones. Collagen-induced release and resting level of serotonin also decreased in platelets from stressed rats. In contrast, stress failed to cause hypoaggregability of washed platelets. Circulating platelet aggregates were detected in stressed rats. From these data, SART stress appears to cause intravascular activation of platelets in spite of in vitro hypofunctions. Alteration in plasma milieu may be associated with stress-induced platelet hypofunctions in PRP.     

Effect of oral administration of prostaglandin E1 on erectile dysfunction

Collagen-related peptides, Gly-Pro-Arg and its analogues, were examined for their inhibitory effects on platelet aggregation induced by the addition of ADP. Human platelet aggregation was suppressed by more than 50% with each of Gly-Pro-Arg and such Gly-Pro-Arg-containing peptides as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Pro-Pro, and Gly-Pro-Arg-Pro-Pro-Pro at a concentration of 0.3 mM. The inhibitory effects of these peptides were about 10 times higher in human PRP than in rat PRP. Other Gly-Pro-Arg analogues such as Sar-Pro-Arg, Gly-Pro-Lys, Gly-Ala-Arg, and Ala-Gly-Pro-Arg had no inhibitory effect at a concentration from 0.1 to 0.8 mM even in human PRP. Intravenous and oral administrations of Gly-Pro-Arg and enzymatic hydrolysates of collagen suppressed the decrease in platelet count for endotoxin-induced DIC in rats. Collagen itself has been regarded as a potent inducer of platelet aggregation, but these findings suggest that collagen-related peptides and enzymatic hydrolysates of collagen prevent platelet aggregation.     

Adhesion of ADP-activated platelets to intact endothelium under stagnation point flow in vitro is mediated by the integrin alphaIIbeta3

As we demonstrated earlier, platelets adhere to intact endothelium provided they are activated and convectively transported against the endothelial surface. To identify the platelet receptors involved we superfused cultured endothelium with activated platelet rich plasma (PRP) by means of the Stagnation Point Flow Adhesio- Aggregometer while blocking various platelet receptors. Inhibition was performed with the tetrapeptide RGDS, the non-peptide Ro-43-8857, or a monoclonal antibody directed against integrin alphaIIbeta3. Platelet deposition was video-recorded and quantified by image analysis. Infusion of RGDS or Ro-43-8857 into ADP-stimulated PRP completely prevented adhesion as well as subsequent aggregation. Interrupting the inhibitor infusion while ADP stimulation persisted, prompted adhesion and aggregation, demonstrating the reversibility of the inhibition. Platelet adhesion was irreversibly blocked by preincubation of the PRP with the moab against alphaIIbeta3. Its specific binding was confirmed by immunoelectron microscopy. Our results suggest that platelet adhesion to intact endothelium is mediated via platelet integrin alphaIIbeta3.     

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.     

Modulatory effect of 8-iso-PGE2 on platelets

1. 8-Iso-PGE2 induced either reversible or irreversible aggregation of platelets in human platelet-rich plasma (PRP) or in the suspension of washed platelets (WP). The values of EC50 for irreversible aggregation in PRP and WP were 4 and 2 microM, respectively. 2. In rabbit PRP, 8-iso-PGE2 (0.1-100 microM) itself did not induce or induced only reversible aggregation. 3. 8-Iso-PGE2 (0.1-20 microM) potentiated adenosine diphosphate-(ADP) induced platelet aggregation in both human and rabbit. The same effect also was found for adrenaline-induced platelet aggregation in rabbit. 4. The lower concentrations (0.2-0.5 microM) of 8-iso-PGE2 decreased, and higher concentrations (1-2 microM) increased platelet aggregating factor- (PAF) induced aggregation in human PRP. In rabbit PRP, 8-iso-PGE2 (0.02-200 microM) had only a decreasing effect on PAF-induced aggregation. 5. The results suggest that low concentrations of 8-iso-PGE2 can amplify or weaken platelet aggregation induced by various aggregatory agents.     

Long term effects of 50 mg acetylsalicylic acid alone and in combination with dipyridamole on platelet function after coronary bypass surgery

In a prospective randomized trial in 42 patients undergoing coronary artery bypass surgery, we analyzed the long term platelet inhibiting effects of 50 mg acetylsalicylic acid (ASA) by itself and in combination with dipyridamole (2 x 200 mg), in comparison with phenprocoumon. Three and six months therapy led to significant inhibition of maximum aggregation induced by collagen 1 microgram/ml in platelet rich plasma (PRP) by more than 50% (p < or = 0.05). In PRP stimulated with 5 micrograms/ml collagen maximum inhibition amounted to nearly 20% (n.s.). The groups treated with ASA/ASA + dipyridamole showed an ADP threshold concentration 2.5 times higher than the group treated with phenprocoumon (p < or = 0.05). After stimulation with collagen 1 microgram/ml and 5 micrograms/ml thromboxane B2 synthesis in vitro in both groups treated with ASA was reduced to 1% of the base line values (p < or = 0.01). Inhibition of aggregation in whole blood appeared evident, but was not statistically significant due to considerable fluctuation of measurement. An additional effect of dipyridamole was not detectable. In conclusion, treatment with 50 mg ADA/d results in a lasting, effective inhibition of aggregation of platelets in patients with coronary artery bypass surgery. There is no synergistic effect of additional dose of 400 mg dipyridamole/d.     

Localization and translocation of MMP-2 during aggregation of human platelets

We have previously shown that human platelets express matrix metalloproteinase-2 (MMP-2) and that the release of this enzyme during platelet activation mediates the ADP- and thromboxane-independent part of aggregation. We have now used immunogold electron microscopy, flow cytometry. Western blot analysis and zymography methods to study the ultrastructural localization of MMP-2 in human washed platelets. Platelet aggregation was stimulated by collagen and the MMP-2 immunoreactivity of platelets was followed during various stages of aggregation. In resting platelets, MMP-2 was randomly distributed in the platelet cytosol without detectable association with platelet granules. Platelet aggregation caused the translocation of MMP-2 from the cytosol to the extracellular space. During the early stages of aggregation, MMP-2 remained in close association with the platelet plasma membrane. We conclude that the interactions of MMP-2 with platelet surface membranes mediate the aggregatory response induced by this enzyme.     

Platelet dysfunction associated with immune-mediated thrombocytopenia in dogs

The effect of antiplatelet antibody on in vitro platelet function was investigated in 15 dogs with immune-mediated thrombocytopenia (ITP). Platelet aggregation was assessed after addition of serum from healthy dogs (n = 5) or dogs with ITP (n = 15) to platelet-rich plasma from a healthy donor dog. The aggregation responses to adenosine diphosphate, thrombin, and collagen/epinephrine were measured as the maximum aggregation observed after 2 minutes. In 13 of 15 dogs with ITP, maximal aggregation was significantly inhibited in response to ADP, thrombin, or collagen/epinephrine. The slope of the aggregation curve was decreased after addition of serum from 9 of 15 patients. A polyclonal rabbit anti-dog platelet antiserum induced inhibition of aggregation with all 3 agonists. Serum from control dogs neither inhibited nor activated platelet aggregation. Aggregation experiments were repeated with all 3 agonists after addition of patient immunoglobulin (Ig)G or IgG from a healthy dog to platelet-rich plasma. The IgG fraction from 9 of 10 dogs with ITP suppressed platelet aggregation. The IgG fraction from polyclonal rabbit anti-dog platelet antiserum inhibited platelet aggregation with all agonists. These results suggest that many canine ITP patients have circulating antibodies that, in addition to causing platelet destruction, may cause platelet dysfunction.     

On the measurement of spontaneous platelet aggregation. The platelet aggregation test III. Methods and first clinical results

A new measuring device was developed for the study of "spontaneous" aggregating activity of thrombocytes. In the photometric platelet aggregation test (PAT III) 0.6 ml of platelet-rich plasma (PRP) are rotated in a disc-shaped cuvette at 20 rpm and 37 degrees C. Changes in optical density of PRP which are induced by the formation of platelet aggregates are continuously registered using a chart recorder. PAT III was developed for the detection of enhanced platelet aggregation, indicating a risk of thrombosis and thromboembolic complications. In 146 healthy individuals a certain percentage showed slight primary aggregation (alpha1) which in some cases was followed by marked aggregation (alpha2) at a certain time (Tr) after the beginning of rotation. The percentage of individuals showing alpha2 increased with age. An increase of plasma pH in the rotating sample, which was caused by diffusion of CO2, was an important conditioning factor for aggregation. The test results depended on the platelet count in PRP. Aggregation curves were suppressed by admixture of erythrocytes and lipid turbidity. The tendency of platelets to aggregate increased within 60-90 min following blood sampling. During this period the interval to the onset of aggregation (Tr) became shorter and the maximum aggregation speed (alpha 2) increased with time. PAT III yielded reproducible results when it was carried out more than 60 min after blood drawing. In a group of 327 diabetic patients "spontaneous" aggregation occurred more frequently in all age groups as compared with the controls. Additional equipment was available for the registration of ADP-, collagen-, or epinephrine-induced aggregation similar to Born's and O'Brien's method. The device can easily be mounted on an Eppendorf photometer without further alterations.     

Red-wine polyphenols and inhibition of platelet aggregation: possible mechanisms, and potential use in health promotion and disease prevention

Correction of uremic platelet serotonin (5-HT) storage pool deficiency is one of the very early hemostatic effects of erythropoietin (Epo) therapy. In this work, platelet 5-HT with relation to primary hemostasis was studied in 15 hemodialysis patients treated with Epo for 8 months. Moreover, effects of ketanserin, a blocker of platelet and vascular smooth muscle cell 5-HT2A receptors, in these patients were followed. The parameters studied were compared with relevant values in healthy controls and in hemodialysis patients not treated with Epo, and remeasured in the long-term Epo patients after a 14-day oral ketanserin trial. Platelet 5-HT content in the eighth month of Epo therapy was not different from the one in untreated patients. Ristocetin- and collagen-induced platelet aggregation were enhanced in comparison with both control groups, as opposed to unaltered response to ADP and arachidonic acid. Fibrinogen concentration was lower than in the untreated group. An inverse correlation between ADP-induced platelet aggregation and the skin bleeding time (r=-0.536, p<0.05) and a positive one between the former and platelet 5-HT (r=0.644, p<0.01) were found. Platelet count correlated positively with both platelet 5-HT (r=0.823, p<0.0002) and ADP-induced platelet aggregation (r=0.596, p<0.02). Ketanserin produced a decrease in ristocetin-induced platelet aggregation, fibrinogen, and prolongation of the bleeding time. The first two of the changes correlated positively with their pre-ketanserin values (r=0.923, p<0.00001 and r=0.839, p< 0.0001, respectively). Post-ketanserin, positive correlations between depressed ristocetin- and arachidonic acid-induced platelet aggregation (r=0.760, p<0.005), and between collagen- and corresponding values of arachidonic acid- (r=0.622, p<0.02), ADP-induced platelet aggregation (r=0.396, p<0.01), and platelet 5-HT (r=0.654, p<0.05) were found. Efficient hemostasis in hemodialysis patients on protracted Epo therapy is, in part, dependent on enhanced platelet aggregability. Correction of platelet 5-HT storage pool deficiency is not evident in this stage but 5-HT still influences complex mechanisms of primary hemostasis. Ketanserin is of anticoagulant value in these patients but its effects must be weighted against possible exacerbation of the anemia.     

Enhancement by ticlopidine of the inhibitory effect on in vitro platelet aggregation of the glycoprotein IIb/IIIa inhibitor tirofiban

We determined the effects of combining the glycoprotein IIb/IIIa inhibitor tirofiban (MK-383, Aggrastat) and ticlopidine on inhibition of ADP-induced platelet aggregation, inhibition of collagen-induced platelet aggregation, and prolongation of bleeding time in 5 healthy male volunteers. The study consisted of 2 periods. In period 1, tirofiban was administered intravenously as a bolus injection at a dose of 5.0 microg/kg for 5 min, then as a continuous infusion at a rate of 0.05 microg/kg/min for 5 h 55 min. In period 2, ticlopidine was given orally at a dose of 200 mg once daily for 4 days, after which tirofiban was administered in the same manner as in period 1, starting 2 h after the last dose of ticlopidine. The percent inhibition of platelet aggregation induced by ADP 5 microM at the end of tirofiban infusion in periods 1 and 2 was 73.6 +/- 2.6 and 87.1 +/- 5.7% (mean +/- SD), respectively. The corresponding values for inhibition of platelet aggregation induced by collagen 2 microg/ml were 45.4 +/- 36.1 and 82.8 +/- 27.0%, respectively. In contrast, the percent inhibition of platelet aggregation induced by ADP and collagen after treatment with ticlopidine alone was 15.8 +/- 20.2 and 2.2 +/- 4.8%, respectively. Compared with tirofiban alone, the combination of tirofiban and ticlopidine significantly enhanced inhibition of platelet aggregation induced by both ADP and collagen (p <0.02 and p <0.012, respectively). The inhibitory effects of ticlopidine alone were not statistically significant. Tirofiban prolonged bleeding time both in period 1 and in period 2. However, tirofiban and ticlopidine combined did not affect bleeding time. Ticlopidine administration did not alter the pharmacokinetics of tirofiban. In conclusion, at the doses of tirofiban and ticlopidine used in this study, the combination of the two drugs enhanced inhibition of platelet aggregation but did not prolong bleeding time, suggesting that appropriate doses of tirofiban can be used concomitantly with ticlopidine with no further safety concerns but with potential additional clinical effects.     

Evidence for altered platelet nitric oxide synthesis in essential hypertension

OBJECTIVES: To determine whether platelet aggregation to collagen was abnormal in patients with essential hypertension and whether nitric oxide donors and inhibitors of nitric oxide synthesis affect platelet aggregation differently in hypertensives compared with healthy controls. DESIGN: Platelet aggregation assays were conducted ex vivo from both hypertensive and normal subjects simultaneously. METHODS: Platelet aggregation in response to collagen was measured in platelet-rich plasma from 16 patients with untreated essential hypertension and 16 healthy volunteers matched for age, sex and smoking habits. The effect of sodium nitroprusside (a nitric oxide donor) and NG-monomethyl-L-arginine (L-NMMA), a specific nitric oxide synthase inhibitor, was studied. RESULTS: In healthy controls L-NMMA caused a marked increase in platelet aggregation, whereas in hypertensive patients a small inhibition of aggregation was seen. This was significantly different from the response seen in normal controls. No difference was seen in the aggregatory response to collagen between hypertensive patients and healthy controls. Sodium nitroprusside caused inhibition of aggregation in hypertensive patients and in controls, but there was no significant difference in the degree of inhibition between the two groups. CONCLUSIONS: We conclude that in platelets from hypertensive patients there is a markedly reduced sensitivity to L-NMMA, which could be explained by a reduction in nitric oxide synthesis.     

Effects of Russell''s viper venom on blood coagulation, platelets and the fibrinolytic enzyme system

Abnormalities of second-wave platelet aggregation were demonstrated in 17 of 33 asthmatic patients in whom drug and diet intake were controlled in the hospital. Mean abnormal responses were significantly greater after epinephrine- (p less than 0.001), adenosine diphosphate-(less than 0.001), collagen- (p = 0.01), and thrombin- (p less than 0.001) induced platelet aggregation in patients with immunologically mediated asthma and serum IgE levels greater than 250 U/ml as compared to patients without immunologic factors and/or normal controls. Mean pollen-specific radioallergosorbent (RAST) binding was also significantly higher in patients with abnormal aggregation as compared to normal platelet responders (p = 0.02). Release of serotonin generally reflected abnormal aggregation patterns in asthmatic patients. Platelet factor 4 release was significantly decreased in the same groups of patients. These results suggest that the allergic state may affect platelet membrane responsiveness to multiple aggregating agents.     

Inhibitory effects of Acanthopanax gracilistylus saponins on human platelet aggregation and platelet factor 4 liberation in vitro

AIM: To study the effects of Acanthopanax gracilistylus var pubescens Li saponins (AGVPS) on human platelet aggregation and platelet factor 4 (PF4) liberation in vitro. METHODS: Human platelet aggregations induced by ADP, adrenaline, and collagen were measured turbidimetrically. The aggregation curve was recorded on a platelet aggregometer and the maximal aggregation rate (ARmax), effective deaggregation rate in 5 min (DR5 min) and lag time (LT) were autocalculated by the built-in microcomputer; PF4 liberation from human platelets stimulated by ADP and collagen was determined by recording the heparin thrombin clotting time (HTCT). Thrombosis was tested by weighing the wet and dry thrombi formed in a siliconized revolving ring. RESULTS: AGVPS inhibited in vitro the ARmax with IC50 of 1.33 (95% confidence limits: 1.09-1.63, ADP-induced), 1.66 (1.54-1.79, adrenaline-induced), and 4.2 g.L-1 (0.6-29, collagen-induced). The DR5 min (on ADP-induced aggregation) and LT (collagen-induced) were also increased as well. Meanwhile, AGVPS 0.63-2.50 g.L-1 prolonged HTCT on ADP- and collagen-stimulated PF4 liberation. At 0.34-1.39 g.L-1, AGVPS reduced the wet and dry weight of thrombi formed in vitro. CONCLUSION: AGVPS inhibits human platelet aggregation, liberation, and thrombosis in vitro, suggesting its possible antithrombotic action in man.     

Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets

S-Nitroso-cysteine (SNC), a putative endothelium-derived relaxing factor, potently inhibited collagen- and arachidonic acid-induced platelet aggregation (IC50=100 nM) and thromboxane A2 (TxA2) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC-induced formation of cGMP but did not reverse inhibition by SNC of collagen- and arachidonic acid-induced platelet aggregation. Combination of ODQ with SQ-29548, a specific platelet TxA2 receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP-independent mechanisms that may involve inhibition of TxA2 synthesis in human platelets.     

Membrane-destabilizing properties of C2-ceramide may be responsible for its ability to inhibit platelet aggregation

We have studied the effects of short-chain ceramides on platelet structure and function. N-Acetylsphingosine (C2-ceramide), a cell-permeable short-chain analogue, and N-acetyldihydrosphingosine (C2-dihydroceramide), which lacks the 4-5 double bond, have been investigated. C2-Ceramide (15 microM) inhibited ADP-induced aggregation by 50% at a platelet concentration of 1.25 x 10(8)/mL, while it took twice that concentration to inhibit aggregation by 50% when the platelet concentration was doubled. This indicates that the effect of C2-ceramide on ADP-induced platelet aggregation depends on the ratio of ceramide to total platelet lipid, with a ratio of 0.2 giving significant inhibition. C2-Ceramide at a ceramide: lipid ratio of 0.2 caused platelets to form fenestrations and pseudopodia which were longer and thinner than those caused by agonists such as ADP or thrombin. C2-Dihydroceramide had no effect on ADP-induced aggregation or platelet morphology at any ceramide:lipid ratio. Platelet lysis was induced by C2-ceramide at higher ceramide:lipid ratios (0.5), whereas C2-dihydroceramide did not induce lysis, suggesting that C2-ceramide is able to destabilize membranes. This was tested directly by assessing whether the ceramides induced leakage of 6-carboxyfluorescein from lipid vesicles. C2-Ceramide caused nearly total leakage of dye from the vesicles at a ceramide:lipid ratio of 10. The leakage caused by C2-dihydroceramide at a ceramide:lipid ratio of 10 was equal to that induced by C2-ceramide at a ratio of 0.2 (approximately 3%). The ability of the ceramides to destabilize membranes was also examined by measuring changes in fluorescence anisotropy of the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into lipid vesicles. C2-Ceramide induced a larger decrease in anisotropy than a detergent (Triton X-100) which is known to lyse membranes. C2-Dihydroceramide did not alter membrane fluidity. The ability of C2-ceramide to cause platelet fenestrations, formation of irregular platelet pseudopodia, platelet lysis, lipid vesicle leakage, and increases in the fluidity of lipid vesicles all suggest that C2-ceramide inhibits platelet aggregation because it destabilizes the platelet membrane. C2-Dihydroceramide did not inhibit platelet aggregation and lacked the nonspecific effects on membranes that C2-ceramide possessed, suggesting that C2-dihydroceramide is not an appropriate control for the nonspecific effects of C2-ceramide.     

Recent advances in antiviral drugs--antiviral agents to HCMV, HHV-6, and HHV-7

As we have previously reported, intraperitoneal injections of NG-nitro-L-arginine methyl ester [L-NAME; a competitive inhibitor of nitric oxide (NO) synthase] before and after the injection of B16 melanoma cells through a tail vein increased experimental pulmonary metastasis, while simultaneous injections of L-arginine (a substrate of NO synthase) at a 20-fold higher dose synergistically increased pulmonary metastasis. Our present study was intended to elucidate the mechanisms by which L-NAME alone or together with L-arginine increases metastasis. Injections of L-NAME decreased the serum concentration of nitrite plus nitrate (metabolites of NO) by about 50%, which was not reversed by simultaneous injections of L-arginine. Injections of L-NAME also decreased the diameter of arterioles and venules by 20-30%, while simultaneous injections of L-arginine did not show any significant effect. When collagen- or ADP-induced platelet aggregation was examined using platelet-rich plasma, injections of L-NAME showed little effects on platelet aggregation, while simultaneous injections of L-arginine rather suppressed platelet aggregation. B16 melanoma cells produced NO in culture, and L-NAME (0.2 mM) decreased NO production without effects on viability. Our results suggest that the increased experimental pulmonary metastasis induced by L-NAME can be ascribed partly to the contraction of arterioles and venules, which is induced by the inhibition of endogenous NO production by L-NAME, and that the synergistic effect of L-arginine on metastasis is related to the inhibition of endogenous NO production through unknown mechanisms.