PROTEIN-PLATELET AND PLATELET-LEUKOCYTE INTERACTION AT MATERIALS IN CONTACT WITH HUMAN BLOOD

The adhesion and activation of platelets and leukocytes at blood-mater ial interfaces was studied by fluorescence microscopy and photometry u sing specific anti-CD antibodies, antiplasma protein antibodies, and t he calcium probe Fura-2. Hydrophilic glass or methylized, hydrophobic glass was prepared and capillary blood was placed as droplets on the s urface in a humified chamber. The adsorption of plasma proteins was mo nitored with FITC-labeled antibodies directed against albumin, IgG, fi brinogen, fibronectin, the v. Willebrand factor, prothrombin/thrombin, and complement factor C3c. The adhesion of platelets was shown by ant i-CD 61 antibodies, specific for this cell type. Adhesion of leukocyte s was measured by staining their DNA with acridine orange. Adhering pl atelets were found after 15 s of blood-material contact on both surfac es. The number of adhering platelets rapidly decreased at the hydrophi lic surface, but remained high for more than 8 min at the hydrophobic surface. Fibrinogen was the dominating protein at the material surface , whereas fibronectin and the v. Willebrand factor were found at the c ell surfaces. Platelet-derived microvesicles were found after 4 and 8 min of blood-material contact. These microvesicles showed intense stai ning with anti-C3c antibodies. Significant numbers of leukocytes (PMN cells) were seen after 2 h of blood-material contact. In other experim ents, granulocytes were isolated and incubated with Fura-2. The supern atant of hirudin-treated blood, exposed to hydrophilic or hydrophobic glass surfaces, was added to the cells and the fluorescence was record ed after emission at 340 and 380 nm. A rapid peak was seen, indicating calcium influx into the cytoplasm. The activating substance was remov ed from the supernatant by filtering it through a 0.1-0.45 mu m Millip ore filter. The blood samples were taken from patients undergoing trea tment with extracorporeal circulation. The samples were incubated with monoclonal antibodies against surface antigens CD-11b, 16, 35, 61 and 62. The fluorescence was measured in a flow cytofluorometer. The PMN cells were shown to be activated rapidly after the onset of oxygenator circulation. (C) 1995 American Vacuum Society.