Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets.
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. αIIbβ3 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.



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.



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.



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.
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.
Peter M
PM
Benz
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.
Hebatullah
H
Laban
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.
Joana
J
Zink
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.
Lea
L
Günther
Centre for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz, Germany.
Ulrich
U
Walter
Department of Cytology and Histology, St. Petersburg State University, St. Petersburg, Russia.|Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia.
Stepan
S
Gambaryan
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.
Karim
0000-0002-1777-1915
K
Dib
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000378:metabolism; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism; D058913:Purinergic Agonists / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D020668:rap GTP-Binding Proteins / Q000378:metabolism*; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D020735:Guanine Nucleotide-Releasing Factor 2; D008840:Microfilament Proteins; D010750:Phosphoproteins; D050737:Proto-Oncogene Proteins c-crk; D058913:Purinergic Agonists; D000098662:Vasodilator-Stimulated Phosphoprotein; D013917:Thrombin; C492136:Rap1b protein, mouse; D020668:rap GTP-Binding Proteins
Crkl; Platelets; Rap1b; VASP; cAMP; cGMP