PROCOAGULANT ACTIVITY AFTER EXPOSURE OF MONOCYTE-DERIVED MACROPHAGES TO MINIMALLY OXIDIZED LOW-DENSITY-LIPOPROTEIN - COLOCALIZATION OF TISSUE FACTOR ANTIGEN AND NASCENT FIBRIN FIBERS AT THE CELL-SURFACE

The role of tissue factor (TF) as nn initiator of the thrombotic compl ications secondary to atherosclerosis has been acknowledged, and in si tu expression of TF activity by monocyte-derived macrophages and lesio n-associated macrophage foam cells has been documented Macrophages exp ress TF activity upon exposure in vitro to either oxidized low density lipoprotein LDL (Ox-LDL) or endotoxin (lipopolysaccharide). This acti vity has been associated with membrane vesicles that apparently are sh ed after procoagulant expression Tbe present study, based upon the cor relative use of an enzyme-linked coagulant assay and three-dimensional multi-antigen, immunogold electron microscopy, reports the ultrastruc tural localization of TF antigen and spatially correlates TF with Ox-L DL binding and the presence of nascent fibrin polymers on the plasma m embrane of cultured macrophages. Pigeon monocyte/macrophages, after a 4-hour induction with lipopolysaccharide (2 mu g/ml) or minimally oxid ized LDI, (50 mu g/ml; thiobarbituric acid reducing substance, 5 to 8 nmol/mg protein) were incubated for 40 minutes in a Tris-buffered medi um containing factors VII, V, X, II, and I before either assaying for coagulant activity or processing for gold-colloid cytochemistry. TF ac tivity, as measured by enzyme-linked coagulant assay peaked 6 hours af ter agonist exposure with lipopolysaccharide and Ox-LDL giving, respec tively, 115- and 60-fold stimulation as compared with control. This ac tivity corresponded to the elaboration of membrane ruffles and microvi lli on the cell surfaces. Through correlative immunogold cytochemistry (15-nm-diameter colloid) and gold-ligand cytochemistry (30-nm-diamete r colloid), TF antigen (83%) and Ox-LDL (78%) were primarily associate d with the membrane ruffles and microvilli. Multi-antigen immunogold c ytochemistry when used in conjunction with ligand-gold cytochemistry d ocumented co-localization of Ox-LDL (22-nm gold), TF antigen (15-nm go ld) and a delicate three-dimensional network of short fibrin fibers th at were decorated in a linear fashion with the immunogold probes (30-n m gold). These results provide evidence that TF antigen is located at selected regions on the cell surfaces. Furthermore, these same regions provide binding sites for agonist uptake and organization sites for f ibrin polymerization. Hypothetically, localized membrane regions could be shed from the cell surface as a means for regulating coagulation p otential.