sq-29548 and arginyl-glycyl-aspartyl-serine

Phospholipase D (PLD), which hydrolyzes phosphatidylcholine to phosphatidic acid (PA) and choline, is present in human platelets. Thrombin and other agonists have been shown to activate PLD but the precise mechanisms of activation and PLDs role in platelet activation remains unclear. We measured thrombin-stimulated PLD activity in platelets as formation of phosphatidylethanol. Since no specific PLD inhibitors exist, we investigated possible roles for PLD in platelets by correlating PLD activity with platelet responses such as thrombin-mediated secretion and F-actin formation (part of platelet shape change). Extracellular Ca2+ potentiated thrombin-stimulated PLD, but did not stimulate PLD in the absence of thrombin. Thrombin-induced PLD activity was enhanced by secreted ADP and binding of fibrinogen to its receptors. In contrast to others, we also found a basal PLD activity. Comparison of time courses and dose responses of platelets with PLD showed many points of correlation between PLD activation and lysosomal secretion and F-actin formation. The finding of different PLD activities suggested that different PLD isoenzymes exist in platelets as reported for other cells. Here we present evidence for the presence of both PLD1 and PLD2 in platelets by use of specific antibodies with immunoblotting and immunohistochemistry. Both isoforms were randomly localized in resting platelets, but became rapidly translocated to the proximity of the plasma membrane upon thrombin stimulation, thus indicating a role for PLD in platelet activation.

Topics: Actins; Adenosine Diphosphate; Antibodies, Monoclonal; Autocrine Communication; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cell Membrane; Fatty Acids, Unsaturated; Glycerophospholipids; Humans; Hydrazines; Isoenzymes; Lysosomes; Oligopeptides; Phospholipase D; Platelet Activation; Platelet Aggregation Inhibitors; Thrombin

Expression of tissue factor (TF) by activated monocytes may initiate thrombotic episodes associated with diseases, such as thrombosis and atherosclerosis. In this study, steps in the regulatory pathways of lipopolysaccharide (LPS)-induced monocyte TF activity and released TNF-alpha in human whole blood were probed for using an array of inhibitors, comprising specific inhibitors of cytosolic phospholipase A(2) (PLA(2)) (AACOCF(3)), secretory PLA(2) (SB-203347), protein kinase (PK) (staurosporine), PKC (GF-109203; BIM), and serine protease (Pefabloc SC), antagonists of thromboxane prostanoid (TP) receptor (R) (SQ-29548), platelet activating factor (PAF) R (BN-52021), leukotriene B(4) R (SC-41930), serotonin R (cyproheptadine), fibronectin/fibrinogen R (RGDS), and finally, creatine phosphate/creatine phosphokinase (CP/CPK) which removes ADP. Whereas when added alone neither of these agents significantly inhibited LPS-induced TF or TNF-alpha, when presented as a reference cocktail comprising all the agents, TF activity and TNF-alpha were reduced by 77% and 49%, respectively. By subsequently testing a series of incomplete inhibitory cocktails equal to the reference except for deleted single agents or combinations of two or three active agents, the inhibitory effect of the reference cocktail could be shown to depend on the presence of the protease inhibitor and the thromboxane A(2) and PAF antagonists.

Topics: Arachidonic Acids; Benzopyrans; Bridged Bicyclo Compounds, Heterocyclic; Creatine Kinase; Cyproheptadine; Diterpenes; Enzyme Inhibitors; Escherichia coli; Fatty Acids, Unsaturated; Ginkgolides; Humans; Hydrazines; Indoles; Lactones; Lipopolysaccharides; Maleimides; Monocytes; Oligopeptides; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Thromboxane; Sulfonamides; Sulfones; Tumor Necrosis Factor-alpha

Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3).

Topics: Adenosine Diphosphate; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Collagen; Creatine Kinase; Cyproheptadine; Diterpenes; Drug Synergism; Fatty Acids, Unsaturated; Ginkgolides; Humans; Hydrazines; In Vitro Techniques; Lactones; Oligopeptides; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphocreatine; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Thrombin

There is mounting evidence that lipid peroxides contribute to pathophysiological processes and can modulate cellular functions. The aim of the present study was to investigate the effects of lipid hydroperoxides on platelet aggregation and arachidonic acid (AA) metabolism. Human platelets, isolated from plasma, were incubated with subthreshold (i.e. non-aggregating) concentrations of AA in the absence or presence of hydroperoxyeicosatetraenoic acids (HPETEs). Although HPETEs alone had no effect on platelet function, HPETEs induced the aggregation of platelets co-incubated with non-aggregating concentrations of AA, HPETEs being more potent than non-eicosanoid peroxides. The priming effect of HPETEs on platelet aggregation was associated with an increased formation of cyclo-oxygenase metabolites, in particular thromboxane A2, and was abolished by aspirin, suggesting an activation of cyclo-oxygenase by HPETEs. It was not receptor-mediated because the 12-HPETE-induced enhancement of AA metabolism was sustained in the presence of SQ29, 548 or RGDS, which blocked the aggregation. These results indicate that physiologically relevant concentrations of HPETEs potentiate platelet aggregation, which appears to be mediated via a stimulation of cyclo-oxygenase activity.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Aspirin; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Deferoxamine; Enzyme Activation; Fatty Acids, Unsaturated; Humans; Hydrazines; Hydrogen Peroxide; Leukotrienes; Lipid Peroxides; Oligopeptides; Peroxides; Platelet Aggregation; Prostaglandin-Endoperoxide Synthases; tert-Butylhydroperoxide; Thromboxane B2; Vitamin E

Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 micromol/L adenosine-diphosphate (ADP) in a dose dependent fashion. The enhancement was not affected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 micromol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 micromol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 micron.mL),. thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin-induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL-3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.

Topics: Adenosine Diphosphate; Blood Viscosity; Bridged Bicyclo Compounds, Heterocyclic; Collagen; Drug Synergism; Epinephrine; Fatty Acids, Unsaturated; Hematopoietic Cell Growth Factors; Hirudins; Humans; Hydrazines; Interleukins; Oligopeptides; Platelet Aggregation; Recombinant Proteins; Serotonin; Stress, Mechanical; Thrombin; Thrombopoietin; Vasopressins