Transglutaminase-mediated oligomerization of the fibrin(ogen) αC domains promotes integrin-dependent cell adhesion and signaling by Alexey M. Belkin, Galina Tsurupa, Evgeny Zemskov, Yuri Veklich, John W. Weisel, and Leonid Medved Blood Volume 105(9):3561-3568 May 1, 2005 ©2005 by American Society of Hematology

Preparation and characterization of αC(FXIII) and αC(tTG) oligomers. Preparation and characterization of αC(FXIII) and αC(tTG) oligomers. (A) SDS-PAGE analysis of the recombinant αC domain fragment and its oligomers. The αC domain (lane 1) was covalently cross-linked with factor XIIIa (lane 2) or tTG (lane 4), and the resulting material was subjected to size-exclusion chromatography to separate high molecular mass fractions containing αC(FXIII) oligomers (lane 3) or αC(tTG) oligomers (lane 5); the right outer lane contains protein markers of the indicated molecular mass. (B-D) Electron microscopy of the rotary shadowed samples of the αC domain fragment (B) and its αC(FXIII) and αC(tTG) oligomers (C-D, respectively). Bar indicates 100 nm. (E-F) Higher magnification and contrast images of small portions of panels C and D, respectively, showing details of the branching polymers. Bar indicates 100 nm. Alexey M. Belkin et al. Blood 2005;105:3561-3568 ©2005 by American Society of Hematology

Oligomerization of the αC domains stimulates RGD-dependent adhesion of endothelial cells via αVβ3, αVβ5, and α5β1 integrins. Oligomerization of the αC domains stimulates RGD-dependent adhesion of endothelial cells via αVβ3, αVβ5, and α5β1 integrins. Quantitative adhesion assays were performed with 5 × 104 HUVECs plated in serum-free DMEM for 20 minutes at 37°C on plastic wells coated with 20 μg/mL αC monomers, αC(FXIII) oligomers, or αC(tTG) oligomers. (A) Effects of oligomerization and RGD-containing peptide on cell adhesion to the αC domain species (□, αC monomers; ▪, αC(FXIII); ▦, αC(tTG)). Cells were plated without treatment or in the presence of 250 μg/mL GRGDSP or control GRGESP peptides. The number of untreated adherent cells on αC monomers was taken as 100%. (B) The role of individual integrins in adhesion to αC monomers and oligomers. Cells were plated without treatment or in the presence of 20 μg/mL blocking antibodies to α5β1 (mAb P1D6; ▨), αVβ5 (mAb P1F6; ▦), or αVβ3 (mAb LM609; ▪). The numbers of untreated cells (□) adherent to αC monomers or αC(FXIII) oligomers were taken as 100%. The results shown in both panels are the means ± standard deviations of 3 independent experiments performed in duplicates. Alexey M. Belkin et al. Blood 2005;105:3561-3568 ©2005 by American Society of Hematology

Binding of purified αVβ3 integrin to immobilized αC monomers and αC(FXIII) or αC(tTG) oligomers. Binding of purified αVβ3 integrin to immobilized αC monomers and αC(FXIII) or αC(tTG) oligomers. Plastic wells were coated with 20 μg/mL αC monomers (□), αC(FXIII) oligomers (▪), or αC(tTG) oligomers (▦). Binding of purified αVβ3 integrin (20 μg/mL) was measured by ELISA with anti-β3 mAb 25E11 followed by secondary goat anti–mouse IgG coupled with peroxidase. Binding to αC monomers was taken as 100%. Results are the means ± standard deviations of 2 independent experiments performed in triplicates. Alexey M. Belkin et al. Blood 2005;105:3561-3568 ©2005 by American Society of Hematology

Oligomerization of the αC domains facilitates endothelial cell spreading. Oligomerization of the αC domains facilitates endothelial cell spreading. A total of 2 × 104 HUVECs in serum-free DMEM were plated at 37°C for indicated periods of time on plastic wells coated with 20 μg/mL αC monomers (A), αC(FXIII) oligomers (B), or αC(tTG) oligomers (C). At different time points of spreading, cells were fixed with 3.7% paraformaldehyde, stained with Coomassie blue, destained, and photographed. Shown are representative photographs of cells 90 minutes after plating on the substrates. Bar = 50 μm. (D) Time-dependent increase in cell spreading on αC monomers () and oligomers (▪, αC(FXIII); ▴, αC(tTG)). The average areas were determined for 120 sparsely plated cells on each substrate. Results are the means ± standard deviations of 2 independent experiments performed in triplicate. Alexey M. Belkin et al. Blood 2005;105:3561-3568 ©2005 by American Society of Hematology

Endothelial cells assemble prominent focal adhesions on αC(FXIII) and αC(tTG) oligomers but not on αC monomers. Endothelial cells assemble prominent focal adhesions on αC(FXIII) and αC(tTG) oligomers but not on αC monomers. HUVECs in serum-free DMEM were plated on 20 μg/mL αC monomers (A-B), αC(FXIII) oligomers (C-D), or αC(tTG) oligomers (E-F) for 2 hours. Paraformaldehyde-fixed, Triton X-100–permeabilized cells were double stained with anti-β3 integrin mAb 25E11 and polyclonal antiphosphotyrosine antibodies, followed by rhodamine-labeled anti–mouse and fluorescein-conjugated anti–rabbit IgG. A clear peripheral staining for αVβ3 integrin and phosphotyrosine was observed at lower magnification in HUVECs on αC oligomers (C,E) but not on monomeric αC domains (A). At higher magnification, well-developed focal contacts containing αVβ3 integrin and phosphotyrosine were detected in HUVECs on αC oligomers (D,F), whereas no distinct focal adhesions were formed on αC monomers (B). Bars indicate 20 μm. Arrowheads mark colocalization of αVβ3 integrin and phosphotyrosine in the peripheral focal contacts of HUVECs on αC oligomers. Alexey M. Belkin et al. Blood 2005;105:3561-3568 ©2005 by American Society of Hematology

Oligomerization of the αC domains increases the amounts of αVβ3, αVβ5, and α5β1 integrins chemically cross-linked to substrate. Oligomerization of the αC domains increases the amounts of αVβ3, αVβ5, and α5β1 integrins chemically cross-linked to substrate. (A) HUVECs in serum-free DMEM were plated for 2 hours on 20 μg/mL αC monomers, αC(FXIII) oligomers, or αC(tTG) oligomers; 5 × 106 adherent cells were cross-linked to the substrates with 2 mM DTSSP in PBS for 30 minutes at 4°C and extracted 4 times for 20 minutes with 0.1% SDS. The cross-linked material was recovered by treating plates for 1 hour at 37°C with a buffer containing 100 mM DTT and 0.1% SDS and then concentrated and analyzed by SDS-PAGE and immunoblotting for the β3, β1, and β5 integrin subunits. Right lanes on the gels contained 10 ng purified αVβ3, αVβ5, and α5β1 integrins. Shown is a representative of 3 experiments. Numbers below the blots refer to the relative amounts of individual integrins, as determined by densitometry and normalized to their amounts cross-linked to αC monomers. (B) Bands corresponding to cellular integrins in panelAwere compared with external standards of purified integrins and then converted to the numbers of ligand-bound integrin receptors per cell. □ indicates αC monomers; ▪, αC(FXIII) oligomers; and ▦, αC(tTG) oligomers. Results are the means ± standard deviations of 3 independent experiments. Alexey M. Belkin et al. Blood 2005;105:3561-3568 ©2005 by American Society of Hematology

Oligomerization of the αC domains amplifies adhesion-dependent activation of FAK and ERK. HUVECs were either kept in suspension or plated in serum-free DMEM for 2 hours on tissue culture plates coated with 20 μg/mL αC monomers or αC(FXIII) oligomers. Oligomerization of the αC domains amplifies adhesion-dependent activation of FAK and ERK. HUVECs were either kept in suspension or plated in serum-free DMEM for 2 hours on tissue culture plates coated with 20 μg/mL αC monomers or αC(FXIII) oligomers. (A) Phosphorylation of FAK tyrosines 397, 577, and 861 was examined by SDS-PAGE and immunoblotting with specific polyclonal antibodies (see “Materials and methods”). Overall tyrosine phosphorylation of FAK was tested by immunoprecipitation of FAK, followed by SDS-PAGE and immunoblotting with polyclonal antiphosphotyrosine antibodies. (B) Adhesion-dependent phosphorylation of ERK was analyzed by immunoblotting with antibodies against dually phosphorylated ERK1/2 and total ERK1/2. Panels A and B are representative of 3 independent experiments for FAK and ERK. Alexey M. Belkin et al. Blood 2005;105:3561-3568 ©2005 by American Society of Hematology