Epi-fluorescence videomicroscopy permits real-time imaging of platelet (plt) adhesion-aggregation to a defined microinjury site of an endothelial cell monolayer (ECM) exposed to flowing blood. The fluorescent label is the TAB murine monoclonal antibody (courtesy of Dr. R.P. McEver) directed against human pit cp HB, together with a fluorescein-conjugated goat F(ab')2 against murine immunoglobulin. The combination assures specificity for pit membranes, yet leaves pit function intact. Bovine aortic ECM, grown on rectangular cover glasses, comprise one wall of a flow chamber mounted on a vertical microscope stage. A 6-0 sterile suture, drawn across the ECM in a direction transverse to flow, creates microinjuries of width 70 ± 15 (mean ± SD). Pit deposition is virtually absent upon intact and confluent regions of the ECM. On microinjury sites and at a shear rate of 270 sec-1, however, computer-enhanced images show pit adherence, aggregation, and embolization. Pretreatment of the ECM with 1.0 mMFC lysine acetyl salicylate, further, leads to a three-fold increase in aggregate length. ECM products inhibitable by aspirin, therefore, modulate adhesion-aggregation in disease and normal states under physiologic flow conditions. The Table shows that nercent coverage of the injury area, and mean aggregate length readily discriminate normal, post-aspirin, and von Willebrand's (vWD's) bloods. Aggregate length is reduced in vWD's blood to a greater degree (p<0.01) than by oral aspirin, while the latter is associated with a paradoxic increase (p<0.01) in single plt adhesion.
Nanoplasmonics-enhanced label-free imaging of endothelial cell monolayer integrity
