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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- "Furthermore, we observed that this selective secretory response of platelets to low-dose collagen was not associated with a sustained rise in intracellular calcium. In a similar manner, the prevention of inflammatory bleeding by platelets was shown previously to be independent of the calcium and diacylglycerol-regulated guanine nucleotide exchange factor 1 (CalDAG-GEF1) , , , a molecule which is central to calcium signalling in platelets . Whether or not the collagen-induced platelet secretory phenotype we described here does actually contribute to the prevention of inflammatory bleeding by platelets however remains to be verified. "
ABSTRACT: Platelets are not only central actors of hemostasis and thrombosis but also of other processes including inflammation, angiogenesis, and tissue regeneration. Accumulating evidence indicates that these "non classical" functions of platelets do not necessarily rely on their well-known ability to form thrombi upon activation. This suggests the existence of non-thrombotic alternative states of platelets activation. We investigated this possibility through dose-response analysis of thrombin- and collagen-induced changes in platelet phenotype, with regards to morphological and functional markers of platelet activation including shape change, aggregation, P-selectin and phosphatidylserine surface expression, integrin activation, and release of soluble factors. We show that collagen at low dose (0.25 µg/mL) selectively triggers a platelet secretory phenotype characterized by the release of dense- and alpha granule-derived soluble factors without causing any of the other major platelet changes that usually accompany thrombus formation. Using a blocking antibody to glycoprotein VI (GPVI), we further show that this response is mediated by GPVI. Taken together, our results show that platelet activation goes beyond the mechanisms leading to platelet aggregation and also includes alternative platelet phenotypes that might contribute to their thrombus-independent functions.PLoS ONE 08/2014; 9(8):e104712. DOI:10.1371/journal.pone.0104712 · 3.23 Impact Factor
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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; 20(9). DOI:10.1016/j.chembiol.2013.07.011 · 6.59 Impact Factor