Human fibrinogen

Fibrinolytic activity of LK was analyzed by a modified fibrin plate assay as previously described [7 (link)]. Agarose (0.2 g) was dissolved in 40 mL of 1x Phosphate Buffered Saline (PBS) by boiling in a microwave and then cooled to approximately 40°C. Prior to the solidification of the gel, 32 mg of human fibrinogen (Sigma-Aldrich), 10 units of human plasminogen (rPeptide LLC, Bogart, GA, USA), and 10 units of human thrombin (BioPharm Laboratories LLC, Riverton, UT, USA) were added to the gel and mixed with gentle swirling to prevent the introduction of bubbles. The mixture was then poured to Petri dishes and allowed to solidify at room temperature. Wells (3 mm in diameter) were subsequently made in the solidified gel. 10 µL of concentrated samples (1 µg/µL) was diluted in 40 µL of 1x PBS buffer, loaded in the wells of the gels, and incubated at room temperature overnight. Standard lumbrokinase (Doctor's Best Inc., Irvine, CA, USA) was used as positive control (10 µg) and elution samples from nonagroinfiltrated leaves were used as negative control. Actual diameters (mm) of clear halo circles were measured. The fibrinolytic activity of rPI239 was calculated in comparison to the standard LK product (18,000 U/mg) as described in our previous study [7 (link)]. Means ± SE (n = 4) were analyzed by a one-way ANOVA test.

Recombinant dispersin (1 μg in 100 μL of PBS – 10 μg/mL) or BSA were immobilized onto microplate wells. Purified MntC protein was used as positive control for fibrinogen cleavage (Salazar et al., 2014 (link)). Coated wells were blocked with 3% BSA diluted in PBS and plasminogen (10 μg/mL) was added. Incubation proceeded for 1 h at 37°C. After washing three times with PBS-T, human fibrinogen (10 μg) or human native vitronectin (1 μg) (Sigma-Aldrich) and plasminogen activator uPA (3U) were added. Reaction mixtures were incubated for 0, 1, or 4 h at 37°C, proteins were separated by 12% SDS-PAGE, and transferred to a nitrocellulose blotting membrane. Membranes were blocked with 5% skimmed milk for 1 h at room temperature with shaking, and the immunodetection was performed as described above for immunoblotting for dispersin detection, using primary antibodies anti-fibrinogen (Cloud-Clone, Katy, TX, United States) and anti-vitronectin (Complement Technology, Tyler, TX, United States) diluted 1:1000 and 1:5000, respectively, in 2.5% skimmed milk in PBS. Fibrinogen and vitronectin degradation products were visualized by ECL Analysis System (GE Healthcare) in the UVITEC Cambridge Image System (Alliance vs. 6.7).

Porphyromonas cell suspensions (10⁷ CFU/reaction), sBHI media controls, or Porphyromonas cell-free supernatants (240 µg of total protein) were incubated with 120 µg of human fibrinogen (Sigma-Aldrich, St. Louis, MO). Reaction mixtures were incubated at 37 °C under anaerobic conditions or in an atmosphere of 5% CO₂ for cell suspension or cell-free supernatants, respectively. Samples from each time point (cell suspensions: 0, 2, 18, 24 h; supernatants: 0, 2, 24, 48 h) were collected, mixed 1:1 with Novex^TM 2X sample buffer (Invitrogen) with dithiothreitol (DTT, Fisher Scientific), heated at 95 °C for 10 min, and separated on Novex^TM 10% Tris-Glycine polyacrylamide pre-cast gels (Invitrogen) at a constant voltage of 180 V. Gels were stained in 0.25% Coomassie brilliant blue R-250 solution (Fisher Scientific) and de-stained in 5% methanol/7.5% acetic acid in distilled water. Fibrinogen degradation was evaluated qualitatively by visualization of fibrinogen α chain (63.5 kDa), β chain (56 kDa), and γ chain (47 kDa) between experimental and control samples over the time-course. Gels are derived from the same experiments and were processed in parallel. Unprocessed scans of fibrinogen degradation protein gels are available in the Supplementary Information (Supplementary Figs. 9, 10).

A total of 1 mL of 0.4% human fibrinogen or 0.2% human fibrin (Sigma-Aldrich, St. Louis, MO, USA) in 20 mM Tris-HCl, pH 6.8 was incubated separately with 0.1 mL of each crude extract (1 μg/μL of protein) at 37 °C. At various time intervals, aliquots of 7.5 μL of the fibrinogen reaction mixture or 12.5 μL for fibrin reaction mixture were taken and mixed with 12.5 μL of denaturing loading buffer (20% glycerol, 8% SDS and 2% β-mercaptoethanol) and incubated for 5 min at 100 °C. The degradation patterns of fibrin(ogen)olysis were analyzed by 10% SDS-polyacrylamide gel according to the method of Laemmli [41 (link)]. The data have been shown as supplementary material (Figure S1).