Thrombin

The melting point (uncorrected) was determined on an apparatus made by Beijing TECH INSTRUMENT CO. LTD. Optical rotations were measured with a Perkin Elmer spectropolarimeter. The UV spectra were measured on a VARIAN SARY 50 spectrophotometer. The IR spectra were recorded using a BRUKER VERTEX 70 spectrometer. The NMR experiments were run on a Bruker AM-400 spectrometer. The HRFABMS data were obtained on a VG 7070-HF spectrometer. Column chromatographic separations were carried out using silica gel H60 and ODS as packing materials. HSGF254 silica gel TLC plates were used for analytical TLC. The HPLC columns consisted of a Welch Material column (XB-C18, 10 μm, 10 × 250 mm), a normal phase chiral column (CHIRALPAK AS-H, 10 μm, 4.6 mm × 250 mm, part no. 20325), and a reversed phase chiral column (CHIRALPAK AD-RH, 10 μm, 4.6 mm × 150 mm, part no. 19724). The automatic coagulative instrument (Sysmex CA-7000), activated partial Thromborel S, Thrombin and Dade Actin Activated Cephaloplastin Reagent were commercial reagents from Siemens Healthcare Diagnostics Products GmbH. The semi-automatic biochemical analyser (MC-4000, Germany).

A total of 75 μL of transfected THP-1 cell suspension was added into 75 μL of platelet suspension and mixed gently. Each group was treated with 100 U/L of thrombin (final concentration; Siemens Healthcare Diagnostics Products, Erlangen, Germany). To observe the effect of NF-kB inhibitors on miRNA-146a expression, BAY11-7082 (1 μmol/L final concentration) was added into the co-culture system (with miRNA-146a mimic negative controls and inhibitor negative controls, but without miRNA-146a mimic or inhibitor) in the presence of 100 U/L of thrombin. An additional blank control group contained a mixture of THP-1 cells, platelets, and saline. Mixtures for each group were incubated at 37°C for 2 h and prepared for subsequent miRNA-146a detection and related assays.

The flow cytometric assessment of platelets was performed using FACSCalibur (Becton Dickinson, Heidelberg, Germany) [12 (link)]. Diluted PRP aliquots (5 × 10⁷/mL) were fixed and stained with FITC-labeled monoclonal surface antibody against glycoproteins (GP) CD41 (GPIIb/IIIa-complex, integrin αIIbβ3), CD42a (GPIX) and CD42b (GPIb) (Coulter, Immunotech, Marseille, France). FITC-labeled anti-vWF (Bio-Rad AbD Serorech, Puchheim, Germany) and Alexa Fluor 488-labeled anti-fibrinogen (Invitrogen, Waltham, MA USA) were used to stain the platelets. In the presence of 1.25 mM Gly-Pro-Arg-Pro (Bachem, Bubendorf, Switzerland) diluted PRP (5 × 10⁷ platelets/mL) was stimulated with different concentrations of thrombin (0, 0.05, 0.1, 0.2, 0.5, and 1 U/mL; Siemens Healthineers, Marburg, Germany) to conduct the CD62 and CD63 expression analyses. Additionally, the platelets were stained with monoclonal FITC-labeled anti-CD62 (P-selectin) and anti-CD63 antibodies (lysosomal membrane associated glycoprotein 3, Immunotech, Marseille, France). Data of patients and controls (day control and 20 independent measurements from 10 controls as mean ± standard error of the mean, SEM) were analyzed using GraphPad Prism software (version 8, San Diego, CA, USA).

Human THP-1 cells were purchased from American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium (Life Technologies, 23400021) containing 10% FBS (Life Technologies, 10099) and incubated at 37°C in a humidified atmosphere containing 5% CO2 (Thermo, 3111). Total of 75 μL THP-1 cell suspension (5×10⁷ cells/L) was added to 75 μL platelet suspension and mixed gently. In control group, the mixture of THP-1 and platelet was treated with saline or isotype antibodies. In platelet activation group, the mixture was administrated only with 100 U/L of thrombin (final concentration, Siemens Healthcare Diagnostics Products GmbH). In papain group, the mixture with 100 U/L of thrombin was treated with 50, 100, and 200U/L papain, respectively. In Cox-2 inhibitor group, NS-398 was added to the mixture of THP-1, thrombin, and platelet. To observe the effect of NF-κB agonist, TNF-α was added to the cocultured system in the presence of 100 U/L of thrombin and 200U/L of papain. The mixture in each group was incubated at 37°C for 30 mins and prepared for the following detection.

Flow cytometry analyses were performed using FACSCalibur (Becton Dickinson, Heidelberg, Germany) (Lahav et al., 2002 (link)). Diluted PRP aliquots (5 × 10⁷ platelets/ml) were fixed and stained with FITC-labeled monoclonal surface antibody against CD41 (GPIIb/IIIa-complex), CD42a (GPIb/IX) and CD42b (GPIb) (Coulter, Immunotech, Marseille, France). FITC-labeled anti-VWF (Bio-Rad AbD Serorech, Puchheim, Germany) and Alexa Fluor 488-labeled anti-fibrinogen (Invitrogen, Waltham, MA United States) was used to stain the platelets. In the presence of 1.25 mM Gly-Pro-Arg-Pro (Bachem, Bubendorf, Switzerland) diluted PRP (5 × 10⁷ platelets/ml) was stimulated with a number of concentrations of thrombin (0, 0.05, 0.1, 0.2, 0.5, and 1 U/ml; Siemens Healthineers, Marburg, Germany) to conduct the CD62 and CD63 expression analyses. Additionally, the platelets were stained with monoclonal FITC-labeled anti-CD62 (P-selectin) and anti-CD63 antibodies (lysosomal membrane-associated glycoprotein 3, LAMP-3; Immunotech, Marseille, France). Data of patients and controls (day control and 20 independent measurements from 10 controls as mean ± standard error of the mean, SEM) were analyzed using GraphPad Prism software (version 8, San Diego, CA, United States).

Fluo-3 AM was acquired from Thermo Fisher Scientific (Waltham, MA, USA). ION NaTRIUM Green-2 AM and ION Potassium Green-2 AM were purchased from ION Biosciences (San Marcos, TX, USA). Convulxin was a kind gift from Prof. K.J. Clementson (Bern, Switzerland) [58 (link)]. Thrombin was purchased from Siemens (Zürich, Switzerland). Cy5 Annexin V and Annexin V Buffer containing calcium (140 mM NaCl, 2.5 mM CaCl₂, 1 mM HEPES, pH 7.4) were from Becton Dickinson (Allschwil, Switzerland). Phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated antibodies for flow cytometric analyses were from either Becton Dickinson or Dako. Dimethyl-sulfoxide (DMSO), calcium ionophore A23187, Annexin-V-FITC FLUOS Staining Kit, eptifibatide acetate and tirofiban hydrochloride monohydrate were acquired from Merck Sigma Aldrich (St. Louis, MO, USA). To inhibit clot formation Gly-Pro-Arg-Pro-OH (GPRP H2935) from Bachem (Bubendorf, Switzerland) was used. Tyrode’s buffer was produced in our laboratory (137 mM NaCl, 2.8 mM KCl, 12 mM NaHCO₃, 1 mM MgCl₂, 0.4 mM NaH₂PO₄, 10 mM HEPES, 3.5 mg/mL bovine serum albumin, 5.5 mM glucose, adjusted to pH 7.4 with NaOH).