Investigation of the co-localization of pd-FX and N-degly-FX with early endosomes in differentiated THP-1 cells and HepG2 cells.

<p>(A) HepG2 cells and (B) THP-1 cells differentiated to macrophages by PMA (see Materials and Methods) were incubated with 10 µg/mL of FX variants for 60 min at 4°C then washed and incubated for 30, 60 and 120 min at 37°C. Nucleus/DNA was stained with DAPI (blue) and FX variants were co-localized by red fluorescence using goat anti-human FX with anti-early endosome-antigen 1 in a proximity ligation assay (PLA)–based method. A representative set of experiments is presented. Fluorescence intensity was quantified using BlobFinder v3.2 software package. Data represent the mean fluorescent intensity ± S.D. of 20–35 cells, obtained from three independent experiments.</p

Identification of cells as main target for FX variants in liver.

<p>Liver sections from mice injected with 20 µg/mouse of pd-FX (C, F), N-degly-FX (B, E) or with PBS as control (A, D). Livers were collected 10 minutes after injection. In a first set of sections (A, B, and C) FX variants are stained with rabbit anti-human FX (Eurogentec followed by highly sensitive polymer-based detection reagent (HRP) (Dako, Trappes, France) and then with diaminobenzamidine. These tissue sections were slightly counterstained with diluted hematoxylin (magnification 200). One representative experiment out of six performed with different mice is shown. (D, E, and F) Merged images of liver sections stained with rabbit anti-human FX followed by Alexa Fluor 488 F(ab’)₂ fragment (Invitrogen) of goat anti-rabbit IgG (H+L) as secondary antibody (green) and with monoclonal rat anti-mouse CD68 (AbD Serotec, Oxford, UK) followed by TRITC-conjugated goat anti-rat immunoglobulins (Southern Biotech, Birmingham, AL, USA) (red). Original magnification 200. One representative experiment out of four performed with different mice is shown.</p

Investigation of ¹²⁵I-pd-FX and ¹²⁵I-N-degly-FX degradation by differentiated THP-1 cells and HepG2 cells, respectively.

<p>(A) ¹²⁵I-pd-FX and ¹²⁵I-N-degly-FX were added to differentiated THP-1 cells and HepG2 cells, respectively, for a 1-hour period at 4°C. Cells were then washed, and incubation was continued at 37°C to initiate endocytosis. At indicated time points, cell lysates and cell supernatants were taken to determine the amount of degraded material. Degraded material is defined as the radioactivity that is soluble in 10% trichloroacetic acid. In all experiments, controls were included to determine the amount of nonspecific degradation in the absence of cells, which routinely was less than 10% of degradation in the presence of cells. Data represent mean ± S.D. of 3 experiments. (B) ¹²⁵I-pd-FX was incubated with differentiated THP-1 cells at indicated time points (1h at 4°C, then for 15′, 30′ and 60′ at 37°C). Cell lysate and supernatant were migrated on SDS-PAGE and results were visualized by autoradiography using PharosFX™ Plus Molecular Imager (BioRad, Hercules, CA, USA).</p

In vivo organs biodistribution and blood recovery of FX variants.

<p>(A) Five minutes after injection of I^{125<i>−</i>}pd-FX, I^{125<i>−</i>}N-degly-FX, rFX, or rFX^{N181A–N191A} in mice, the relative amount of radioactivity associated to blood after withdrawal was compared to the total amount injected and called recovery. Mice were injected with I^{125<i>−</i>}pd-FX (B), I^{125<i>−</i>}N-degly-FX (C), rFX (D), or with rFX^{N181A–N191A} (E). Associated radioactivity to several organs was counted at selected time points and the relative amount of radioactivity compared to the total amount injected was calculated. Three mice were used for each time point. Data represent mean triplicate values ± S.D.</p

Binding and internalization of pd-FX by differentiated THP-1 and N-degly-FX by HepG2 cells.

<p>(A) HepG2 cells and (B) THP-1 cells differentiated to macrophages by PMA (see Materials and Methods) were incubated with 10 µg/mL of pd-FX, N-degly-FX or with PBS as control (CT) for 1 h at 4°C. Then, after washing cells were incubated at 37°C for 15 and 30 min. Nucleus/DNA was stained with DAPI (blue) and FX variants were visualized by red fluorescence using mouse monoclonal and rabbit polyclonal antibodies both anti-human FX in a proximity ligation assay (PLA)–based method. Fluorescence intensity was quantified using BlobFinder v3.2 software package. Data represent the mean fluorescent intensity ± S.D. of 20–35 cells, obtained from three independent experiments.</p

Decreased pd-FX levels upon gadolinium chloride treatment.

<p>Mice were treated with saline or GdCl₃, and 24 hours after treatment ¹²⁵I-pd-FX or ¹²⁵I-N-degly-FX was administered. Then, 15 minutes later plasma samples were taken and the associated radioactivity was counted. The values obtained were compared to the total amount injected and expressed as percent. At the same time point, mice were anesthetized, sacrificed and their liver was taken and washed with PBS. The associated radioactivity was compared to the total amount injected. Data represent mean ± S.D. of 3 experiments.</p

LAIR-2/Fc interferes with VWF binding to collagen.

<p>Microtiter wells were coated with 50 µg/ml collagen I (<i>panel</i> A) or collagen III (<i>panel</i> B) and subsequently incubated with 0.1 µg/ml purified plasma-derived VWF in the presence of increasing concentrations of LAIR-1/Fc (open circles), LAIR-2/Fc (closed circles) or SIRL-1/Fc (squares). VWF binding was detected with horseradish-conjugated polyclonal anti-human VWF antibodies and 3-3′-5-5′-tetramethylbenzidine. Data are representative of 3 independent experiments.</p

Platelets do not express LAIR-1 or LAIR-2.

<p><i>Panel</i> A: Flowcytometric analysis of washed platelets (2×10E5/µl), unstimulated or stimulated for 5 min at RT with 10 µM TRAP or 5000 nM PMA. Upper panels represent staining for CD62L, lower panels represent LAIR-1 staining. <i>Panel</i> B: Western blots containing whole platelet lysates and controls were incubated with antibodies against LAIR-1 (<i>lanes 1–3</i>), LAIR-2 (<i>lanes 4–8</i>) or GpVI (<i>lane 9</i>). <i>Lane 1:</i> purified LAIR-1/Fc; <i>lane 2:</i> purified LAIR-2/Fc; <i>lane 3:</i> whole platelet lysate; <i>lane 4:</i> purified LAIR-1/Fc; <i>lane 5:</i> purified LAIR-2/Fc; <i>lane 6:</i> conditioned medium of non-transfected human 293T cells; <i>lane 7:</i> conditioned medium of human 293T cells secreting LAIR-2; <i>lane 8:</i> whole platelet lysate; <i>lane 9:</i> whole platelet lysate.</p

LAIR-2/Fc inhibits adhesion of platelets to collagen under flow conditions.

<p><i>Panel</i> A: Collagen type III-coated coverslips were perfused with whole blood in the absence or presence of 100 µg/ml soluble LAIR-1/Fc, LAIR-2/Fc or SIRL-1/Fc. Representative pictures are shown. Perfusion was performed at a shear rate of 300 s⁻¹ (upper panels) or 1500 s⁻¹ (lower panels). Percentages below figures indicate percentage surface-coverage for each individual photo. <i>Panels</i> B & C: Quantitative representation of surface coverage of collagen type III-coated coverslips that were perfused in the absence or presence of 100 µg/ml soluble LAIR-1/Fc, LAIR-2/Fc or SIRL-1/Fc at 300 s⁻¹ (<i>panel</i> B) or 1500 s⁻¹ (<i>panel</i> C). Data represent mean±SD of three independent perfusions. <i>Panel</i> D: Dose dependent inhibition of surface platelet coverage in the presence of LAIR-2/Fc at shear rates of 300 s⁻¹ (open symbols) or 1500 s⁻¹ (closed symbols). *: <i>p</i><0.005.</p

LAIR-2/Fc inhibits collagen but not TRAP-induced platelet aggregation.

<p>Aggregation of platelet rich plasma (PRP) in response to collagen was measured using an optical aggregometer. <i>Panel</i> A: Platelet aggregation in response to 50 µM TRAP alone (PBS) or in the presence of 100 µg/ml LAIR-1/Fc, LAIR-2/Fc or SIRL-1/Fc. <i>Panel</i> B: Platelet aggregation in response to collagen (1 µg/ml) alone (PBS) or in the presence of 100 µg/ml LAIR-1/F, LAIR-2/Fc or SIRL-1/Fc. <i>Panel</i> C: Platelet aggregation in response to collagen (1 µg/ml) alone (PBS) or in the presence of 0.01 µg/ml, 0.1 µg/ml or 1.0 µg/ml LAIR-2/Fc. <i>Panel</i> D: Platelet aggregation in response to 0.5 µg/ml, 1 µg/ml, 2 µg/ml or 4 µg/ml collagen in the presence of 1.0 µg/ml LAIR-2/Fc.</p