Thrombin agonists

The release of NO from the endothelium is induced by various chemical substances, including acetylcholine polypeptides such as substance P, bradykinin, and arginine vasopressin histamine ATP/ADP a2-adrenoceptor agonists thrombin and Ca2+ iono-phores. NO formed in response to mechanical stimuli like shear stress or transmural pressure plays an important role in maintaining basal blood flow. Endothelial NO causes vasodilatation, decreased... 

In some cases, receptor inactivation, e.g., of the V2 vasopressin receptor, is mediated by agonist-induced enzymatic cleavage of the GPCR. This nonendocytic proteolysis is promoted by a plasma membrane-associated metalloprotease. Proteinase-activated receptors (PARs) such as the thrombin receptor also follow a distinctly different pathway. PARs require the enzymatic cleavage of their N terminus, and the newly generated N terminus activates the receptor. Once... 

GEA-3175 is more stable than GEA-3162 in vitro but still retains its biological activity [95]. The release of NO and NO2 by GEA 3175 was increased 140-fold in the presence of human plasma, as analyzed by ozone chemiluminescence [94]. GEA 3175 inhibited agonist-induced platelet aggregation and induced a more than 4-fold increase in platelet cGMP without affecting cAMP levels [94]. Thrombin-stimulated rises in the cytosolic free Ca2+ concentration and secretion were dose-dependently inhibited by GEA 3175. GEA 3175 showed a reduced capacity to inhibit platelet aggregation of uremic platelets compared to controls [96]. 

Apart from inducing vascular damage via infiltration and degranulation of various blood cells, PAF and TNF exert also direct effects in the endothelium. In vitro, both substances cause contraction of endothelial cells, which may partially account for the increased vascular permeability and plasma extravasation observed in many species following PAF or TNF administration [311]. While it has been known for some time that endothelial cells produce PAF when stimulated with various agonists such as thrombin, it has recently been established that TNF and IL-1 also induce cultured endothelial cells to synthesize PAF, the majority of which remains associated with the cells [317]. 

Platelets can be activated by a variety of agents including the physiologic agonists ADP, thromboxane A2, epinephrine, collagen, and thrombin. Platelet activation is generally associated with a change in platelet shape (except for epinephrine-induced platelet activation) from discs to spiny spheres with pseudopodia. Platelet pseudopod formation is dependent on actin polymerization in the activated platelets. The interaction of actin filaments with myosin, mediated by calcium (9), facilitates platelet contractile activity (e.g., clot retraction). 

McLaughlin, J. N., Shen, L., Holinstat, M., Brooks, J. D., Dibenedetto, E., and Hamm, H. E. (2005). Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1. / Biol. Chem. 280, 25048-25059. 

At this point, we had assembled a preliminary model of the human thrombin receptor that contained all three extracellular loops linked to the seven TM domains, as well as the N-terminus out to residue 75. Docking studies with agonist peptide SFLLRN, and additional mutagenesis experiments, were carried out in parallel. Measurement of the effects of site-specific mutations in human PAR-1 identified several key amino acids of the receptor as being important for activation (Table 1),... 

The effects of the mutations on functional responses were determined on thrombin receptors that were expressed in Xenopus oocytes. Agonist-stimulated calcium efflux with human a-thrombin (10 nM) and SFLLRNP-NH2 (40 pM), as described previously,29 was employed to determine the functional responsiveness of the receptor. 

Figure 9. Hydrophobic cluster involving Phe-182 (magenta), Leu-340 (orange), Tyr-337 (red), and Phe-339 (magenta) of the human thrombin receptor complexed with the Phe residue (white) of the agonist peptide.

Overall, our results provide a working hypothesis for the molecular recognition that occurs between the human thrombin receptor (PAR-1) and its agonist peptide SFLLRN. Similar features may contribute to the interactions of the X. laevis homologue of PAR-1 with TFRIFD and of human PAR-2 with SLIGKV, although... 

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