Perfusion of anticoagulated blood over artery cross-sections at high shear results in platelet adhesion which requires plasma von Willebrand factor (vWf) and is localized to the adventitial layer of the vessel wall. We investigated whether the incapability of the media to mediate platelet accretion results from the lack of vWf binding. Von Willebrand factor binding to collagen-coated microplate wells is measured by indirect ELISA. Binding to frozen porcine artery cross-sections or collagen preparations, exposed to normal pig plasma, is visualized by indirect particle-immunofluorescence using goat anti-mouseIgG-oated fluorescent beads. Tissue sections or protein targets exposed to plasma of pigs with severe von Willebrand disease are used as negative controls. Alternatively, porcine vWf-coated fluorescent beads or bovine serum albumin-coated beads are incubated on the sections to locate vWf binding sites in the tissue. Sections of polymerized collagen fibrils bind vWf from normal pig plasma, as demonstrated by the high number of goat anti-mouse-lgG-coated beads compared to the few beads found after exposure to vWf deficient plasma. The binding of vWf to collagen sections is completely inhibited by Wl-23, a monoclonal anti-vWf antibody, that also inhibits vWf binding to collagen in the quantitative microplate assay. Binding of plasma vWf to artery sections is localized to the adventitia, as indicated by the selective distribution of the goat anti-mouse-lgG-coated beads leaving the media essentially devoid of beads. The binding of plasma vWf to adventitia in this assay is completely inhibited by Wl-23. Porcine vWf-coated beads also bind preferentially to the adventitia. Transmission electron microscopy of artery sections shows significant amounts of banded collagen fibrils in the media, although collagen fibrils are thicker and more abundant in the adventitia. Therefore, the reduction of vWf binding and platelet adhesion is not commensurate with a quantitative reduction in fibrillar collagen content. We conclude that vWf-dependent platelet adhesion is localized to the adventitial layer of the arterial wall because this layer contains the components necessary for plasma vWf binding. We speculate that in the media, fibrillar matrix polymers are complexed in such a way as to shield the vWf binding sites, or that media matrix is chemically different from that found in the adventitia, being defective in vWf binding.
|Original language||English (US)|
|Issue number||11 PART II|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology