Results: Effect of rTM EGF3-6 mutants on breast cancer cell invasion
We sought to determine which substrate of the thrombin/TM complex is responsible for the anti-
metastatic effects of TM. To do so we generated mutants of TM that can either support only TAFI
activation or only PC activation. Using site-directed mutagenesis, Wang et al. previously determined
residues of TM that are crucial for thrombin/TM-mediated activation of TAFI and PC. Mutagenesis
of V340/D341A abolishes TM cofactor activity for TAFI activation. Mutagenesis of F376A inhibits
activation of PC by thrombin/TM. We generated these mutants in the context of a truncated variants
of rTM, EGF3-6, since the minimum essential structure of TM necessary for activation of TAFI is
the c-loop of EGF-3 to EGF6. To verify the effects of the mutations in these novel rTM constructs,
TAFI and PC were activated by thrombin in the presence of the rTM variants and activity assays
were completed to assess activity of each enzyme. The mutants behaved as previously published and
rTM EGF3-6 behaved similar to the TM control (data not shown). Cell invasion is a measure of
pro-metastatic behaviour and therefore we examined the effect of the rTM EGF3-6 mutants on breast
cancer cell invasion. Treatment of MDA-MB-231 cells with rTM EGF3-6 resulted in a significant
increase in cell invasion. Conversely, treatment of SUM149 cells with rTM EGF3-6 did not affect
cell invasion. Treatment of both MDA-MB-231 and SUM149 cells with rTM EGF3-6 V340A/D341A
(the mutant that does not support TAFI activation) increased cell invasion. Furthermore, rTM EGF3-
6 F376A (the mutant that does not support PC activation) significantly decreased MDA-MB-231
invasion when compared to rTM EGF3-6 WT. However, this effect was not observed in SUM149
cells. These results suggest that the thrombin cofactor ability of TM affects cell invasion.
References
1. Komnenov D, Scipione CA, Bazzi ZA, Garabon JJ, Koschinsky ML, Boffa MB: Pro-
inflammatory cytokines reduce human TAFI expression via tristetraprolin-mediated mRNA
destabilisation and decreased binding of HuR. Thromb Haemost 2015
2. Boffa MB, Koschinsky ML: Curiouser and curiouser: recent advances in measurement of
thrombin-activatable fibrinolysis inhibitor (TAFI) and in understanding its molecular genetics, gene
regulation, and biological roles. Clin Biochem 2007
3. Williams CB, Yeh ES, Soloff AC: Tumor-associated macrophages: unwitting accomplices in
breast cancer malignancy. NPJ breast cancer 2016
4. Kaftan O, Kasapoglu B, Koroglu M, Kosar A, Yalcin SK: Thrombin-activatable fibrinolysis
inhibitor in breast cancer patients. Med Princ Pract 2011
5. Wang W, Nagashima M, Schneider M, Morser J, Nesheim M: Elements of the primary structure
of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation. J Biol
Chem 2000
6. Binette TM, Taylor FB, Jr., Peer G, Bajzar L: Thrombin-thrombomodulin connects
coagulation and fibrinolysis: more than an in vitro phenomenon. Blood 2007