The First EGF Domain of Coagulation Factor IX Increases PAR1 Distributionin Lipid Rafts and Modulates the Response to Thrombin in Endothelial Cells

Abstract

The first epidermal growth factor 1 (EGF-1) domain of coagulation factor IX (FIX) contains a CXDXXXXYXCXC consensus amino acid sequence, which reportedly induces endocytosis and clustering of lipid rafts in adhesive cells. Endocytosis and lipid rafts regulate signaling via receptors on the plasma membrane. In this study, weinvestigated the effect of FIX EGF-1 domains on intracellular signaling by thrombin (FIIa) in endothelial cells.Recombinant EGF-1 protein of FIX (EGF-FIX) alone did not induce ROCK-dependent phosphorylation ofmyosin light chain (MLC) in human umbilical vein endothelial cells (HUVECs). On the other hand, a high concentration (100 ng/ml) of FIIa increased phosphorylated MLC, and EGF-FIX enhanced it by 1.3-fold. Interestingly, low concentrations (0.3 ng/ml) of FIIa suppressed the phosphorylation of MLC, and the combination oflow-concentration FIIa and EGF-FIX promoted MLC phosphorylation. Methyl-beta-cyclodextrin (MβCD), whichinhibits the formation of lipid rafts, suppressed the phosphorylation of MLC regardless of the FIIa concentration,whereas EGF-FIX attenuated the effects of MβCD. EGF-FIX increased the distribution of the FIIa receptor(PAR1) in the lipid raft fraction. Active FIX could modulate intracellular signaling by other molecules in endothelial cells via its EGF domain.