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

Authors


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/AFITC) or with R-phycoerythrin (JON/APE) and used for flow cytometric analysis of mouse platelets.

Results

Although JON/AFITC bound to resting and activated platelets, virtually no binding of the larger JON/APE to resting platelets was detectable. However, strong binding of JON/APE occurred on platelet activation in a dose-dependent manner. Binding of JON/APE required extracellular free calcium and was irreversible, thereby stabilizing the high affinity conformation of αIIbβ3.

Conclusion

JON/APE is the first tool for direct assessment of integrin αIIbβ3 activation in mice. Furthermore, JON/AFITC and JON/APE 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. © 2002 Wiley-Liss, Inc.

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