Regulation of αIIbβ3 Function in Human B Lymphocytes

Weiwei Qi,Elwyn Loh,Gaston Vilaire,Joel S Bennett

doi:10.1074/jbc.273.24.15271

Weiwei Qi, Elwyn Loh + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.24.15271

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Abstract

We studied the function of the platelet integrin alphaIIb beta3 using a B lymphocyte model in which alphaIIb beta3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alphaIIb beta3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alphaIIb beta3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galphai, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alphaIIb beta3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alphaIIb beta3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galphai, protein kinase C, and the actin cytoskeleton.

Highlights

Exposure of a binding site for ligands such as fibrinogen and von Willebrand factor on the platelet integrin ␣IIb␤3 is a prerequisite for platelet aggregation [1]
To study the process of ␣IIb␤3 activation in vitro, we have developed a model system in which wild-type and mutant ␣IIb␤3 expressed in GM1500 cells, an Epstein-Barr virus-transformed human B lymphocyte line, can be induced to interact with soluble and immobilized fibrinogen by the phorbol ester phorbol 12-myristate 13-acetate (PMA)1 [3, 4]
Introduction of a Functional formyl peptide chemoattractant receptor (fPR) into GM1500 Cells—Previously, we demonstrated that the phorbol ester PMA induces the adherence of B lymphocytes expressing ␣IIb␤3 to immobilized fibrinogen [3]

Summary

Introduction

Exposure of a binding site for ligands such as fibrinogen and von Willebrand factor on the platelet integrin ␣IIb␤3 is a prerequisite for platelet aggregation [1]. To determine the effect of PKC inhibitors on fMLP-stimulated ␣IIb␤3 function in human B cells, we incubated transfected GM1500 cells overnight with nanomolar concentrations of either the high affinity PKC inhibitor BIM I [17] or the low affinity inhibitor BIM V [18] and measured agonist-stimulated lymphocyte adherence to fibrinogen. To determine whether tyrosine phosphorylation regulates ␣IIb␤3 function in lymphocytes, we incubated transfected GM1500 cells overnight with micromolar concentrations of the tyrosine kinase inhibitor genistein [20] and measured agonist-stimulated lymphocyte adherence to fibrinogen.

Results

Conclusion