Evaluation of Non-Proteolytic Functions of Murine Alternatively Spliced Tissue Factor.

Aside from hemostatic maintenance, Tissue Factor (TF) also plays a major role in such pathophysiological processes as thrombogenesis and cancer progression. TF protein occurs naturally in two forms: full length TF (flTF), a well–studied integral membrane glycoprotein that serves as an obligatory enzymatic co-factor of the serine protease FVIIa, and alternatively spliced TF (asTF), which lacks a transmembrane domain and can be secreted. asTF has a unique 40 amino acid C-terminus and can be detected, alongside flTF, in organized arterial thrombi (Bogdanov et al, Nat Med 2003 Apr; 9(4):458-62). Following the discovery of human asTF, the murine form of asTF (masTF) was identified and characterized (Bogdanov et al, J Thromb Haemost. 2006 Jan;4(1):158-67). Like human asTF, masTF lacks a transmembrane domain due to the exclusion of exon 5 from the primary transcript during its splicing, possesses a unique 93 amino acid C-terminus, and exhibits minimal coagulant potential. Most recently, hasTF was discovered to induce cell adhesion and angiogenesis via integrin ligation, independent of FVIIa, PAR-2 cleavage and/or any other proteolytic events (van den Berg et al, Proc Natl Acad Sci U.S.A. 2009 Nov 17;106(46):19497-502). It has yet to be investigated whether masTF exhibits non-proteolytic biologic activity analogous to that of hasTF. As murine models comprise the preferred in vivo platform in preclinical cardiovascular and cancer research, it is highly warranted to ascertain these possible functional properties of masTF. In this study, we performed an initial set of experiments to address this issue.

N-terminally His-tagged recombinant masTF mature protein was generated in E. Coli, purified, and assessed by Coomassie staining; masTF's identity was successfully verified by western blotting. Analogously to hasTF, masTF induced adhesion of murine endothelial cells (bEnd.3) in a time-dependent fashion. A 15-fold increase over BSA (n = 3; p < 0.0001) was observed as early as 1 hour after the experiment's onset; at 4 hours, bEnd.3 cells exposed to masTF displayed a 37-fold increase over BSA (p < 0.0005). We noted that masTF also induced bEnd.3 cells to display characteristic endothelial morphology, whereas BSA did not. We subsequently used a wound healing assay to ascertain whether masTF promotes directional migration of bEnd.3 cells, and used VEGF (100 ng/mL) as a positive control to assess the degree of potency. Per wound, the area of complete closure generated by masTF (50 nM) was ∼4.9-fold greater than that of the vehicle (n = 3; p < 0.0001), an effect similar to that elicited by VEGF (area of closure ∼7-fold greater than that of the vehicle; p < 0.0001). Microarray analysis of masTF-treated bEnd.3 cells (Affymetrix Gene 1.0 ST platform) revealed upregulation of the genes encoding multiple CXC chemokines, with CXCL2, CXCL10, and CXCL1 topping the list (R = 1.42, 1.37, and 1.32, respectively). Notably, expression of the major adhesion molecule VCAM-1 was also upregulated (R = 1.3), suggesting that masTF may promote interactions between murine endothelial cells and leukocytes. Indeed, we found that stimulating bEnd.3 cells with 50 nM masTF caused J774A.1 cells (murine monocytes/macrophages) to adhere with a 75% increased affinity (n = 3; p = 0.0001 vs. vehicle) when exposed to orbital shear conditions. Depletion of masTF from the medium by Ni-charged beads and denaturation by heat eliminated increased monocyte adhesion, while addition of polymyxin B and non-charged beads had no effect, confirming that the observed biologic phenomena were elicited by the masTF protein.

We report, for the first time, that murine asTF appears to possess non-proteolytic biologic properties analogous to those of human asTF, which indicates that the alternatively spliced TF may be a general cell agonist eliciting changes in gene expression via integrin ligation. We are currently investigating whether the endothelial surface molecules interacting with masTF, as well as the intracellular signaling pathways activated by masTF in endothelial cells, are analogous to those engaged by hasTF.

No relevant conflicts of interest to declare.