Reconstitution of receptors and GTP-binding regulatory proteins (G Proteins) in Sf9 Cells: a direct evaluation of selectivity in receptor.G protein coupling

The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. α, β1, and γ2 subunits). Membranes were incubated with [35S]guanosine 5′-(3-O-thio)triphosphate (GTPγS) ± an agonist, and the amount of radiolabel bound to the α subunit was quantitated following immunoprecipitation. When expressed without receptor (but with β1γ2), the G protein subunits αz, α12, and α13 did not bind appreciable levels of [35S]GTPγS, consistent with a minimal level of GDP/[35S]GTPγS exchange. In contrast, the subunits αs and αq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPγS to αz but not to α12, α13, or αs. Binding to αz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPγS binding to αz and more robust increases in binding to αq, α12, and α13. Binding of [35S]GTPγS to αs was strongly enhanced only by the activated β1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13

Thrombin, phorbol ester, and cAMP regulate thrombin receptor protein and mRNA expression by different pathways

Human mesangial cells have been used to study the regulation of thrombin receptor protein and mRNA expression during cross-talk between different signal transduction pathways. Persistent activation of thrombin receptor by thrombin led to homologous down-regulation of thrombin receptor protein. However, thrombin receptor mRNA expression was not affected, suggesting that increased receptor degradation is responsible for homologous down-regulation. Chronic activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and of adenylylcyclase by prostaglandin E1 (PGE1) resulted in heterologous down-regulation of thrombin receptor protein. In contrast to thrombin, PMA and PGE1 reduced in parallel thrombin receptor mRNA levels to 51% and 24% of control, respectively, indicating that heterologous down-regulation of thrombin receptor protein is, at least in part, due to inhibition of receptor mRNA expression. The mechanisms of heterologous down-regulation of thrombin receptor protein have been studied in detail and compared to homologous down-regulation. PMA-induced down-regulation was completely blocked by GF 109 203 X, an inhibitor of protein kinase C. However, the loss of thrombin receptor induced by thrombin was not prevented by GF 109 203 X, indicating that homologous regulation is not dependent on protein kinase C activation. The heterologous effect of PGE1 was mimicked by 8-bromo-cAMP, isobutylmethylxanthine, and forskolin, suggesting that an increase in intracellular cAMP level is involved in heterologous regulation. Interestingly, heterologous down-regulation induced by PGE1 seems not to require previous internalization of thrombin receptor. These data indicate that thrombin receptor protein and mRNA expression can be regulated in homologous and heterologous ways by different mechanisms

Collagen surfaces to measure thrombus formation under flow: possibilities for standardization

Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Thrombin increases the adhesion of washed human platelets to collagen

Thrombin stimulates the adhesion of washed human platelets to fibrillar collagen. This phenomenon occurs also when platelets, before thrombin stimulation, are resuspended in the presence of prostaglandin E 1 to minimize the release reaction. Enzymatic activity of thrombin is not necessary for the enhancement of platelet adhesiveness, since phenylmethylsulphonylfluoride inhibited thrombin is effective in this respect. Detachment of thrombin from thrombin treated platelets by the use of hirudin restores normal platelet adhesiveness to collagen

Nitridergic platelet pathway activation by hementerin, a metalloprotease from the leech Haementeria depressa

Hementerin (HT) is an 80 kDa fibrino(geno)lytic metalloprotease, purified from saliva of the leech Haementeria depressa. in the present report, the effect of HT on several functional parameters of human platelets was assessed. HT inhibited platelet aggregation and ATP release induced by different agonists such as ADP, adrenaline, collagen, thrombin, and arachidonic acid. HT did neither modify the expression of platelet glycoproteins (Ib, IIbIIIa, IaIIa, IV) nor intraplatelet fibrinogen levels, whereas it markedly decreased CD62P and CD63 levels after the stimulation with thrombin. HT significantly increased thrombininduced platelet Ca2+ intracellular levels, cGMP content and nitric oxide synthase (NOS) activity. the effect of HT on platelet aggregation was reversed by two NOS inhibitors, N-omega-Nitro-L-arginine methyl ester and 2 N-G-Nitro-L-arginine. in summary, these results indicate that HT is an effective inhibitor of human platelet aggregation, presumably through activation of the platelets nitridergic pathway.Inst Butantan, Lab Bioquim & Biofis, BR-05503900 São Paulo, BrazilAcad Nacl Med Buenos Aires, IIHEMA, Buenos Aires, DF, ArgentinaUniv Buenos Aires, Fac Med, Dept Bioquim Humana, Buenos Aires, DF, ArgentinaUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilWeb of Scienc