piperidines and arginyl-glycyl-aspartyl-serine

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA(2) metabolism ETYA affect both these parameters. Thus, we aimed to define the role of p38MAPK/cytosolic phospholipase A(2) (cPLA(2)) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA(2) activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B(2) formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 µM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover, the incubation of platelets with 2-AG led to the phosphorylation of cPLA(2) and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK, abolished both cPLA(2) phosphorylation and activation. In addition SR141716 strongly impaired cPLA(2) phosphorylation, arachidonic acid release and thromboxane B(2) formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion, data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA(2) pathway could play a relevant role in this complicated process.

Topics: Apyrase; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Endocannabinoids; Glycerides; Humans; Imidazoles; In Vitro Techniques; Indomethacin; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Phospholipases A2, Cytosolic; Phosphorylation; Piperidines; Pyrazoles; Pyridines; Reactive Oxygen Species; Rimonabant; Signal Transduction

The pharmacological properties of YM-57029 [4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino]acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A ((S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 microg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Benzamidines; Biotinylation; Bleeding Time; Blood Platelets; Collagen; Cricetinae; Dogs; Dose-Response Relationship, Drug; Fibrinogen; Fibrinolytic Agents; Guinea Pigs; Haplorhini; Humans; Mice; Microspheres; Oligopeptides; Piperazines; Piperidines; Platelet Adhesiveness; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Rabbits; Rats; Stress, Mechanical

Topics: Animals; Benzodiazepines; Caspase 3; Caspases; Cells, Cultured; Coumarins; Enzyme Activation; Fibrinolytic Agents; Heart; Myocardium; Oligopeptides; Piperidines; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Rats; Rats, Sprague-Dawley

In this study, we characterised the mechanisms of Rac GTPase activation in human platelets stimulated by two physiological agonists, either thrombin, acting through membrane receptors coupled to heterotrimeric G-proteins, or collagen which is known to mobilise a tyrosine kinase-dependent pathway. Both agonists induced a rapid activation of Rac that was not significantly affected by the inhibition of integrin alpha(IIb)beta(3) engagement. Using pharmacological inhibitors, we found that phospholipase C activation and calcium mobilisation were essential for platelet Rac activation by either thrombin or collagen whereas protein kinase C inhibition was without effect. In contrast to Rac, Cdc42 activation was independent of phospholipase C activation, indicating that the two GTPases are differently regulated. We also found that phosphoinositide 3-kinase was not required for Rac activation in response to thrombin but was involved in its activation by collagen.

Topics: Benzylamines; Blood Platelets; Calcium; cdc42 GTP-Binding Protein; Cells, Cultured; Chromones; Collagen; Enzyme Activation; Enzyme Inhibitors; Epoprostenol; Estrenes; Humans; Indoles; Morpholines; Oligopeptides; p21-Activated Kinases; Phosphatidylinositol 3-Kinases; Piperidines; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Kinase C; Protein Serine-Threonine Kinases; Pyrrolidinones; rac GTP-Binding Proteins; Thiazoles; Thrombin; Type C Phospholipases