The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment

Abstract

Thrombin, the terminal serine protease in the coagulation cascade, is a proinflammatory molecule in vivo and induces endothelial activation in vitro. The cellular signaling mechanisms involved in this function are unknown. The role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in thrombin-induced chemokine production was studied. Phosphorylation of both p38 MAPK and its substrate, ATF-2, was observed in human umbilical vein endothelial cells (HUVECs) stimulated with thrombin, with a maximum after 5 minutes of stimulation. Using the selective p38 MAPK inhibitor SB203580, there was a significant decrease in thrombin-induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) protein production and messenger RNA steady-state levels. In addition, SB203580 decreased IL-8 and MCP-1 production induced by the thrombin receptor-1 agonist peptide (TRAP), suggesting functional links between the thrombin G protein-coupled receptor and the p38 MAPK pathway. Furthermore, endothelial activation in the presence of SB203580 decreased the chemotactic activity of thrombin-stimulated HUVEC supernatant on neutrophils and monocytic cells. In contrast, the p42/p44 MAPK pathway did not appear to be involved in thrombin-or TRAP-induced endothelial chemokine production, because there was no reduction in the presence of the p42/ p44-specific inhibitor PD98059. These results demonstrate that the p38 rather than p42/44 MAPK signaling pathway plays an important role in thrombininduced endothelial proinflammatory activation and suggest that inhibition of p38 MAPK may be an interesting target for anti-inflammatory strategies in vascular diseases combining thrombosis and inflammation. IntroductionThrombin, a well-known procoagulant molecule, activates platelet aggregation and processing of fibrinogen into fibrin. 1 Three different thrombin receptors, belonging to the protease-activated receptor (PAR-1, -3, -4) family, have been identified on different cells. [2][3][4] PAR-2, another member of this family, does not seem to be activated by thrombin but, rather, by trypsin. 4 Following enzymatic cleavage of PAR-1, thrombin exhibits pleiotropic effects on cells. Notably, thrombin acts as a proliferation-inducing factor for smooth muscle cells and fibroblasts in vitro. 5 In addition, thrombin appears to be a potent proinflammatory molecule in vivo and in vitro. 6 Indeed, thrombin participates in the different steps of leukocyte-endothelium interactions by inducing in vitro endothelial expression of leukoendothelial adhesion molecules. Thrombin has been shown to induce P-selectin expression through a protein synthesis-independent mechanism and E-selectin as well as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression through gene transcription and protein synthesis. [7][8][9][10] Thus, thrombin may participate in the initial phase of rolling mediated by selectins, followed by firm leukoendothelial adhesion mediated by members of the immunoglobulin superfamily. 11 Thrombin also favors l...

Conformational Rearrangement in C-Reactive Protein Is Required for Proinflammatory Actions on Human Endothelial Cells

“…mCRP stimulated rapid phosphorylation of this kinase, and the specific p38 MAPK inhibitor SB203580 markedly inhibited HCAEC responses to mCRP, although the inhibition was incomplete. These results are consistent with those observed with SB203580 on thrombininduced endothelial chemokine production and ICAM-1 expression, 32,33 and suggest involvement of other intracellular signaling mechanism(s). Unlike in neutrophils, 18,19 mCRP does not appear to activate the MAPK kinase and phosphatidylinositol 3-kinase pathways in HCAECs, because there was no reduction in the presence of PD98059 and wortmannin.…”

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration

“…A large body of evidence suggests that thrombin and its receptors (PARs), particularly PAR1, play a role in endothelial barrier disruption, promoting the sticking of leukocytes and monocytes/macrophages to the endothelium and their infiltration into the subendothelial space (4,6,20,21), which are the hallmarks of atherogenesis (57). An enhanced expression of PAR1 in the regions of inflammation associated with macrophage influx, smooth muscle cell proliferation, and mesenchymal-like intimal cells further supports a role for its involvement in atherosclerosis (58 -60).…”

“…Inflammation appears to be the major factor underlying the atherogenesis, and it may be initiated with endothelial cell dysfunction, leading to leukocyte adhesion, ROS production, and low density lipoprotein (LDL) oxidation (2,3). Several studies have demonstrated that the recruitment of leukocytes and monocytes/macrophages to the site of vascular injury is dependent on the production of cytokines and adhesion molecules by the dysfunctional endothelium, resulting in their adherence to the endothelium and their subsequent transendothelialization (4,5). In addition, the injured/dysfunctional endothelium may expose the underlying vascular matrix to the flowing blood, which can lead to activation of platelets and production of thrombin (6,7).…”

Cell‐derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII–dependent mechanism

“…Platelet-derived microparticles transport arachidonic acid to endothelial cells and induce the expression of cyclooxygenase 2 and the increased production of prostacyclin (43), enhance the interaction between monocytes and endothelial cells (44), and enhance leukocyte aggregation and their accumulation on the endothelium (45). Furthermore, thrombin, factor Xa, and fibrin degradation products are all potent inducers of the inflammatory response (46)(47)(48)(49)(50). Hence, the presence of microparticles in synovial fluid may facilitate not only coagulation, but also inflammation via direct and indirect mechanisms.…”