Materials
Bovine thrombin was from Enzyme Research Laboratories (South Bend, IN). Lysine-sepharose 4B was from GE Healthcare (Piscataway, NJ). Pefabloc® SC (4- (2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) was from Fluka, Sigma-Aldrich (Buchs, Switzerland). Tissue-type plasminogen activator and plasmin were from American Diagnostica (Stamford, CT). Human albumin and benzamidine were from Sigma Chemical Company (St Louis, MO). Albumin antibody conjugated to peroxidase was from Dako Corporation (Carpinteria, CA). The substrate 3,3′-diaminobenzidine (DAB) was from Thermo Scientific (Rockford, IL). The LabTek chambers and Alexa Fluor 488 were purchased from Invitrogen, Nalge Nunc International (Rochester, NY).
Blood collection
Blood was collected in citrate (1 volume of 0.13 M trisodium citrate and 9 volumes of blood), the first 3 ml of blood discarded, and centrifuged twice at 2000 g for 10 min. The platelet poor plasma obtained (PPP) was supplemented with benzamidine 10 mM (final concentration), except the plasma to be used for fibrinolysis experiments, aliquoted and kept at −80 °C until use. Routine coagulation tests were performed with citrated plasma on coagulation analyzer STA Compact®, Stago, France. Fibrinogen level was determined by Clauss (Laboratoire Stago, Asnière, France) and clot weight method [11]. Antigenic fibrinogen concentration was measured by a latex immunoassay (Liaphen Fibrinogen, Hyphen BioMed, France).
Mutation analysis
Blood was collected in 0.5 M ethylenediaminetetraacetate tetrasodium salt (EDTA Na4) (50:1). Genomic DNA was isolated using the Invisorb Spin Blood Mini Kit (Invitek GmbH, Berlin, Germany) according to the manufacturer’s protocol. Sequences comprising all exons and exon-intron boundaries from the three fibrinogen genes: FGA, FGB, and FGG were amplified by the polymerase chain reaction (PCR) according to standard protocols. After purification of the PCR products using the Invisorb Spin PCRapid Kit® (Invitek, Berlin, Germany), direct DNA cycle sequencing was performed, applying the Big Dye kit from Applied Biosystems (Foster City, CA), according to the manufacturer’s recommendations.
Fibrinogen purification
Fibrinogen was purified essentially as described elsewhere with modifications [12]. Plasma samples were thawed and supplemented with 1 mM Pefabloc® and 5 mM EDTA (final concentrations). Plasma was depleted of plasminogen by passing through a lysine-sepharose 4B column, and then fibrinogen was purified by precipitation (×3) with 25%-saturation ammonium sulphate, pH 7.5. This fibrinogen fraction also contained co-purifying fibronectin, factor XIII and vW factor. The precipitate was dissolved in 0.3 M NaCl, dialyzed against the same solution, and stored at −80 °C until used.
The integrity of the purified fibrinogen was analyzed by sodium dodecylsulfate-polyacilamide gel electrophoresis (SDS-PAGE) on 8% gel. The coagulability of the purified fibrinogen was 96% and 93% and the yield 43 and 27%, control and patient, respectively.
Fibrinogen degradation
Fibrinogen was incubated with plasmin as described [13] with minor modifications. Purified fibrinogen (0.9 mg/ml, in TBS) was incubated with plasmin (18 μg/ml, in TBS) in the presence of 1 mM CaCl2 or 5 mM EDTA at 37 °C at different incubation times (15, 30 min and 4 h), quenched with 2% SDS-DTT (v:v) sample buffer and immediately boiled. The zero time sample contained no plasmin. The fibrinogen degradation products were analyzed in a 6% gel SDS-PAGE under non reducing conditions.
Western blotting
In order to detect fibrinogen-albumin complexes, Western blotting was performed under non reduced conditions essentially as described [14]. Briefly, purified fibrinogens (5 μg) and human albumin (5 μg) were loaded in a 5% gel SDS-PAGE, and electroblotted onto nitrocellulose [15]. The membrane was incubated for 2 h with anti-human albumin antibody conjugated to peroxidase (1:1000). The cross-reacting bands were detected with 0.6% 3, 3’diaminobenzidine (DAB), 3% cobalt chloride and 3% hydrogen peroxide.
Activated factor XIII (FXIIIa) fibrin cross-linking
The kinetics of fibrin cross-linking was examined essentially as described elsewhere [14]. Fibrin was cross-linked by the endogenous factor XIIIa that precipitated together with fibrinogen during the purification process. Purified fibrinogen (1 mg/ml) was clotted with 1 U/ml of thrombin and 5 mM CaCl2. The reactions were quenched at different time points (0, 2, 5, 15 min and 1, 4, 24 h) with 2% SDS-DTT and analyzed in a 8% gel SDS-PAGE.
Fibrin polymerization
The kinetics of fibrin formation were studied in plasma and purified fibrinogen [16]. Briefly, 100 μl fresh plasma or 0.5 mg/ml purified fibrinogen in 50 mM Tris, 0.15 M NaCl, pH 7.4 (TBS) were dispensed in a 96-well plate. Then 10 μl of 1 unit/ml bovine thrombin - 20 mM CaCl2 (final) was added to plasma or 5 units/ml bovine thrombin and 5 mM CaCl2 to fibrinogen solution. The changes in optical density (OD) were recorded every 15 s over 1 h at 350 nm in a Tecan Infinite® M 200. The polymerizations were done in three different experiments in triplicate. The lag time (s), slope (mOD/s) and final turbidity (mOD) were calculated from each curve and averaged.
Fibrinolysis
The method was performed as described by Carter et at 2007 [17] with minor modifications. The PPP aliquots without benzamidine were used. Twenty five μl of PPP were aliquoted in a 96-well plate, then 75 μl of tPA 166 ng/ml diluted in 20 mM Hepes, 0.15 M NaCl, pH 7.4 was added. Clotting was initiated by adding 50 μl of thrombin-CaCl2 (0.03 U/ml and 9 mM, respectively).The OD changes were recorded at 350 nm every 15 s over 1.5 h in a TECAN® infinite 2 M microplate reader. The fibrinolysis was done at least three times in triplicate. The time to degrade 50% of the clot (T50%) was calculated from the time elapsed from half the value of the maximum absorbance of the polymerization to half-the value decreased of the maximum absorbance of the lysis curve branch. The rate of clot degradation (slope) was calculated in the descending part of the curve and the absolute value reported.
Direct plasma mass spectrometry
Plasma was precipitated with saturated (NH4)2SO4 (25%, final), and the precipitate washed (2×) with 25% saturated (NH4)2SO4. The pellet was dissolved in 8 M urea, 30 mM dithiothreitol, 50 mM Tris–HCl, pH 8.0 and left 3 h at 37 °C. The reduced sample was injected into an Agilent 6230 Accurate-Mass electrospray time-of-flight (TOF) mass spectrometry system [18]. A Poroshell 300SB C3 (2.1 × 75 mm) column was used with an acetonitrile gradient and profile data was collected. Multi charged spectral envelopes were deconvoluted using maximum entropy processing and BioConfirm software with an isotope width of 15 Da.
Clots biophysical characterization
In order to characterize some biophysical parameters of the clot structure, the elastic modulus, the Darcy constant (Ks), and fibrin network imaging by confocal microscopy were performed. For these experiments a healthy man with plasma fibrinogen concentration similar to the patient was chosen as a control.
Elastic modulus
The fibrin elastic modulus (EM) was measured in the hemostasis analyzer system (HAS) Hemodyne® (Richmond, VA). Briefly, 700 μL of plasma was placed in the plastic cone and incubated for 1 min at 37 °C. Then 50 μL of a thrombin - CaCl2 solution (1.3 U/ml and 25 mM final concentrations; respectively) was gently and carefully mixed in. The increase in the EM was recorded every 1 min over a 30 min period. Each sample was run in triplicate in three independent experiments. The EM (kdyne/cm2) reported corresponds to the averaged EM value that was reached at 30 min.
Permeation
Permeation through plasma clots was recorded essentially as described elsewhere [19]. The clotting conditions used were 1 U/ml of thrombin and 20 mM CaCl2 (final concentrations). The clots were left for 2 h in a moist environment at 37 °C in order to fully polymerize. The buffer percolated through the columns was TBS. Nine clots of each sample were run per experiment (n = 3) and one measurement per clot was taken.
The Darcy constant (Ks) was calculated using the following equation [20]:
$$ \mathrm{Ks}=\mathrm{QL}\upeta /\mathrm{tAP} $$
Where Q = volume of the buffer (in cm3), having a viscosity η of 0.01(poise), flowing through the column of height L (cm) and area A (cm2) in a given time (s) under a hydrostatic pressure P (dyne/cm2).
Confocal microscopy
The experiments were done essentially as described elsewhere [16]. Briefly, clots were formed inside the eight wells LabTek chambers (Invitrogen, Nalge Nunc International, Rochester, NY). Plasma samples were mixed with Alexa Fluor 488-labeled fibrinogen (10 μg/315 μl final sample volume), then clotted with thrombin-CaCl2 solution (0.3 U/ml and 20 mM, respectively, final concentration). The clots were left for 2 h in a moist environment at 37 °C in order to fully polymerize.
The fibrin clots were observed in a Nikon Eclipse TE 2000 U laser scanning confocal microscopy (LSCM), with an argon ion laser (473 nm excitation and 520/540 nm for emission). The objective used was Plan APO VC 60X water immersion with a work distance of 0.27. The acquisition pinhole was set to 60 μm. Image analyses were done as described [21]. A z-stack of 60 slice was use for construct a 3D projection of 30 μm thick (0.5 μm/slice) were done. Five image by clot (212 × 212 μm) for each experiment (control and patient) were accomplished. Two diagonal lines, a horizontal and a vertical were drawn on the volumetric image of the stack using the Olympus FV10-ASW 2.1 software for obtain the pseudocolor perfil by line. Line graphs were used to calculate density (picks/μ) and diameter of fibers (μm) with Origin Pro 8 software.
Dynamic fibrin clot growth
The spatio temporal dynamics of fibrin clot formation in real time was assessed in plasma by measuring light scattering over 30 min every 15 s using a Thrombodynamics Analyser System (HemaCore, Moscow, Russia) as previously described [21]. Briefly, plasma coagulation is activated when it is brought in contact with tissue factor coated on a plastic cuvette. The clot formation begins on the activator and propagates into the bulk of plasma in which no TF is present. Images are analyzed computationally to measure lag time, initial and stationary growth rate, size at 30 min and clot density. Based on the plots of clot size versus time, the initial velocity of clot growth is measured as the mean slope over the 2–6 min period (characterizing the VIIa-TF pathway) and the stationary velocity of clot growth is measured as the mean slope over the 15–25 min period [22].