Human fibrin

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).

Bovine fibrinogen, human fibrinogen, human fibrin, bovine thrombin, human thrombin, plasmin, azocasein, tris (hydroxymethyl) aminomethane (Tris), tetramethyl-ethylenediamine (TEMED), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, phenylmethanesulfonyl fluoride (PMSF), ethylenediaminetetra-acetic acid (EDTA), and ethylene glycol tetraacetic acid (EGTA) were purchased from Sigma-Aldrich Co. (Burlington, NJ, USA). The protein markers used for SDS-PAGE were purchased from Fermentas Co. (Burlington, MA, USA). Sephadex resin was purchased from GE Healthcare Co. (Boston, MA, USA). The substrates for the amidolytic assay were purchased from Diapharma (West Chester, OH, USA). Other reagents were of special grade and were purchased commercially.