Recombiplastin 2g

Each reagent was obtained from the indicated sources: calcium chloride anhydrous (Sigma-Aldrich, Darmstadt, Germany), RPMI 1640 (Corning^®, Manassas, VA, USA), PBS without calcium and magnesium (Euroclone, Pero, Italy), trypsin solution in HBSS without calcium and magnesium (Corning^®, Manassas, VA, USA), recombinant human t-plasminogen activator (BioLegend^®, San Diego, CA, USA), human glu-plasminogen native protein (Thermofisher, Milan, Italy). RecombiPlasTin 2G (Instrumentation Laboratory, Milan, Italy), containing recombinant tissue factor supplemented with synthetic phospholipids, was reconstituted according to manufacturer’s instructions before each experiment.

Human citrated plasma (0.5 mL) was incubated in the absence or presence of 2 µM thrombin aptamers or dabigatran for 2 h. PT time was measured using RecombiPlasTin 2G for quantitative determination in human citrated plasma of PT and Fibrinogen on an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory (Bedford, MA, USA). To assess the activity of the aptamers in a dose-dependent manner in whole blood, human blood samples were collected in citrate-treated tubes. The blood samples were then incubated at room temperature for 2 h in the absence or presence of varying concentrations of the thrombin aptamers. To measure the Prothrombin Time (PT) on the ACLTOP coagulation analyzer, plasma was obtained by centrifuging the samples at 1500× g for 10 min at 4 °C. For the dose dependence study, concentrations of 1 µM, 2 µM, 3 µM, and 4 µM of AYA1809002 and AYA1809004 were utilized.

Citrated blood collected from a donor was incubated in the absence or presence of 1 µM AYA1809002 at room temperature. After 2 h incubation, the indicated concentration of the reverse complement strand was added to the citrated blood sample. After an additional incubation time of 2 h, plasma was obtained through centrifugation. Factor II activity was measured using human plasma immunodepleted of Factor II for the quantitative determination of Factor II activity in citrated plasma based on the prothrombin time (PT) assay, for which an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory (Bedford, MA, USA) was used. The PT time was measured using RecombiPlasTin 2G for quantitative determination in human citrated plasma of PT and Fibrinogen on an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory (Bedford, MA, USA). As a control, 1 µM of dabigatran was incubated with the citrated blood.

Specific activity of FX zymogens was measured using aPTT and PT clotting assays. In the aPTT assay, 1 part of the FX sample pre-diluted in Tris-BSA buffer was added to 1 part of FX-deficient plasma (Affinity Biologicals, Inc.), then mixed with one part of contact trigger and lipid reagent (SynthaSil, Instrumentation Laboratory) followed by 180 s incubation at 37 ^oC. Coagulation was initiated by the addition of one part of CaCl₂ to two parts of the reaction mixture, after which the clotting time was measured by monitoring clot absorbance at 671 nm on a BioTek Synergy Neo2 reader (Agilent Technologies, Inc.).
In the PT assay, FX sample and FX-deficient plasma were incubated for 60 s at 37 ^oC. Coagulation was triggered by the addition of two parts of PT reagent (RecombiPlasTin 2 G, Instrumentation Laboratory) to one part of the plasma mixture, and clotting time was measured in a microplate reader. Calibration curves consisted of FX with known activity (Enzyme Research Laboratories).

Seroma samples were centrifuged at 2500 rpm for 10 minutes. After removal of the supernatant, Papanicolaou-stained smears were prepared from the buffy coat of the cell deposit. On the smears a detailed evaluation of cytomorphological parameters including cellularity, size of atypical cells, nuclear details (presence of nucleoli, nuclear shape), cytoplasmic details (amount, presence of vacuoles), presence of mitoses, necrosis, apoptotic bodies, and the background appearances (serous, fibrinous, necrotic, hematic), was performed.
In addition to the preparation of conventional smears, fluids with an abundant cell deposit were subjected to cell block preparation by mixing the remaining cell sediment with 200μl of human plasma. Subsequently, 2 drops of thromboplastin (RecombiPlasTin 2G, Instrumentation Laboratory, Bredford, MA, USA) were added and mixed. The mixture was allowed to stand for 2 minutes. The resultant clot was placed in a cassette, fixed with buffered formalin and embedded in paraffin (FFPE). Cell blocks were sectioned at 2μm thickness and stained with Haematoxylin and Eosin.

Routine coagulation tests and D-dimer concentration were measured using an ACL TOP 700 coagulometer (Instrumentation Laboratory, Bedford, MA, USA) in platelet-poor plasma (PPP), obtained by centrifugation at 1750g for 15min. The following assays were performed: APTT (SynthASil, Instrumentation Laboratory, Bedford, MA, USA), prothrombin (RecombiPlasTin 2G, Instrumentation Laboratory, Bedford, MA, USA), fibrinogen concentration (QFA Thrombin, Instrumentation Laboratory, Bedford, MA, USA), D-dimer (HemosIL D-dimer HS, Instrumentation Laboratory, Bedford, MA, USA).

Commercial reagents in a validated setup using an automated coagulometer (ACLT op 500, Instrumentation Laboratory, Tokyo, Japan) for aPTT and PT (Syemex CA-500); fibrinogen (RecombiPlasTin 2G, Instrumentation Laboratory) tests were performed as previously described [26 (link)]. Normal values for these assays were based on a previous study [8 (link)]. The canine plasma samples were tested by the Vcheck D-dimer diagnostic assay (Bionote Ltd., Hwaseong-si, Republic of Korea). D-dimer concentrations were performed on a Bionote Vcheck V2400 analyzer (Bionote Ltd., Hwaseong-si, Republic of Korea).