Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody.

BACKGROUND: Integrin alphaIIbbeta3 mediates platelet adhesion and aggregation and plays a crucial role in thrombosis and hemostasis. alphaIIbbeta3 is expressed in a low affinity state on resting platelets. Upon platelet activation, alphaIIbbeta3 shifts to a high affinity conformation that efficiently binds its ligands. On human platelets, the high affinity conformation of alphaIIbbeta3 is detected by the monoclonal antibody (mAb), PAC-1. However, a reagent with binding specificity to high affinity mouse alphaIIbbeta3 has not been described so far.
METHODS: A novel rat mAb directed against mouse alphaIIbbeta3 (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 alphaIIbbeta3.
CONCLUSION: JON/A(PE) is the first tool for direct assessment of integrin alphaIIbbeta3 activation in mice. Furthermore, JON/A(FITC) and JON/A(PE) provide the first examples of fluorescent antibody derivatives with identical antigenic specificity that allow the discrimination between the resting and the activated state of an integrin. Copyright 2002 Wiley-Liss, Inc.