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Tissue factor and IL8 production by P‐selectin‐dependent platelet–monocyte aggregates in whole blood involves phosphorylation of Lyn and is inhibited by IL10

Summary.  Background: P‐selectin and CD40L expressed by activated platelets induce tissue factor (TF) and inflammatory cytokines in monocytes, but little is known of the cellular signaling pathways involved. The anti‐inflammatory cytokine IL10 reduces atherosclerotic plaque formation. Objectives: To evaluate the importance of P‐selectin upon platelet–monocyte aggregate (PMA) formation in thrombin receptor activator peptide (TRAP) stimulated whole blood, the P‐selectin–P‐selectin glycoprotein ligand (PSGL)‐1‐induced cellular signaling pathway, and the effects of IL10 on these functions. Methods: TF, IL8, and monocyte chemotactic protein‐1 (MCP‐1) production, PMAs and phosphorylation of Lyn were analyzed in whole blood, purified monocytes, and vitamin D3‐differentiated U‐937 cells stimulated with TRAP or P‐selectin with or without IL10. Anti‐P‐selectin or anti‐CD40L antibodies (Abs), Src‐kinases inhibitors, SU6656 or PP2, were added in some experiments. Results: TRAP and P‐selectin increased TF, IL8, and MCP‐1 mRNA in whole blood and purified monocytes. Anti‐P‐selectin Ab reduced TRAP‐induced PMA formation by 80 ± 2% (P = 0.001) and production of TF (P = 0.04) and IL8 (P = 0.01). IL10 and SU6656 had no effect on PMA formation, although both significantly reduced TF (P = 0.002 and P = 0.02) and IL8 (P = 0.009 and P = 0.001) mRNA upon TRAP and P‐selectin stimulation. Induced Lyn phosphorylation in monocytes was diminished by SU6656 (P = 0.02), anti‐P‐selectin Ab (P = 0.02), and IL10 (P = 0.03) upon TRAP or P‐selectin stimulation. These results were confirmed in the vitamin D3‐differentiated U‐937 cells. Conclusions: The formation of PMAs in whole blood was P‐selectin‐dependent in the long term. P‐selectin–PSGL‐1‐induced TF and IL8 expression through Lyn phosphorylation, and part of the inhibitory effect of IL10 depends on reduced phosphorylation.