Thrombin is a multifunctional serine protease involved in a number of pathophysiological processes that include blood clotting, inflammation, repair processes and tumor metastasis. In brain, Thrombin regulates the viability of neurons and astrocytes by increasing survival under conditions of hypoglycemia and oxidative stress and inducing apoptosis under other conditions. Thrombin is also chemotactic for macrophages and mitogenic for smooth muscle cells, fibroblasts, and astrocytes and induces secretion of growth factors and cytokines from fibroblasts and smooth muscle cells. Most of the Thrombin-mediated effects are preceded by morphological changes in cells that follow activation of PARs (Protease-Activated Receptors) (Ref.1).
PARs are a unique class of heterotrimeric, transmembrane GPCRs (G-Protein Coupled Receptors) 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. PARs are found in a wide variety of cell types, including platelets, endothelial cells, fibroblasts, monocytes, T-Cell lines, osteoblast-like cells, smooth muscle cells, neurons, glial cells, and in certain tumor cell lines (Ref.2). To date, four PARs have been identified with distinct N-terminal cleavage sites and tethered ligand pharmacology, out of which, PAR1, PAR3, and PAR4 are activated by Thrombin, whereas PAR2 is activated by trypsin and tryptase but not by Thrombin. While PAR1 and PAR4 are direct cellular targets of Thrombin (PAR1 is the most potent and PAR4 functions as a low affinity thrombin receptor that is activated in conditions where high concentrations of Thrombin are achieved), PAR3 seems to play a role in the activation of other PARs but does not itself transduce a signal directly (Ref.3). A variety of G-Proteins can be coupled to the activated PARs (PAR1 and PAR4) and this determines the pluripotent nature of the cellular responses to Thrombin (Ref.4). PARs activate signaling of members of the G-Alpha12/13, G-AlphaQ and G-AlphaI families and hence to a host of intracellular effectors leading to activation of PLC (Phospholipase-C), generation of IP3 (Inositol Trisphosphate), increase of intracellular Ca2+, activation of PKC (Protein Kinase-C) and MAPKs (Mitogen-Activated Protein Kinases) (Ref.5). PARs increase intracellular Ca2+ levels by activating PLC and this PAR-mediated increase in intracellular Ca2+ has been linked to coupling of the intracellular loop of PARs to G-AlphaQ (Ref.3). The activated PLC hydrolyzes PIP2 (PhosphatidylInositol-4,5-Bisphosphate) to IP3 (Inositol Trisphosphate) and DAG (Diacylglycerol), which are responsible for Ca2+ release from intracellular stores and activation of various conventional or classic PKC isoforms (cPKC-Alpha, -Beta1, -Beta2, and -Gamma) respectively. These events mediate the cellular functions of granule secretion, platelet aggregation and angiogenesis (Ref.6).
Signaling by PAR1 and PAR4 through G-Alpha12/13 pathways couples to Rho
signaling and mediate several Thrombin responses. The activated small GTP-binding protein RhoA translocates to the membrane, interacts with ROCK (Rho-Associated Coiled-Coil-Containing Protein Kinase), and activates PAR-induced MLC (Myosin Light Chain) phosphorylation required for cytoskeletal reorganization, stress-fiber formation, permeability, migration in endothelial cells, platelet aggregation and endothelial cell barrier dysfunction (Ref.7). Disruption of the endothelial barrier or stimulation of vascular cells by inflammatory cytokines expose tissue factor, setting in motion a cascade that ultimately generates the protease Thrombin. Thrombin promotes blood clotting by converting fibrinogen to fibrin and by stimulating platelets through proteolytic activation of PARs on the surface of platelets. PARs also utilize G-Alpha12/13 to activate Src, resulting in the phosphorylation of SHC (SH2 Containing Protein) and the subsequent activation of MAPK pathway. Recent findings indicate the participation of C-tail (Cytoplasmic tail) of PARs to be important for the regulation of cytoskeletal changes (Ref.8).
MAPKs are activated by Thrombin through GN-AlphaI-coupled signaling leading to proliferation and mitogenesis in various cell types. The G-AlphaI class also stimulates the inhibition of AC (Adenyl Cyclase) and hence, cAMP (Cyclic Adenosine-3,5-Monophosphate). Phosphotyrosine residues within the intracellular domain of the activated receptor interact with the adaptor protein SHC that in turn recruits GRB2 (Growth Factor Receptor-Bound Protein-2)-SOS resulting in increased rate guanine-nucleotide exchange by the monomeric G-protein Ras. This initiates binding of Raf1 isoforms to the plasma membrane for activation by Ras and downstream activation of MEK1 (MAPK/ERK kinase), the direct activator of MAPKs. Activation of MAPKs that comprise the ERK1 (p44MAPK) and ERK2 (p42MAPK) (Extracelluar Signal Regulated Protein Kinase) plays a crucial role in regulating cellular proliferation and differentiation signals from the cell surface to the nucleus (Ref.5). PI3K (Phosphatidylinositol-3-Kinase) 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 (p70S6K inase), and Akt/PKB (Protein Kinase-B), two important regulators of cell survival and mitogenesis (Ref.3). Thrombin stimulates binding of NF-KappaB, p65 homodimers via a PI3K and PKC-Delta-dependent pathway; and, binding of GATA2 through a PI3K and PKC-Zeta-dependent signaling cascade culminating in VCAM1 (Vascular Adhesion Molecule-1) expression. ICAM1 (Intercellular Adhesion Molecule-1) gene is also expressed in a PI3K-PKC-Delta-NF-KappaB dependent manner. Increased expression of these adhesion molecules (ICAM1 and VCAM1) along with tissue factor not only further promotes the coagulation process and the binding and aggregation of platelets but also facilitate the rapid adherence of neutrophils, monocytes and lymphocytes to the endothelial cell layer (Ref.6).
Thrombin plays a central role in normal and abnormal hemostatic processes (Ref.9). Thrombin and PAR activation have been implicated in several cardiovascular diseases, other disease states, such as pulmonary fibrosis, acute lung injury, and neurological disorders and in ischemic cell death during brain insult and post-traumatic hyperexcitibility and seizure. All these facts suggest the blockade of the Thrombin signaling through PARs may be a potential site of therapeutic intervention. Arterial thrombosis underlies most cases of unstable angina and myocardial infarction. PAR activation is involved in the dilation of arteries during inflammation through the action of Thrombin on endothelial cells and in platelet activation by Thrombin during clotting. Complete enzymatic inhibition of Thrombin may also result in prolonged bleeding (Ref.3). Clinical, laboratory, histopathological and pharmacological evidence support the fact that cancer patients often suffer from a systemic activation of blood coagulation. Additionally, Thrombin has been shown to promote tumor progression and metastasis in animals (Ref.4). The recognition that Thrombin plays an important role in angiogenesis has also implicated a role for PARs in tumor formation and metastasis suggesting a possible therapeutic use for PAR blockade in some forms of cancer (Ref.3).