The International Journal of Biochemistry & Cell Biology 40 (2008) 517–529 Available online at www.sciencedirect.com Prothrombin fragments containing kringle domains induce migration and activation of human neutrophils Andr´ ea Mariano-Oliveira a , Marta S. De Freitas a , Robson Q. Monteiro b , Christina Barja-Fidalgo a,∗ a Departamento de Farmacologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro 87 fds, Vila Isabel, Rio de Janeiro, 20551-030 RJ, Brazil b Instituto de Bioqu´ ımica M´ edica, Centro de Ciˆ encias da Sa ´ ude, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Received 22 June 2007; received in revised form 29 August 2007; accepted 3 September 2007 Available online 16 September 2007 Abstract The cross-talk between inflammatory and coagulation cascades has been demonstrated. Prothrombin processing releases the protease domain (thrombin) along with two catalytically inactive kringle-containing derivatives: prothrombin fragments 1 (F1) and 2 (F2). It is well established that thrombin is able to trigger an inflammatory response but the possible effects of prothrombin fragments on leukocyte functions are still unknown. In this report, we demonstrate for the first time that both F1 and F2 prothrombin fragments, interfere with intracellular functional signaling pathways to modulate human neutrophil migration. In addition, we show that thrombin, fragment 1 and fragment 2 induce human neutrophil chemotaxis. The effect of fragment 2, but not fragment 1, was partially inhibited by pertussis toxin, an inhibitor of G i -signaling. The pre-treatment of cells with fragment 2 inhibited thrombin-induced chemotaxis, while both fragments impaired neutrophil migration induced by interleukin-8. F1 and F2 increased the expression and activation of G-protein-coupled receptor kinase-2, which has emerged as a key effector in the desensitization of chemokine receptors. In parallel, prothrombin fragments activated extracellular signal-regulated kinase 1/2, stimulating its phosphorylation and nuclear translocation, and induced inhibitor of kappa-B phosphorylation and degradation followed by nuclear factor-kappa B translocation to nucleus. Furthermore, both prothrombin fragments induced interleukin-8 gene expression in human neutrophils. These findings suggest that the interference with neutrophil signaling and function, caused by kringle-containing prothrombin fragments may desensitize these cells to respond to further activation by thrombin and interleukin-8 during inflammatory and coagulation responses. © 2007 Elsevier Ltd. All rights reserved. Keywords: Neutrophil; Prothrombin fragments; Kringle domain; Chemotaxis; GRK-2 1. Introduction During inflammation, an effective neutrophil response to chemotactic agents depends on their ability ∗ Corresponding author. Tel.: +55 21 2587 6398; fax: +55 21 2587 6808. E-mail address: barja-fidalgo@uerj.br (C. Barja-Fidalgo). to leave the circulating blood and transmigrate along a chemotactic gradient towards the site of injury (Baggiolini, 1998). Leukocyte migration is regulated by signaling mechanisms activated by chemokines, which act through specific G-protein-coupled receptors (GPCRs) (Proudfoot, 2002). IL-8 is a potent chemokine produced by neutrophils during inflammation that involved in neutrophil chemotaxis, in the respiratory burst, granule release, cell adhesion and survival 1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2007.09.002