UHRA does not bind or incorporate into purified fibrin, blood clots, or other clot components. Clots for confocal microscopy were prepared as described in the supplemental Data (confocal microscopy of fibrin and blood clots). Briefly, human fibrinogen 3 mg/mL, of which 2% was Alexa Fluor 546–fibrinogen, was mixed with UHRA or protamine (both conjugated with Alexa Fluor 488), and clotting was initiated with thrombin 2.5 National Institutes of Health Units/mL and 3 mM CaCl2. (A) The top panel (red channel) shows confocal micrographs of fibrin clots formed from Alexa Fluor 546–labeled fibrinogen. The middle panel (green channel) shows confocal micrographs of protamine or UHRA localization within fibrin clots. Fibers of clots (i-k) formed in the presence of protamine exhibit green fluorescence, indicating binding of protamine to fibrin fibers. Green signal is absent in clots formed in the presence of UHRA (l-n), suggesting no binding or incorporation of UHRA into fibrin fibers. The lower panel (merge) shows overlay of images from the red and green channels confirming the colocalization of fibrin and protamine. Scale bar, 10 μm. (B) Magnified images from the white boxes shown in the red channel. Visual inspection of clots (II-IV) formed in the presence of Alexa Fluor 488–labeled protamine reveals different fibrin architecture (presence of more compact and round fibrin (ogen) aggregates indicated by white arrowheads) compared with the control clot (I). No architectural differences are observed for clots formed in the presence and absence of UHRA (V-VII). Scale bar, 2μm. (C) Normalized raw integrated density (sum of all pixel intensities normalized to polycation-free buffer) of the green channel analyzed using FIJI. Depicted are mean values ± SD with 10 or more randomly acquired images analyzed per condition. The P values were determined using the unpaired t test. (***P < .001). (D) Representative confocal micrographs of whole blood clots formed in the presence of UHRA or protamine. No green fluorescence is observed in blood clots formed in the presence of UHRA. This shows that UHRA does not interact or incorporate into the clot components. In contrast, fluorescence from protamine containing blood clot fibers and platelet aggregates/clumps show binding of protamine to these clot components. Scale bar, 10 µm.
