Flow cytometric detection of activated mouse integrin αIIbβ3 with a novel monoclonal antibody

Abstract

Background Integrin αIIbβ3 mediates platelet adhesion and aggregation and plays a crucial role in thrombosis and hemostasis. αIIbβ3 is expressed in a low affinity state on resting platelets. Upon platelet activation, αIIbβ3 shifts to a high affinity conformation that efficiently binds its ligands. On human platelets, the high affinity conformation of αIIbβ3 is detected by the monoclonal antibody (mAb), PAC-1. However, a reagent with binding specificity to high affinity mouse αIIbβ3 has not been described so far. Methods A novel rat mAb directed against mouse αIIbβ3 (JON/A) was generated and characterized. JON/A was conjugated with fluorescein isothiocyanate (JON/A^FITC) or with R-phycoerythrin (JON/A^PE) and used for flow cytometric analysis of mouse platelets. Results Although JON/A^FITC bound to resting and activated platelets, virtually no binding of the larger JON/A^PE to resting platelets was detectable. However, strong binding of JON/A^PE occurred on platelet activation in a dose-dependent manner. Binding of JON/A^PE required extracellular free calcium and was irreversible, thereby stabilizing the high affinity conformation of αIIbβ3. Conclusion JON/A^PE is the first tool for direct assessment of integrin αIIbβ3 activation in mice. Furthermore, JON/A^FITC and JON/A^PE provide the first examples of fluorescent antibody derivatives with identical antigenic specificitiy that allow the discrimination between the resting and the activated state of an integrin. Cytometry 48:80–86, 2002.