Protein–platelet and platelet–leukocyte interaction at materials in contact with human blood

Abstract

The adhesion and activation of platelets and leukocytes at blood–material interfaces was studied by fluorescence microscopy and photometry using specific anti-CD antibodies, antiplasma protein antibodies, and the calcium probe Fura-2. Hydrophilic glass or methylized, hydrophobic glass was prepared and capillary blood was placed as droplets on the surface in a humified chamber. The adsorption of plasma proteins was monitored with FITC-labeled antibodies directed against albumin, IgG, fibrinogen, fibronectin, the v. Willebrand factor, prothrombin/thrombin, and complement factor C3c. The adhesion of platelets was shown by anti-CD 61 antibodies, specific for this cell type. Adhesion of leukocytes was measured by staining their DNA with acridine orange. Adhering platelets were found after 15 s of blood–material contact on both surfaces. The number of adhering platelets rapidly decreased at the hydrophilic 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 cell surfaces. Platelet-derived microvesicles were found after 4 and 8 min of blood–material contact. These microvesicles showed intense staining with anti-C3c antibodies. Significant numbers of leukocytes (PMN cells) were seen after 2 h of blood–material contact. In other experiments, granulocytes were isolated and incubated with Fura-2. The supernatant of hirudin-treated blood, exposed to hydrophilic or hydrophobic glass surfaces, was added to the cells and the fluorescence was recorded after emission at 340 and 380 nm. A rapid peak was seen, indicating calcium influx into the cytoplasm. The activating substance was removed from the supernatant by filtering it through a 0.1–0.45 μm Millipore filter. The blood samples were taken from patients undergoing treatment 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.