Figure 7.
Figure 7. UHRA reverses anticlotting activity of UFH with no adverse effect on lung ultrastructure or clot morphology. Neutralization of UFH by UHRA or protamine was studied by activated partial thromboplastin time assay in heparinized human plasma. (A) UHRA neutralizes UFH (4 IU/mL) over a wide range of concentrations. Conversely, excess protamine impairs clotting. (B) Morphology of clots formed after neutralization of UFH with UHRA or protamine analyzed by SEM. Clot micrographs obtained in the presence of UHRA revealed normal morphology in comparison with the buffer control clot. Clot images at original magnification ×5000 are depicted. Scale bar, 5 μm. (C) Thickness of clot fibers was measured from clot micrographs using ImageJ. Fibers for size analysis were selected by probing 4 different spots in each image. Data are mean ± SE (n = 30 fibers, measured from 2 images of each clot). Fibrin fibers formed in the presence of 75 µg/mL protamine are significantly thicker than those in the buffer control clot (1-way ANOVA followed by a Dunnett post hoc test; ****P < .0001). Size of clot fibers obtained after neutralizing UFH with UHRA at all tested concentrations is comparable to buffer control. (D) In vivo neutralization of UFH activity were studied in mice by injecting UFH 200 IU/kg intravenously (via tail-vein), followed by UHRA (10 mg/kg) or protamine (5 mg/kg). aPTT confirms neutralization of UFH activity by antidotes. (E-F) Histopathological sections of lungs after heparin neutralization were obtained from buffer and UHRA-treated groups, which were comparable. However, in the protamine-treated group, significant damage to lung alveolar structure was observed. Lungs from the heparin-only group showed mild hemorrhage (presence of red blood cells in alveolar space). Scale bar, 50 μm. In magnified images: scale bar, 20 μm. Hematoxylin and eosin stain for panels E-F. A, alveolar space; AD, alveolar disruption; AH, alveolar hemorrhage. (G) Relative cumulative distribution of lung alveolar area. Depicted are values measured from 144 images from the lungs of 8 mice (2 mice per treatment group). Measurements confirm enhancement of alveolar area in the protamine-treated group compared with the buffer control.

UHRA reverses anticlotting activity of UFH with no adverse effect on lung ultrastructure or clot morphology. Neutralization of UFH by UHRA or protamine was studied by activated partial thromboplastin time assay in heparinized human plasma. (A) UHRA neutralizes UFH (4 IU/mL) over a wide range of concentrations. Conversely, excess protamine impairs clotting. (B) Morphology of clots formed after neutralization of UFH with UHRA or protamine analyzed by SEM. Clot micrographs obtained in the presence of UHRA revealed normal morphology in comparison with the buffer control clot. Clot images at original magnification ×5000 are depicted. Scale bar, 5 μm. (C) Thickness of clot fibers was measured from clot micrographs using ImageJ. Fibers for size analysis were selected by probing 4 different spots in each image. Data are mean ± SE (n = 30 fibers, measured from 2 images of each clot). Fibrin fibers formed in the presence of 75 µg/mL protamine are significantly thicker than those in the buffer control clot (1-way ANOVA followed by a Dunnett post hoc test; ****P < .0001). Size of clot fibers obtained after neutralizing UFH with UHRA at all tested concentrations is comparable to buffer control. (D) In vivo neutralization of UFH activity were studied in mice by injecting UFH 200 IU/kg intravenously (via tail-vein), followed by UHRA (10 mg/kg) or protamine (5 mg/kg). aPTT confirms neutralization of UFH activity by antidotes. (E-F) Histopathological sections of lungs after heparin neutralization were obtained from buffer and UHRA-treated groups, which were comparable. However, in the protamine-treated group, significant damage to lung alveolar structure was observed. Lungs from the heparin-only group showed mild hemorrhage (presence of red blood cells in alveolar space). Scale bar, 50 μm. In magnified images: scale bar, 20 μm. Hematoxylin and eosin stain for panels E-F. A, alveolar space; AD, alveolar disruption; AH, alveolar hemorrhage. (G) Relative cumulative distribution of lung alveolar area. Depicted are values measured from 144 images from the lungs of 8 mice (2 mice per treatment group). Measurements confirm enhancement of alveolar area in the protamine-treated group compared with the buffer control.

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