Figure 1.
Figure 1. UHRA does not bind to human fibrinogen or influence fibrin polymerization. For fluorometric experiments, (A) a fixed concentration of fibrinogen (0.25 µM) in 10 mM phosphate-buffered saline containing 137 mM NaCl (pH 7.4) was incubated with either UHRA or polyethyleneimine (PEI) at 37°C for 5 minutes. The solution was then excited at 280 nm, and emission spectra from 300 to 400 nm were recorded to follow the effect of increasing PEI concentrations on the tryptophan fluorescence emission of fibrinogen. PEI quenched the fluorescence signal in a concentration-dependent fashion. (B) The effect of increasing UHRA concentration on the tryptophan fluorescence emission of fibrinogen. The intensity of the fluorescence signal remained unchanged even in the presence of 1000 µg/mL UHRA, indicating minimal interaction of UHRA with fibrinogen. The depicted fluorescence spectra are the average of 3 independent experiments. Error values are small and not shown for data presentation clarity. The insets show the quenching effect of UHRA or PEI on the fibrinogen fluorescence. Data in the inset are mean ± standard deviation (n = 3). Experimental settings were as in A. (C) CD experiments were conducted by incubating UHRA or PEI with fibrinogen (0.4 µM) in 20 mM sodium phosphate (pH 7.4) at 37°C for 5 minutes; the equilibrated sample was then scanned from 190 to 260 nm. PEI at 500 or 1000 µg/mL caused significant changes in the secondary structure of fibrinogen. CD spectra shown are baseline-corrected using mean protein-free control scans (n = 2). (D) CD spectra of the fibrinogen–UHRA mixture were acquired as in C. No significant change in ellipticity at 208 and 222 nm was observed. (E) A binding isotherm was obtained using ITC by titrating UHRA with fibrinogen. The upper panel depicts the raw data (power signal), and the lower panel shows the integrated areas corresponding to each injection, normalized to the moles of UHRA injected into fibrinogen solution and plotted as a function of molar ratio (UHRA/fibrinogen). The raw heat data of UHRA titration into fibrinogen solution showed very weak endothermic peaks. This confirmed that UHRA did not bind to fibrinogen. (F) The final turbidity of mature fibrin clots produced from purified fibrinogen in the presence of UHRA or protamine was measured. Even at 500 µg/mL, UHRA did not alter the final turbidity relative to the polycation-free control. In comparison, significant increases in final turbidity are observed as protamine concentration is increased to 50 µg/mL (****P < .0005). All experiments were performed in triplicate. Results are expressed as the mean ± SE of 9 measurements from 3 independent experiments. Unpaired 2-tailed t tests were performed to determine significance, with P < .05 indicating a statistically significant change. Details of ITC and fibrin polymerization experiments are described in the supplemental Data (pages 6-7).

UHRA does not bind to human fibrinogen or influence fibrin polymerization. For fluorometric experiments, (A) a fixed concentration of fibrinogen (0.25 µM) in 10 mM phosphate-buffered saline containing 137 mM NaCl (pH 7.4) was incubated with either UHRA or polyethyleneimine (PEI) at 37°C for 5 minutes. The solution was then excited at 280 nm, and emission spectra from 300 to 400 nm were recorded to follow the effect of increasing PEI concentrations on the tryptophan fluorescence emission of fibrinogen. PEI quenched the fluorescence signal in a concentration-dependent fashion. (B) The effect of increasing UHRA concentration on the tryptophan fluorescence emission of fibrinogen. The intensity of the fluorescence signal remained unchanged even in the presence of 1000 µg/mL UHRA, indicating minimal interaction of UHRA with fibrinogen. The depicted fluorescence spectra are the average of 3 independent experiments. Error values are small and not shown for data presentation clarity. The insets show the quenching effect of UHRA or PEI on the fibrinogen fluorescence. Data in the inset are mean ± standard deviation (n = 3). Experimental settings were as in A. (C) CD experiments were conducted by incubating UHRA or PEI with fibrinogen (0.4 µM) in 20 mM sodium phosphate (pH 7.4) at 37°C for 5 minutes; the equilibrated sample was then scanned from 190 to 260 nm. PEI at 500 or 1000 µg/mL caused significant changes in the secondary structure of fibrinogen. CD spectra shown are baseline-corrected using mean protein-free control scans (n = 2). (D) CD spectra of the fibrinogen–UHRA mixture were acquired as in C. No significant change in ellipticity at 208 and 222 nm was observed. (E) A binding isotherm was obtained using ITC by titrating UHRA with fibrinogen. The upper panel depicts the raw data (power signal), and the lower panel shows the integrated areas corresponding to each injection, normalized to the moles of UHRA injected into fibrinogen solution and plotted as a function of molar ratio (UHRA/fibrinogen). The raw heat data of UHRA titration into fibrinogen solution showed very weak endothermic peaks. This confirmed that UHRA did not bind to fibrinogen. (F) The final turbidity of mature fibrin clots produced from purified fibrinogen in the presence of UHRA or protamine was measured. Even at 500 µg/mL, UHRA did not alter the final turbidity relative to the polycation-free control. In comparison, significant increases in final turbidity are observed as protamine concentration is increased to 50 µg/mL (****P < .0005). All experiments were performed in triplicate. Results are expressed as the mean ± SE of 9 measurements from 3 independent experiments. Unpaired 2-tailed t tests were performed to determine significance, with P < .05 indicating a statistically significant change. Details of ITC and fibrin polymerization experiments are described in the supplemental Data (pages 6-7).

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