PAR1 activation initiates integrin engagement and outside-in signalling in megakaryoblastic CHRF-288 cells

• Published in: Biochimica et biophysica acta Vol. 1450; no. 3; pp. 265 - 276
• Main Authors: Cichowski, Karen, Orsini, Michael J, Brass, Lawrence F
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 08.07.1999
• Subjects: Blood Platelets - metabolism; Calcium - metabolism; Cell Adhesion; Cell Cycle Proteins; Cell Line; Fibrinogen - metabolism; Humans; Indoles - pharmacology; Maleimides - pharmacology; Peptide Fragments - pharmacology; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Platelet Glycoprotein GPIIb-IIIa Complex - metabolism; Protein Binding; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-vav; Receptor, PAR-1; Receptors, Thrombin - metabolism; Signal Transduction; Tetradecanoylphorbol Acetate - pharmacology; Thrombin - pharmacology; Bis-IM, bisindoylmaleimide-I; BSA, bovine serum albumin; PKC, protein kinase C; cPLA 2, cytoplasmic phospholipase A 2; RGDS, Arg-Gly-Asp-Ser; ERK2, p42 MAPK; PMA, phorbol myristate acetate
• ISSN: 0167-4889; 0006-3002; 1879-2596; 1878-2434
• DOI: 10.1016/S0167-4889(99)00065-8

To better understand the means by which cells such as human platelets regulate the binding of the integrin α IIbβ 3 to fibrinogen, we have examined agonist-initiated inside-out and outside-in signalling in CHRF-288 cells, a megakaryoblastic cell line that expresses α IIbβ 3 and the human thrombin receptor, PAR1. The results show several notable similarities and differences. (1) Activation of PAR1 caused CHRF-288 cells to adhere and spread on immobilized fibrinogen in an α IIbβ 3-dependent manner, but did not support the binding of soluble fibrinogen or PAC-1, an antibody specific for activated α IIbβ 3. (2) Direct activation of protein kinase C with PMA or disruption of the actin cytoskeleton with low concentrations of cytochalasin D also caused CHRF-288 cells to adhere to fibrinogen. (3) Despite the failure to bind soluble fibrinogen, activation of PAR1 in CHRF-288 cells caused phosphoinositide hydrolysis, arachidonate mobilization and the phosphorylation of p42 MAPK, phospholipase A 2 and the Rac exchange protein, Vav, all of which occur in platelets. PAR1 activation also caused an increase in cytosolic Ca 2+, which, when prevented, blocked adhesion to fibrinogen. (4) Finally, as in platelets, adhesion of CHRF-288 cells to fibrinogen was followed by a burst of integrin-dependent (‘outside-in’) signalling, marked by FAK phosphorylation and a more prolonged phosphorylation of p42 MAPK. However, in contrast to platelets, adhesion to fibrinogen had no effect on Vav phosphorylation. Collectively, these observations show that signalling initiated through PAR1 in CHRF-288 cells can support α IIbβ 3 binding to immobilized ligand, but not the full integrin activation needed to bind soluble ligand. This would suggest that there has been an increase in integrin avidity without an accompanying increase in affinity. Such increases in avidity are thought to be due to integrin clustering, which would also explain the results obtained with cytochalasin D. The failure of α IIbβ 3 to achieve the high affinity state in CHRF-288 cells was not due to the failure of PAR1 activation to initiate a number of signalling events that normally accompany platelet activation nor did it prevent at least some forms of outside-in signalling. However, at least one marker of outside-in signalling, the augmentation of Vav phosphorylation seen during platelet aggregation, did not occur in CHRF-288 cells.