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

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

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

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

Recently, mice made deficient in growth arrest-specific gene 6 product (Gas6) or in which Gas6 gene expression was inhibited were shown to have platelet dysfunction and to be less susceptible to thrombosis. The aim of this study was to define and characterize the relevant Gas6 receptor or receptors involved in platelet function.. Using RT-PCR and Western blot analysis we found that mer was the predominantly expressed subtype in mouse and human platelets, whereas axl and rse were not detected. We generated mer-deficient mice by targeted disruption of the mer receptor gene. Platelets derived from mer-deficient mice had decreased platelet aggregation in responses to low concentrations of collagen, U46619, and PAR4 thrombin receptor agonist peptide in vitro. However, the response to ADP was not different from wild-type platelets. Knockout of the mer gene protected mice from collagen/epinephrine-induced pulmonary thromoembolism and inhibited ferric chloride-induced thrombosis in vivo. Tail bleeding times, coagulation parameters, and peripheral blood cell counts in mer-deficient mice were similar to wild-type mice.. Our data provide the first evidence that mer, presumably through activation by its ligand Gas6, participates in regulation of platelet function in vitro and platelet-dependent thrombosis in vivo.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Axl Receptor Tyrosine Kinase; Blood Coagulation Tests; Blood Platelets; Blotting, Western; c-Mer Tyrosine Kinase; Chlorides; Collagen; Epinephrine; Female; Ferric Compounds; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligopeptides; Oncogene Proteins; Platelet Aggregation; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptors, Thrombin; Reverse Transcriptase Polymerase Chain Reaction; Thromboembolism

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