Alteplase actilyse

Human α-thrombin was obtained from CellSystems (St. Katharinen, Germany). Argatroban (Argatra^®) was obtained from Mitsubishi Pharma (Düsseldorf, Germany). The 3’-biotinylated DNA-aptamer HD1-22 was synthesized and purified from Microsynth (Balgach, Switzerland). The fluorogenic peptide substrate I-1560 (Thrombin, Boc-Asp(OBzl)-Pro-Arg-AMC) was purchased from Bachem (Weil am Rhein, Germany). Alteplase (Actilyse^®) was purchased from Boehringer Ingelheim (Biberach, Germany).

Apixaban (provided by Pfizer Inc., United States; material no. 1151519, batch no. ABA8622) was initially dissolved in dimethyl sulfoxide, and subsequently diluted with PBS to a concentration of 2.5 mg ml⁻¹. Diluted Apixaban was stored aliquoted at −20°C and was not re-frozen once thawed. Concentration of Apixaban was selected to be in line with clinically relevant dosing for the secondary prevention of thrombotic events in patients with atrial fibrillation, which is 250 ng ml⁻¹ (Artang et al., 2017 (link)).
Alteplase (Actilyse, provided by Boehringer-Ingelheim International GmbH, Germany; Z. Nr. 1-24,717) was dissolved in distilled water to a concentration of 1 mg ml⁻¹ and was stored aliquoted at -20°C (not re-frozen once thawed); after which it was further diluted with PBS. The final concentration of alteplase was also selected to be in line with clinically relevant dosing indicated for patients with ischemic stroke (1.3 mg L⁻¹), according to the manufacturer’s instructions and supporting pharmacokinetic data (Acheampong and Ford, 2012 (link)).

Clots were formed from 30% PNP or FDP supplemented with cryoprecipitate, RiaSTAP^® (CSL Behring GmbH, Marburg, Germany) or FibCLOT^® (LFB, Les Ulis, France) (0.5–2 mg/mL) in the presence of 16 mM phospholipids (Rossix, Mölndal, Sweden) ± 300 pM tPA (Alteplase (Actilyse^®) Boehringer Ingelheim International GmbH, Ingelheim am Rhein, Germany) in Tris buffered saline with Tween-20 (TBST; 10 mM Tris, 140 mM NaCl and 0.01% Tween-20 pH 7.4). Clotting was initiated with CaCl₂ (10.6 mM) and thrombin (0.1 U/mL, Sigma Aldrich Sigma-Aldrich, Darmstadt, Germany), and absorbance readings were taken every min for 4 h at 37 °C in a in a FLX-800 plate reader (Biotek Instruments, Winooski, VT, USA).