Ultrastructural analysis of the distribution of von Willebrand factor and fibrinogen in platelet aggregates formed in the PFA-100 (TM)

The PFA-100(TM) is a new apparatus used to detect platelet dysfunction irt vitro, Anticoagulated blood flows under constant pressure through a capilla ry, and across an aperture that pierces a membrane coated with collagen and either epinephrine or ADP, Through their ability to adhere and aggregate, platelets occlude the orifice and the closure time is a test of platelet fu nction. Using electron microscopy and immunogold staining, we have analyzed the ultrastructure of platelet aggregates formed within the aperture and t hat are responsible for the occlusion, Standard electron microscopy showed that the aggregates formed on both collagen-epinephrine and collagen-ADP ca rtridges presented the same morphological features. The aggregates were exc lusively composed of platelets, some of which were degranulated. Degranulat ion was particularly intense at the periphery of the aggregate where platel ets were often totally devoid of secretory organelles. Immunogold staining on ultrathin frozen sections with polyclonal antibodies, allowed us to eval uate the distribution of adhesive proteins such as fibrinogen and von Wille brand factor (VWF) within the aggregate. The latter was found to be abundan t in the intercellular spaces between adjoining platelets. Although fibrino gen was also present, its labeling was less intense suggesting that vWF is the major protein implicated in the platelet-platelet interactions in the a ggregates formed in the PFA-100(TM) system. This may be because of the high shear rate that occurs across the aperture which suggests that the PFA-100 (TM) is particularly sensitive for detecting abnormalities of vWF-platelet interactions.