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.
Bergmeier
Wolfgang
W
Department of Molecular Oncology, General Surgery, Witten/Herdecke University, Wuppertal, Germany.
Schulte
Valerie
V
Brockhoff
Gero
G
Bier
Ulrich
U
Zirngibl
Hubert
H
Nieswandt
Bernhard
B
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