Cytoplasmic Tail of Tissue Factor(TF): Role in TF Trafficking to Microparticles.

Introduction: Tissue factor (TF) is a transmembrane protein that is primarily extracellular. It has a 21 amino acid cytoplasmic tail, which is not essential for coagulation but may have a role in signal transduction. We studied the activity of deletion variants of TF, transfected into a pancreatic cancer line (MiaPaCa-2), which lacks TF RNA or protein.

Methods: TF variants: lctTF (lacking cytoplasmic tail), sTF(soluble, lacking transmembrane and cytoplasmic domains), wtTF (wild type) were transfected into MiaPaCa-2 cells. Microparticles were prepared from serum-free conditioned media(CM) by ultracentrifugation at 100,000g. Presence of TF antigen was detected by immunoblot using a monoclonal antibody to TF, 10H10. Tissue factor activity (TFA) was assessed by recalcification time.

Results: Immunoblot confirmed the presence of TF in the cell lysates of MiaPaCa-2 cells transfected with wtTF or lctTF. In the sTF MiaPaCa-2 cells, TF was not detected in the cell lysates, but was present in CM. TF was present in CM of lctTF-transfected cells, but only trace antigen was observed in CM of wtTF cells. TF was microparticle-associated in the CM collected from lctTF cells, but not in the sTF cells. The TF protein made by sTF cells was not functional in the CM as demonstrated by no difference in TFA as compared to untransfected MiaPaCa-2 cells (>350 sec vs 351 sec). The CM from lctTF MiaPaCa-2 cells shortened clotting time by over 50% (159 sec vs 351 sec), while wtTF MiaPaCa-2 cells did not (349 sec vs 351 sec). The microparticles from lctTF had the most TFA when compared to wtTF or untransfected cells (107 sec vs 194 sec vs 436 sec). In the CM from cells with lctTF, TFA was exclusively in the microparticle section and absent in the supernatant (107 sec vs 440 sec).

Conclusions: Wild type TF remains primarily cell associated. In contrast, a higher fraction of the lctTF was released into the CM and remained microparticle associated. TF protein that lacks the transmembrane portion is soluble but not microparticle-associated and does not have procoagulant activity. Lack of the cytoplasmic tail results in increased generation of functional, microparticle-associated TF compared with the wild-type protein. These data suggest the cytoplasmic tail may influence the trafficking of functional TF to the microparticle fraction.