Fig 1.
Effect of free fatty acids on the kinetics of thrombin-mediated fibrin generation.
Fibrinogen was clotted with thrombin in the presence of sodium oleate or stearate. The course of clot formation was monitored by measuring the absorbance at 340 nm and T50 was defined as the time needed to reach the half-maximal turbidity (illustrated in the Inset for clotting in the absence of any additive in red, in the presence of 200 μM stearate in green or oleate in blue as mean with continuous lines ± 1SEM with dotted lines, n = 5). Relative T50 values are presented in green for stearate and blue for oleate (T50 measured in the absence of additives is considered to be 1) as mean (symbols) and SEM (red bars), n = 5. Lines represent the optimal fit to a ratio of empirical polynomial functions with a degree of 2 for the power coefficient in both the numerator and denominator functions (Curve Fitting Tool 3.5.3 of Matlab 2016a).
Fig 2.
Effect of free fatty acids on the kinetic parameters of thrombin on a small peptide- substrate.
Boc-VPR-AMC at 6 different concentrations adjusted to match the range of 0.5Km to 5Km for each modulator concentration was added to thrombin and different concentrations of sodium oleate (A) or stearate (B) (three parallel series of each substrate concentrations were measured for each fatty acid concentration). The release of amido-methylcoumarin was measured continuously and the progress curves were analyzed to estimate the kinetic parameters (kp, Km) according to the procedures described in the Materials and methods. The estimated synthetic parameter pairs within the 95% confidence region are shown by green symbols and the pairs out of this region are presented in blue (the numbers indicate the concentration of the fatty acid in μM, for which the respective data set was generated in the Monte-Carlo simulation, +Alb indicates the presence of 100 μM albumin). The best estimate from the Monte-Carlo simulation is indicated by a red circle. The exact numerical values are presented in Table 1. Insets show progress curves (mean with continuous lines ± 1SEM with dotted lines, n = 3) at 80 μM Boc-VPR-AMC and different sodium stearate or oleate concentrations (the arrows indicate the initiation of the reaction with thrombin injection into the substrate).
Table 1.
Kinetic parameters of thrombin in the presence of free fatty acids.
Fig 3.
Effect of free fatty acids on thrombin autodigestion.
Thrombin was incubated in the presence or absence of sodium oleate or stearate at 37°C for 72 hours. Samples were treated with a non-reducing denaturing buffer and polyacrylamide gel electrophoresis was performed, followed by visualization of protein bands by silver staining. Samples run on different gels are separated by vertical white lines. Lanes 1 and 2: Thrombin incubated in the absence of modulators for 0 min and 72 hours. Lanes 3 and 4: Thrombin incubated in the presence of oleate or stearate for 72 hours.
Fig 4.
Effect of free fatty acids on fibrin fiber diameters.
Fibrinogen premixed with sodium stearate (A) or oleate (B) at the indicated concentrations was clotted with thrombin. Clots were processed for SEM imaging and fibrin fiber diameters were measured and analyzed as detailed in Materials and methods. Bars contain representative images of fibrin with the indicated additive (scale bar = 1 μm) and the median values (bottom—top quartiles) of the diameter distributions are shown above each bar. The height of the bars indicates the relative change in fiber diameter as percentage of the median values in the absence of additives. Asterisk indicates statistical significance at p<0.001 according to Kuiper’s test in comparison to pure fibrin.
Fig 5.
Effect of free fatty acids on the course of rigidity changes during clot formation.
Fibrinogen premixed with sodium oleate or stearate was clotted with thrombin in the measurement gap of a cone-and-plate type oscillation rheometer and an oscillatory strain was imposed on the samples. Representative curves of storage (G') and loss (G'') moduli for each clot type are presented, all rheological parameters with statistics are shown in Table 2.
Fig 6.
Effect of free fatty acids on the gel/fluid transition of fibrin clots.
Fibrin clots were prepared in the measurement gap of a cone-and-plate type oscillation rheometer as detailed in Materials and methods. Thereafter, stepwise increasing shear stress (τ) was applied to the clot and dynamic viscosity (η) was determined. The abrupt fall in viscosity to 0 indicates the gel/fluid transition of the fibrin clots (τflow in Table 2). Two representative curves are shown for each clot type.
Table 2.
Effect of free fatty acids on the viscoelasticity of fibrin clots.