α2 antiplasmin

Ethacrynic acid was obtained from Sigma Aldrich (St. Louis, MO). Human plasma thrombin, α₂-antiplasmin, and FXIIIa were obtained from Haematologic Technologies (Essex Junction, VT). N,N-dimethyl-casein, dansyl-cadaverine, and dithiothreitol (DTT) were also from Sigma Aldrich. Dabigatran, rivaroxaban, and AntiF11 (mouse monoclonal antibody from Abnova™) for plasma studies as well as Coomassie Brilliant Blue for gel electrophoresis were from Fisher Scientific (Waltham, MA). Fibrinogen was from Haematologic Technologies. Stock solution of FXIIIa was prepared in 50 mM Tris–HCl, 1 mM CaCl₂, 100 mM NaCl, 0.1% PEG8000, 0.02% Tween80, and 2 mg/mL N,N–dimethylcasein. For the clotting assays, pooled normal human plasma was purchased from George King Bio-Medical (Overland Park, KS). APTT reagent containing ellagic acid, thromboplastin‒D (PT reagent), and 25 mM solution of CaCl₂ were purchased from Thermo Fisher Scientific. All experiments in this paper were repeated at least two times. For molecular modeling studies, initial structure of FXIIIa (4kty.pdb) was prepared by removing the crystallographic water molecules and adding hydrogen atoms consistent with the physiologic pH of 7 using Maestro 12.4.1. Docking studies were carried out by generating a non-covalent pose using Glide (Schrodinger Suite 2020), and then, by using the covalent docking program (Schrodinger Suite 2020).

Following antibodies were obtained from indicated distributors: anti-annexin A2 (rabbit monoclonal, (CST D11G2, Danvers, MA), anti-annexin A2 (mouse monoclonal 610068, BD biosciences), anti-caveolin-1 (rabbit polyclonal Sc-894, Santa Cruz), anti-cavin-1 (anti-PTRF, rabbit polyclonal AP7421a, Abgent, San Diego, CA), anti-S100A10 (mouse monoclonal 4E7E10, CST), anti-phosphotyrosine (mouse monoclonal 4G10, Merck Millipore, Darmstadt, Germany), anti-eNOS (mouse monoclonal 610296, BD Biosciences), anti-p-Src (Y418, BS4176) and anti-p-Src (Y529, BS4729) (rabbit polyclonal, Bioworld Technology, St. Louis Park, MN), Donkey anti-rabbit IgG HRP and anti-mouse IgG HRP (Promega). OLA (Wako), EPA (Nu-Chek Prep, Elysian, MN) and DHA (Cayman Chemicals, Ann Arbor, MI) were obtained from indicate distributors. Aprotinin (Wako), α-2-antiplasmin (Haematologic Technologies Essex Junction VT), 4-(2-Aminomethyl) benzensulfonyl fluoride (AEBSF, nacalai tesque, Kyoto Japan), chymostatin (Sigma-Aldrich, St. Louis, MO) and Gö6976 (LC Laboratories, Woburn, MA) were obtained from indicated distributors. Other reagents not specified were obtained from Wako.