Tiplaxtinin

Human recombinant PAI-1, human recombinant tPA single chain and human thrombin were purchased from Stago BNL (Asnières sur Seine, FR). Tiplaxtinin was obtained from Axon Medchem (Groningen, NL). Annonacin and annonacinone were isolated from the seeds of Annona muricata L. and rolliniastatin-2 was isolated from the seeds of Annona squamosa L., using previously described procedure30 (link). Isoannonacin and isorolliniastatin-2 were obtained by semisynthesis under basic conditions, as described49 . The substrate Spectrozyme-tPA was acquired from American Diagnostica (Stamford, CT, USA). Thromboelastography was performed on a Haemonetics TEG 5000. For in vivo studies, recombinant tissue-plasminogen activator (rtPA): Actilyse^® was from Boehringer (Ingelheim, Germany), Argatroban and Arganova^® from Mitsubishi Pharma Europe Ltd (London, UK), and Rhodamine 6G and ferric chloride from Sigma (St Louis, MO, USA). Buffer used for all experiments was PBS containing 20 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA with 0.1% PEG-8000 and 0.1% BSA.

In all the experiments, we used a preformed water-soluble complex of corticosterone (Cort) and 2-hydroxypropyl-β-cyclodextrin (#C174, Sigma, USA) [26 (link)–28 (link)]. In mice, Cort (2 mg/kg in a volume of 0.1 ml/10 g body weight) or vehicle (NaCl 0.9%) was administered i.p. immediately after the acquisition of fear conditioning in order to mimic the effect of intense trauma [7 (link)]. Cort was used at 100 and 1000 nM on rat PC12 cells and at 10 and 1000 nM on rat hippocampal slices [26 (link)–28 (link)]. Millipore (USA) provided the recombinant human BDNF (CAS Nb 218441-99-7, #GF029, 100 ng/side) and tPA inhibitor, stable recombinant mutant of human PAI-1 (CAS Nb: 140208-23-7, #528208, ranging from 30 to 240 ng/side) [28 (link), 37 (link)]. The small-molecule inhibitor of PAI-1 activity tiplaxtinin (PAI-039; CAS Nb: 393105-53-8, #1383, 5 ng/side) was provided by Axon MEDCHEM (The Netherlands).