Thromborel s

The contribution of platelets to local thrombin generation was evaluated in a cell-based model of thrombin generation primed by platelets, using a fluorogenic assay (Technoclone GmBH, Austria) as previously described^{11 (link)}. Briefly, this cell-based model consists of isolated platelets at 1 × 10⁶ platelets/µL from controls or treated blood samples resuspended in Hanks' balanced salt solution, with platelets or plasma always from the same participant. Thrombin generation was initiated by the addition of 1.1 pM tissue factor exposed on phospholipids micelles (Thromborel^®S, Dade Behring, Marburg GmbH, Germany), and a fluorogenic substrate that also contains CaCl₂ to favor the activation of coagulation mechanisms. The fluorescence generated was evaluated at a wavelength of 390 nm/450 nm (excitation/emission) for 90 min (at intervals of 1 min) and fluorescence units were analyzed with the Thermo Fluoroskan Ascent Software (Technoclone GmbH). Parameters assessed in our studies were lag time (min), maximum thrombin peak (nM), time to achieve this peak (min), and the total amount of thrombin generated as the area under the curve (arbitrary units).

Bovine trypsin was purchased from Roche (Mannheim, Germany) and human plasma kallikrein (PKa) was purified in the Laboratory of Chemistry and Protein Function using the protocol established by Prof. Dr. Maria Luiza Vilela Oliva and colleagues in 1982 [14 ].
The substrates Boc-Glu(Obzl)-Ala-Arg-AMC.HCl, Bz-DL-Arg-pNA.HCl and H-D-Pro-Phe-Arg-pNa were purchased from Bachem (Bubendorf, Switzerland). Thromborel S, thrombin, and Dade actin-activated cephaloplastin were obtained from Dade Behring (Marburg, Germany), and heparin was obtained from Roche (liquenime^®). Rose Bengal (3,4,5,6 tetrachloro-2,4,5,7-tetraiodofluorescein) and rhodamine 6G were purchased from Sigma Aldrich (Saint Louis, MO, USA). ADP, arachidonic acid, and collagen I were obtained from Chrono-log (Pennsylvania, CA, USA).

Prothrombin time, aPTT and INR, preoperative and postoperative Hb values were
obtained from blood samples. Prothrombin time test measures how long it takes
for a clot to form in a blood sample; and it was determined by coagulometric
method using Thromborel S (Dade Behring OUHP G29) kit, at 405 nm wavelength with
an optical reader. Activated partial thromboplastin time was determined using
Pathromtin SL (Dade Behring OQGS G17) kit, coagulometric method, at 405 nm
wavelength with an optical reader10 (link),11 (link). An INR is a type of calculation based
on PT test results. Hemoglobin was detected in EDTA tubes by using blood counter
(Beckman Coulter HMX California/USA) device.