Abstract
Thrombin stimulation of the T leukemic cell line Jurkat induced a transient increase in [Ca2+]i. Proteolytic activity of the enzyme was required for this effect since diisopropyl fluorophosphate-thrombin failed to increase [Ca2+]i. Furthermore, hirudin and anti-thrombin III inhibited the thrombin-induced [Ca2+]i rise in Jurkat T cells. A synthetic thrombin receptor agonist peptide (TRP) of 7 residues (SFLLRNP) was found to be as effective as thrombin for [Ca2+]i mobilization, and both agonists induced Ca2+ release exclusively from internal stores. Thrombin stimulated tyrosine phosphorylation of several proteins of molecular mass 40, 42, 70, 120, and 130 kDa. There was a good correlation between thrombin-induced tyrosine phosphorylation of the latter three proteins and Ca2+ mobilization. Thrombin and TRP also caused translocation of protein kinase C from the cytosol to the plasma membrane. As a likely consequence of these events, thrombin activated the nuclear factor NF-kB. Several cell lines of hematopoietic origin including the leukemic T cell line HPB.ALL and the erythroleukemic cell line K562 were responsive to thrombin, whereas others such as THP1, a myelomonocytic cell line, and BL2, a Burkitt lymphoma were refractory to thrombin or TRP stimulation. The magnitude of the thrombin response in the different cell types paralleled the expression of the thrombin receptor mRNA. We found that activation of Jurkat T cells by a combination of phytohemagglutinin and phorbol 12-myristate 13-acetate led to a dramatic inhibition of thrombin receptor mRNA expression and to a concomitant loss of the thrombin response. Finally, we demonstrate that thrombin and TRP enhanced CD69 expression and interleukin 2 production induced by T cell receptor cross-linking in both Jurkat T cells and peripheral blood lymphocytes. These findings highlight the role of thrombin as a potential regulator of T lymphocyte activation.
Highlights
A synthetic thrombin receptor agonist peptide ceptor belonging to the seven-transmembranedomain receptor (TRP)of 7 residues (SFLLRNP) was fountdo be as effec- family has been cloned by expression in Xenopus oocytes (Vuet tive as thrombin for [Ca2+Ii mobilizationa,nd both ago- a l . , 1991a; Rasmussen et al, 1991)
Thrombin Acts as a n Accessory Signal and Synergizes with TCR Cross-linking to Induce CD69 Expression andIL2 Production-T cell activation is reflected by changes in gene expression
To determine whether activation of the thrombin receptor has functional consequences on gene expression, we examined the effect of thrombin on CD69 expression and IL2 al., 1983) and mitogenic for mousesplenocytes (Chen et al, 1976), information concerning the effects of thrombin on defined populations of T lymphocyteshas been lacking
Summary
Following a IO-min was pretreatedfor 1h in the presenocfea 2-fold excess of either hirudin incubation, the reaction was stoppeadt 0 "C and thecells were pelleted or anti-thrombin I11 before Ca2+ determination. CD69 ExpressionJurkat T cells (106/ml) were incubatedin RPMI, was enriched on DEAF, ion-exchange columns for both fractions 5% fetal calf serum with or without 10 pg/ml immobilized anti-CD3 and measureda s previously described using histone HIIISas substrate mAb (IOT3, Immunotech, Marseille, Francei)n thepresence or absence (Tanti et al, 1989). Assays were performedfor 30 min at 20 "C with 100 pl of the indicated fraction in a volume of 250 pl of 20 mM Tris/HCl buffer (pH 7.5) containing [y-3'PlATP (5 pCi; 50 p ~ ) 1,0 mM of 3 unitsiml thrombin, 100 p~ TRP, or a suboptimal concentration of PMA (0.5 ng/ml)for 18h at 37 "C.
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