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.


CONCLUSIONS
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.
Department of Molecular Oncology, General Surgery, Witten/Herdecke University, Wuppertal, Germany.
Wolfgang
W
Bergmeier
Valerie
V
Schulte
Gero
G
Brockhoff
Ulrich
U
Bier
Hubert
H
Zirngibl
Bernhard
B
Nieswandt
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000494:pharmacology; D004306:Dose-Response Relationship, Immunologic; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D051379:Mice; D008807:Mice, Inbred BALB C; D010974:Platelet Aggregation / Q000187:drug effects / Q000276:immunology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D013047:Specific Pathogen-Free Organisms
D000911:Antibodies, Monoclonal; D019039:Platelet Glycoprotein GPIIb-IIIa Complex