Three PAR2-activating proteases (trypsin, matriptase, thrombin) each induced TF protein synthesis over 24 h in HTECs in a concentration-dependent manner (Figures 1ACF)
Three PAR2-activating proteases (trypsin, matriptase, thrombin) each induced TF protein synthesis over 24 h in HTECs in a concentration-dependent manner (Figures 1ACF). antibody for TF. In summary, activation of PAR2 on HTEC stimulates synthesis and secretion of TF that induces blood clotting, and this is Clonixin attenuated by PAR2 antagonism. Thrombin-induced TF synthesis is at least partly mediated by PAR1 transactivation of PAR2. These findings reveal how underlying hemostatic imbalances might increase thrombosis risk and subsequent chronic fibrin deposition in the kidneys of patients with CKD and suggest PAR2 antagonism as DKK2 a potential therapeutic strategy for intervening in CKD progression. prior to storage. The inhibitors used in this study (PAR2: GB88 10 M or I-191 10 M; PAR1: vorapaxar 4 M) were added 30 min prior to peptide or protease activator. Extracellular Vesicle Analysis Initially to characterize the tissue factor secreted into the medium by 2F treated cells, the conditioned medium was concentrated 10-fold using either a Nanosep Centrifugal Device with a 3 kDa (OD003C34) or 100 kDa (OD100C35) molecular weight cut off membrane (Pall Melbourne Australia). If the secreted TF was 45 kDa we expected TF to pass through a 100 kDa membrane but be retained by a 3 kDa cut-off membrane. However, if secreted TF was larger than 100 kDa we expected it to be retained. For further analysis of EVs differential centrifugation was used. The conditioned medium was consecutively centrifuged at 300 (sediments microvesicles and apoptotic bodies EVs, size C 0.1C1.0 m), and 200,000 (sediments exosomes, size C 30C100 nm). Quantitative Real-Time PCR (Used in the Supplementary Figure) The method used for TF Clonixin real-time quantitative PCR (qPCR) Clonixin has been previously reported (Vesey et al., 2013). Total RNA was isolated using a RNeasy Mini Kit (Qiagen, Crawley United Kingdom) according to manufacturers instructions. RNA was reverse transcribed using Superscript III (ThermoFisher Scientific, Mt Waverley, Australia) and an oligo (dT) primer. cDNA from various cell samples were amplified by qPCR with specific primers as reported previously (Vesey et al., 2013). Relative gene expression was quantified using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, United States) on an Applied Biosystems Prism 7000 sequence detector. Amplification cycles proceeded as follows: 50C for 2 min and 95C for 10 min, follows by 40 cycles of 95C for 15 s and 50C for 1 min. cDNA levels at the linear phase of amplification were compared relative to expression of 18S or HRPT. Western Blot Analysis Western blot analysis was performed on protein lysate from cells grown to confluence in six-well plates. A total of 2 mL of medium was used per well. Clonixin At the end of the experiment, the medium was harvested, centrifuged at 900 and stored at ?80C. Cells were washed twice with ice-cold PBS and lysed with RIPA buffer (Sigma-Aldrich Cat # R0278), containing a protease inhibitor cocktail (Sigma-Aldrich, Cat. # P8340) and the phosphatase inhibitor NaF (10 mM) in 150 L of lysis buffer per well. Cells were further disrupted by sonication, cell debris pelleted by centrifugation (13,000 version 4 software (TASTM) was used for data analysis to capture four important parameters; R time (min), K time (min), -angle (degrees) and maximum amplitude (MA) value (mm). The R value represents the time until the first evidence of a clot; K value is the time from the end of R until the clot reaches 20 mm, representing the speed of clot formation. The angle is the tangent of the curve made as K is reached and MA reflects clot strength. Each reaction mix was prepared in a purpose-made disposable cup (Cat #. 6211, Haemoscope Corporation, Niles, IL, United States) with a maximum volume of 360 L. Whole citrated blood was a component of all the assays and was kept at a constant volume of 320 L. The order in which the other components were added was as follows; 1. Cell medium (20 L), 2. Calcium (final concentration 20 mM), 3. Citrated whole blood (320 L). This gives an excess of calcium over citrate. The citrated blood was inverted 5 times (10C15 s) prior to addition to the TEG cup. The addition of citrated blood starts the clotting Clonixin time (R time). TEG analysis was carried out 37C. Statistical Analysis All studies were performed in at least triplicate from HTEC cultures obtained from at least three separate human being donors unless normally indicated. Each experiment contained internal settings originating from the same tradition.