Calcium Ionophore-Induced Tissue Factor (TF) Decryption Induces TF Immobilization Into Lipid Rafts and Negative Regulation of TF Procoagulant Activity.

Abstract 1131

Cell exposed tissue factor (TF), the physiologic initiator of blood coagulation, is normally expressed in a low procoagulant, or cryptic conformation, and requires activation, or decryption, to fully exhibit its procoagulant potential. TF decryption is not fully understood and multiple decrypting mechanisms have been proposed including phosphatidylserine (PS) exposure, TF monomerization, association with lipid rafts and redox modulation of TF. Calcium ionophores have been extensively used as TF decrypting agents, and both PS-dependent and independent mechanisms have been associated with ionophore-induced TF decryption. In the present study we analyzed the changes that occur in the lateral mobility of cell exposed TF during calcium ionophore-induced decryption, using a TF chimera with monomeric yellow fluorescent protein (YFP-TF). The YFP-TF expressed by endothelial cells (EC) retains TF procoagulant activity, is mainly exposed on the cell surface and can be decrypted similarly with endogenous TF by the calcium ionophore ionomycin. We analyzed the changes in TF membrane mobility during decryption using live cell imaging of YFP-TF expressed in EC. Fluorescence recovery after photobleaching (FRAP) analysis revealed a decreased mobility of TF in EC treated with the decrypting agent ionomycin. The YFP-TF fluorescence in the region of interest was more easily bleached in ionomycin–treated cells as compared with controls. The observed maximum recovery (R_max) of YFP-TF fluorescence in the bleached region of interest was significantly higher in control cells (80.84% recovery) as compared with ionomycin treated EC (39.29% recovery). These correlated with a decrease in YFP-TF mobile fraction from 50% for the control cells to 18% for the ionomycin treated EC. The lateral diffusion of the YFP-TF mobile fraction was similar between the two conditions, with halftime of fluorescence recovery of 7.69 sec in ionophore-treated cells and 10.69 sec in controls. These results suggest an immobilization of YFP-TF during decryption, which can be achieved by either lipid raft translocation or cytoskeleton floating. Similar to previous observations where TF cytoplasmic domain did not influence TF decryption, deletion of the TF cytoplasmic domain did not affect the lateral mobility of YFP-TF in FRAP analysis. To analyze decryption-induced changes in TF association with lipid domains, membrane fractions were isolated on a discontinuous Opti-Prep density gradient. Ionomycin treatment induced YFP-TF translocation from higher density, non-raft membrane fractions toward higher-buoyancy, raft fractions. Furthermore, the observed TF translocation into lipid rafts occurs without the formation of the quaternary complex with coagulation factors FVIIa, FXa and tissue factor pathway inhibitor (TFPI), as previously described. To address the functional modulation of TF procoagulant potential in response to lipid raft translocation, cell membrane cholesterol was either depleted with methyl-β-cyclodextrine (MβCD) or supplemented from an aqueous mixture of cholesterol-MβCD. Membrane cholesterol depletion decrypted TF in EC, likely through PS exposure, while also enhancing the procoagulant potential of ionomycin-decrypted TF. In contrast, cholesterol supplementation decreases the procoagulant potential of ionomycin-decrypted TF. Taken together, these observations support the model of tonic inhibition of TF procoagulant activity by the lipid raft environment. In conclusion, by live cell imaging we show that TF membrane mobility changes during calcium-ionophore induced decryption resulting in an immobilization of TF in lipid rafts. The immobilization is not influenced by the cytoplasmic domain of TF and does not require the formation of the TF-FVIIa-FXa-TFPI quaternary complex. Translocation into lipid rafts provides tonic inhibition of TF procoagulant potential and, as a consequence, we show for the first time that decrypting agents can also initiate negative regulation of TF procoagulant function. This negative feedback loop may help convert the decrypted TF back to its cryptic, low coagulant form.