Thrombin Signaling

Thrombin is a multifunctional serine protease involved in a number of pathophysiological processes that include blood clotting, inflammation, repair processes and tumor metastasis. Most of the thrombin-mediated effects are preceded by morphological changes in cells that follow activation of PARs. PARs are a unique class of heterotrimeric, transmembrane GPCR activated by serine proteases that cleave specific regions of the extracellular NH2 terminus of the molecule to unmask a new NH2 terminus that serves as a tethered ligand docking intramolecularly with the body of the receptor to effect transmembrane signaling. The tethered ligand, by binding to other extracellular domains on the PAR molecule, stimulates G-protein dependent signaling. PAR1 and PAR4 are direct cellular targets of thrombin; PAR3 seems to play a role in the activation of other PARs but does not itself transduce a signal directly. A variety of G-proteins are coupled to the activated PARs, and this determines the pluripotent nature of the cellular responses to thrombin. PARs activate signaling of members of the Gα12/13, Gαq and Gαi families resulting in activation of PLC, generation of IP3, increase of intracellular Ca2+, and activation of PKC and MAPKs. Activated PLC hydrolyzes PIP2 to IP3 and DAG, which are responsible for Ca2+ release from intracellular stores and activation of various conventional or classic PKC isoforms, respectively. These events mediate the cellular functions of granule secretion, platelet aggregation and angiogenesis. Signaling by PAR1 and PAR4 through Gα12/13 pathways couples with Rho molecules to mediate several thrombin responses. The activated RhoA translocates to the membrane, interacts with ROCK, and activates MLC phosphorylation, resulting in cytoskeletal reorganization, stress-fiber formation, cell permeability changes, endothelial cell migration, platelet aggregation and endothelial cell barrier dysfunction. Thrombin promotes blood clotting by converting fibrinogen to fibrin and by stimulating platelets through proteolytic activation of PARs on the surface of platelets. Gαi inhibits adenyclate cyclase and cAMP production. PARs also utilize Gα12/13 to activate Src, resulting in the phosphorylation of SHC and the subsequent activation of the Ras-Raf-MEK/ERK signaling cascade. Activation of ERK1/2 MAPKs play a crucial role in regulating cellular proliferation and differentiation signals from the cell surface to the nucleus. PI3K plays important roles in thrombin-mediated regulation of cytoskeletal structure, cell motility, cell survival, and mitogenesis and, also in some cell types, functions as an intermediate in activation of ERKs.In human airway smooth muscle cells and in pulmonary artery fibroblasts, PI3K is implicated in PAR-mediated activation of p70S6K and Akt. Thrombin stimulates binding of NF-κB p65 homodimers via a PI3K and PKC-δ-dependent pathway; as well as promoting GATA2-dependent transcription of VCAM through a PI3K and PKC-ζ-dependent signaling cascade. ICAM1 gene is also expressed in a PI3K-PKC-δ-NF-κB dependent manner. Increased expression of adhesion molecules ICAM1 and VCAM1 and tissue factor not only further promotes the coagulation process and the binding and aggregation of platelets, but also facilitates the rapid adherence of neutrophils, monocytes and lymphocytes to the endothelial cell layer.