Novel non-peptide fibrinogen receptor antagonists. 1. Synthesis and glycoprotein IIb-IIIa antagonistic activities of 1,3,4-trisubstituted 2-oxopiperazine derivatives incorporating side-chain functions of the RGDF peptide

Based on the lead tetrapeptide RGDF, two possible non-peptide glycoprotein (GP) IIb-IIIa antagonists possessing an (S)-2-oxopiperazine-3-acetic acid moiety as a scaffold incorporating the indispensable Asp fragment were prepared, and (S)-4-[[trans-[4-(guanidinomethyl)-cyclohexyl]carbonyl]glycyl]-2- oxopiperazine-1,3-diacetic acid, 1a, was identified as a potential lead. A series of 3-substituted 2-oxopiperazine-1-acetic acids bearing the Arg-Gly equivalent at the 4-position were prepared and evaluated for their ability to prevent platelet aggregation and for their binding affinity for the GP IIb-IIIa receptor purified from human HEL cells. (S)-4-[(4-Amidinobenzoyl)glycyl]-3-[(methoxycarbonyl)methyl]- 2-oxopiperazine-1-acetic acid, 9 (TAK-029), inhibited in vitro human platelet aggregation with an IC50 value of 0.03 microM and GP IIb-IIIa-fibrinogen binding with an IC50 value of 0.49 nM. The [4-(2-aminoethyl)benzoyl]glycyl derivative 26 showed activity comparable to that of 9 (IC50 = 0.093 microM, guinea pig platelet aggregation assay). Compound 9 dose-dependently inhibited ex vivo platelet aggregation in guinea pigs (0.03 and 0.1 mg/kg, i.v.), and long-lasting inhibition of platelet aggregation was observed upon oral administration of 9 (3 mg/kg) to guinea pigs. On the other hand, the activity of 26 disappeared within 1 h after a dose of 1 mg/kg (i.v.). Compound 9 may therefore be useful in the clinical treatment of arterial thrombotic diseases.     

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.     

Inhibition of platelet-vessel wall interactions by thromboxane receptor antagonism in a human in vitro system: potentiation of antiplatelet effects of aspirin

BACKGROUND: Pharmacological inhibition of arachidonic acid metabolism has proven therapeutically useful in the prevention of cardiovascular events. METHODS: We have investigated the ability of Bay u 3405, a synthetic thromboxane antagonist, to interfere with platelet aggregation and arachidonic acid metabolism. The antiplatelet action was also analysed in a perfusion system in which vascular subendothelium was exposed to circulating human blood (10 min; shear rate = 800 s-1). Platelet interactions were morphometrically analysed and results compared with those obtained in studies with blood from donors taking aspirin (acetylsalicylic acid, ASA) (500 mg day-1). The additional effect of Bay u 3405 on the antiplatelet action of ASA was also evaluated. RESULTS: Bay u 3405 caused a dose-dependent inhibition of platelet aggregation induced by U46619 with a maximal effect at concentrations > or = 0.01 microgram mL-1. Higher concentrations (> or = 0.05 micrograms mL-1) also inhibited aggregations induced by ADP or collagen. Bay u 3405 did not interfere with platelet arachidonic acid metabolism. In perfusion studies, Bay u 3405 (0.01 microgram mL-1) significantly decreased the total surface of the vessel covered by platelets (%CS = 18.7 +/- 1.09 vs. 24.4 +/- 1.94; P < 0.05) and the formation of large aggregates %T = 7.5 +/- 0.87 vs. 19.3 +/- 1.61; P < 0.01). ASA treatment reduced platelet aggregate formation (%T = 13.7 +/- 2.06; P < 0.05) but did not affect the total surface covered by platelets. The in vitro addition of Bay u 3405 to blood from ASA-treated donors further reduced the formation of large aggregates (%T = 2.7 +/- 0.79; P < 0.01 vs. ASA). CONCLUSIONS: In vitro effect of Bay u 3405 on platelet function were superior to those observed with ASA. The thromboxane antagonism antagonism provided by Bay u 3405 further enhanced the inhibition of platelet aggregate formation found after ASA treatment.     

Effects of MK-447 on thrombin-induced aggregation, secretion of ATP, and [Ca2+]i mobilization in rabbit platelets

AIM: To study the effects of MK-447 on aggregation release reaction and intracellular calcium mobilization by thrombin. METHODS: Aggregation and release reaction were assessed by light transmission and ATP content in rabbit citrate platelet-rich plasma (PRP), and cytosolic-free calcium was measured by fluorescence and imaging. RESULTS: MK-447 (2-aminomethyl-4-t-butyl-6-iodophenol hydrochloride) induced a decrease in light transmission (DLT), so called platelet shape change, without detectable aggregation and secretion of ATP, and increased intracellular calcium concentration ([Ca2+]i) slightly in washed single platelet loaded with Fura 2, the peak value being about 160 nmol.L-1. These effects were not inhibited by egtazic acid 3 mmol.L-1 or indometacin 3 mumol.L-1. The pretreatment of PRP with MK-447 700 mumol.L-1 reduced the DLT by thrombin, potentiated and enhanced thrombin-induced aggregation and secretion of ATP in a concentration-dependent manner. Thrombin-induced [Ca2+]i mobilization (peak value: 369 +/- 45 nmol.L-1) was further enhanced by the administration of MK-447 at 2 min before the addition of thrombin, and the peak value reached 623 +/- 121 nmol.L-1 (P < 0.01). CONCLUSION: MK-447-induced platelet shape change was involved in intracellular calcium release in this preparation. MK-447 enhanced thrombin-induced aggregation and release reaction and these effects of MK-447 on aggregation and release reaction by thrombin might result from the synergistic effect of intracellular calcium mobilization.     

The clinical course of asymptomatic mesenteric arterial stenosis

The activation of rabbit platelets by rabbit plasma clots, and the inhibition of clot-associated thrombin by heparin:antithrombin III, recombinant hirudin (rHV2Lys47) and argatroban, a low molecular weight thrombin inhibitor, was studied. Plasma clots caused the aggregation of platelets suspended in a plasma-free medium as assessed by single platelet counting, and by scanning electron microscopy (platelet aggregates present on the clot surface). Platelet aggregation, induced by clot-associated thrombin, was inhibited by argatroban with an IC50) of 14 +/- 3 nM compared to an IC50) of 12 +/- 2 nM when human thrombin in solution titrated to give the same decrease in the platelet count as plasma clots was used. rHV2Lys47 also inhibited aggregation induced by clot-associated thrombin with an IC50 of 1.6 +/- 0.4 nM compared to 1.6 +/- 0.5 nM with thrombin in solution. Heparin was less active against clot-associated thrombin (IC50) = 69 +/- 9 mU/ml) than against thrombin in solution (IC50 = 15 +/- 5 mU/ml). This study shows that plasma clot-bound thrombin activates platelets and that direct-acting thrombin inhibitors such as argatroban and rHV2Lys47 are more effective than heparin:antithrombin III in inhibiting this phenomenon.     

Competitive inhibition of phospholipase A2 activity by the platelet-activating factor antagonist Ro 19-3704 and evidence for a novel suppressive effect on platelet activation

The compound Ro 19-3704 [3-4(R)-2-(methoxycarbonyl) oxy-3-(octadecylcarbamoyl)oxy-propoxy butylthiazolium iodide], initially described as an antagonist of platelet-activating factor, is reported here to directly inhibit rabbit platelet phospholipase (PL) A2 activity, with an IC50 value of 4 to 7 microM. Classical Michaelis-Menten analysis showed that inhibition was reversible and competitive, inasmuch as apparent Km values increased in the presence of Ro 19-3704 (from 0.2-0.4 to 2 microM), whereas Vmax values remained constant (200 +/- 20 nmol/min/10(9) cells). Ro 19-3704 inhibited platelet aggregation, PLA2 release and thromboxane B2 formation induced by thrombin (0.25 U/ml), with IC50 values of 8, 15 and below 5 microM, respectively. Aggregation and PLA2 release by arachidonic acid (100 microM) were also inhibited, but thromboxane B2 formation was unaffected, indicating that Ro 19-3704 does not inhibit cyclooxygenase. Platelet activation by collagen (5 micrograms/ml), the thromboxane mimetic U46619 ([15(S)-hydroxy-11,9(epoxymethano)-prosta-5Z,13E-dienoic acid] 1 microM) and low concentrations of thrombin (0.05-0.1 U/ml) was also inhibited by Ro 19-3704. Inhibition of platelet activation was reversible, suggesting that its suppressive effect was not due to cytotoxicity. Finally, Ro 19-3704 did not stimulate cyclic AMP formation or inhibit phosphodiesterase activity. Ro 19-3704 is a competitive inhibitor of PLA2 activity, and is also endowed with a potent suppressive effect on platelet activation induced by different agonists.     

The effect of halofenate or halofenate free acid on human, rat and guinea pig platelet aggregation

Halofenate free acid (HFA), the major metabolite of the hypolipemic agent halofenate, blocked the secondary phase of human platelet aggregation induced by ADP, epinephrine, or thrombin; higher concentrations of clofibrate free acid (CFA) were required to produce similar inhibitory effects on platelet aggregation. HFA and CFA inhibited collagen-induced aggregation of human, rat, or guinea pig platelets. Halofenate orally administered to rats caused inhibition of collagen-induced aggregation when plasma levels of HFA exceeded 300 mug/ml, a clinically achievable human plasma concentration. The platelet inhibitory effects of clofibrate administration were less than those observed with halofenate administration.     

Effects of suramin on human platelet aggregation and Ca2+ mobilization induced by thrombin and other agonists

The purpose of this study was to investigate the effect of suramin, a polyanionic napthalene sulfonic acid, on human platelet aggregation and Ca2+ mobilization induced by various agonists. Our results show that suramin completely inhibited aggregation by thrombin, platelet activating factor (PAF), alkyllysophosphatidic acid (ALPA), or arachidonic acid in a concentration-dependent manner. The IC50 values of suramin for inhibition of aggregation by PAF, arachidonic acid, and thrombin were 76.7, 239, and 1.49 microg/ml, respectively. Ca2+ mobilization induced by thrombin was inhibited by suramin with an approximate IC50 value of 20 microg/ml. This concentration of suramin had no effect on PAF or oleic acid-induced Ca2+ mobilization. The mechanism by which suramin inhibits aggregation is not clear, but our results suggest that suramin inhibits the ligand-receptor interaction.     

Synthesis and preliminary pharmacological investigation of some 2-[4-(dialkylaminoalkoxy)phenyl]benzotriazoles and their N-oxides

A set of 2-[4-(dialkylaminoalkoxy)phenyl]benzotriazoles and corresponding N-oxides was prepared. In a preliminary pharmacological investigation concerning some of these compounds, several in vitro and in vivo activities were shown. At concentrations in the range of 3-10 microM all tested compounds strongly inhibited (50-100%) the guinea pig ileum contractions induced either electrically or by means of several agonists; of particular interest was the antagonism to leukotriene D4. Compound 5b inhibited platelet aggregation induced by thromboxane A2, PAF and ADF (but not by arachidonic acid) and increased the bleeding time in mice. Compounds 5b and 6b protected mice from potassium cyanide hypoxia and exerted anti-hypercholesterolemic action; the first compound produced a ratio between HPL and total serum cholesterol concentrations below 0.92, thus indicating a potential anti-atherogenic activity.     

Effects of very low dose and enteric-coated acetylsalicylic acid on prostacyclin and thromboxane formation and on bleeding time in healthy subjects

OBJECTIVE: Low dose acetylsalicylic acid (ASA) is widely used as an anti-aggregatory agent in the primary and secondary prevention of cardiovascular diseases. In an effort to spare prostacyclin formation and to reduce gastrointestinal side-effects, both very low doses and enteric-coated formulations of ASA have been introduced. However, it still remains unclear whether these different formulations and dosages are equally effective with respect to inhibition of platelet aggregation and thromboxane A2 (TXA2) formation. METHODS: In a randomized study, we therefore investigated the effects of 100 mg ASA plain (p), 100 mg ASA enteric-coated (ec) and 40 mg ASA (p) to 36 healthy male subjects given for 7 days on platelet aggregation and endogenous prostanoid formation rates. Platelet aggregation and platelet TXB2 release in platelet rich plasma (PRP) and serum TXB2 and 6-keto-PGF1alpha levels were determined at baseline and after 7 days of each medication. The urinary metabolites of TXA2 (2,3-dinor-TXB2) and prostacyclin (2,3-dinor-6-keto-PGF1alpha) were measured by gas chromatography/tandem mass spectrometry in 24-h-urines at baseline and on day 7 of each medication. RESULTS: Collagen-induced platelet aggregation was 73.1+/-1.6% of maximal aggregation at baseline. It was inhibited by 68.9%, 58.6% and 24.0% by ASA 100 mg plain, 100 mg enteric-coated, and 40 mg plain on day 7, respectively. Platelet TXB2 release was 11592.0+/-367.5 pg x ml(-1) PRP. It was inhibited by 90.1%, 86.5%, and 55.2% by ASA 100 mg plain, 100 mg enteric-coated, and 40 mg plain, respectively. Serum TXB2 was almost completely reduced on day 7 by 100 mg ASA, but not by 40 mg ASA; serum 6-keto-PGF1alpha was slightly, but significantly reduced in all three groups. Urinary 2,3-dinor-TXB, excretion was 196.0+/-41.5 pg x mg(-1) creatinine at baseline. It was reduced by 80.3% and 79.1% by ASA 100 mg plain and enteric-coated, respectively (each P < 0.05 versus baseline), but only by 55.4% by ASA 40 mg plain (P < 0.05 versus both formulations of ASA 100 mg). CONCLUSIONS: Our present data show that the plain and enteric-coated formulations of 100 mg ASA are equally effective in inhibiting platelet aggregation, platelet thromboxane production, and urinary 2,3-dinor-TXB2 excretion rates. In contrast, a very low dose of 40 mg ASA was significantly less effective in inhibiting these indices of platelet activation in healthy human subjects. ASA enteric-coated 100 mg may be a useful alternative to 100 mg ASA (p) in patients with gastrointestinal side-effects, whereas 40 mg ASA (p) may be too low to inhibit sufficiently platelet activity in patients with cardiovascular diseases in whom platelet activity is increased.     

Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis"

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.     

Difference of (Ca2+)i movements in platelets stimulated by thrombin and TRAP: the involvement of alpha(IIb)beta3-mediated TXA2 synthesis

This study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of alpha(IIb)beta3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with alpha(IIb)beta3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM 13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that alpha(IIb)beta3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with alpha(IIb)beta3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to alpha(IIb)beta3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.     

Percutaneous nephroscopy in the diagnosis of renal pelvic filling defect: a report of two cases

Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B2 formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Synthesis and antiplatelet evaluation of alpha-methylene-gamma-butyrolactone bearing 2-methylquinoline and 8-hydroxyquinoline moieties

In a search for inhibitors of platelet aggregation, some alpha-methylene-gamma-butyrolactones bearing 2-methylquinoline and 8-hydroxyquinoline moieties were synthesized and evaluated for antiplatelet activities against thrombin (Thr)-, arachidonic acid (AA)-, collagen (Col)-, and platelet-activating factor (PAF)-induced aggregation in washed rabbit platelets. With the exception of 2-[[2,3,4,5-tetrahydro-4-methylene-5-oxo-2-(4-phenylphenyl)-2 -furanyl]methoxy]-8-hydroxyquinoline (8f), these alpha-methylene-gamma-butyrolactones completely inhibited the platelet aggregation induced by AA and Col. The 2-methylquinoline derivatives were also active against Thr- and PAF-induced aggregation, while their 8-hydroxyquinoline counterparts were relatively inactive.     

Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase

The kinase inhibitors SB 203580 and PD 98059 have been reported to be specific inhibitors of the 38- and 42/44-kDa mitogen-activated protein kinase (MAPK) pathways, respectively. In this study, the two inhibitors were found to decrease platelet aggregation induced by low concentrations of arachidonic acid, suggesting that they also interfere with the metabolism of arachidonic acid to thromboxane A2. In support of this, SB 203580 and PD 98059 inhibited the conversion of exogenous [3H]arachidonic acid to [3H]thromboxane in intact platelets. Measurement of platelet cyclooxygenase-1 activity following immunoprecipitation revealed that SB 203580 and PD 98059 are direct inhibitors of this enzyme. Both compounds were shown to inhibit purified cyclooxygenase-1 and -2 by a reversible mechanism. In addition, SB 203580 (but not PD 98059) inhibited platelet aggregation induced by prostaglandin H2 and the conversion of prostaglandin H2 to thromboxane A2 in intact platelets. SB 203580 also inhibited this pathway in platelet microsome preparations, suggesting a direct inhibitory effect on thromboxane synthase. These results demonstrate that direct effects of the two kinase inhibitors on active arachidonic acid metabolites have to be excluded before using these compounds for the investigation of MAPKs in signal transduction pathways. This is of particular relevance to studies on the regulation of cytosolic phospholipase A2 as these two MAPKs are capable of phosphorylating cytosolic phospholipase A2, thereby increasing its intrinsic activity.     

Cyclopiazonic acid and thapsigargin induce platelet aggregation resulting from Ca2+ influx through Ca2+ store-activated Ca2+-channels

The effects of cyclopiazonic acid and thapsigargin, selective inhibitors of the endoplasmic reticulum Ca2+-ATPase pump, on the platelet aggregation were investigated using washed rat platelets prepared by chromatography on Sepharose 2B columns. In Ca2+-free medium, cyclopiazonic acid and thapsigargin did not induce aggregation, but in the presence of 1 mM Ca2+, platelet aggregation was induced in a concentration-dependent manner. Cyclopiazonic acid- and thapsigargin-induced platelet aggregation was blocked by 1 mM Ni2+ but not by 100 microM indomethacin or 1 microM nifedipine. In aequorin-loaded platelets, cyclopiazonic acid and thapsigargin caused sustained elevation of the cytosolic Ca2+ concentration, an effect which was blocked by Ni2+, a non-selective Ca2+ channel blocker and SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride), a putative receptor-operated Ca2+ channel antagonist. The above results indicated that both cyclopiazonic acid and thapsigargin induced platelet aggregation and elevation of cytosolic Ca2+ concentration, that extracellular Ca2+ was essential for cyclopiazonic acid- and thapsigargin-induced platelet aggregation, and that platelet aggregation may be associated with Ca2+ influx through Ca2+ store-activated Ca2+ channels.     

Inhibition of platelet function by A02131-1, a novel inhibitor of cGMP-specific phosphodiesterase, in vitro and in vivo

The effect of A02131-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl thieno (3,2-c)pyrazole], a cGMP-specific phosphodiesterase (PDE) inhibitor, on platelet function was investigated. The compound was found to inhibit the aggregation of and adenosine triphosphate (ATP) release from human platelet-rich plasma and washed platelets that were induced by aggregation inducing drugs such as arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), adenosine diphosphate (ADP) and A23187, and the inhibitory effect was concentration-dependent. A02131-1 also disaggregated the performed platelet aggregates induced by these inducers. Thromboxane B2 (TXB2) formations caused by collagen, PAF, ADP, and A23187 were inhibited by A02131-1 at concentrations that did not affect the AA-induced formation of TXB2 and prostaglandin D2 (PGD2). A02131-1 suppressed both the generation of inositol 1,4,5-triphosphate (IP3) and the increase of intracellular Ca2+ concentration stimulated by these aggregation inducers. A02131-1 was shown to increase the cAMP and cGMP levels in platelets and the extent was found to be dependent on concentration as well as time. A02131-1 increased the cAMP level much more slowly than the cGMP level. Activities of adenylate cyclase, guanylate cyclase, and PDEs (type I and III) were not altered by A02131-1. However, the activity of cGMP-specific PDE (type V) was inhibited by A02131-1. The antiplatelet aggregation activity and the effect on raising cAMP level of A02131-1 were both potentiated by prostaglandin E1 (PGE1). In the mouse tail bleeding test, A02131-1 was clearly shown to be more effective than dipyridamole in prolonging the tail bleeding time of conscious mice. These data indicate that A02131-1 is a cGMP-specific PDE (type V) inhibitor in human platelets.     

Inhibition of collagen-induced platelet activation by arachidonyl trifluoromethyl ketone

Collagen-induced platelet activation is associated with, and markedly potentiated by, the release of arachidonic acid and its subsequent conversion to thromboxane A2. The precise mechanism of arachidonic acid release is unknown. An inhibitor of isolated cytosolic phospholipase A2 (cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), was used to examine the role that cPLA2 plays in this process. AACOCF3 inhibited platelet aggregation in response to collagen and arachidonic acid but not to thrombin, calcium ionophore, phorbol ester, or a thromboxane mimetic. Thromboxane formation stimulated by thrombin or collagen was inhibited by AACOCF3. However, AACOCF3 did not inhibit collagen-induced [14C]arachidonic acid release. These data are consistent with the inhibitory effects of AACOCF3 on collagen-induced aggregation involving an action on the conversion of arachidonic acid to thromboxane.     

Pharmacologic modulation of autonomic tone: implications for the diabetic patient

The inhibitory effects of nitroglycerin (NTG) on platelet function and the mechanisms of inhibition have been studied in vitro, but not in vivo. Therefore, we have investigated the effects of NTG on platelet function in eight patients undergoing orthopaedic surgery. Simultaneous measurements of platelet aggregation and change in intracellular calcium concentrations were performed in Fura-2 loaded platelets using thrombin as a stimulator. Intraplatelet concentrations of cyclic 3',5'-guanine monophosphate (cGMP) were measured by radioimmunoassay, and the concentration of nitrite ion was also measured. Continuous i.v. infusion of NTG 4-8 micrograms kg-1 min-1 significantly inhibited platelet aggregation and the increase in intracellular Ca2+ concentration (first phase, mean 439.9 (SEM 68.7) vs 210.6 (38.7) nmol litre-1; second phase, 154.4 (19.8) vs 106.7 (18.0) nmol litre-1). The concentration of cGMP (from 0.633 (0.098) to 1.764 (0.578) pmol/10(9) platelets) and the concentration of nitrite ion (from 532.6 (17.6) to 724.4 (34.8) nmol litre-1) also increased significantly after infusion of NTG. The NTG concentration in plasma was of the order of 10(-8) mol litre-1. We have demonstrated that in vivo, NTG increased intraplatelet cGMP concentrations and inhibited platelet function; one mechanisms of this effect is likely to be related to nitric oxide liberation from NTG bioconversion.     

Modulation of thrombin-induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol-protease inhibitory sequence of kininogens and S-(3-nitro-2-pyridinesulfenyl)-cysteine

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High-molecular-mass kininogen (HK) contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is thought to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2(Npys, 3-nitro-2-sulfenylpyridine), P1, corresponding to the thiol-protease-binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin-induced platelet-shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.