title: Novel pharmacological inhibitors demonstrate the role of the tyrosine kinase Pyk2 in adhesion and aggregation of human platelets.
abstract: Pyk2 is a Ca2+-regulated kinase predominantly expressed in neuronal and in haematopoietic cells. Previous studies on Pyk2-null mice have demonstrated that Pyk2 plays a crucial role in platelet activation and thrombus formation, thus representing a possible target for antithrombotic therapy. Very limited information is available about the role of Pyk2 in human platelets, mainly because of the lack of specific pharmacological inhibitors. In this work, we have tested two novel Pyk2 inhibitors, PF-4594755 and PF-4520440, to validate their specificity and to investigate their ability to modulate platelet activation. Both molecules were able to efficiently block Pyk2 activity in human and mouse platelets stimulated with thrombin or with the Ca2+-ionophore. In wild-type murine platelets, PF-4594755 and PF-4520440 reduced thrombin-induced aggregation to the level observed in Pyk2 knockout platelets, but did not affect aggregation induced by GPVI stimulation. Importantly, neither compounds affected the residual thrombin-induced aggregation of Pyk2-null platelets, thus excluding possible off-target effects. In human platelets, PF-4594755 and PF-4520440 significantly reduced aggregation stimulated by thrombin, but not by the GPVI agonist convulxin. Both inhibitors reduced platelet adhesion on fibrinogen and prevented Akt phosphorylation in adherent cells, indicating that Pyk2 regulates PI3K and cell spreading downstream of integrins in human platelets. Finally, the Pyk2 inhibitors significantly inhibited thrombus formation upon blood perfusion on immobilized collagen under arterial flow rate. These results demonstrate that PF-4594755 and PF-4520440 are specific inhibitors of Pyk2 in intact platelets and allowed to reliably document that this kinase plays a relevant role in human platelet activation.
authors.affiliation: Gianni F. Guidetti, Department of Biology and Biotechnology, Laboratory of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy, Tel.: +39 0382 987241, Fax: +39 0382 987240, E-mail: gianni.guidetti@unipv.it.
authors.forename: Gianni Francesco
authors.initials: GF
authors.lastname: Guidetti
authors.forename: Marta
authors.initials: M
authors.lastname: Zarà
authors.forename: Ilaria
authors.initials: I
authors.lastname: Canobbio
authors.forename: Caterina
authors.initials: C
authors.lastname: Visconte
authors.forename: Giada
authors.initials: G
authors.lastname: Di Nunzio
authors.forename: Mauro
authors.initials: M
authors.lastname: Torti
mesh_terms: D000818:Animals; D001792:Blood Platelets; D051418:Focal Adhesion Kinase 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D015539:Platelet Activation; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation
chemical_list: D019869:Phosphatidylinositol 3-Kinases; D051418:Focal Adhesion Kinase 2
keywords: PI3K; Pyk2; pharmacological inhibitors; platelet adhesion; platelet aggregation