PMID- 27620165 OWN - NLM STAT- MEDLINE DCOM- 20170206 LR - 20181113 IS - 1478-811X (Electronic) IS - 1478-811X (Linking) VI - 14 IP - 1 DP - 2016 Sep 13 TI - Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets. PG - 21 LID - 10.1186/s12964-016-0144-z [doi] LID - 21 AB - BACKGROUND: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. alphaIIbbeta3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo. METHODS: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins. RESULTS: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide (NO) was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) Protein Kinase A (PKA) -mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl. CONCLUSIONS: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation. FAU - Benz, Peter M AU - Benz PM AD - Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University and DZHK (German Centre for Cardiovascular Research) partner site Rhine-Main, 60590, Frankfurt, Germany. FAU - Laban, Hebatullah AU - Laban H AD - Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University and DZHK (German Centre for Cardiovascular Research) partner site Rhine-Main, 60590, Frankfurt, Germany. FAU - Zink, Joana AU - Zink J AD - Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University and DZHK (German Centre for Cardiovascular Research) partner site Rhine-Main, 60590, Frankfurt, Germany. FAU - Gunther, Lea AU - Gunther L AD - Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University and DZHK (German Centre for Cardiovascular Research) partner site Rhine-Main, 60590, Frankfurt, Germany. FAU - Walter, Ulrich AU - Walter U AD - Centre for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz, Germany. FAU - Gambaryan, Stepan AU - Gambaryan S AD - Department of Cytology and Histology, St. Petersburg State University, St. Petersburg, Russia. AD - Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia. FAU - Dib, Karim AU - Dib K AUID- ORCID: 0000-0002-1777-1915 AD - Centre for Experimental Medicine, Medical Biology Center (MBC) building, Queen's University of Belfast, Third floor, 97 Lisburn Road, BT9 7BL, Belfast, Northern Ireland, UK. k.dib@qub.ac.uk. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20160913 PL - England TA - Cell Commun Signal JT - Cell communication and signaling : CCS JID - 101170464 RN - 0 (Cell Adhesion Molecules) RN - 0 (Guanine Nucleotide-Releasing Factor 2) RN - 0 (Microfilament Proteins) RN - 0 (Phosphoproteins) RN - 0 (Proto-Oncogene Proteins c-crk) RN - 0 (Purinergic Agonists) RN - 0 (vasodilator-stimulated phosphoprotein) RN - EC 3.4.21.5 (Thrombin) RN - EC 3.6.1.- (Rap1b protein, mouse) RN - EC 3.6.5.2 (rap GTP-Binding Proteins) SB - IM MH - Animals MH - Blood Platelets/drug effects/*metabolism MH - Cell Adhesion Molecules/genetics/*metabolism MH - Cells, Cultured MH - Guanine Nucleotide-Releasing Factor 2/metabolism MH - Humans MH - Mice MH - Microfilament Proteins/genetics/*metabolism MH - Phosphoproteins/genetics/*metabolism MH - Protein Binding MH - Proto-Oncogene Proteins c-crk/metabolism MH - Purinergic Agonists/pharmacology MH - Thrombin/pharmacology MH - rap GTP-Binding Proteins/*metabolism PMC - PMC5020514 OTO - NOTNLM OT - Crkl OT - Platelets OT - Rap1b OT - VASP OT - cAMP OT - cGMP EDAT- 2016/09/14 06:00 MHDA- 2017/02/07 06:00 PMCR- 2016/09/13 CRDT- 2016/09/14 06:00 PHST- 2016/03/11 00:00 [received] PHST- 2016/09/06 00:00 [accepted] PHST- 2016/09/14 06:00 [entrez] PHST- 2016/09/14 06:00 [pubmed] PHST- 2017/02/07 06:00 [medline] PHST- 2016/09/13 00:00 [pmc-release] AID - 10.1186/s12964-016-0144-z [pii] AID - 144 [pii] AID - 10.1186/s12964-016-0144-z [doi] PST - epublish SO - Cell Commun Signal. 2016 Sep 13;14(1):21. doi: 10.1186/s12964-016-0144-z.