Retention of leukocytes and platelets by thrombi, in vivo, or immobilized PCA, ex vivo. Standard light microscopy (PTAH stain) demonstrated the increased presence of trapped leukocytes in thrombi that developed for 60 minutes in the chamber region of the thrombogenic device. A representative section (A) shows the distribution of leukocytes in and around the thrombus that formed on the wall of a thrombus chamber. Thrombi were rich in fibrin and platelets, attracted leukocytes to their surface regions, and had relatively fewer red blood cells than the same volume of blood clot. For comparison of histology, blood that was clotted for 60 minutes before fixation is also shown in a typical section (B). Polystyrene 24-well plates were coated with PCA or thrombin for 3 hours followed by incubation with heat-inactivated FBS for 20 minutes. Purified leukocytes (2 × 106/mL) were placed on surface-immobilized PCA or thrombin (α-Thr) for 1 hour at 37°C in the absence (▪) or presence (□) of EDTA (5 mM) (C). To quantify the degree of adherent leukocytes, surfaces were washed with HBSS, fixed with paraformaldehyde, and imaged via phase-contrast microscopy. To quantify platelet-PCA interactions (D), fluorescently labeled platelets (plt; 2 μM DiOC6) were reconstituted with RBCs and perfused over PCA-coated coverslips at 300 s−1 for 3 minutes in the absence or presence of the anti-GPIb mAb 6D1 (20 μg/mL). Platelet rolling (transient platelet interactions lasting > 500 ms) was recorded in real time using fluorescence microscopy.
