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

To better understand the means by which cells such as human platelets regul ate the binding of the integrin alpha(IIb)beta(3) to fibrinogen, we have ex amined agonist-initiated inside-out and outside-in signalling in CHRF-288 c ells, a megakaryoblastic cell line that expresses alpha(IIb)beta(3) and the human thrombin receptor, PAR1. The results show several notable similariti es and differences. (1) Activation of PAR1 caused CHRF-288 cells to adhere and spread on immobilized fibrinogen in an alpha(IIb)beta(3)-dependent mann er, but did not support the binding of soluble fibrinogen or PAC-1, an anti body specific for activated alpha(IIb)beta(3) (2) Direct activation of prot ein kinase C with PMA or disruption of the actin cytoskeleton with low conc entrations of cytochalasin D also caused CHRF-288 cells to adhere to fibrin ogen. (3) Despite the failure to bind soluble fibrinogen, activation of PAR 1 in CHRF-288 cells caused phosphoinositide hydrolysis, arachidonate mobili zation and the phosphorylation of p42(MAPK), phospholipase A(2) and the Rac exchange protein, Vav, all of which occur in platelets. PAR1 activation al so caused an increase in cytosolic Ca2+, which, when prevented, blocked adh esion to fibrinogen. (4) Finally, as in platelets, adhesion of CHRF-288 cel ls to fibrinogen was followed by a burst of integrin-dependent ('outside-in ') signalling, marked by FAK phosphorylation and a more prolonged phosphory lation of p42(MAPK). However, in contrast to platelets, adhesion to fibrino gen had no effect on Vav phosphorylation. Collectively, these observations show that signalling initiated through PAR1 in CHRF-288 cells can support a lpha(IIb)beta(3) binding to immobilized ligand, but not the full integrin a ctivation needed to bind soluble ligand. This would suggest that there has been an increase in integrin avidity without an accompanying increase in af finity. Such increases in avidity are thought to be due to integrin cluster ing, which would also explain the results obtained with cytochalasin D. The failure of alpha(IIb)beta(3) to achieve the high affinity state in CHRF-28 8 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 on e marker of outside-in signalling, the augmentation of Vav phosphorylation seen during platelet aggregation, did not occur in CHRF-288 cells. (C) 1999 Elsevier Science B.V. All rights reserved.