CalDAG-GEFI is at the nexus of calcium-dependent platelet activation
ABSTRACT The importance of the second messengers calcium (Ca(2+)) and diacylglycerol (DAG) in platelet signal transduction was established more than 30 years ago. Whereas protein kinase C (PKC) family members were discovered as the targets of DAG, little is known about the molecular identity of the main Ca(2+) sensor(s). We here identify Ca(2+) and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a critical molecule in Ca(2+)-dependent platelet activation. CalDAG-GEFI, through activation of the small GTPase Rap1, directly triggers integrin activation and extracellular signal-regulated kinase-dependent thromboxane A(2) (TxA(2)) release. CalDAG-GEFI-dependent TxA(2) generation provides crucial feedback for PKC activation and granule release, particularly at threshold agonist concentrations. PKC/P2Y12 signaling in turn mediates a second wave of Rap1 activation, necessary for sustained platelet activation and thrombus stabilization. Our results lead to a revised model for platelet activation that establishes one molecule, CalDAG-GEFI, at the nexus of Ca(2+)-induced integrin activation, TxA(2) generation, and granule release. The preferential activation of CalDAG-GEFI over PKC downstream of phospholipase C activation, and the different kinetics of CalDAG-GEFI- and PKC/P2Y12-mediated Rap1 activation demonstrate an unexpected complexity to the platelet activation process, and they challenge the current model that DAG/PKC-dependent signaling events are crucial for the initiation of platelet adhesion.
- SourceAvailable from: Ilaria Canobbio
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- "The dissection of the molecular mechanism for PI3K contribution to integrin a IIb b 3 inside-out activation pointed to a role for this enzyme in the regulation of the small GTPase Rap1b. The activation of Rap1b induced by platelet stimulation by different agonists largely depends on Gi-signaling stimulated by the binding of ADP, released from platelet granules, to the P2Y12 receptor (Crittenden et al., 2004; Lova et al., 2003, 2002; Stefanini et al., 2009; Woulfe et al., 2002). The pharmacological inhibition of PI3K suppressed Gi-induced Rap1b activation (Lova et al., 2003; Woulfe et al., 2002). "
ABSTRACT: Blood platelets are anucleated circulating cells that play a critical role in hemostasis and are also implicated in arterial thrombosis, a major cause of death worldwide. The biological function of platelets strongly relies in their reactiveness to a variety of extracellular agonists that regulate their adhesion to extracellular matrix at the site of vascular injury and their ability to form rapidly growing cell aggregates. Among the membrane receptors expressed on the cell surface, integrins are crucial for both platelet activation, adhesion and aggregation. Integrin affinity for specific ligands is regulated by intracellular signaling pathways activated in stimulated platelets, and, once engaged, integrins themselves generate and propagate signals inside the cells to reinforce and consolidate platelet response and thrombus formation. Phosphatidylinositol 3-Kinases (PI3Ks) have emerged as crucial players in platelet activation, and they are directly implicated in the regulation of integrin function. This review will discuss the contribution of PI3Ks in platelet integrin signaling, focusing on the role of specific members of class I PI3Ks and their downstream effector Akt on both integrin inside-out and outside-in signaling. The contribution of the PI3K/Akt pathways stimulated by integrin engagement and platelet activation in thrombus formation and stabilization will also be discussed in order to highlight the possibility to target these enzymes in effective anti-thrombotic therapeutic strategies. Copyright © 2015 Elsevier Ltd. All rights reserved.Advances in Biological Regulation 06/2015; DOI:10.1016/j.jbior.2015.06.001
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- "Increasing concentrations of either WWL91 (Figure 5A) or JW480 (Figure 5B) potently blocked RAP1 activation in response to collagen at concentrations similar to the IC 50 for JW480-dependent inhibition of aggregation. PKC activation is the second major node responsible for aIIbb3 activation in platelets (Cifuni et al., 2008; Stefanini et al., 2009). As with RAP1, JW480 blocked PKC activation in response to collagen (Figure 5C). "
ABSTRACT: A comprehensive knowledge of the platelet proteome is necessary for understanding thrombosis and for envisioning antiplatelet therapies. To discover other biochemical pathways in human platelets, we screened platelets with a carbamate library designed to interrogate the serine hydrolase subproteome and used competitive activity-based protein profiling to map the targets of active carbamates. We identified an inhibitor that targets arylacetamide deacetylase-like 1 (AADACL1), a lipid deacetylase originally identified in invasive cancers. Using this compound, along with highly selective second-generation inhibitors of AADACL1, metabolomics, and RNA interference, we show that AADACL1 regulates platelet aggregation, thrombus growth, RAP1 and PKC activation, lipid metabolism, and fibrinogen binding to platelets and megakaryocytes. These data provide evidence that AADACL1 regulates platelet and megakaryocyte activation and highlight the value of this chemoproteomic strategy for target discovery in platelets.Chemistry & biology 08/2013; DOI:10.1016/j.chembiol.2013.07.011 · 6.59 Impact Factor
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- "A number of events involved in platelet activation are largely dependent on intracellular calcium levels ([Ca 2+ ] í µí± ) , and elevation of [Ca 2+ ] í µí± is considered to be a prerequisite for platelet aggregation. The final common event of platelet aggregation is the binding of fibrinogen to integrin í µí»¼ IIb í µí»½ 3 . "
ABSTRACT: A number of reagents that prevent thrombosis have been developed but were found to have serious side effects. Therefore, we sought to identify complementary and alternative medicinal materials that are safe and have long-term efficacy. In the present studies, we have assessed the ability of chlorine e6 (CE6) to inhibit ADP-induced aggregation of rat platelets and elucidated the underlying mechanism. CE6 inhibited platelet aggregation induced by 10 µM ADP in a concentration-dependent manner and decreased intracellular calcium mobilization and granule secretion (i.e., ATP and serotonin release). Western blotting revealed that CE6 strongly inhibited the phosphorylations of PI3K, Akt, c-Jun N-terminal kinase (JNK), and different mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (ERK1/2) as well as p38-MAPK. Our study also demonstrated that CE6 significantly elevated intracellular cAMP levels and decreased thromboxane A2 formation in a concentration-dependent manner. Furthermore, we determined that CE6 initiated the activation of PKA, an effector of cAMP. Taken together, our findings indicate that CE6 may inhibit ADP-induced platelet activation by elevating cAMP levels and suppressing PI3K/Akt activity. Finally, these results suggest that CE6 could be developed as therapeutic agent that helps prevent thrombosis and ischemia.Evidence-based Complementary and Alternative Medicine 08/2013; 2013:569160. DOI:10.1155/2013/569160 · 1.88 Impact Factor