Plasma was supplemented with 200 U/mL aprotinin, 1mM phenylmethylsulfonyl fluoride (PMFS), 5mM etilendiaminotetracetic acid disodium salt (EDTA Na2), and applied to a Lysine-Sepharoseâ (Health Care, USA) column in order to remove plasminogen . Then fibrinogen was precipitated with b-alanine, essentially as described . Briefly, vitamin K- dependent proteins were adsorbed first with 2.4 mg/mL MgSO4 and then twice with 90 g/L BaSO4. The supernatant was precipitated thrice with 6M b-alanine (2.7M, final), with gentle stirring during 30 min at room temperature (RT), then centrifuged at 2000g for 30 min at 4ºC. Each time the pellet was dissolved with Tris-saline buffer (TSB) pH 7.4 (50mM Tris, 0.15M NaCl) supplemented with 0.1M epsilon aminocaproic acid (EACA). The pellet of the last precipitation was dissolved in TSB pH 7.4 and dialyzed overnight against the same buffer. The clottability of purified protein was >90%. The purified fibrinogen was depleted of plasminogen, contaminated mainly with FXIII, and the integrity of the fibrinogen chains was confirmed by SDS/PAGE.
Fibrin polymerization on the surface of HMEC-1 cell at different fibrinogen γ’ concentrations
MCDB131 medium (GIBCO, USA) supplemented with 10% foetal bovine serum (FBS) (GIBCO, USA), 100 U/mL penicillin (GIBCO, USA), 100 µg/mL streptomycin (GIBCO, USA), 200mM L-glutamine (GIBCO, USA), and 10 ng/mL epidermal growth factor (Invitrogen, USA) was used to suspend and cultivate HMEC-1 cells.
The cells (100,000 cells/well) were seeded on 96-well microtiter plate (Thermo Scientific Nunc, USA), and cultivated overnight essentially as described elsewhere .
Clot formation on the top of HMEC-1 monolayer:
In an eppendorf tube were added 143 µL purified fibrinogen 1 mg/mL (3% fibrinogen g/g´, quantified by ELISA ), 1 mg/mL 10% and 30% fibrinogen g/g´ (prepared by mixing commercial g/g and g/g´ fibrinogen, Enzyme Research Laboratories, USA), 40 µL MCDB 131 medium without supplements, and 17.5 µL bovine thrombin (Sigma, USA) – CaCl2 (1 nM and 2 mM, respectively, final concentrations), and the mixture immediately transferred on the top of the cells or directly to the bottom of the well (control clots without cells). The optical density (OD) was read every 2 min at 350 nm in an Infinite 200M (Tecan, Vienna, Austria). Experiments were performed each time in triplicate in three independent experiments. The kinetic of fibrin formation was characterized by measuring the lag time (min), slope (mOD/min) and final turbidity (mOD).
HMEC-1 uPA and PAI 1 secretion under different stimulation conditions
HMEC-1 cells were seeded (100,000 cells/well) on 96 wells plates. When the culture reached approximately 80% confluence, 100 mL of fibrinogen solution containing 3% and 30% of fibrinogen g´, clotted with thrombin-CaCl2 (1nM - 2mM, respectively, final) was gently added on the cell surface. The plates were kept for 30 min in the incubator (5% CO2 in a moist environment), then 200 mL/well of supplemented medium was added and incubated again for 12 h. The control conditions were the cells cultivated during 12 h in the presence of medium with or without 10% FBS, or with thrombin – CaCl2 (1nM - 2mM, respectively).
The plates were centrifuged at 1000g x10 min and the supernatants (SN) were collected, aliquoted and stored at -80°C. The cells were lysed essentially as described . The uPA and PAI 1 concentrations in supernatants and cell lysates were measured using uPA and PAI 1 ELISA kits (Sekisui-Diagnostic, Germany), according to the manufacturer´s recommendations. The ODs obtained were normalized dividing by the OD of the control condition of cells cultivated during 12 h without FBS. Each condition was tested in triplicate in three independent experiments.
Determination of uPA receptor (uPAR) in HMEC-1
A citoELISA was performed in order to determine if HMEC-1 cells express uPAR. The assay is based on the assumption that if uPAR was present it will bind uPA, and uPA will cleave plasminogen to plasmin, and plasmin will cleave the Lys-pNA bond of S2251 substrate. Briefly, HMEC-1 cells were seeded (100,000 cells/well) on 96 well plates. When the culture reached approximately 80% confluence, the monolayer was washed three times with phosphate buffered saline (PBS) + 4 mg/mL albumin. Then, the monolayer of cells were incubated with 10 and 20nM uPA (American Diagnostica inc. USA) for 1 h at 37ºC. After incubation, the cells were washed three times with PBS-albumin, 50 mL 0.5 mM plasminogen (American Diagnostica inc. USA) and 50 mL of 0.6 mM S-2251 (Chromogenix, Italy) were added. The plate was immediately transferred to a TECAN Infinite 200 M, and OD was read at 405 nm each 30 s during 1 h. The assay was performed in three independent experiments by duplicate.
Fibrin interaction with HMEC-1
The procedure used was essentially as described previously . Briefly, HMEC-1 cell membrane was stained with 4mM di-8-anepps (Molecular Probes, USA), and washed three times with 50mM Tris, 0.15M NaCl, pH 7.4 (supplemented with 20% MCDB-131 and 200mM glutamine). Clots were formed on the cell surface by mixing 1 mg/mL fibrinogen (3% and 30% fibrinogen g/g´), supplemented with Alexa Fluor® 488 (Molecular Probes, USA)-conjugated fibrinogen, and thrombin-CaCl2 (1nM and 1mM, respectively, final). Polymerization was allowed to proceed for 2 h in the culture incubator, and then the clot structure on the surface of HMEC-1 was visualized using a Nikon Eclipse TE 2000-U laser microscope (with a 488 nm Argon or 543 nm HeNe laser). Image acquisition and processing were done as described elsewhere . The quantification of the images was not included, since the measurement of fibrin density and width did not reflect the differences observed by LSCM.
Results are expressed as mean ± standard error (SE). A two-way ANOVA was performed, with factor 1 as the experiment: clots without cells vs. clots with cells and factor 2, the different g/g´ fibrinogen concentrations. The effects of the different conditions used on uPA and PAI 1 secretion: controls and different g/g´ fibrinogen concentrations were analyzed by a one-way ANOVA, after a Shapiro-Wilk test for normality, Levene test for variance homogeneity, and a posteriori Tukey test. Furthermore, two way ANOVA analysis was performed in order to compare the differences between uPA and PAI 1 secretion at the different conditions used, and in the lysed cells. The SPSS 17.0 for windows (SPSS, Chicago, IL), and Origin Pro 8.1 software were used. A p <0.05 was considered statistically significant.