The role of galectin-3 and galectin-3-binding protein in venous thrombosis

Abstract

Galectin-3-binding protein (gal3bp) and its receptor/ligand, galectin-3 (gal3), are secreted proteins that initiate signaling cascades in several diseases, and recent human proteomic data suggest they may play a role in venous thrombosis (VT). We hypothesized that gal3bp and gal3 may promote VT. Using a mouse stasis model of VT, we found that gal3bp and gal3 were localized on vein wall, red blood cells, platelets, and microparticles, whereas leukocytes expressed gal3 only. Gal3 was dramatically increased during early VT and gal3bp:gal3 colocalized in the leukocyte/endothelial cell interface, where leukocytes were partially attached to the vein wall. Thrombus size correlated with elevated gal3 and interleukin-6 (IL-6) vein wall levels. Recombinant gal3 promoted VT and increased vein wall IL-6 mRNA. Although recombinant gal3 restored the VT size in gal3(-/-) mice, it had no effect on IL6(-/-) mice, suggesting that gal3:gal3bp promotes VT through IL-6. Moreover, significantly fewer activated neutrophils were present in the gal3(-/-) vein walls. In a group of human patients, elevated circulating gal3bp correlated with acute VT. In conclusion, gal3bp:gal3 play a critical role in VT, likely via IL-6 and PMN-mediated thrombotic mechanisms, and may be a potential biomarker in human VT.

Figure 1

The effects of VT on Gal3BP and gal3 in blood-circulating elements (systemic) or thrombus and vein wall (Local). Western blot and quantification: western blot analysis of gal3BP and gal3 in RBCs (n = 1 per band), WBCs (n = 5 per band), PLTs (n = 3 per band), procoagulant microparticles (n = 3 per band), thrombus (n = 1 per band), and vein wall (n = 1 per band) collected from non-VT (–) (n = 21) or VT (+) (n = 25) mice 48 hours postthrombosis. Actin was used as a loading control. Quantification of blot intensity relative to loading control is shown below. gal3BP, galectin 3–binding protein; gal3, galectin 3; gal3m, gal3 monomer; gal3MD, gal3 multimer dimer; gal3MT, gal3 multimer trimer; gal3d, gal3 degradation product; IVC, vein wall; MP, procoagulant microparticles.

Figure 2

The effects of VT on Gal3BP and gal3 in vein wall and thrombus (local). Gene expression and Histology. (A) Analysis of gal3BP and gal3 in vein wall by qRT-PCR: gal3BP and gal3 mRNA isolated from the IVC (vein wall) of non-VT (n = 8) or 48 hours VT (n = 12) WT mice were quantified using qRT-PCR. Gene expression was quantified relative to β-actin. (B) H&E and IHC for gal3. H&E stain and IHC for gal3 in both non-VT and VT conditions. Arrows indicate positive stained cells. Of note, the number of gal3 positive cells were increased on VT condition.

Figure 3

Gal3 correlation with IVC+thrombus weights: time course. (A) IVC or IVC+thrombus weight 3 hours, 6 hours, or 48 hours (n = 5 per time point) postligation, and in non-VT mice (n = 5). (B) Expression of gal3 in the vein wall of non-VT and VT mice 3 hours, 6 hours, or 48 hours postligation. (C) Correlation between gal3 gene expression in the IVC and IVC+thrombus weight. VT, venous thrombosis; IVC, inferior vena cava.

Figure 4

Gal3/IL-6 and IL-6/CCL2 correlations in VT. (A) Gal3 and IL-6 gene expression in the IVC of non-VT (n = 4) and 48 hours VT (n = 3) mice. Shown below is the correlation between gal3 and IL-6 gene expression in individual mice. (B) IL-6 and CCL2 gene expression in the IVC of non-VT (n = 4), 48 hours VT (n = 3), and 48 hours VT mice that were treated with recombinant human gal3 (n = 4). Shown below is the correlation between IL-6 and CCL2 expression in individual mice.

Figure 5

The effect of recombinant gal3 (rGal3) on 48 hours IVC+thrombus weight in WT, Gal3KO, and IL-6KO mice. Columns from left to right: IVC or 48 hours postligation IVC+TW in non-VT (black column, n = 4), WT (white, n = 10), rGal3-treated WT (white, n = 4), Gal3KO (gray, n = 7), rGal3-treated Gal3KO (gray, n = 4), IL-6KO (gray with dots, n = 7), and rGal3-treated IL-6KO (gray with dots, n = 3) mice.

Figure 6

Leukocyte infiltration into the vein wall of WT and Gal3KO mice. (A) Representative pictures of 48 hours postthrombosis initiation showing leukocyte infiltration in C57Bl/6 mice and Gal3KO mice (original magnification ×20). (B) Quantification of leukocyte migration into the vein walls of WT and Gal3KO mice at 3 hours, 6 hours, and 48 hours (n = 5 per time point) post-IVC ligation, and non-VT (n = 5) were examined under high-power oil-immersion light microscopy. Note that in Gal3KO mice, leukocyte extravasation was significantly depressed compared with WT mice at 48 hours after thrombosis was initiated, the time point used for most of the experiments described in this manuscript.

Figure 7

Gal3BP and gal3 at the lumen/IVC interface. Non-VT images: Top raw (A-D); VT images 48 hours after thrombosis was initiated from an area where there was not thrombus/vein wall contact, middle raw (E-H); and VT images 48 hours after thrombosis was initiated from an area where there was thrombus/vein wall fusion, bottom raw (I-L). Note that vein wall gal3 and gal3bp are abundant in areas where the thrombus is not contacting the vein wall (F-G), but disappear in areas where the thrombus contacts the vein wall (J-K). Red blood cell fluorescence is predominately nonspecific autofluorescence. The IVC lumen is indicated with an asterisk in DAPI images. White arrows indicate WBCs. (A,E,I) Nuclear DAPI staining. (B,F,J) Gal3bp localization (FITC). (C,G,K) Gal3 localization (Texas Red). (D,H,L) Merge of images.

Figure 8

Gal3BP and gal3-mediated leukocyte interactions with the IVC. Sockets distribution of gal3bp appears to be interacting with gal3 rich leukocytes (H,L). Red blood cell fluorescence is predominately nonspecific autofluoresence. The IVC lumen is indicated with an asterisk in DAPI images. Yellow arrows indicate WBC that is not attached to the vein wall. White arrows indicate gal3bp sockets on the vein wall. (A,E,I) Nuclear DAPI staining. (B,F,J) Gal3bp localization (FITC). (C,G,K) Gal3 localization (Texas Red). (D,H,L) Merge of images.