Kunitz 3-Selective but Not Kunitz 2-Selective TFPI Inhibitors Cause Release of Tfpiα from Endothelial Cells: Insights into the Release Mechanism
Authors: Peterson Julie A. , Augustsson Cecilia , Maroney Susan A , Heibroch Petersen Helle , Hilden Ida , Mast Alan , Zwifelhofer Wes 
Published: October 2019

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that regulates early blood coagulation events and contains 3 Kunitz-type serine protease domains (K1-3). TFPI inhibitors are being developed to treat hemophilia. However, an inhibitory aptamer to the K3 domain of TFPI has been found to produce an increase in plasma TFPI with associated bleeding. Therefore, understanding the mechanisms causing release of TFPI from endothelium is important for further development of these drugs. Two major isoforms of TFPI, TFPIα and TFPIβ, are generated by human endothelial cells. Both isoforms contain K1 and K2. TFPIβ attaches to the cell surface via a C-terminal glycosylphosphatidyl inositol (GPI) anchor. TFPIα is a soluble protein with an additional Kunitz domain (K3) and a basic C-terminal tail present in human plasma. It has been found that plasma TFPIα promptly increases 2- to 4-fold following heparin infusion. Treatment of cultured human endothelial cells with heparin also promptly increases TFPIα 2- to 4-fold in culture media, suggesting that the basic C-terminal tail of TFPIα interacts with cell surface glycosaminoglycans (GAGs) and that this association is disrupted by heparin, releasing TFPIα into plasma or media. However, cell surface TFPI can be almost entirely removed by treatment with phosphatidylinositol phospholipase C, an enzyme that removes GPI-anchored proteins from the cell surface, indicating that only the GPI-anchored TFPIβ is present at the surface. This suggests that heparin may cause release of TFPIα from an intracellular pool. Thus, the mechanism of heparin-induced TFPIα release from endothelium remains unresolved.

METHODS: Wild type (WT) and aerolysin-resistant (AR) Ea.hy926 cells, which lack GPI-anchored proteins, including TFPIβ, but produce normal amounts of TFPIα, were used to examine how heparin and anti-TFPI antibodies induce release of TFPI. For this, WT or AR cells were incubated for 15 minutes at room temperature or 4°C, with either heparin, 0.3 M NaCl, anti-K2 or anti-K3 antibody. Surface TFPI was detected with an anti-K2 antibody using flow cytometry. TFPIα released into culture media was measured with a TFPIα-specific ELISA. Brefeldin A, an inhibitor of protein transport from endoplasmic reticulum to Golgi, was used to block TFPIα secretion. Real time PCR was used to measure changes in TFPI mRNA.

RESULTS: WT cells express surface TFPI that can be detected with an anti-K2 antibody using flow cytometry and which is not altered by exposure of cells to heparin or anti-K3 antibody. AR cells do not have TFPI on their surface that is detectable by flow cytometry. Incubation of WT or AR cells with heparin, 0.3 M NaCl, or anti-K3 antibody produced an approximately 4-fold increase of TFPIα released into culture media. This was not observed when cells were incubated with anti-K2 antibody. To determine if heparin induces release of TFPIα from inside the cell, they were incubated for 15 minutes with heparin 4 times, in the presence or absence of Brefeldin A. During the fourth 15 minute treatment the amount of TFPIα secreted into the media was equal in heparin-treated or control cells. The cells were then allowed to incubate for an additional hour in the presence or absence of heparin. During this time, heparin-treated cells secreted TFPIα at a 2-fold faster rate than cells in buffer only (p=0.036). This difference was not observed in cells treated with Brefeldin A, which reduced TFPIα secretion by almost 5-fold (p=0.005). TFPIα transcript levels were not different between the heparin- and non-heparin-treated cells.

CONCLUSION: TFPIα is released from Ea.hy926 cells by heparin and an anti-K3 antibody, both of which bind to the C-terminal region of TFPIα, while an anti-K2 antibody has been shown not to cause release of TFPIα from the cells. Indirect evidence suggests that there is a pool of TFPIα associated with cell surface GAGs and that this pool is released by agents that disrupt this interaction. However, the presence of TFPIα on the cell surface cannot be directly demonstrated using flow cytometry. Cells stripped of heparin-releasable TFPIα continue to secrete it into the media at a faster rate in the presence of heparin. Heparin does not alter transcription of TFPIα. Instead, it appears to enhance its secretion, perhaps by diverting it from storage in an intracellular pool.

Disclosures

Augustsson: Novo Nordisk: Employment. Heibroch Petersen:Novo Nordisk: Employment. Hilden:Novo Nordisk: Employment. Mast:Novo Nordisk: Research Funding; Siemens: Honoraria.