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

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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.

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