15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and Hemorrhage

The arachidonic acid (AA) cascade plays a significant role in platelet aggregation. AA released from membrane phospholipids is metabolized by cyclooxygenase (COX) pathway to thromboxane A

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Blood Coagulation Disorders, Inherited; Blood Platelets; Calcium; Disease Susceptibility; Genetic Predisposition to Disease; GTP Phosphohydrolases; Hemorrhage; High-Throughput Nucleotide Sequencing; Humans; Inositol 1,4,5-Trisphosphate; Mutation; Pedigree; Phospholipase C beta; Platelet Aggregation; Prostaglandin-Endoperoxide Synthases; Signal Transduction; Thromboxane A2

Chronic hemorrhagic diathesis in patients showing normal levels of plasmatic clotting factors strongly suggests for congenital platelet disorders. We report on a pediatric patient (male, 3 years, D1) with mild bleeding. A sibling (D2), his mother (D3) and father (D4) were included for laboratory investigation. Platelet counts in D1, D2 and D4 indicated mild thrombocytopenia (100 Gpt/L). D1 and D3 platelets showed significantly diminished aggregation response on arachidonic acid and U46619 stimulation. Immunostaining for platelet proteins on blood smears of D1 and D2 indicated defects in ß1-tubulin. Exon sequencing of

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Arachidonic Acid; Blood Platelet Disorders; Blood Platelets; Child, Preschool; Female; Hemorrhage; Hemorrhagic Disorders; Heterozygote; Humans; Male; Mutation; Phenotype; Platelet Count; Receptors, Thromboxane A2, Prostaglandin H2; Thrombocytopenia; Tubulin

Acquired and inherited bleeding disorders may present in the neonatal period with devastating lifelong effects. Diagnosing bleeding disorders in the neonatal population could aid in preventing and treating the associated complications. However, currently available platelet function testing is limited in neonates, owing to difficulties in obtaining an adequate blood volume, a lack of normal reference ranges, and an incomplete understanding of the neonatal platelet functional phenotype.. To develop small-volume, whole blood platelet function assays in order to quantify and compare neonatal and adult platelet function.. Peripheral blood was obtained from healthy, full-term neonates at 24 h of life. Platelet activation, secretion and aggregation were measured via flow cytometry. Platelet adhesion and aggregation were assessed under static and flow conditions. As compared with adult platelets, peripheral neonatal platelet P-selectin expression and integrin glycoprotein IIbIIIa activation were significantly reduced in response to the G-protein-coupled receptor (GPCR) agonists thrombin receptor activator peptide-6 (TRAP-6), ADP, and U46619, and the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway agonists collagen-related peptide (CRP) and rhodocytin. Neonatal platelet aggregation was markedly reduced in response to TRAP-6, ADP, U46619, CRP and rhodocytin as compared with adult platelets. The extents of neonatal and adult platelet adhesion and aggregate formation under static and shear conditions on collagen and von Willebrand factor were similar.. As compared with adult platelets, we found that neonatal platelet activation and secretion were blunted in response to GPCR or ITAM agonists, whereas the extent of neonatal platelet adhesion and aggregate formation was similar to that of adult platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Adult; Blood Platelets; C-Reactive Protein; Cell Separation; Flow Cytometry; Glycoproteins; Hemorrhage; Humans; Immunoreceptor Tyrosine-Based Activation Motif; Infant, Newborn; Lectins, C-Type; Oligopeptides; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Platelet Function Tests; Platelet Glycoprotein GPIb-IX Complex; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, G-Protein-Coupled; Signal Transduction

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Arachidonic Acid; Blood Platelets; Cells, Cultured; Cyclooxygenase 1; Genetic Association Studies; Hemorrhage; Heterozygote; Humans; Male; Platelet Aggregation; Platelet Function Tests; Polymorphism, Single Nucleotide; Postoperative Complications; Recurrence; Thromboxanes; Transcriptome

In this study, we report BF066, a novel adenine derivative, inhibits platelet activation and thrombosis via the adenosine receptor (A(2A)) activation and phosphodiesterase (PDE) inhibition. BF066 inhibits platelet aggregation and ATP releasing induced by multiple platelet agonists in a dose-dependent manner. The inhibition of BF066 on ADP-induced aggregation is potentiated by adenosine and can be dramatically antagonized by the A(2A) antagonist SCH58261. BF066 also inhibits the PDE activity and platelet spreading on fibrinogen. In FeCl(3)-injured mouse mesenteric arterial thrombosis model, BF066 prevents thrombus formation effectively, similar to clopidogrel. Intriguingly, at dose achieving similar antithrombotic effect compared to clopidogrel, BF066 does not increase bleeding significantly. Taken together, these results suggest that BF066 may be an effective and safe antiplatelet agent targeting both PDE and A(2A). Considering the successful use of combined antiplatelet therapy, BF066 may be further developed as a novel dual target antiplatelet agent.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenine; Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Arachidonic Acid; Arterioles; Chlorides; Collagen; Ferric Compounds; Fibrinogen; Hemorrhage; Humans; Immobilized Proteins; Mice; Mice, Inbred C57BL; Oligopeptides; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Platelet Aggregation; Platelet Aggregation Inhibitors; Receptor, Adenosine A2A; Thrombin; Thrombosis

Thromboxane A(2) receptor (TXA(2)R) abnormality appears to dominantly disturb platelet function.. To reveal a molecular genetic defect in a patient with TXA(2)R abnormality and investigate the mechanism for the impaired response to TXA(2).. The proband (OSP-2, PT) was a 7-year-old Japanese girl, suffering from repeated mucocutaneous bleeding.. U46619 (2.5 and 10 μm)-induced platelet aggregation was remarkably impaired in the proband and her father. Immunoblots showed that TXA(2)R expression levels in their platelets were approximately 50% of controls, and nucleotide sequence analysis revealed that they were heterozygous for a novel mutation, c.167dupG in the TXA(2)R cDNA. Expression studies using Chinese hamster ovary (CHO) cells indicated that the mutation is responsible for the expression defect in TXA(2)R. We then examined α(IIb)β(3) activation by employing an initial velocity analysis and revealed that U46619 failed to induce a sustained α(IIb)β(3) and Rap1B activation in the proband. In addition, platelet secretion as monitored by P-selectin expression was markedly impaired in response to U46619 but not to ADP. The interaction between secreted ADP and P2Y(12) has been shown to play a critical role in the sustained α(IIb)β(3) activation (Kamae et al. J Thromb Haemost 2006; 4: 1379). As expected, small amounts of exogenous ADP (0.5 μm) partially restored the sustained α(IIb)β(3) activation induced by U46619.. Our present data strongly suggest that the impaired platelet activation in response to U46619 in the heterozygous subject for the TXA(2)R mutation is, at least in part, as a result of the decrease in ADP secretion.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blood Platelets; Child; CHO Cells; Cricetinae; Cricetulus; Female; Hemorrhage; Heterozygote; Humans; Male; Mutation; Parents; Platelet Aggregation; Platelet Function Tests; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, G-Protein-Coupled; Receptors, Thromboxane A2, Prostaglandin H2

In the present study, we investigated the role of the central cholinergic system in mediating the pressor effect of intracerebroventricularly administrated U-46619, a thromboxane A2 (TxA2) analog, in hemorrhaged hypotensive rats. Hemorrhage was performed by withdrawing a total volume of 2.1 ml of blood per 100 g body weight over a period of 10 min. Intracerebroventricular (i.c.v.) injection of U-46619 (0.5, 1, 2 micro g) produced a dose- and time-dependent increase in arterial pressure and reversed the hypotension of this condition. Hemorrhage caused small increases in extracellular hypothalamic acetylcholine and choline levels. Intracerebroventricular administration of U-46619 (1 micro g) further increased the levels of extracellular acetylcholine and choline by 57% and 41%, respectively. Pretreatment with SQ-29548 (8 mug; i.c.v.), a selective TxA2 receptor antagonist, completely abrogated the effects of subsequent injection of U-46619 (1 mug; i.c.v.) on arterial pressure and extracellular acetylcholine and choline levels. Pretreatment with mecamylamine (50 micro g; i.c.v.), a cholinergic nonselective nicotinic receptor antagonist, attenuated the pressor effect of U-46619 (1 micro g, i.c.v.) in hemorrhaged rats whereas pretreatment with atropine (10 micro g; i.c.v.), a cholinergic nonselective muscarinic receptor antagonist, had no effect. Interestingly, pretreatment of rats with methyllycaconitine (10 micro g; i.c.v.) or alpha-bungarotoxin (10 micro g; i.c.v.), selective antagonists of alpha-7 subtype nicotinic acetylcholine receptors (alpha7nAChRs), partially abolished the pressor effect of U-46619 (1 micro g; i.c.v.) in the hypotensive condition. Pretreatment with a combination of mecamylamine plus methyllycaconitine or mecamylamine plus alpha-bungarotoxin attenuated the reversal effect of U-46619, but only to the same extent as pretreatment with either antagonist alone. In conclusion, i.c.v. administration of U-46619 restores arterial pressure and increases posterior hypothalamic acetylcholine and choline levels by activating central TxA2 receptors in hemorrhaged hypotensive rats. The activation of central nicotinic cholinergic receptors, predominantly alpha7nAChRs, partially acts as a mediator in the pressor responses to i.c.v. injection of U-46619 under these conditions.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; alpha7 Nicotinic Acetylcholine Receptor; Animals; Blood Pressure; Bridged Bicyclo Compounds, Heterocyclic; Cholinergic Fibers; Disease Models, Animal; Dose-Response Relationship, Drug; Extracellular Fluid; Fatty Acids, Unsaturated; Hemorrhage; Hydrazines; Hypotension; Hypothalamus, Posterior; Injections, Intraventricular; Male; Neural Pathways; Nicotinic Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Nicotinic; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2; Time Factors; Vasoconstrictor Agents

1. In the present study, we aimed to determine the involvement of brain thromboxane A2 (TXA2) in blood pressure decreases evoked by acute and/or graded haemorrhage in rats. 2. Sprague-Dawley rats were used throughout the study. Acute haemorrhage was achieved by withdrawing a total volume of 2.1 and 2.5 mL blood/100 g bodyweight over a period of 10 min. A microdialysis study was performed in a hypothalamic area to measure extracellular TXA2 levels. Graded haemorrhage was conducted successively by withdrawing carotid arterial blood (0.55 mL/100 g bodyweight) over a 10 s period four times (S1-S4) at 5 min intervals. Furegrelate (125, 250 and 500 microg), a TXA2 synthase inhibitor, was injected intracerebroventricularly (i.c.v.) 60 min before acute or graded haemorrhage was initiated. U-46619 (0.5, 1 and 2 microg, i.c.v.), a synthetic TXA2 analogue, was administered 5 min before acute haemorrhage (2.1 mL/100 g bodyweight). 3. Acute haemorrhage produced a severe and long-lasting decrease in blood pressure and had a tendency to increase heart rate. Both haemorrhage protocols (2.1 or 2.5 mL/100 g) generated similar approximate twofold increases in extracellular hypothalamic TXA2 levels. Intracerebroventricular furegrelate (250 microg) pretreatment completely blocked the TXA2 increases induced by acute haemorrhage. Furegrelate administration (100, 250 and 500 microg, i.c.v.) attenuated the fall in arterial pressure evoked by acute haemorrhage and caused significant increases in heart rate at all doses injected. 4. Graded haemorrhage progressively lowered arterial pressure and increased plasma vasopressin and adrenaline levels in the last period. Furegrelate-injected rats were greatly resistant to the hypotensive effect of haemorrhage for all degrees of blood removed. Plasma adrenaline and vasopressin levels were significantly elevated in furegrelate-pretreated rats compared with the saline-treated group during S2-S3 and S4, respectively. U-46619 administration caused small but statistically significant decreases in arterial pressure induced by haemorrhage. 4. The results show that acute hypotensive haemorrhage increases extracellular hypothalamic TXA2 levels. The increase in brain endogenous TXA2 levels involves a decrease in blood pressure evoked by haemorrhage because the blockade of TXA2 synthesis by furegrelate pretreatment attenuated the haemorrhagic hypotension. Increases in plasma adrenaline and vasopressin levels may mediate this effect.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Benzofurans; Blood Pressure; Disease Models, Animal; Epinephrine; Heart Rate; Hemorrhage; Hypotension; Hypothalamus; Injections, Intraventricular; Male; Rats; Rats, Sprague-Dawley; Thromboxane A2; Thromboxane-A Synthase; Time Factors; Vasoconstrictor Agents; Vasopressins

In the present study, we investigated the cardiovascular effects of centrally injected U-46619, a thromboxane A(2) (TXA(2)) analog, and the central and peripheral mechanisms of these effects in hemorrhagic shock conditions. Hemorrhage was performed by withdrawing a total volume of 2.1 ml of blood/100 g body weight over a period of 10 min. Injections were made into the lateral cerebral ventricle (LCV), nucleus tractus solitarius (NTS), rostral ventrolateral medulla (RVLM) and paraventricular nucleus of hypothalamus (PVN). U-46619 (0.1, 1 and 2 microg) increased blood pressure and reversed hypotension in hemorrhagic shock. The pressor effect was dose- and time-dependent in all investigated brain areas. Heart rate changes were not significantly different in all groups. Pretreatment of rats with an injection of SQ-29548 (4 or 8 microg), a TXA(2) receptor antagonist, into the LCV, NTS, RVLM and PVN completely blocked the pressor effect of U-46619 (1 microg) injected into respective brain areas. Hemorrhage itself increased plasma adrenaline, noradrenaline, vasopressIN levels and renin activity. U-46619 (1 microg) injected into the LCV, PVN, RVLM and NTS produced additional increases in these hormone levels and in renin activity. Intravenous pretreatments of rats with prazosin (0.5 mg/kg), an alpha(1)-adrenoceptor antagonist, [beta-mercapto-beta,beta-cyclopentamethylenepropionyl(1), O-Me-Tyr(2),Arg(8)]- vasopressin (10 microg/kg), a vasopressin V(1)-receptor antagonist, or saralasin (250 microg/kg), an angiotensin II receptor antagonist, in hemorrhaged rats partially blocked the pressor response to U-46619 (1 microg) injected into the LCV, PVN, RVLM and NTS. Results show that centrally administered U-46619, a TXA(2) analog, increases blood pressure and reverses hypotension in hemorrhagic shock. Activation of central TXA(2) receptors mediates the pressor effect of the drug. Furthermore, the increases in plasma adrenaline, noradrenaline, vasopressin levels and renin activity are involved in these effects.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adrenergic alpha-1 Receptor Antagonists; Angiotensin II Type 1 Receptor Blockers; Animals; Antidiuretic Hormone Receptor Antagonists; Blood Pressure; Brain; Bridged Bicyclo Compounds, Heterocyclic; Catecholamines; Fatty Acids, Unsaturated; Heart Rate; Hemodynamics; Hemorrhage; Hydrazines; Hypotension; Injections; Injections, Intraventricular; Male; Medulla Oblongata; Paraventricular Hypothalamic Nucleus; Rats; Rats, Sprague-Dawley; Renin; Shock, Hemorrhagic; Solitary Nucleus; Thromboxane A2; Vasoconstrictor Agents; Vasopressins

Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice.. Mice lacking (-/-) or expressing half-levels (+/-) of the other major platelet collagen receptor, integrin alpha2beta1, were injected with the anti-GP VI antibody JAQ1 and analyzed on day 5. Anti-GP VI treatment resulted in a marked hemostatic defect in alpha2-/- or alpha2+/- mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti-GP VI-treated wild-type but not alpha2-/- or alpha2+/- platelets to collagen. This process required the simultaneous activation of the G(q) and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti-GP VI-treated or GP VI/Fc receptor gamma-chain-deficient but not control mice.. Anti-GP VI therapy may result in defective hemostasis in patients with reduced alpha2beta1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti-GP VI-based therapeutics in the prevention of cardiovascular disease.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Antibodies, Monoclonal; Aspirin; Bleeding Time; Collagen; Drug Evaluation, Preclinical; Drug Synergism; Fibrinolytic Agents; GTP-Binding Protein alpha Subunits, G12-G13; GTP-Binding Protein alpha Subunits, Gq-G11; Hemorrhage; Hemostasis; Integrin alpha2beta1; Mice; Mice, Knockout; Platelet Activation; Platelet Adhesiveness; Platelet Membrane Glycoproteins; Receptors, Thromboxane A2, Prostaglandin H2; Signal Transduction; Thrombosis

Among the prostanoids, thromboxane (TX) A(2) is a potent stimulator of platelets, whereas prostaglandin (PG) I(2) inhibits their activation. The roles of PGE(2) in the regulation of platelet function have not been established, however, and the contribution of PGE(2) in hemostasis and thromboembolism is poorly understood. The present study was intended to clarify these roles of PGE(2) by using mice lacking the PGE(2) receptor subtype 3 (EP(3)(-/-) mice).. Expression of mRNAs for EP(3) in murine platelets was confirmed by quantitative reverse transcription-polymerase chain reaction. PGE(2) and AE-248, a selective EP(3) agonist, showed concentration-dependent potentiation of platelet aggregation induced by U46619, a TXA(2) receptor agonist, although PGE(2) alone could not induce aggregation. PGE(2) and AE-248 increased cytosolic calcium ion concentration ([Ca(2+)](i)), and AE-248 inhibited the forskolin-induced increase in cytosolic cAMP concentration ([cAMP](i)), suggesting G(i) coupling of EP(3). The potentiating effects of PGE(2) and AE-248 on platelet aggregation along with their effects on [Ca(2+)](i) and [cAMP](i) were absent in EP(3)(-/-) mice. In vivo, the bleeding time was significantly prolonged in EP(3)(-/-) mice. Moreover, when mice were challenged intravenously with arachidonic acid, mortality and thrombus formation in the lung were significantly reduced in EP(3)(-/-) mice.. - PGE(2) potentiated platelet aggregation induced by U46619 via EP(3) by increasing [Ca(2+)](i), decreasing [cAMP](i), or both. This potentiating action of PGE(2) via EP(3) is essential in mediating both physiological and pathological effects of PGE(2) in vivo.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blood Platelets; Dinoprostone; Disease Susceptibility; Dose-Response Relationship, Drug; Drug Synergism; Female; Gene Expression; Hemorrhage; Male; Mice; Mice, Mutant Strains; Platelet Aggregation; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP3 Subtype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Thromboembolism

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Blood Platelets; Calcium; Clot Retraction; Collagen; Cyclic AMP; Epinephrine; Fibrinogen; Hemorrhage; Humans; Indomethacin; Kinetics; Male; Middle Aged; Platelet Activating Factor; Platelet Aggregation; Platelet Membrane Glycoproteins; Prostaglandin Endoperoxides, Synthetic; Reference Values; Thrombin; Vasoconstrictor Agents; von Willebrand Factor