Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets.

The glycoprotein P-selectin is a cell adhesion molecule of stimulated platelets and endothelial cells, which mediates the interaction of these cells with neutrophils and monocytes. It is a membrane component of cell storage granules, and is a member of the selectin family which includes E-selectin and L-selectin. P-selectin recognizes both lineage-specific carbohydrate ligands on monocytes and neutrophils, including the Lewis x antigen, sialic acid, and a protein component. In inflammation and thrombosis, P-selectin may mediate the interaction of leukocytes with platelets bound in the region of tissue injury and with stimulated endothelium. To evaluate the role of P-selectin in platelet-leukocyte adhesion in vivo, the accumulation of leukocytes within an experimental thrombus was explored in an arteriovenous shunt model in baboons. A Dacron graft implanted within an arteriovenous shunt is thrombogenic, accumulating platelets and fibrin within its lumen. These bound platelets express P-selectin. Here we show that antibody inhibition of leukocyte binding to P-selectin expressed on platelets immobilized on the graft blocks leukocyte accumulation and inhibits the deposition of fibrin within the thrombus. These results indicate that P-selectin is an important adhesion molecule on platelets, mediating platelet-leukocyte binding in vivo, that the presence of leukocytes in thrombi is mediated by P-selectin, and that these leukocytes promote fibrin deposition.

P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities.

P-selectin on platelets and endothelial cells and E-selectin on endothelial cells are leukocyte receptors that recognize lineage-specific carbohydrates on neutrophils and monocytes. The proposed ligands for these receptors contain the Le(x) core and sialic acid. Since other investigators have shown that both E-selectin and P-selectin bind to sialylated Le(x), we evaluated whether E-selectin and P-selectin recognize the same counter-receptor on leukocytes. The interaction of HL60 cells with Chinese hamster ovary (CHO) cells expressing P-selectin or E-selectin was studied. To determine whether a protein component is required in addition to sialyl Le(x) for either P-selectin or E-selectin recognition, HL60 cells or neutrophils were digested with proteases, including chymotrypsin, elastase, proteinase Glu-C, ficin, papain, or thermolysin. Cells treated with these proteases bound E-selectin but not P-selectin. Fucosidase or neuraminidase treatment of HL60 cells markedly decreased binding to both E-selectin- and P-selectin-expressing CHO cells. Growth of HL60 cells in tunicamycin inhibited the ability of these cells to support P-selectin-mediated binding and, to a lesser extent, E-selectin-mediated binding. Purified P-selectin inhibited CHO:P-selectin binding to HL60 cells, but incompletely inhibited CHO:E-selectin binding to HL60 cells. However, purified soluble E-selectin inhibited CHO:P-selectin and CHO:E-selectin binding to HL60 cells equivalently and completely. COS cells, unable to bind to E-selectin or P-selectin, bound E-selectin but not P-selectin upon transfection with alpha-1,3-fucosyltransferase or alpha-1,3/1,4-fucosyltransferase. Similarly, LEC 11 cells expressing sialyl Le(x) bound E-selectin- but not P-selectin-expressing CHO cells. Sambucus nigra lectin, specific for the sialyl-2,6 beta Gal/GalNAc linkage, inhibited P-selectin but not E-selectin binding to HL60 cells. Although sialic acid and Le(x) are components of the P-selectin ligand and the E-selectin ligand, these results indicate that the ligands are related, having overlapping specificities, but are structurally distinct. A protein component containing sialyl Le(x) in proximity to sialyl-2,6 beta Gal structures on the P-selectin ligand may contribute to its specificity for P-selectin.

Fibrin coating of bladder tumor cells (T24) is not protective against LAK cell cytotoxicity.

Certain evidence indicates that tumor cells in the circulation may be enshrouded with a coat of fibrin. It has been suggested that this fibrin coat protects tumor cells from attack by the immune system. This study compared the interaction of lymphokine activated killer (LAK) cells with tumor cells alone and with fibrin coating. LAK cell killing of cultured human bladder tumor cells (T24) was measured by a 4-hour chromium release assay. Tumor cells (3 x 10(6] were incubated with Na51CrO4 for 2 hours at 37 degrees C and 5% CO2 in serum-free medium. After washing, one half of the cells were coated with fibrin by exposure to recalcified platelet-poor plasma. Fibrin coating was confirmed by immunofluorescence with anti-human-fibrinogen-fluorescein-conjugated antibodies. LAK cells were prepared from peripheral blood lymphocytes by incubation with interleukin-2 at a concentration of 1000 units of interleukin-2/1 ml serum-free medium/1 million cells for 5 days at 37 degrees C, 5% CO2. Five thousand tumor cells with or without fibrin were incubated with varying concentrations of either LAK or peripheral blood lymphocytes (10,000 to 100,000 cells). After 4 hours the supernatants were harvested and counted in a gamma counter for 1 minute. Over a range of effector-to-target cell ratios of 10:1 to 100:1 (LAK to T24), no difference was seen in percentage of specific lysis for T24 alone versus fibrin-coated T24 cells. At a ratio of 100:1 (LAK to T24), percentage of specific lysis was 83.3% versus 87.7% for uncoated and coated T24 cells, respectively. This suggests that fibrin coating of tumor cells is insufficient to provide protection from LAK cell killing.