Protein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in a resting state. Here we show that in thrombin-and collagen-stimulated platelets, PKA is activated by cAMP-independent mechanisms involving dissociation of the catalytic subunit of PKA (PKAc) from an NFB-IB␣-PKAc complex. We demonstrate mRNA and protein expression for most of the NFB family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with IB␣, and conversely, IB␣ was also co-immunoprecipitated with PKAc. This interaction was significantly reduced in thrombin-and collagen-stimulated platelets. Stimulation of platelets with thrombin-or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of IB␣, disruption of an IB␣-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkB␣ phosphorylation, PKAIkB␣ dissociation, and VASP phosphorylation, and potentiated integrin ␣IIb␤3 activation and the early phase of platelet aggregation. We conclude that thrombin and collagen not only cause platelet activation but also appear to fine-tune this response by initiating downstream NFB-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.Platelets are small anucleate cells derived from megakaryocytes in the bone marrow, in a process in which megakaryocyte cytoplasmic extensions into microvessels are sheared from their transendothelial stems by flowing blood (1-2). Platelets play a key role in the normal homeostatic process through their ability to rapidly adhere to activated and/or injured endothelium and subendothelial matrix proteins (platelet adhesion), and to other activated platelets (platelet aggregation). Many factors bind to specific platelet receptors and regulate signaling pathways, which promote or inhibit platelet adhesion, aggregation, and secretion. In vivo, circulating platelets are continually exposed to a variety of activating factors including collagen, fibrinogen, ADP, von Willebrand Factor (vWF), thrombin, and thromboxane (3-5), as well as inhibitory factors such as endothelial-derived nitric oxide (NO), prostacyclin (PG-I 2 ), and ADPase (3, 5-6). A strong equilibrium between the two opposing processes of platelet stimulation and inhibition is thought to be essential for normal platelet and vascular function. An impairment of this equilibrium will promote either thrombotic or bleeding disorders.In the initial steps of platelet activation, the platelet receptor glycoproteins (GP) 3 1b and GPVI interact with extracellular matrix (ECM) proteins, causing platelets to tether and roll on the injured endothelium or subendothelial ECM (5). Stimulation of these receptors triggers intracellular signaling cascades that activate integrin ␣IIb␤3 and induce the release of secondary mediators like A...

p38 MAP kinase in human platelets is activated by platelet agonists including thrombin, thromboxane A 2 (TxA 2 ), ADP, and others. However, both upstream mechanisms of p38 MAP kinase activation, and their downstream sequelae, are presently controversial and essentially unclear. Certain studies report sequential activation of cGMP-dependent protein kinase (PKG) and p38/ERK pathways by platelet agonists, leading to integrin activation and secretion, whereas others establish an essential role of Src/ERKmediated TxA 2 generation for fibrinogen receptor activation in human platelets. Here, we show that ADP secreted from platelet-dense granules, and subsequent activation of P2Y 12 receptors, as well as TxA 2 release are important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation did not significantly contribute to calcium mobilization, P-selectin expression, ␣IIb␤3 integrin activation, and aggregation of human platelets in response to thrombin. Finally IntroductionIn vivo, circulating platelets are continually exposed to both adhesive and/or activating factors (fibrinogen, ADP, von Willebrand factor [VWF], thrombin, TxA 2 , etc), as well as inhibitory factors such as endothelium-derived nitric oxide (NO), prostacyclin (PG-I 2 ), and ADPase. 1 Most of these activating and inhibitory molecules bind to specific platelet receptors and stimulate signaling pathways that promote or inhibit platelet adhesion, aggregation, and secretion. A central role among platelet-activating factors is played by ADP, which induces multiple platelet responses and potentiates platelet aggregation caused by other agonists. 2,3 ADP is released from platelet-dense granules upon activation by agonists such as thrombin and collagen, and binds to purinergic receptors (P2Y 1 , P2Y 12 , P2X 1 ) to reinforce platelet aggregation and thrombus formation. 2,[4][5][6] Recently, new mechanisms for agonist-induced platelet activation and secretion were proposed to involve sequential activation of cGMP-dependent protein kinase (PKG)/p38/integrin or Akt/ eNOS/sGC/cGMP/PKG secretion, respectively, 7-9 whereas others indicate an essential role of Src/ERK-mediated TxA 2 generation for fibrinogen receptor activation in human platelets. 10 We 11,12 and others 10,13 were unable to reproduce the reported ERK activation by PKG. However, both the upstream mechanisms of p38 activation, and their downstream effects, are presently controversial and unclear. Here, we show that ADP secreted from platelet-dense granules, and subsequent activation of P2Y 12 receptors, as well as TxA 2 secretion are important mediators upstream of thrombin-evoked p38 activation. Furthermore, PKG activation does not stimulate, but rather inhibits, thrombin-evoked p38 activation, which alone has no significant effect on platelet stimulation/aggregation. Materials and methodsAnalysis of P-selectin expression, ␣IIb␤3 activation, aggregation, and intracellular calcium measurement Platelets were isolated from whole blood obtained from healthy volunteer...

AimPrimary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet.Materials and MethodsIn HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes.ResultsIn HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished.ConclusionsPTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement.