Caveolae optimize tissue factor–Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor

Abstract

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that prevents intravascular coagulation through inhibition of factor Xa (fXa) and the tissue factor (TF)-factor VIIa complex (TF-fVIIa). Localization of TFPI within caveolae enhances its anticoagulant activity. To further define how caveolae contribute to TFPI anticoagulant activity, CHO cells were co-transfected with TF and membrane associated TFPI targeted to either caveolae (TFPI-GPI) or to bulk plasma membrane (TFPI-TM). Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent insoluble membrane fractions, while TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-FVIIa than TFPI-TM, as measured in both amidolytic and plasma clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIβ, and HUVECs, did not affect their fXa inhibition but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms, and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.

Protein S Is a Cofactor for Platelet and Endothelial Tissue Factor Pathway Inhibitor-α but Not for Cell Surface–Associated Tissue Factor Pathway Inhibitor

“…PS cofactor activity toward cell surface–associated TFPI containing K3 was assessed using CHO cells expressing a GPI-anchored form of TFPI containing all 3 Kunitz domains (K1K2K3). 36 PS did not enhance the initial or final rate of FXa inhibition by these cells (Figure 5A). To verify that PS is capable of interacting with this form of TFPI, the cells were treated with PIPLC, and the FXa inhibitory activity of the supernatants was assessed (Figure 5B and 5C).…”

Comparison of the inhibitory activities of human tissue factor pathway inhibitor (TFPI)α and TFPIβ

“…The data presented here suggest that expression of TFPIβ on endothelial cells, where it is available at a high local concentration to inhibit the potentially detrimental TF‐mediated coagulation and/or cell signaling events that occur during inflammation, is optimized to dampen intravascular TF activity,. The localization of TFPIβ within lipid rafts/caveolae in endothelial cells has been shown to further enhance its TF‐inhibitory activity .…”

“…CHO‐TF cells were then transfected with a neomycin‐resistant plasmid containing human TFPIβ to produce CHO‐TF/TFPIβ cells. Cells were prepared for flow cytometry as previously described . To verify the presence of a GPI‐anchor, transfected CHO‐TF/TFPIβ cells were treated with 1 U mL −1 PIPLC for 1 h at 37 °C , and analyzed by flow cytometry.…”

Biology of tissue factor pathway inhibitor

“…121 In addition, disruption of these membrane microdomains with methyl-b-cyclodextran results in decreased inhibitory activity. 121 The TF-FVIIa-FXa ternary complex, apart from its role in coagulation, initiates signaling pathways through proteolytic activation of the protease activated receptors 1 and 2 (PAR1 and PAR2). 123 Studies of the ability of TFPI to inhibit the signaling activity of the ternary complex have yielded mixed results.…”