Plasmin

Two independent reviewers conducted electronic literature searches in 3 electronic databases (PubMed, Embase, and Cochrane library database) as well as a manual search to identify eligible clinical studies from inception to Nov 2022. Search terms used were: “Agents, Antifibrinolytic,” “Antifibrinolysin,” “Antifibrinolysins,” “Antifibrinolytics” “Antifibrinolytic,” “Antifibrinolytic Agent,” “Agent, Antifibrinolytic,” “Plasmin Inhibitors,” “Inhibitors, Plasmin,” “Antiplasmins” “Antiplasmin,” “Plasmin Inhibitor,” “Inhibitor, Plasmin, “spine surgery,” “spinal surgery,” “spine,” “lumbar surgery,” “thoracic surgery,” and “cervical surgery.” Reference lists in studies, reviews, and previous meta-analyses were checked to identify any initially omitted studies. In accordance with the abstract review, 2 investigators independently reviewed all titles, abstracts, and full texts of articles that were potentially eligible.

Oxidized proteins were quantified using a Western blot assay (OxyBlot Kit, Chemicon International, Temacula, CA, USA). Briefly, 25 µg of total protein from platelets obtained from PRP was denatured by adding 12% SDS and 10% β-mercaptoethanol followed by incubation at room temperature by 15 min. Proteins were then separated in 12% SDS-PAGE and transferred to PVDF membranes, blocked with blocking/dilution buffer, and incubated with rabbit primary anti-2,4-dinitrophenylhydrazone (2,4-DNP, 1:150) for 1 h at 18-25 °C with gentle shaking. Later, the membrane was rinsed three times with 1X PBS-T and incubated with a secondary antibody (1:300) in blocking dilution buffer for 1 h at 18 °C with gentle shaking. The membrane was then rinsed three times with 1X PBS-T. Blots were washed and protein was revealed using BM Chemiluminescence Kits (Roche Diagnostics, Indianapolis, IN, USA). Densitometric analysis was performed with the Kodak 1D 3.5 image analyzer (Eastman Kodak Co., Rochester NY, USA).
Zymography for Detecting Plasmin, MMP2, and MMP3MMP2 and MMP3 enzymatic activity was evaluated by zymography assays of total protein from platelet homogenates or plasma obtained from PRP as described previously [21 (link)]. Briefly, 30 µg of total protein from platelets or plasma was loaded onto 10% SDS-PAGE gel containing either 1 mg/mL gelatin for plasmin and MMP2 (Bio-Rad Laboratories, Hercules, CA, USA) or 2 mg/mL casein for MMP3; after electrophoresis, the SDS was removed from the gel by incubation in Triton X-100 at room temperature and samples were subsequently washed to remove the detergent and incubated at 37 °C for 48 h in a development buffer (BRIJ35, Sigma-Aldrich, St. Louis, MO, USA) [18 (link)]. Casein was used as a substrate for MMP3 with development buffer containing 0.1 mol/L glycine at pH 8.3 [21 (link)]. This gel was stained with the Coomassie technique, followed by destaining in the same solution without dye. Proteinase activity was detected as unstained bands on a blue background representing areas of gelatin digestion. Degradation bands were observed at 66 kDa (MMP2), 80 kDa (MMP9), and 55 kDa (MMP3). Gels were digitized and analyzed using the Kodak 1D 3.5 image analyzer (Eastman Kodak Co., Rochester NY, USA) and represented in area (relative units).