Bacterial LPS induces rapid thrombocytopenia, hypotension, and sepsis. Although growing evidence suggests that platelet activation plays a critical role in LPS-induced thrombocytopenia and tissue damage, the mechanism of LPS-mediated platelet activation is unclear. In this study, we show that LPS stimulates platelet secretion of dense and α granules as indicated by ATP release and P-selectin expression, and thus enhances platelet activation induced by low concentrations of platelet agonists. Platelets express components of the LPS receptor-signaling complex, including TLR (TLR4), CD14, MD2, and MyD88, and the effect of LPS on platelet activation was abolished by an anti-TLR4-blocking Ab or TLR4 knockout, suggesting that the effect of LPS on platelet aggregation requires the TLR4 pathway. Furthermore, LPS-potentiated thrombin- and collagen-induced platelet aggregation and FeCl3-induced thrombus formation were abolished in MyD88 knockout mice. LPS also induced cGMP elevation and the stimulatory effect of LPS on platelet aggregation was abolished by inhibitors of NO synthase and the cGMP-dependent protein kinase (PKG). LPS-induced cGMP elevation was inhibited by an anti-TLR4 Ab or by TLR4 deficiency, suggesting that activation of the cGMP/protein kinase G pathway by LPS involves the TLR4 pathway. Taken together, our data indicate that LPS stimulates platelet secretion and potentiates platelet aggregation through a TLR4/MyD88- and cGMP/PKG-dependent pathway.

Abstract-Adenosine has been reported to stimulate or inhibit the release of angiogenic factors depending on the cell type examined. To test the hypothesis that differential expression of adenosine receptor subtypes contributes to endothelial cell heterogeneity, we studied microvascular (HMEC-1) and umbilical vein (HUVEC) human endothelial cells. Based on mRNA level and stimulation of adenylate cyclase, we found that HUVECs preferentially express A 2A adenosine receptors and HMEC-1 preferentially express A 2B receptors. Neither cells expressed A 1 or A 3 receptors. The nonselective adenosine agonist 5Ј-N-ethylcarboxamidoadenosine (NECA) increased expression of interleukin-8 (IL-8), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in HMEC-1, but had no effect in HUVECs. In contrast, the selective A 2A agonist 2-p-(2-carboxyethyl)phenylethylamino-NECA (CGS 21680) had no effect on expression of these angiogenic factors. Key Words: adenosine receptors Ⅲ vascular endothelium Ⅲ angiogenesis Ⅲ vascular endothelial growth factor Ⅲ interleukin-8 T he purine nucleoside adenosine is an intermediate catabolite of adenine nucleotides. Adenosine serves as an autocoid in situations when oxygen supply is decreased or energy consumption is increased. Under these conditions, adenosine is released into the extracellular space and signals to restore the balance between local energy requirements and energy supply. Endothelial cells interact with adenosine mechanisms in many different ways. Endothelial cells are known to have a very active adenosine metabolism, characterized by a large capacity for uptake and release of the nucleoside, 1,2 and can be an important source of adenosine released during ischemia. 3 Conversely, adenosine may modulate endothelial function via activation of cell membrane receptors. The precise nature of the interaction between adenosine receptor subtypes and endothelial cells and their role in the regulation of endothelial function is not completely understood.Adenosine receptors belong to the G protein-coupled 7 transmembrane superfamily of cell surface receptors and include A 1 , A 2A , A 2B , and A 3 subtypes. Endothelial cells are known to express adenosine receptors, but there are conflicting reports on the presence and the role of specific adenosine receptor subtypes. For example, human umbilical vein endothelial cells (HUVECs) were reported to express either A 1 , 4 A 2A , 5,6 A 2B , 7 or A 3 6 adenosine receptors, depending on the functional end-point studied and pharmacological tools used. Coexpression of more than one adenosine receptor subtype has been reported also in endothelial cells 8,9 ; it is not clear, however, if and how coexpressed receptors interact. Furthermore, endothelial cells from different blood vessels are heterogenous, and it is possible that diverse endothelial cells show differential expression of adenosine receptor subtypes.The functional role of adenosine receptors in endothelial cells also remains unclear. Adenosine-induced vasodilation h...

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT 4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT 7 receptor. Expression of 5-HT 7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT 7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT 7 receptors significantly increased neurite length, whereas stimulation of 5-HT 4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT 7 R/G12 and 5-HT 4 R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

Integrins mediate cell adhesion to the extracellular matrix and transmit signals within the cell that stimulate cell spreading, retraction, migration, and proliferation. The mechanism of integrin outside-in signaling has been unclear. We found that the heterotrimeric guanine nucleotide-binding protein (G protein), Gα13, directly bound to the integrin β3 cytoplasmic domain, and that Gα13-integrin interaction was promoted by ligand binding to the integrin αIIbβ3 and by guanosine triphosphate (GTP)-loading of Gα13. Interference of Gα13 expression or a myristoylated fragment of Gα13 that inhibited interaction of αIIbβ3 with Gα13 diminished activation of protein kinase c-Src and stimulated the small GTPase RhoA, consequently inhibiting cell spreading and accelerating cell retraction. We conclude that integrins are non-canonical Gα13-coupled receptors that provide a mechanism for dynamic regulation of RhoA.

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