Page 225 - Haematologica - Vol. 105 n. 6 - June 2020

FHL2 modulates thrombosis formation
(Figure 3A). FHL2 expression decreased the luciferase activity of the WT TF-promoter reporter (Figure 3B). TF promoter constructs with either a disrupted NFκB response element or a mutated AP-1 element exhibited lower basal luciferase activity (Figure 3B) and FHL2-medi- ated inhibition of luciferase activity of the WT-promoter (61%) was stronger than that of the NFκB- or AP-1-mutat- ed promoter (52% and 43%, respectively) (Figure 3B). These data suggest that FHL2 affects the transcriptional activity of both NFκB and AP-1 to downregulate TF-pro- moter activity in these cells. To assess whether activation of EC affects cellular localization of FHL2, we stimulated EC with TNF-α (50 ng/mL) and observed enhanced nuclear localization, which may contribute to the changed modulation of transcription factor activity by FHL2 (Online Supplementary Figure S4). To determine which domain of FHL2 is essential to inhibit TF-promoter activi- ty, deletion mutants of FHL228 (Figure 3C) were tested in TF-promoter luciferase assays. The first one and a half LIM-domain (LIM0-1) already exhibited partial inhibition (41%) with the LIM0-2 variant having similar effects, while LIM0-3 (55% inhibition) and FHL2 were more potent inhibitors of TF-promoter activity (Figure 3D). Of note, overexpression of individual LIM domains of FHL2 had no effect on TF promoter activity (data not shown) indi- cating that at least one and a half LIM domains of FHL2 are required to elicit decreased TF-promoter activity.
FHL2 physically interacts with tissue factor
To investigate whether inhibition of TF by FHL2 is also attributable to this latter’s physical interaction with TF, we performed co-immunoprecipitation experiments. Full- length TF co-immunoprecipitated efficiently with HA- tagged FHL2 from whole cell extracts using an anti-HA antibody (Figure 4A), whereas ∆CT-TF, which lacks the intracellular cytoplasmic TF domain, failed to bind FHL2 (Figure 4A), indicating that FHL2 requires the relatively short cytoplasmic tail of TF for its interaction. Pull-down assays using FHL2 deletion mutants (Figure 3C) revealed that all variants of FHL2 bind TF (Figure 4B). It is known that FHL2 can enhance protein stability and to measure
the effect of FHL2 on TF protein stability, we exposed cells to cycloheximide and determined the half-life of full- length TF and ∆CT-TF mutant proteins. The ∆CT-TF vari- ant was more stable than full-length TF, but TF protein levels were not influenced by FHL2 (Figure 4C, D). We concluded that FHL2 interacts with TF but does not affect TF protein stability.
FHL2 modulates tissue factor procoagulant activity
Having established that FHL2 interacts with TF and reg- ulates TF expression in EC and SMC, we sought to exam- ine the impact of FHL2 on the activity of TF in cultured EC and murine and human SMC, by assessing the potential of these cells to generate factor Xa (FXa). Knockdown of FHL2 in HUVEC and HMEC-1 resulted in enhanced TNFα-induced FXa generation compared to control (Figure 4E, F). In a similar fashion, FHL2-deficient mouse SMC showed higher ionomycin-induced FXa generation than WT SMC (Figure 4G). Ectopic expression of FHL2 resulted in a modest decrease of TNFα-induced FXa gen- eration compared to that of control-transduced cells (Figure 4H). Of note, following FHL2 knockdown in HMEC-1, we found, in the same cell lysates, that TF pro- tein levels were increased 1.5-fold and that FXa generation was increased even more, 3-fold (Online Supplementary Figure S5A, B). These data indicate that FHL2 may regulate TF activity partly by binding the intracellular domain of TF and by modulating TF gene expression. Figure 5 is a schematic summary of our data.
Genetic variation in the FHL2 gene is associated with venous thrombosis in humans
Having established that FHL2 reduces TF activity in EC and SMC, we reasoned that genetic variation in FHL2 may affect venous thrombosis in man. We therefore analyzed 18 FHL2 SNP and venous thrombosis risk in the INVENT consortium (Table 1). We observed that the FHL2 poly- morphism rs4851770 was associated with thrombosis risk with a P-value of 2x10-4, even after correction for multiple testing. No association was found between other tested SNP in the FHL2 gene and risk of venous thrombosis
Figure 5. Schematic representation of FHL2 function in the modulation of tissue factor activity. The left panel shows the normal situation in which FHL2 inhibits tissue factor (TF) expression in activated endothelial cells (EC) and smooth muscle cells (SMC), whereas the right panel represents the effect of FHL2 deficiency, which results in enhanced TF expression and increased activity.
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