The role of protein disulfide isomerase in the post-ligation phase of ß3 integrin-dependent cell adhesion.

ABSTRACT

INTRODUCTION: Protein disulfide isomerase (PDI) catalyzes disulfide bond exchange. It is crucial for integrin-mediated platelet adhesion and aggregation and disulfide bond exchange is necessary for αIIbß3 and αvß3 activation. However, the role of disulfide bond exchange and PDI in the post-ligation phase of αIIbß3 and αvß3 mediated cell adhesion has yet to be determined. METHODS: To investigate a possible such role, we expressed wild type (WT) human αIIb and either WT human ß3, or ß3 harboring single or double cysteine to serine substitutions disrupting Cys473-Cys503 or Cys523-Cys544 bonds, in baby hamster kidney (BHK) cells, leading to expression of both human αIIbß3 and a chimeric hamster/human αvß3. Adhesion to fibrinogen-coated wells was studied in the presence or absence of bacitracin, a PDI inhibitor, with and without an αvß3 blocker. RESULTS: Flow cytometry showed WT and mutant αIIbß3 expression in BHK cells and indicated that mutated αIIbß3 receptors were constitutively active while WT αIIbß3 was inactive. Both αIIbß3 and αvß3 integrins, WT and mutants, mediated adhesion to fibrinogen as shown by reduced but still substantial adhesion following treatment with the αvß3 blocker. Mutated αIIbß3 integrins disrupted in the Cys523-Cys544 bond still depended on PDI for adhesion as shown by the inhibitory effect of bacitracin in the presence of the αvß3 blocker. Mutated integrins disrupted in the Cys473-Cys503 bond showed a similar trend. CONCLUSIONS: PDI-mediated disulfide bond exchange plays a pivotal role in the post-ligation phase of αIIbß3-mediated adhesion to fibrinogen, while this step in αvß3-mediated adhesion is independent of disulfide exchange.