Regulated Guanine Nucleotide Exchange Factor Research Articles

Human CalDAG-GEFI deficiency increases bleeding and delays αIIbβ3 activation

AbstractAffinity regulation of integrin αIIbβ3 for fibrinogen by inside-out signaling plays a critical role in hemostasis. Calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) was identified as a Rap1-activating molecule, and its role in inside-out αIIbβ3 activation was established in CalDAG-GEFI–deficient mice. However, little information regarding CalDAG-GEFI in human platelets is available. Here, we report a 16-year-old girl with CalDAG-GEFI deficiency who has been suffering from severe bleeding tendency. Although talin and kindlin-3 were normally detected, CalDAG-GEFI was undetectable in her platelets by western blotting. Genetic analysis revealed compound heterozygous CalDAG-GEFI mutations, Lys309X and Leu360del, which were responsible for CalDAG-GEFI deficiency. The functional analysis demonstrated impaired αIIbβ3 activation by various agonists except for phorbol myristate acetate, normal calcium mobilization, and impaired Rap1 activation, which were consistent with the phenotype of CalDAG-GEFI–deficient mice. Despite substantial αIIbβ3 activation at high agonist concentrations, she had severe bleeding tendency. Further functional analysis demonstrated markedly delayed αIIbβ3 activation velocity and decreased shear-induced thrombus formation. Contrary to CalDAG-GEFI–deficient mice, which showed integrin-dependent neutrophil functional abnormality, neutrophil β2 integrin activation was not impaired in the patient. Our results demonstrate the critical role of CalDAG-GEFI in rapid αIIbβ3 activation of human platelets.

Differential regulation of platelet inhibition by cGMP- and cAMP-dependent protein kinases

Background Under normal conditions circulating platelets are maintained in a resting state by the paracrine signaling molecules, prostacyclin (PGI2) and nitric oxide (NO), released from vascular endothelial cells. PGI2 and NO trigger the synthesis of cAMP and cGMP, respectively, which in turn leads to the activation of Protein Kinase A (PKA) and Protein Kinase G (PKG). These cyclic nucleotide-dependent protein kinases phosphorylate numerous substrate proteins involved in several aspects of platelet activation. Both, PKA and PKG can phosphorylate the same substrates including VASP, LASP, RAP1GAP2, Rap1B, HSP27, and some others. Only few substrates are known to be specific for PKA (PDE3) and PKG (PDE5) [1,2]. Here we report the identification and characterization of a new PKA specific substrate, CalDAG-GEFI (calcium and diacylglycerol regulated guanine nucleotide exchange factor I), a guanine exchange factor for Rap1b in platelets.

Critical Role of CalDAG-GEFI In FCγRIIa-Dependent Platelet Activation and Thrombosis

AbstractAbstract 3196Immune-mediated thrombosis is a major cause of death in autoimmune diseases and contributes to complications in drug treatments (e.g. Heparin Induced Thrombocytopenia). The major receptor on platelets for immunoglobulin-mediated activation is FcγRIIA. FcγRIIA signals through an immunoreceptor tyrosine-based activation motif (ITAM) that leads to phospholipase C activation, which induces the release of calcium and diacylglycerol (DAG). In our previous work, we identified CalDAG-GEFI (calcium and DAG regulated guanine nucleotide exchange factor I) as a key component of collagen/ITAM-mediated platelet activation. In the current study, we evaluated if CalDAG-GEFI is a potential target for the intervention with FcγRIIA receptor dependent, immune-mediated thrombosis.Mice transgenic for the human FcγRIIA (hFCR) and deficient for CalDAG-GEFI-/- (hFCR/CDGFI-/-) were generated. In vitro, aggregation of hFCR/CDGFI-/- platelets required 50–100-fold higher concentrations of anti-CD9 antibodies than hFCR/WT controls. In comparison, inhibition of P2Y12, the target of clopidogrel, shifted the dose response curve for anti-CD9 in hFCR/WT platelets by only ∼2-fold. In addition to their aggregation defect, hFCR/CDGFI-/- platelets were characterized by markedly impaired Rap1 activation.To assess the role of CalDAG-GEFI in FcγRIIA -mediated thrombosis in vivo, we developed a model of antibody-mediated thrombosis, were we injected mice with an Alexa-750 labeled antibody against GPIX and extracted the lungs to visualize pulmonary thrombosis on a LICOR scanner. Anti-GPIX induced pulmonary thrombosis in hFCR mice but not in WT animals. Pretreatment with clopidogrel did not provide a substantial protection from thrombosis in hFCR mice. In contrast, no pulmonary thrombi were observed in hFCR/CDGFI-/- mice.Together, our studies are the first to highlight the importance of CalDAG-GEFI downstream of platelet Fc-receptor/ITAM signaling and suggest CalDAG-GEFI as a powerful new target in the intervention of immune-mediated thrombosis. Disclosures:No relevant conflicts of interest to declare.

The Signaling Molecule CalDAG-GEFI Represents a Novel Target for Antithrombotic Therapy.

Abstract 1077Poster Board I-99 Background/Objective:The signaling molecule Ca2+ and diacylglycerol regulated guanine nucleotide exchange factor I (CalDAG-GEFI) plays a crucial role in the immediate, Ca2+ dependent activation of Rap1 and intergrin αaIIbβ3 in stimulated platelets. Our previous studies demonstrated that signaling by protein kinase C and the Gi-coupled receptor for ADP, P2Y12, provides an alternative pathway that facilitates the delayed but prolonged activation of Rap1. P2Y12 inhibitors such as clopidogrel bisulfate (Plavix) are currently considered the gold standard for the prevention and treatment of thrombotic complications. The aims of the study were to: 1) evaluate CalDAG-GEFI signaling as a potential new target for antithrombotic therapy and 2) to compare the contribution of CalDAG-GEFI and P2Y12 signaling to thrombus formation in pre-clinical models of models of thrombotic disease. Methods/Results:Thrombus formation was compared in: wildtype (WT), CalDAG-GEFI-/-, WT treated with clopidogrel (clopidogrel was administered orally 24h and 3h prior to the experiment at a dosage of 0,075mg/g bodyweight) and CalDAG-GEFI-/- mice treated with clopidogrel. Thrombosis was studied in three different models: 1) laser-induced thrombosis in the microcirculation of the cremasteric muscle (thrombin-dependent, localized), tissue factor-induced pulmonary thromboembolism (thrombin-dependent, systemic) and FeCl3-induced thrombosis in the mesentery (collagen/thrombin-dependent, localized). As shown previously, clopidogrel treatment significantly reduced but did not abolish laser-induced thrombus formation in WT mice. Laser induced thrombosis was completely inhibited in arterioles of CalDAG-GEFI -/- mice, while significant thrombus formation was observed in venules. Clopidogrel treatment of CalDAG-GEFI-/- mice further reduced venous thrombus formation, providing in vivo confirmation that platelet aggregation in the absence of CalDAG-GEFI requires P2Y12 signaling. In line with these findings, we observed that CalDAG-GEF-/- mice were partially protected from tissue factor-induced pulmonary thromboembolism when compared to clopidogrel-treated CalDAG-GEF-/- mice. In the model of FeCl3-induced thrombosis, CalDAG-GEFI-/- platelets adhered to the damaged vascular wall but failed to form thrombi in both venules and arterioles. In contrast clopidogrel-treated WT mice were protected from FeCl3-induced vessel occlusion but continuously formed embolizing thrombi of considerable size that contained activated platelets. This phenomenon was detectable in both arterioles and venules and might be of clinical relevance. Conclusion:Our studies identify CalDAG-GEFI as a promising new target for antiplatelet therapy. CalDAG-GEFI inhibition will have a strong antithrombotic effect as it is a critical component of the near-immediate platelet response to exposed extracellular matrix and/or soluble agonists. However, backup by PKC/P2Y12 signaling should allow for the formation of small but stable mural thrombi, especially under low flow conditions as found in veins and in venules, thus limiting the risk of bleeding complications. Disclosures:Poncz:Diagnostica Stago: Patents & Royalties.

Interactome of Transforming Growth Factor-β Type I Receptor (TβRI): Inhibition of TGFβ Signaling by Epac1

Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. Type I TGFbeta receptor (TbetaRI) is the key receptor for initiation of intracellular signaling by TGFbeta. Here we report proteomics-based identification of proteins that form a complex with TbetaRI. Using 2D-GE and MALDI TOF mass spectrometry, we identified 16 proteins that specifically interacted with a GST-fused TbetaRI Thr204Asp construct with constitutively active serine/threonine kinase. We confirmed interactions of the receptor with cAMP regulated guanine nucleotide exchange factor 1 (Epac1), beta-spectrin, PIASy, and beta-catenin proteins using immunoblotting. Interaction of the receptor with Epac1 required intact kinase activity of TbetaRI but was not affected by deletion of cAMP-binding domain of Epac1. TGFbeta1-induced C-terminal phosphorylation of Smad2 was inhibited in vivo and in vitro in the presence of Epac1. Epac1 inhibited also TGFbeta1/TbetaRI-dependent transcriptional activation, as evaluated by luciferase reporter assays. We observed that expression of Epac1 counteracted TGFbeta/TbetaRI-dependent decrease of cell adhesion and TGFbeta/TbetaRI-induced stimulation of cell migration. Thus, we have reported novel TRI-interacting proteins and have shown that Epac1 inhibited TGFbeta-dependent regulation of cell migration and adhesion.

The Ras-GRF1 exchange factor coordinates activation of H-Ras and Rac1 to control neuronal morphology.

The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRFdeltaN, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRFdeltaC, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRFdeltaC binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size.

P-Rex1 Is a Primary Rac2 Guanine Nucleotide Exchange Factor in Mouse Neutrophils

Leukocyte chemoattractants regulate many leukocyte functions, including leukocyte chemotaxis, via the Rho family of small GTPases that include RhoA, Cdc42, and Rac. Previous work has revealed mechanisms by which chemoattractants regulate RhoA and Cdc42 in mouse neutrophils, but the mechanisms for regulation of Rac remain unclear even though Rac is important for neutrophil functions. Here, we characterized P-Rex1, a Gbetagamma and PIP(3)-regulated guanine nucleotide exchange factor that was initially identified as a Rac activator in response to chemoattractants, for its roles in the regulation of Rac activity and neutrophil functions. We generated a mouse line in which the P-Rex1 gene is disrupted and found that P-Rex1 deficiency did not significantly affect Rac1 activation but diminished Rac2 activation in response to a chemoattractant fMLP in mouse neutrophils. This preference for Rac2 may partially result from the apparent higher affinity of P-Rex1 for Rac2 than for Rac1 because P-Rex1 was more readily immunoprecipitated with Rac2(S17N) than Rac1(S17N). In addition, P-Rex1 deficiency significantly attenuated fMLP-induced F actin formation and superoxide production without affecting LPS- or PMA-induced production. Furthermore, P-Rex1 deficiency caused a chemotactic defect that is primarily attributed to a reduction in the migration rate rather than directionality.

Non-kinase second-messenger signaling: new pathways with new promise.

Intercellular signaling by growth factors, hormones and neurotransmitters produces second messenger molecules such as cyclic adenosine monophosphate (cAMP) and diacylglycerol (DAG). Protein Kinase A and Protein Kinase C are the principal effector proteins of these prototypical second messengers in certain cell types. Recently, novel receptors for cAMP and DAG have been identified. These proteins, designated EPAC (Exchange Protein directly Activated by cAMP) or cAMP-GEF (cAMP regulated Guanine nucleotide Exchange Factor) and CalDAG-GEF (Calcium and Diacylglycerol regulated Guanine nucleotide Exchange Factor) or RasGRP (Ras Guanine nucleotide Releasing Protein) are able to mediate some of the physiologic effects of the second messengers in a protein-kinase-independent fashion. These proteins are exchange factors for Ras family GTPases that operate in pathways that run parallel to the classic kinase-dependent pathways. The rapidly emerging recognition of the functions of these "non-kinase" effectors in diverse processes such as insulin secretion, thymocyte development, asthma and malignant transformation creates new opportunities for discovery and identifies potential new therapeutic targets.