Abstract
The ligand binding activity of the platelet integrin alpha IIb beta 3 is initiated by agonist-generated intraplatelet signals. We studied this process in vitro by expressing recombinant alpha IIb beta 3 in Epstein-Barr virus-immortalized B lymphocytes. We found that phorbol ester stimulation induced the adhesion of lymphocytes expressing alpha IIb beta 3 to immobilized fibrinogen. Moreover, replacement of the transmembrane and cytoplasmic domains of the alpha and beta subunits of alpha IIb beta 3 with those of alpha L beta 2 significantly increased adherence, whereas replacement of only the cytoplasmic domains significantly decreased adherence. This suggests that transmembrane segments are involved in the agonist-induced modulation of alpha IIb beta 3 activity. Similar results were seen when the alpha IIb beta 3 activation-dependent monoclonal antibody PAC-1 was substituted for immobilized fibrinogen. We also found that the adherence of lymphocytes expressing beta 3 with either of the two alpha IIb/alpha L chimeras was similar to that of cells expressing alpha IIb beta 3, whereas the adherence of cells expressing alpha IIb with either of the two beta 3/beta 2 chimeras was substantially decreased, suggesting that the identity of the cytoplasmic domain of beta 3, but not of alpha IIb, is critical for alpha IIb beta 3 function. This report indicates that B lymphocytes contain signal transduction pathways involving protein kinase C that can increase the ligand binding activity of alpha IIb beta 3 and demonstrates the utility of these cells as an expression system for the study of agonist-stimulated alpha IIb beta 3 function.
Highlights
From the :j:Hematology-Oncology Division, Hospital of the University of Pennsylvania, the l1Division of Hematology, Children's Hospital of Philadelphia, and the Departments of Medicine and Pediatrics, the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
We found that phorbol ester stimulation induced the adhesion of lymphocytes expressing a1Ib/33 to immobilized fibrinogen
Intracellular signals initiated by platelet agonists convert the platelet integrin alIb,B3 from a functionally inactive to a functionally active conformation, enabling it to interact with ligands such as fibrinogen [2]
Summary
Hematology-Oncology Division, BRB1, Rm. 1005a, 422 Curie Blvd., Philadelphia, PA 19014. The nature of the signaling pathways, their targets on the cytoplasmic domains of O'IIbf, and the mechanism by which the signals are transmitted across the molecule are largely unknown. Like O'IIbf in platelets, the ligand binding activity of integrins in leukocytes is enhanced by agonists [1]. It has been reported that agonists up-regulate the ligand binding activity oflymphocyte O'vf33 [8]. We hypothesized that it might be possible to generate signals capable of activating O'IIbf in lymphocytes and tested this hypothesis by expressing recombinant O'IIbf in Epstein-Barr virus (EBV)-immortalized B lymphocytes. Because of the possibility that recognition of O'IIbf by the signal transduction pathways in lymphocytes might require the presence of the cytoplasmic and transmembrane domains of O'Lf32, we expressed chimeras of O'IIbf and O'Lf32 in these cells
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