Analysis of adenoviral attachment to human platelets.
BACKGROUND
Systemic adenoviral (Ad) vector administration is associated with thrombocytopenia. Recently, Ad interaction with mouse platelets emerged as a key player determining liver uptake and platelet clearance. However, whether Ad can activate platelets is controversial. Thus, in vitro analysis of Ad attachment to platelets is of interest.


METHODS
We developed a direct flow cytometry assay to specifically detect Ad particles adherent to human platelets. The method was pre-validated in nucleated cells. Blocking assays were employed to specifically inhibit Ad attachment to platelets. Platelet activation was analyzed using annexin v flow cytometry.


RESULTS
We found in vitro that Ad binding to human platelets is synergistically enhanced by the combination of platelet activation by thrombin and MnCl2 supplementation. Of note, Ad binding could activate human platelets. Platelets bound Ad displaying an RGD ligand in the fiber knob more efficiently than unmodified Ad. In contrast to a previous report, CAR expression was not detected on human platelets. Integrins appear to mediate Ad binding to platelets, at least partially. Finally, alphaIIbbeta3-deficient platelets from a patient with Glanzmann thrombasthenia could bind Ad 5-fold more efficiently than normal platelets.


CONCLUSIONS
The flow cytometry methodology developed herein allows the quantitative measurement of Ad attachment to platelets and may provide a useful in vitro approach to investigate Ad interaction with platelets.
Shimony
Nilly
N
Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel. nillys@pob.huji.ac.il
Elkin
Gregory
G
Kolodkin-Gal
Dror
D
Krasny
Lina
L
Urieli-Shoval
Simcha
S
Haviv
Yosef S
YS
D000256:Adenoviridae / Q000378:metabolism / Q000502:physiology*; D001792:Blood Platelets / Q000378:metabolism / Q000821:virology*; D002460:Cell Line; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D006801:Humans; D016023:Integrins / Q000378:metabolism; D013915:Thrombasthenia / Q000503:physiopathology; D053585:Virus Attachment
D016023:Integrins