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

Multiple factors are involved in thrombus formation and require complex and highly therapeutic strategies. Platelet activation plays a critical role in the genesis of acute coronary syndromes involving not only platelets but also endothelial cells, leucocytes and erythrocytes. Angiotensin II (Ang II) is a vasoconstrictor that could participate in the thrombotic process. Platelets also express Ang II AT1 type receptors on their surface. Losartan is a non-peptidic inhibitor of AT1 receptors. It has been demonstrated that losartan reduced platelet aggregation induced by the thromboxane A2 (TXA2) analogue U46619. This effect was not observed with the losartan metabolite EXP 3174. The effect of losartan was assessed in binding studies in which losartan competitively inhibited the binding of [3H]U46619 to platelets in a dose-dependent manner. Irbesartan also inhibits the TXA2 receptor in platelets, an effect that was not obtained with the active form of candesartan, CV11974, and with valsartan. These results suggest that the structural requirements necessary to antagonize the TXA2/PGH2 platelet receptor may be different from those involved in AT1 receptor antagonism. The in vivo relevance of the in vitro findings has been confirmed by the fact that in vivo administration of losartan decreases P-selectin expression in platelets obtained from stroke-prone spontaneously hypertensive rats.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Angiotensin Receptor Antagonists; Animals; Antihypertensive Agents; Benzimidazoles; Biphenyl Compounds; Coronary Disease; Coronary Thrombosis; Humans; Losartan; P-Selectin; Platelet Activation; Platelet Aggregation; Rats; Rats, Inbred SHR; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Thromboxane; Tetrazoles; Thrombosis; Thromboxane A2; Valine; Valsartan

A novel benzimidazole derivative, nstpbp5185, was discovered through in vitro and in vivo evaluations for antiplatelet activity. Thromaboxane receptor (TP) is important in vascular physiology, haemostasis and pathophysiological thrombosis. Nstpbp5185 concentration-dependently inhibited human platelet aggregation caused by collagen, arachidonic acid and U46619. Nstpbp5185 caused a right-shift of the concentration-response curve of U46619 and competitively inhibited the binding of 3H-SQ-29548 to TP receptor expressed on HEK-293 cells, with an IC50 of 0.1 µM, indicating that nstpbp5185 is a TP antagonist. In murine thrombosis models, nstpbp5185 significantly prolonged the latent period in triggering platelet plug formation in mesenteric and FeCl3-induced thrombi formation, and increased the survival rate in pulmonary embolism model with less bleeding than aspirin. This study suggests nstpbp5185, an orally selective anti-thrombotic agent, acting through blockade of TXA2 receptor, may be efficacious for prevention or treatment of pathologic thrombosis.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arachidonic Acid; Aspirin; Benzimidazoles; Blood Platelets; Calcium; Collagen; Humans; In Vitro Techniques; Male; Mice; Mice, Inbred ICR; Microvessels; P-Selectin; Platelet Aggregation; Pulmonary Embolism; Receptors, Thromboxane A2, Prostaglandin H2; Thrombosis; Thromboxane A2

Snake venom is a natural substance that contains numerous bioactive proteins and peptides, nearly all of which have been identified over the last several decades. In this study, we subjected snake venom to enzymatic hydrolysis to identify previously unreported bioactive peptides. The novel peptide ACH-11 with the sequence LTFPRIVFVLG was identified with both FXa inhibition and anti-platelet aggregation activities. ACH-11 inhibited the catalytic function of FXa towards its substrate S-2222 via a mixed model with a Ki value of 9.02 μM and inhibited platelet aggregation induced by ADP and U46619 in a dose-dependent manner. Furthermore, ACH-11 exhibited potent antithrombotic activity in vivo. It reduced paralysis and death in an acute pulmonary thrombosis model by 90% and attenuated thrombosis weight in an arterio-venous shunt thrombosis model by 57.91%, both at a dose of 3 mg/kg. Additionally, a tail cutting bleeding time assay revealed that ACH-11 did not prolong bleeding time in mice at a dose of 3 mg/kg. Together, our results reveal that ACH-11 is a novel antithrombotic peptide exhibiting both FXa inhibition and anti-platelet aggregation activities, with a low bleeding risk. We believe that it could be a candidate or lead compound for new antithrombotic drug development.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Agkistrodon; Amino Acid Sequence; Animals; Anticoagulants; Blood Coagulation Tests; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa Inhibitors; Female; Hydrolysis; Male; Mice; Molecular Weight; Peptides; Platelet Aggregation; Platelet Aggregation Inhibitors; Rabbits; Rats; Snake Venoms; Thrombosis

Flavonoids exert both anti-oxidant and anti-platelet activities in vitro and in vivo. Pentamethylquercetin (PMQ), a polymethoxylated flavone derivative, has been screened for anti-carcinogenic and cardioprotective effects. However, it is unclear whether PMQ has anti-thrombotic effects. In the present study, PMQ (20 mg/kg) significantly inhibited thrombus formation in the collagen- epinephrine- induced acute pulmonary thrombosis mouse model and the ferric chloride-induced carotid injury model. To explore the mechanism, we evaluated the effects of PMQ on platelet function. We found that PMQ inhibited platelet aggregation and granule secretion induced by low dose agonists, including ADP, collagen, thrombin and U46619. Biochemical analysis revealed that PMQ inhibited collagen-, thrombin- and U46619-induced activation of Syk, PLCγ2, Akt, GSK3β and Erk1/2. Therefore, we provide the first report to show that PMQ possesses anti-thrombotic activity in vivo and inhibited platelet function in vitro, suggesting that PMQ may represent a potential therapeutic candidate for the prevention or treatment of thrombotic disorders.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blood Platelets; Mice; Mice, Inbred C57BL; Platelet Aggregation Inhibitors; Quercetin; Thrombin; Thrombosis

RhoA is an important regulator of platelet responses downstream of Gα13 , yet we still know little about its regulation in platelets. Leukemia-associated Rho guanine-nucleotide exchange factor (GEF [LARG]), a RhoA GEF, is highly expressed in platelets and may constitute a major upstream activator of RhoA. To this end, it is important to determine the role of LARG in platelet function and thrombosis.. Using a platelet-specific gene knockout, we show that the absence of LARG results in a marked reduction in aggregation and dense-granule secretion in response to the thromboxane mimetic U46619 and proteinase-activated receptor 4-activating peptide, AYPGKF, but not to adenosine diphosphate. In a ferric chloride thrombosis model in vivo, this translated into a defect, under mild injury conditions. Importantly, agonist-induced RhoA activation was not affected by the absence of LARG, although basal activity was reduced, suggesting that LARG may play a housekeeper role in regulating constitutive RhoA activity.. LARG plays an important role in platelet function and thrombosis in vivo. However, although LARG may have a role in regulating the resting activation state of RhoA, its role in regulating platelet function may principally be through RhoA-independent pathways, possibly through other Rho family members.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Blood Platelets; Cell Degranulation; Chlorides; Crosses, Genetic; Ferric Compounds; Gene Knockout Techniques; Mice; Mice, Knockout; Oligopeptides; Organ Specificity; Platelet Activation; Platelet Aggregation; rho GTP-Binding Proteins; Rho Guanine Nucleotide Exchange Factors; rhoA GTP-Binding Protein; Thrombosis

Thromboxane A2 (TXA2) can induce the platelet aggregation and lead to thrombosis. This will cause the low-reflow phenomenon after ischemic stroke and aggravate the damage of brain issues. Therefore, it is potential to develop the drugs inhibiting TXA2 pathway to treat cerebral ischemia.. This study aims to prove the protective effect of N2 (4-(2-(1H-imidazol-1-yl) ethoxy)-3-methoxybenzoic acid) on focal cerebral ischemia and reperfusion injury through platelet aggregation inhibition.. Middle cerebral artery occlusion/reperfusion (MCAO/R) is used as the animal model. Neurological deficit score, Morris water maze, postural reflex test, Limb-use asymmetry test, infarct volume, and water content were performed to evaluate the protective effect of N2 in MCAO/R rats. 9, 11-dieoxy-11α, 9α-methanoepoxyprostaglandin F2α (U46619) or adenosine diphosphate (ADP) was used as the inducer of platelet aggregation.. N2 can improve the motor function, learning and memory ability in MCAO/R rats while reducing the infarct volume. N2 can inhibit TXA2 formation but promote PGI2, and can inhibit platelet aggregation induced by U46619 and ADP. Further, N2 inhibits thrombosis with a minor adverse effect of bleeding than Clopidogrel. In conclusion, N2 can produce the protective effect on MCAO/R brain injury through inhibiting TXA2 formation, platelet aggregation and thrombosis.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Arteriovenous Shunt, Surgical; Blood Coagulation; Brain; Brain Ischemia; Edema; Enzyme-Linked Immunosorbent Assay; Epoprostenol; Female; Imidazoles; Male; Maze Learning; Platelet Aggregation; Rats; Rats, Sprague-Dawley; Stroke; Thrombosis; Thromboxane A2; Vanillic Acid

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenine; Adenosine Triphosphate; Animals; Arachidonic Acid; Collagen; Cyclic GMP; Guanylate Cyclase; Humans; Iridoids; MAP Kinase Signaling System; Mice; Oxadiazoles; Phospholipase C gamma; Plant Extracts; Platelet Activation; Protein Kinase C; Quinoxalines; Thrombin; Thromboembolism; Thrombosis

The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function.. Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo.. We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa.. Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function may prove beneficial in the search for new antithrombotic therapies.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Arachidonic Acids; Blood Platelets; Calcium; Cannabis; Collagen; Dronabinol; Endocannabinoids; Fibrinogen; Humans; Integrin beta3; Intracellular Signaling Peptides and Proteins; Phosphorylation; Platelet Activation; Platelet Aggregation; Platelet Membrane Glycoprotein IIb; Polyunsaturated Alkamides; Protein-Tyrosine Kinases; Receptor, Cannabinoid, CB2; Receptor, PAR-1; Signal Transduction; Syk Kinase; Thrombosis

The effects of phosphoinositide-dependent protein kinase 1 (PDK1), a master kinase in the phosphoinositide 3-kinase/Akt pathway, on platelet activation are unknown. Accordingly, platelet-specific PDK1-deficient mice were characterized to elucidate the platelet-related function(s) of PDK1. We found that PDK1 deficiency caused mild thrombocytopenia. The aggregation of PDK1(-/-) platelets was diminished in response to low levels of thrombin, U46619, and adenosine 5'-diphosphate. Further results demonstrated that PDK1 regulates thrombin-induced platelet activation by affecting αIIbβ3-mediated outside-in signaling. This result provided an explanation for the diminished spreading of PDK1(-/-) platelets on immobilized fibrinogen (Fg) and the decreased rate of clot retraction in platelet-rich plasma (PRP) containing PDK1(-/-) platelets. PDK1 deficiency diminished agonist-induced Akt Ser473 phosphorylation and thoroughly abolished Akt Thr308 and Gsk3β Ser9 phosphorylation in response to agonist treatment and platelet spreading, respectively. A Gsk3β inhibitor fully restored the aggregation of PDK1(-/-) platelets in response to low levels of thrombin, normal spreading of PDK1(-/-) platelets on Fg, and normal clot retraction in PRP containing PDK1(-/-) platelets. Those results indicated that Gsk3β is one of the major downstream effectors of PDK1 in thrombin-induced platelet activation and αIIbβ3-mediated outside-in signaling. In addition, in vivo data demonstrated that PDK1 is an important regulator in arterial thrombosis formation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Arteries; Blood Platelets; Clot Retraction; Mice; Mice, Inbred C57BL; Mice, Knockout; Platelet Activation; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Thrombin; Thrombosis; Vasoconstrictor Agents

Platelets are critically important for primary haemostasis and the major players in thrombotic vascular occlusion. Platelets are activated by agonists, such as thrombin and collagen-related peptide as well as second-wave mediators including thromboxane A2 via different intracellular signaling pathways resulting in degranulation, aggregation and thrombus formation. Platelet activation is paralleled by phosphorylation and activation of p38 MAPK. The limited specificity of hitherto known p38 MAPK inhibitors precluded safe conclusions on the precise role of p38 MAPK in the regulation of platelet function. The present study examined the impact of Skepinone-L, a novel and highly selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), on platelet activation and thrombus formation.. Experiments were performed in freshly isolated human platelets. Protein phosphorylation was quantified by Western blotting, thromboxane B2 synthesis by enzyme immunoassay, ATP release by ChronoLume luciferin assay, cytosolic Ca(2+) concentration by Fura-2 fluorescence-measurements, platelet aggregation by a light transmissions measurement and in vitro thrombus formation by a flow chamber.. Skepinone-L (1 μM) virtually abrogated the phosphorylation of platelet p38 MAPK substrate Hsp27 following stimulation with CRP (1 μg/ml), thrombin (5 mU/ml) or thromboxane A2 analogue U-46619 (1 μM). Furthermore, Skepinone-L significantly blunted activation-dependent platelet secretion and aggregation following threshold concentrations of CRP, thrombin and thromboxane A2 analogue U-46619. Skepinone-L did not impair platelet Ca(2+) signaling but prevented agonist-induced thromboxane A2 synthesis through abrogation of p38 MAPK-dependent phosphorylation of platelet cytosolic phospholipase A2 (cPLA2). Skepinone-L further markedly blunted thrombus formation under low (500-s) and high (1700-s) arterial shear rates.. The present study discloses a powerful inhibiting effect of p38 MAPK-blocker Skepinone-L on platelet activation and thrombus formation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Blood Platelets; Calcium; Carrier Proteins; Dibenzocycloheptenes; Fura-2; HSP27 Heat-Shock Proteins; Humans; p38 Mitogen-Activated Protein Kinases; Peptides; Phospholipases A2; Phosphorylation; Platelet Activation; Platelet Aggregation; Protein Kinase Inhibitors; Shear Strength; Thrombin; Thrombosis; Thromboxane B2

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a tumor vascular disrupting agent under clinical trials as an adjacent antitumor agent. DMXAA is structurally similar to flavone-8-acetic acid (FAA), an old tumor vascular disrupting agent with antiplatelet and antithrombotic effects. In contrast to FAA, which causes bleeding in tumor patients, no bleeding has been reported in patients receiving DMXAA. Whether DMXAA also affects platelet function is not clear.. To determine the effects of DMXAA on platelet function and explore the underlying mechanisms.. DMXAA concentration-dependently inhibited human platelet aggregation and ATP release induced by U46619, arachidonic acid, ADP, collagen, or ristocetin. Furthermore, DMXAA inhibited phosphorylation of Erk1/2 and Akt downstream of thromboxane A2 signaling inhibition. DMXAA also inhibited human platelet phosphodiesterase. The antiplatelet effects were further confirmed using mice administered DMXAA intravenously. DMXAA dramatically inhibited thrombus formation in FeCl3 -injured mouse mesenteric arterial thrombus model and laser-injured mouse cremaster arteriole thrombus model. Notably, at a dose exhibiting antithrombotic effects similar to those of clopidogrel in mice, DMXAA did not significantly increase bleeding.. For the first time, we found that tumor vascular disrupting agent DMXAA has potent antiplatelet and antithrombotic effects without any bleeding diathesis. As DMXAA inhibits platelet activity with safe profile, DMXAA could be used as an efficacious and safe antiplatelet drug.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Animals; Antineoplastic Agents; Humans; MAP Kinase Signaling System; Mice; Phosphodiesterase Inhibitors; Phosphorylation; Platelet Activation; Platelet Aggregation Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Thrombosis; Thromboxane A2; Thromboxane-A Synthase; Xanthones

The purpose of this study is to investigate the potential in vivo antiplatelet and thromboprotective properties of the antihypertensive drug losartan in mice.. Aggregometry studies were performed on platelets obtained from mice administered losartan for 5 days, via tail vein to examine the ex vivo effects (dose dependence) of this agent and to select an appropriate dose for the in vivo studies. Next, the tail bleeding time test and the time for occlusion in a carotid artery injury thrombosis model (ferric chloride) were also performed to assess the in vivo effects of losartan treatment.. These data indicate that the antihypertensive agent losartan exerts dose-dependent inhibition of the thromboxane receptor-mediated (U46619/agonist)-induced platelet aggregation (ex vivo), whereas it produced no detectable effects on aggregation triggered by adenosine diphosphate or the thrombin receptor activating peptide 4. Findings from the in vivo analysis revealed that tail bleeding time of losartan-treated mice was not different from vehicle-treated mice. On the other hand, in the carotid artery injury thrombosis model, it was found that the losartan-treated mice had significantly longer time for occlusion in comparison with those treated with vehicle control.. These findings provide evidence that administration of the antihypertensive drug losartan into live mice produces thromboxane A(2) receptor-specific antiplatelet effects. Furthermore, interestingly, this antiplatelet activity appears to translate into thromboprotective properties, without resulting in a bleeding phenotype. Consequently, aside from its potential use as an antithrombotic agent, losartan's chemistry may provide a "blueprint" for designing or repurposing novel derivatives which may have the potential to serve as an antiplatelet and thromboprotective agents but are deprived of the usually concomitant bleeding adverse effects.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Antihypertensive Agents; Arachidonic Acid; Blood Coagulation; Carotid Artery Injuries; Losartan; Mice; Mice, Inbred C57BL; Platelet Aggregation; Platelet Aggregation Inhibitors; Receptors, Thrombin; Receptors, Thromboxane A2, Prostaglandin H2; Thrombosis

During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A(2), recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patient's platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye-loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y(1)/P2Y(12), TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donor's pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y(1) inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subject's platelet phenotype allowed prediction of blood function under flow, essential for identifying patient-specific risks, drug responses, and novel genotypes.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Blood Coagulation; Blood Platelets; Calcium; Crotalid Venoms; HEK293 Cells; Humans; Lectins, C-Type; Male; Microfluidic Analytical Techniques; Models, Biological; Platelet Activating Factor; Platelet Function Tests; Predictive Value of Tests; Receptors, Thromboxane; Reference Values; Signal Transduction; Thrombosis; Vasoconstrictor Agents

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

Atherosclerosis has an important inflammatory component. Macrophages accumulating in atherosclerotic arteries produce prostaglandin E(2) (PGE(2)), a main inflammatory mediator. Platelets express inhibitory receptors (EP(2), EP(4)) and a stimulatory receptor (EP(3)) for this prostanoid. Recently, it has been reported in ApoE(-/-) mice that PGE(2) accumulating in inflammatory atherosclerotic lesions might contribute to atherothrombosis after plaque rupture by activating platelet EP(3), and EP(3) blockade has been proposed to be a promising new approach in anti-thrombotic therapy. The aim of our investigation was to study the role of PGE(2) in human atherosclerotic plaques on human platelet function and thrombus formation. Plaque PGE(2) might either activate or inhibit platelets depending on stimulation of either EP(3) or EP(4), respectively. We found that the two EP(3)-antagonists AE5-599 (300 nM) and AE3-240 (300 nM) specifically and completely inhibited the synergistic effect of the EP(3)-agonist sulprostone on U46619-induced platelet aggregation in blood. However, these two EP(3)-antagonists neither inhibited atherosclerotic plaque-induced platelet aggregation, GPIIb/IIIa exposure, dense and alpha granule secretion in blood nor reduced plaque-induced platelet thrombus formation under arterial flow. The EP(4)-antagonist AE3-208 (1-3 μM) potentiated in combination with PGE(2) (1 μM) ADP-induced aggregation, demonstrating that PGE(2) enhances platelet aggregation when the inhibitory EP(4)-receptor is inactivated. However, plaque-induced platelet aggregation was not augmented after platelet pre-treatment with AE3-208, indicating that plaque PGE(2) does not stimulate the EP(4)-receptor. We found that PGE(2) was present in plaques only at very low levels (15 pg PGE(2)/mg plaque). We conclude that PGE(2) in human atherosclerotic lesions does not modulate (i.e. stimulate or inhibit) atherothrombosis in blood after plaque rupture.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Abortifacient Agents, Nonsteroidal; Animals; Apolipoproteins E; Blood Platelets; Carotid Stenosis; Dinoprostone; Female; Humans; Male; Mice; Mice, Knockout; Naphthalenes; Phenylbutyrates; Plaque, Atherosclerotic; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, Prostaglandin E, EP3 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Rupture, Spontaneous; Thrombosis; Vasoconstrictor Agents

Clinical use of selective inhibitors of cyclooxygenase (COX)-2 appears associated with increased risk of thrombotic events. This is often hypothesised to reflect reduction in anti-thrombotic prostanoids, notably PGI(2), formed by COX-2 present within endothelial cells. However, whether COX-2 is actually expressed to any significant extent within endothelial cells is controversial. Here we have tested the effects of acute inhibition of COX on platelet reactivity using a functional in vivo approach in mice.. A non-lethal model of platelet-driven thromboembolism in the mouse was used to assess the effects of aspirin (7 days orally as control) diclofenac (1 mg.kg(-1), i.v.) and parecoxib (0.5 mg.kg(-1), i.v.) on thrombus formation induced by collagen or the thromboxane (TX) A(2)-mimetic, U46619. The COX inhibitory profiles of the drugs were confirmed in mouse tissues ex vivo. Collagen and U46619 caused in vivo thrombus formation with the former, but not latter, sensitive to oral dosing with aspirin. Diclofenac inhibited COX-1 and COX-2 ex vivo and reduced thrombus formation in response to collagen, but not U46619. Parecoxib inhibited only COX-2 and had no effect upon thrombus formation caused by either agonist.. Inhibition of COX-1 by diclofenac or aspirin reduced thrombus formation induced by collagen, which is partly dependent upon platelet-derived TXA(2), but not that induced by U46619, which is independent of platelet TXA(2). These results are consistent with the model demonstrating the effects of COX-1 inhibition in platelets, but provide no support for the hypothesis that acute inhibition of COX-2 in the circulation increases thrombosis.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aspirin; Collagen; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Diclofenac; Disease Models, Animal; Isoxazoles; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Thrombosis

Platelet aggregation in the blood vessel causes thrombosis. Therefore, inhibitors of platelet aggregation promise to be preventive or therapeutic agents of various vascular diseases, including myocardial infarction and stroke. In the present study, we found that hericenone B had a strong anti-platelet activity and it might be a novel compound for antithrombotic therapy possessing a novel mechanism. Prior to this study, we examined anti-platelet aggregation activity of ethanol extracts of several species of mushrooms, and found that extract of Hericium erinaceus potently inhibited platelet aggregation induced by collagen. Therefore, we first fractionated the ethanol extract of H. erinaceus to identify the active substances. The anti-platelet activity of each fraction was determined using washed rabbit platelets. As a result, an active component was isolated and identified as hericenone B. Hericenone B selectively inhibited collagen-induced platelet aggregation, but it did not suppress the aggregation induced by U46619 (TXA₂ analogue), ADP, thrombin, or adrenaline. Furthermore, hericenone B did not inhibit arachidonic acid- or convulxin (GPVI agonist)-induced platelet aggregation. Therefore, hericenone B was considered to block collagen signaling from integrin α2/β1 to arachidonic acid release. Moreover, we found that collagen-induced aggregation was inhibited by hericenone B in human platelets, similar to in rabbit platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Arachidonic Acid; Basidiomycota; Biological Products; Blood Platelets; Collagen; Crotalid Venoms; Epinephrine; Humans; Indoles; Lectins, C-Type; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Rabbits; Signal Transduction; Thrombin; Thrombosis

TRA-418, a compound with both thromboxane A2 receptor (TP receptor) antagonistic and prostacyclin receptor (IP receptor) agonistic activities, was synthesised in our laboratory as a new antithrombotic agent. In this study, we examined the effects of TRA-418 on platelet-leukocyte interactions in human whole blood. Platelet-leukocyte interactions were induced by U-46619 in the presence of epinephrine (U-46619 + epinephrine) or with thrombin receptor agonist peptide 1-6 (TRAP). Platelet-leukocyte interactions were assessed by flow cytometry, with examination of both platelet-neutrophil and platelet-monocyte complexes. In a control experiment, the TP receptor antagonist SQ-29548 significantly inhibited the induction of platelet-leukocyte complexes by the combination of U-46619 and epinephrine, but not TRAP-induced formation of platelet-leukocyte complexes. Conversely, the IP receptor agonist beraprost sodium inhibited platelet-leukocyte complex formation induced by both methods, although the IC50 values of beraprost sodium for U-46619 + epinephrine were at least 10-fold greater than for TRAP. Under such conditions, TRA-418 inhibited both U-46619 + epinephrine-induced and TRAP-induced platelet-leukocyte complex formation in a concentration-dependent manner, in a similar range. These results suggest that TRA-418 exerts its inhibitory effects on platelet-leukocyte interactions by acting as a TP receptor antagonist as well as an IP receptor agonist in an additive or synergistic manner. These inhibitory effects of TRA-418 on formation of platelet-leukocyte complexes suggest the compound is beneficial effects as an antithrombotic agent.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Cell Communication; Cell Separation; Cells, Cultured; Epinephrine; Epoprostenol; Fatty Acids, Unsaturated; Flow Cytometry; Humans; Hydrazines; Leukocytes; Oxazines; Peptide Fragments; Platelet Activation; Receptors, Epoprostenol; Receptors, Thromboxane A2, Prostaglandin H2; Thrombosis

While proper platelet function is a vital component of hemostasis, their inappropriate activation contributes to thrombotic disorders. One pathway for platelet activation involves their synthesis of the lipid mediator thromboxane A₂ (TXA₂). Although TXA₂ acts by binding to a seven-transmembrane receptor (i.e., the prostanoid TP receptor) that participates in the genesis of thrombosis, currently, there are no antagonists available for clinical use. Since the only available drug targeting this pathway (aspirin) is associated with inherent limitations/serious side effects, developing prostanoid TP receptor antagonists is clearly warranted. To this end, we have previously employed the "repurposing old drugs for new uses" approach to identify prostanoid TP receptor antagonists and showed that the antidiabetic agent glybenclamide selectively inhibited human platelet prostanoid TP receptors (in vitro). On this basis, we hypothesized that glybenclamide exhibits in vivo antiplatelet potential, and therefore, may protect against thrombosis development. Using murine platelets, it was found that glybenclamide injections: 1) inhibited platelet aggregation induced by the prostanoid TP receptor agonist U46619 and the TXA₂ precursor arachidonic acid, under ex vivo experimental settings, concentration-dependently; 2) lacked any detectable effects on aggregation stimulated by ADP, or the thrombin receptor activating-peptide 4; 3) impaired hemostasis by prolonging tail bleeding time; and 4) delayed the development of occlusive thrombi in a carotid artery injury model. Taken together, these findings indicate that glybenclamide does indeed exert, ex vivo and in vivo, prostanoid TP receptor-dependent inhibitory effects on platelet function. Thus, glybenclamide has the potential to be applied in the management of thromboembolic disorders.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arachidonic Acid; Bleeding Time; Carotid Artery Thrombosis; Dose-Response Relationship, Drug; Drug Repositioning; Fibrinolytic Agents; Glyburide; Hemostasis; Hypoglycemic Agents; Mice; Mice, Inbred C57BL; Osmolar Concentration; Platelet Aggregation; Platelet Aggregation Inhibitors; Receptors, Thromboxane; Thromboembolism; Thrombosis; Time Factors

Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP(3)) and intracellular calcium mobilization suggesting inappropriate activation of PLCgamma2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ( -/- ) platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA(2) analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ( -/- ) mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCgamma2 activation in platelets and that this is critical for thrombus formation in vivo.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Blood Platelets; Calcium; Lectins, C-Type; Mice; Mice, Knockout; Phospholipase C gamma; Platelet Activation; Platelet Aggregation; Platelet Membrane Glycoproteins; Poly I-C; rac1 GTP-Binding Protein; Thrombosis

Platelets stably interact with collagen via glycoprotein (GP)VI and alpha2beta1integrin. With alpha2-null mice, we investigated the role of alpha2beta1 in thrombus formation and stability in vivo and in vitro. Using a FeCl(3)-induced thrombosis model, in arteries from alpha2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects, while the thromboxane A(2) analogue U46619 was borderline effective in suppressing the embolisation in alpha2-null mice. In vitro, perfusion of alpha2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from alpha2-null mice. We conclude that integrin alpha2beta1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A(2) release. The increased embolization in alpha2-null mice may argue against the use of alpha2beta1 integrin inhibitors for antithrombotic therapy.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aspirin; Chlorides; Collagen; Ferric Compounds; Integrin alpha2beta1; Mice; Mice, Knockout; Thromboembolism; Thrombosis; Thromboxane A2

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

The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in thrombus formation. We used p38alpha heterozygous (p38alpha+/-) mice and used ferric chloride (FeCl3)-induced carotid artery injury as a model of thrombus formation. The time to thrombotic occlusion induced by FeCl3 in p38alpha+/- mice was prolonged compared to that in wild-type (WT) mice. Platelets prepared from p38alpha+/- mice showed impairment of the aggregatory response to a low concentration of U46619, a thromboxane A2 analogue. Furthermore, platelets prepared from p38alpha+/- mice and activated by U46619 were poorly bound to fibrinogen compared with those from WT mice. Both the expression and activity of tissue factor induced by FeCl3 in WT mice were higher than those in p38alpha+/- mice. These results suggest that p38 plays an important role in thrombus formation by regulating platelet function and tissue factor activity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alleles; Animals; Blood Coagulation; Blood Platelets; Chlorides; Ferric Compounds; Fibrinogen; Heterozygote; Homozygote; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; p38 Mitogen-Activated Protein Kinases; Placenta; Platelet Adhesiveness; Protein Binding; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Thrombosis; Thromboxane A2; Time Factors; Vasoconstrictor Agents

The synthesis and biological activity of novel 1-phenylsulfonyl-4- phenylsulfonylaminopyrrolidine analogues are described. All compounds were produced through modification of the substituent formally corresponding to the 1,3-dioxane ring system and the omega-octenol side chain of thromboxane A(2) (TXA(2)), in reference to the structure of Daltroban. Several compounds were found to be potent TXA(2) receptor antagonists. Compound 51a was the most effective inhibitor of 9,11-epoxymethano PGH(2) (U-46619)-induced rat aortic strip contraction (IC(50)=0.48 nM).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta; Disease Models, Animal; Dogs; Guinea Pigs; Haplorhini; Humans; Male; Mice; Muscle Contraction; Muscle, Smooth, Vascular; Platelet Aggregation; Pulmonary Embolism; Pyrrolidines; Rabbits; Rats; Receptors, Thromboxane; Structure-Activity Relationship; Sulfonamides; Thrombosis; Vasoconstrictor Agents

We studied the antithrombotic activity of 2-acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), a novel nitric oxide (NO)-releasing aspirin derivative, in vivo in different animal models of platelet-dependent and independent pulmonary thromboembolism and compared it with that of aspirin. NCX 4016 protected mice from death induced by the intravenous (i.v.) injection of collagen plus epinephrine, of 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F(2alpha) (U46619) and of thrombin while aspirin was only active against collagen plus epinephrine. The drop in platelet count and number of lung emboli were reduced by NCX 4016 more effectively than aspirin. NCX 4016 protected mice also from mechanical pulmonary embolism (i.v. injection of hardened rat red blood cells) while aspirin was ineffective. In rabbits, NCX 4016 significantly reduced the accumulation of [111In]oxine-labeled platelets in the pulmonary vasculature induced by collagen and by thrombin while aspirin produced reductions which were significant only versus collagen. In conclusion, NCX 4016 exerts a more pronounced antithrombotic activity than aspirin in vivo in two different animal species, largely due to a deeper inhibitory effect on platelets. NCX 4016 may represent a better antithrombotic agent than aspirin.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aspirin; Disease Models, Animal; Dose-Response Relationship, Drug; Injections, Intravenous; Lung; Male; Mice; Nitroarginine; Platelet Aggregation Inhibitors; Platelet Count; Pulmonary Artery; Pulmonary Embolism; Rabbits; Thrombin; Thrombosis

This study was carried out to determine whether early failure of infrainguinal bypass grafts is associated with increased expression of platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors. A prospective correlation of preoperative platelet TXA2/PGH2 receptor-mediated activity with lower extremity graft patency was sought.. Twenty-five patients who underwent infrainguinal bypass surgery for limb salvage were studied at an inpatient academic tertiary referral center and Department of Veterans Affairs Medical Center. Outcome measures were primary graft patency rate at 3 months, platelet TXA2/PGH2 receptor activity by equilibrium binding with 125I-BOP, and aggregation to the TXA2-mimetic U46619.. Preoperative platelet TXA2/PGH2 receptor density was higher (Bmax, 3100 +/- 1300 vs 1500 +/- 1100 sites/platelet [mean +/- SD]; p = 0.004) in the five patients who had graft thrombosis within 3 months. The EC50 for U46619 was lower (26 +/- 6 nmol/L vs 57 +/- 30 nmol/L; p < 0.05) in these patients as well, confirming the functional effect of the increased receptor density. Early graft thrombosis was more likely in patients with a platelet TXA2/PGH2 receptor density greater than 3000 sites/platelet (odds ratio, 76; 95% confidence interval, 3.9 to 1500) or an EC50 for U46619 less than 30 nmol/L (odds ratio, 16; 95% confidence interval, 1.4 to 180).. Elevated platelet TXA2/PGH2 receptor levels and enhanced sensitivity of platelet aggregation to TXA2 predict early arterial graft thrombosis. Specific TXA2/PGH2 receptor antagonism may prevent one of the mechanisms that contributes to early graft occlusion.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aged; Blood Platelets; Female; Graft Occlusion, Vascular; Humans; Male; Peripheral Vascular Diseases; Platelet Aggregation; Prospective Studies; Prostaglandins H; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Thrombosis; Vascular Patency

An experimental model of acute thrombosis was developed in pentobarbital- anesthetized ferrets. A 10-min anodal electrical stimulation of 1 mA was delivered to the external surface of the carotid artery while measuring carotid blood flow (CBF). This produced an occlusive thrombus in all vehicle-treated ferrets within 41 +/- 3 min with an average weight of 8 +/- 1 mg (n = 7). These thrombi were enriched in both platelets and fibrin and were adherent at the site of transmural vascular injury as determined by light and electron microscopy. To determine the model's sensitivity to antiplatelet drugs, aspirin or a thromboxane (TxA2) receptor antagonist (ifetroban) were administered 15 min before electrical stimulation. Thrombus weight was reduced 58% by aspirin (10 mg/kg, i.v.) and 74% by ifetroban (1 mg/kg + 1 mg/kg per hr, i.v.). Both drugs also improved CBF and decreased vascular occlusion. Ferrets were more sensitive than rats to aspirin's inhibition of collagen-induced platelet aggregation as determined ex vivo in whole blood. Separate in vitro platelet aggregation studies revealed species differences in reactivity to U-46619 (TxA2 receptor agonist) and collagen in the order of human > ferret > rat, with relatively lesser variations in ADP responses. These studies identify the ferret as a useful species for evaluating antithrombotic drugs in a model in which aspirin is efficacious.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aspirin; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Carotid Arteries; Carotid Artery Injuries; Collagen; Disease Models, Animal; Electric Stimulation; Ferrets; Fibrin; Humans; In Vitro Techniques; Male; Microscopy, Electron; Microscopy, Electron, Scanning; Oxazoles; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Prothrombin Time; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Species Specificity; Thrombosis; Thromboxane A2; Vasoconstrictor Agents

We have developed a photochemical model to induce thrombotic occlusion of the guinea-pig femoral artery. Using this model, we investigated the effect of cholesterol feeding on arterial occlusion time in the guinea-pig. Animals were divided into two groups, one on standard diet and the other on standard diet containing 0.5% cholesterol for 3 weeks. The time for femoral artery occlusion was significantly shorter (p < 0.05) in cholesterol fed animals as compared to the control group. In vitro collagen-, U-46619- (a thromboxane A2 adenosine diphosphate analogue) and (ADP)-induced platelet aggregation responses in whole blood in cholesterol-fed animals were increased 13-, 10- and 4-fold, respectively. U-46619- and collagen-induced washed platelet aggregation responses were also significantly enhanced by cholesterol feeding (p < 0.01). Further, TXA2 generation by collagen-stimulated washed platelets in cholesterol-fed animals increased similar to the platelet aggregation responses. However, platelet-activating factor (PAF)-induced platelet aggregation in whole blood was relatively unaffected by cholesterol feeding. 11-dehydro TXB2 levels in plasma were increased significantly by cholesterol feeding. Our observations suggest that increased plasma TXA2 level and platelet aggregation response to TXA2 and stimulated TXA2 synthesis in platelets play a role in enhanced arterial occlusion in cholesterol fed guinea-pigs.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Arterial Occlusive Diseases; Cholesterol, Dietary; Collagen; Femoral Artery; Guinea Pigs; Male; Photochemistry; Platelet Activating Factor; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Thrombosis; Thromboxane A2; Time Factors

The purpose of this study was to examine whether the blockade of thromboxane A2 (TxA2)/prostaglandin H2 (PGH2) receptor by the selective TxA2/PGH2 receptor antagonist KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]-6,11- dihydrodibenz[b,e]oxepine-2-carboxylate monohydrate, CAS 127166-41-0) is effective in enhancing tissue-type plasminogen activator (tPA)-induced thrombolysis and preventing reocclusion in a model of femoral artery thrombosis in anesthetized dogs. The thrombus was formed by inserting a copper coil into the femoral artery. Sodium heparin (100 U/kg i.v.) was administered shortly after the formation of thrombus. All dogs received i.v. tPA at a dose of 20 micrograms/kg/min starting 60 min after the formation of the occlusive thrombus for up to 60 min if necessary, to achieve reperfusion. After 30 min of thrombotic occlusion, the animals received vehicle (Group I, controls, n = 9) or KW-3635 (Group II, 0.3 mg/kg bolus i.v. + 0.3 mg/kg/h infusion, n = 9; Group III, 1 mg/kg bolus i.v. + 1 mg/kg/h infusion, n = 9) and the infusion of either vehicle or KW-3635 was continued thereafter throughout the experiment. The time to reperfusion in Group I was 37.3 +/- 5.2 min, while those in Group II and Group III were 25.3 +/- 6.2 min (p greater than 0.05) and 17.3 +/- 3.1 min (p less than 0.05), respectively. Reocclusion occurred within 4 h in 100% of Group I, whereas the incidence of reocclusion was reduced to 67% in Group II and to 0% in Group III. These data suggest that endogenous TxA2 generation is involved in lysis and rethrombosis during thrombolytic therapy by tPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arterial Occlusive Diseases; Benzimidazoles; Benzoxepins; Dogs; Female; Femoral Artery; Fibrinolytic Agents; Male; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Recurrence; Thrombosis; Thromboxane A2; Tissue Plasminogen Activator

We studied the effects of thromboxane-receptor antagonism and thromboxane synthetase inhibition in a thrombotic model of sudden death in mice. Intravenous injection of arachidonic acid (AA; 80 mg/kg) or the prostaglandin-endoperoxide analog U-46,619 (2.3 mg/kg) results in sudden death in approximately 90% of the animals. Pretreatment with the thromboxane receptor antagonist SQ-29,548 (0.3-10 mg/kg) protects dose-dependently against AA and U-46,619-induced sudden death. In contrast, CGS-13,080, a thromboxane synthetase inhibitor, shows a dose-dependent beneficial effect in AA-induced sudden death only. Although PTA2 has partial thromboxane agonistic properties in the rabbit, it protected the mice against AA-induced sudden death, thus demonstrating TxA2 antagonistic properties in this species. These data emphasize the importance of thromboxane A2 as a major mediator of arachidonic acid-induced sudden death and the effectiveness of thromboxane-receptor antagonists in endoperoxide-induced sudden death.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Bicyclic Monoterpenes; Bridged Bicyclo Compounds, Heterocyclic; Death, Sudden; Fatty Acids, Unsaturated; Fibrinolytic Agents; Hydrazines; Imidazoles; Injections, Intravenous; Male; Mice; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Pyridines; Receptors, Cell Surface; Receptors, Prostaglandin; Receptors, Thromboxane; Thrombosis; Thromboxane A2; Thromboxane-A Synthase

BM 13.177 (4-[2-(benzenesulfonamido)-ethyl]-phenoxyacetic acid) is a representative of a new class of sulfonamidophenylcarboxylic acids which possess platelet-inhibitory and anti-thrombotic activity and inhibits the contraction of rabbit aorta stimulated by PG endoperoxides and TXA2. BM 13.177 5 mg/kg body weight p.o. protected rabbits from arachidonate-induced sudden death and greater than or equal to 10 mg/kg dose-dependently reduced the experimental thrombus formation induced in the rabbit aorta by perivascular administration of silver nitrate. In guinea-pigs, the collagen-induced bronchoconstriction was inhibited in a dose- and time-dependent fashion. The formation of TXA2 and the TXA2-induced platelet aggregation and smooth muscle contraction are probably crucial events in these experimental models. The protective effect of BM 13.177 may, therefore, be due to the TXA2-antagonizing effect of BM 13.177, which has been conclusively demonstrated in human platelets (PATSCHEKE and STEGMEIER, Thrombosis Res., 33, 277-288 (1984). The antagonism of TXA2 is supported by the observation that BM 13.177 also specifically inhibits the contraction of isolated arterial strips from rabbits which were stimulated with the thromboxane A2 mimetic U 46619. Schild-plot with a slope close to unity suggests a competitive type of antagonism. BM 13.177 exhibited neither anti-inflammatory nor ulcer-inducing activity of cyclooxygenase inhibitors. Furthermore it did not block the TXB2 formation in spontaneously clotting blood from rabbits and did not inhibit the release of prostacyclin-like activity from rabbit aortas. The lack of toxicological effects in long-term toxicity studies in rat and dog, together with the absence of objective and subjective side effects in the first human studies have encouraged us to initiate clinical trials in order to evaluate the therapeutic benefit of this new approach in humans.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Epoprostenol; Female; Fibrinolytic Agents; Guinea Pigs; Male; Muscle Contraction; Muscle, Smooth, Vascular; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Rabbits; Sulfonamides; Thrombosis; Thromboxane A2; Thromboxane B2; Thromboxanes

The effects of (i) the exogenous arachidonic acid (AA), (ii) stable prostaglandin endoperoxide analogue--U46619, and (iii) cyclooxygenase inhibitor--aspirin on the interaction of platelets with a surface coated with fibrillar calf skin collagen were studied using scanning electron microscopy. AA and U46619 stimulate massive spreading of platelets (on the collagen substrate and formation of surface-bound multilayer (thrombi-like) aggregates. The stimulation of spreading and formation of thrombi-like aggregates by AA correlate with the thromboxane A2 (TXA2) synthesis in platelets. Unlike AA, U46619 induces these processes without transformation into TXA2 and stimulation of its synthesis in platelets. Cyclooxygenase inhibitor--aspirin prevents the AA-induced platelet spreading, formation of the surface-bound thrombi-like aggregates, and TXA2 synthesis. In the absence of soluble platelet inducers, aspirin inhibits the substrate-induced spreading, but doesn't affect the initial attachment of nonactivated platelets to the collagen substrate.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Arachidonic Acid; Arachidonic Acids; Aspirin; Blood Platelets; Collagen; Humans; Microscopy, Electron, Scanning; Platelet Aggregation; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Thrombosis; Thromboxane A2; Thromboxanes