Flow-based measurements of von Willebrand factor (VWF) function: binding to collagen and platelet adhesion under physiological shear rate.
BACKGROUND
VWF circulates in plasma as a series of heterogeneous multimers, mediating platelet tethering, translocation and finally adhesion to areas of injured endothelium under physiological high arterial blood flow. VWF-platelet binding requires conformational changes in VWF, which are induced by immobilization and shear. Because of unavailability of a simple flow-based measurement system, VWF activity assays are generally performed under static conditions. We describe an easily reproducible in vitro flow-chamber model using commercially available flow devices to examine VWF-collagen binding and VWF-mediated platelet adhesion under physiological flow conditions.


METHODS
The collagen surface of the flow-chamber was analyzed by atomic force microscopy. Collagen-bound VWF was characterized by multimer analysis and multi labelling immunofluorescence detection of exposed GPIb binding domains. Platelet adhesion was captured by time-lapse microscopy.


RESULTS
The described flow-chamber system facilitates multimer analysis of collagen-bound VWF, whereas all VWF multimers bound to collagen under physiological low to high shear rates. Multi labelling immunofluorescence detection exhibited exposed GPIb binding domains co-localized with VWF molecules. VWF-dependent platelet adhesion using time-lapse microscopy showed values comparable to experiments done with whole blood, and platelet adhesion was dependent on the VWF concentration.


CONCLUSIONS
The established flow-chamber model represents an easy-to-set-up and customized tool for the characterization of VWF-binding to collagen as well as the determination of VWF-dependent platelet adhesion under defined flow conditions in real-time.
Fuchs
Birte
B
Octapharma Research & Development, Molecular Biochemistry Department, Berlin, Germany.
Budde
Ulrich
U
Schulz
Andrea
A
Kessler
Craig M
CM
Fisseau
Claudine
C
Kannicht
Christoph
C
D001792:Blood Platelets / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism*; D006801:Humans; D010973:Platelet Adhesiveness / Q000502:physiology*; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000378:metabolism*
D014841:von Willebrand Factor; D003094:Collagen