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


OBJECTIVE
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 D(3)-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 D(3)-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.
Christersson
C
C
Department of Cardiology, Uppsala University Hospital, Uppsala, Sweden. christina.christersson@akademiska.se
Johnell
M
M
Siegbahn
A
A
D001792:Blood Platelets / Q000378:metabolism*; D023201:CD40 Ligand / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D016753:Interleukin-10 / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008954:Models, Biological; D009000:Monocytes / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D013925:Thromboplastin / Q000378:metabolism*; D020298:U937 Cells; D019061:src-Family Kinases / Q000378:metabolism*
D016207:Cytokines; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D016753:Interleukin-10; D023201:CD40 Ligand; D013925:Thromboplastin; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases