Meta-Biol

• Pathways

Thrombin activates proteinase activated receptors (PARs) that signal through heterotrimeric G proteins of the G12/13 and Gq families, thereby connecting to a host of intracellular signaling pathways. Thrombin activates PARs by cleaving an N-terminal peptide that then binds to the body of the receptor to effect transmembrane signaling. Intermolecular ligation of one PAR molecule by another can occur but is less efficient than self-ligation. A synthetic peptide of sequence SFLLRN, the first six amino acids of the new N-terminus generated when thrombin cleaves PAR1, can activate PAR1 independent of protease and receptor cleavage. PARs are key to platelet activation. Four PARs have been identified, of which PARs 1 ,3 and 4 are substrates for thrombin. In humans PAR 1 is the predominant thrombin receptor followed by PAR4 which is less responsive to thrombin. PAR 3 is not considered important for human platelet responses as it is minimally expressed, though this is not the case for mouse. PAR2 is not expressed in platelets. In mouse platelets, Gq is necessary for platelet secretion and aggregation in response to thrombin but is not necessary for thrombin-triggered shape change. G13 appears to contribute to platelet aggregation as well as shape change in response to low concentrations of thrombin but to be unnecessary at higher agonist concentrations; G12 appears to be dispensable for thrombin signaling in platelets. G alpha (q) activates phospholipase C beta thereby triggering phosphoinositide hydrolysis, calcium mobilization and protein kinase C activation. This provides a path to calcium-regulated kinases and phosphatases, GEFs, MAP kinase cassettes and other proteins that mediate cellular responses ranging from granule secretion, integrin activation, and aggregation in platelets. Gbeta:gamma subunits can activate phosphoinositide-3 kinase and other lipid modifying enzymes, protein kinases, and channels. PAR1 activation indirectly leads to activation of cell surface 'sheddases' that liberate ligands for receptor tyrosine kinases, providing a link between thrombin and receptor tyrosine kinases involved in cell growth and differentiation. The pleiotrophic effects of PAR activation are consistent with many of thrombin's diverse actions on cells.

Platelet activation begins with the initial binding of adhesive ligands and of the excitatory platelet agonists (released or generated at the sites of vascular trauma) to cognate receptors on the platelet membrane (Ruggeri 2002). Intracellular signaling reactions then enhance the adhesive and procoagulant properties of tethered platelets or of platelets circulating in the proximity. Once platelets have adhered they degranulate, releasing stored secondary agents such as ADP, ATP, and synthesize thromboxane A2. These amplify the response, activating and recruiting further platelets to the area and promoting platelet aggregation. These amplify the response, activating and recruiting further platelets to the area and promoting platelet aggregation. Adenosine nucleotides signal through P2 purinergic receptors on the platelet membrane. ADP activates P2Y1 and P2Y12, which signal via both the alpha and gamma:beta components of the heterotrimeric G-protein (Hirsch et al. 2001, 2006), while ATP activates the ionotropic P2X1 receptor (Kunapuli et al. 2003). Activation of these receptors initiates a complex signaling cascade that ultimately results in platelet activation, aggregation and thrombus formation (Kahner et al. 2006). Integrin AlphaIIbBeta3 is the most abundant platelet receptor, with 40 000 to 80 000 copies per resting platelet, acting as a major receptor for fibrinogen and other adhesive molecules (Wagner et al. 1996). Activation of AlphaIIbBeta3 enhances adhesion and leads to platelet-platelet interactions, and thus aggregation (Philips et al. 1991). GP VI is the most potent collagen receptor initiating signal generation, an ability derived from its interaction with the FcRI gamma chain. This results in the phosphorylation of the gamma-chain by non-receptor tyrosine kinases of the Src family (1). The phosphotyrosine motif is recognized by the SH2 domains of Syk, a tyrosine kinase. This association activates the Syk enzyme, leading to activation (by tyrosine phosphorylation) of PLC gamma2 (2). Thrombin is an important platelet agonist generated on the membrane of stimulated platelets. Thrombin acts via cell surface Protease Activated Receptors (PARs). PARs are G-protein coupled receptors activated by a proteolytic cleavage in an extracellular loop (Vu, 1991) (3). Activated PARs signal via G alpha q (4) and via the beta:gamma component of the G-protein (5). Both stimulate PLC giving rise to PIP2 hydrolysis and consequent activation of PI3K (6). PLCgamma2 activation also gives rise to IP3 (7) which stimulates the IP3 receptor (8) leading to increased intracellular calcium. Platelet activation further results in the scramblase-mediated transport of negatively-charged phospholipids to the platelet surface. These phospholipids provide a catalytic surface (with the charge provided by phosphatidylserine and phosphatidylethanolamine) for the tenase complex (formed by the activated forms of the blood coagulation factors factor VIII and factor I).

Hemostasis is a physiological response that culminates in the arrest of bleeding from an injured vessel. Under normal conditions the vascular endothelium supports vasodilation, inhibits platelet adhesion and activation, suppresses coagulation, enhances fibrin cleavage and is anti-inflammatory in character. Under acute vascular trauma, vasoconstrictor mechanisms predominate and the endothelium becomes prothrombotic, procoagulatory and proinflammatory in nature. This is achieved by a reduction of endothelial dilating agents: adenosine, NO and prostacyclin; and by the direct action of ADP, serotonin and thromboxane on vascular smooth muscle cells to elicit their contraction (Becker et al. 2000). The chief trigger for the change in endothelial function that leads to the formation of a haemostatic thrombus is the loss of the endothelial cell barrier between blood and extracellular matrix components (Ruggeri 2002). Circulating platelets identify and discriminate areas of endothelial lesions; here, they adhere to the exposed sub endothelium. Their interaction with the various thrombogenic substrates and locally generated or released agonists results in platelet activation. This process is described as possessing two stages, firstly, adhesion - the initial tethering to a surface, and secondly aggregation - the platelet-platelet cohesion (Savage & Cattaneo et al. 2001). Three mechansism contribute to the loss of blood following vessel injury. The vessel constricts, reducing the loss of blood. Platelets adhere to the site of injury, become activated and aggregate with fibrinogen into a soft plug that limits blood loss, a process termed primary hemostasis. Proteins and small molecules are released from granules by activated platelets, stimulating the plug formation process. Fibrinogen from plasma forms bridges between activated platelets. These events initiate the clotting cascade (secondary hemostasis). Negatively-charged phospholipids exposed at the site of injury and on activated platelets interact with tissue factor, leading to a cascade of reactions that culminates with the formation of an insoluble fibrin clot.