Real-Time Imaging of Platelet-Initiated Plasma Clot Formation and Lysis Unveils Distinct Impacts of Anticoagulants

Figures

• 

  Fig. 1. Activated platelets potentiate thrombin generation and thrombin-activatable fibrinolysis inhibitor (TAFI) activation. The representative data show changes in turbidity during the formation and lysis of clots in plasma with and without platelets ( A–C ) and during thrombin generation ( D–F ). Assays were conducted with an activated TAFI inhibitor (TAFIaI: orange lines) or anti-thrombomodulin-neutralizing antibody (aTM Ab; blue lines), in addition to the control without any inhibitor or antibody (black lines). ( G ) The relative endogenous thrombin potential (ETP)-60 of the platelet-free control was calculated for each group (control: black boxes; TAFIaI: orange boxes; aTM Ab: blue boxes), using six to eight samples from three individuals. The data are shown as median and interquartile ranges and were analyzed using a nonparametric multiple comparison test, specifically the Shirley–Williams method (** p < 0.01). ( H ) The relationship between ETP-60 and lysis time (control: black circles; TAFIaI: orange circles; aTM Ab: blue circles) was analyzed using Spearman's correlation coefficient by rank test ( rs : * p < 0.05, ** p < 0.01). The different groups are represented by the…

• 

  Fig. 2. Anticoagulant-induced reduction of thrombin activity leads to shorter lysis time due to inadequate thrombin-activatable fibrinolysis inhibitor (TAFI) activation. The representative data show changes in turbidity ( A, B ) and thrombin generation ( C, D ) in platelet-containing plasma treated with different concentrations of dabigatran (Dab 25, 50, 100, and 200 nM; represented by blue lines in A , C ) or rivaroxaban (Riv 125, 250, 500, and 750 nM; represented by pink lines in B , D ). The black lines indicate the absence of anticoagulants (control). The thicker pink and blue lines indicate higher anticoagulant concentrations. In panel E , the clotting time (upper panel) and lysis time (lower panel) are shown for the control and different concentrations of anticoagulants in platelet-containing plasma. These data include 15 samples from three individuals. The data are presented as the median and interquartile ranges, with or without activated TAFI (TAFIa) inhibitor, indicated by closed and open bars, respectively. Statistical analysis was performed using the Mann–Whitney U-test (# p < 0.05, ## p < 0.01). The dose-dependent effect of anticoagulants in the absence of TAFIa inhibitor…

• 

  Fig. 3. Correlation between coagulation and fibrinolysis parameters in platelet-containing plasma treated with different classes of anticoagulants. The correlations among relative endogenous thrombin potential (ETP)-60, clotting time, and lysis time in platelet-containing plasma treated with dabigatran ( A–C ) or rivaroxaban ( D–F ) were analyzed using Spearman's correlation coefficient (shown as rs in graphs) by a rank test (15 samples from three individuals, * p < 0.05, ** p < 0.01).

• 

  Fig. 4. Real-time imaging analysis of clot formation and dissolution in platelet-containing plasma. We used a confocal laser scanning microscope (TCS SP8; Leica Microsystem GmbH), equipped with a 20× (NA 0.8) objective lens and a stage top incubator (Tokai Hit Co., Ltd.), maintaining samples at a temperature of 37°C. Coagulation and fibrinolysis of platelet-containing plasma were initiated by tissue factor and tissue-type plasminogen activator, respectively. Trace amounts of Alexa Fluor (AF) 488-labeled fibrinogen (green) and AF 568-labeled plasminogen (magenta) were added for visualization. Representative sequentially overlaid images are shown. The dense fibrin network at the activated platelets indicates the heterogeneity of the fibrin network structure. ( A ) Control (2, 10, 20, 30, 40, and 50 minutes after the start of video capture). ( B ) 200 nM dabigatran (16, 18, 20, 25, 30, and 36 minutes after the start of video capture). ( C ) 500 nM rivaroxaban (7, 10, 13, 15, 20, and 25 minutes after the start of video capture).

• 

  Fig. 5. Changes in fluorescence intensity of Alexa Fluor (AF) 488-labeled fibrinogen (fbg-488) and AF 568-labeled plasminogen (plg-568) in the dense fibrin regions. ( A ) Fluorescence intensities of both fbg-488 and plg-568 in the central region of five concentric circles placed in the dense fibrin regions were separately measured using ImageJ software. The changes in fluorescence intensity are shown as solid lines (fig-488) and dotted lines (plg-568) without anticoagulants (control, black), or with 500 nM rivaroxaban (Riv 500, pink lines) or 100/200 nM dabigatran (Dab 100/Dab 200, light blue/blue). Fibrin fiber appearance time ( B ) and plasminogen accumulation time ( C ) were determined by measuring the time, as described in the “Fluorescence Intensity Analysis” section of the Materials and Methods. The fluorescence intensities of both fbg-488 and plg-568 were separately measured in five concentric circles within the dense fibrin network region. Statistical analysis was performed using the Mann–Whitney U test (## p < 0.01 vs. control). The dose-dependent effect of the anticoagulants was analyzed using the Shirley–Williams method, a nonparametric multiple comparison test (** p < 0.01)…

• 

  Fig. 6. Propagation of fibrin network formation and plasminogen accumulation from the central regions in the dense fibrin to the periphery. The fluorescence intensities of both Alexa Fluor (AF) 488-labeled fibrinogen (fbg-488) and AF 568-labeled plasminogen (plg-568) were separately measured in five concentric circles within the dense fibrin network region. The average intensities were determined at the center and in the four outer ring-shaped areas. The green lines of fbg-488 ( A ) and the red lines of plg-568 ( C ) are thicker and closer to the center of the concentric circles. Without anticoagulants: control; dabigatran: Dab; rivaroxaban: Riv. ( B ) The relative fluorescence intensity of fbg-488 in the outer regions compared with the maximum intensity in the center is shown. A thicker green color indicates concentric circles close to the center (C1), which become lighter green as they move toward the periphery (C2–C4). The decrease in intensity (the intensity relative to C0 from C1 to C4) with increasing distance from the center was analyzed using the Shirley–Williams method, a nonparametric multiple comparison test (8–13 areas from three experiments, ** p < 0.01). ( D ) The delay in…

• 

  Fig. 7. Spatial modulation of thrombin activity influences thrombin-activatable fibrinolysis inhibitor (TAFI) activation and plasminogen accumulation dynamics. The activation of the coagulation cascade, initiated at the surface of activated platelets, results in the formation of an uneven fibrin network with a heterogeneous distribution of thrombin activity (shown by the green curve in control). Dabigatran (Dab), functioning as a direct thrombin inhibitor within the fluid, reduces thrombin activity, particularly in the protruding part of the green line, leading to uniform thrombin activity and a more homogenous fibrin network. In contrast, rivaroxaban (Riv) selectively impedes activated factor X-dependent catalysis of thrombin generation on activated platelet surfaces, resulting in a green curve (representing thrombin activity) with a structure similar to that of the control but with reduced amplitude. This leads to the formation of a more heterogeneous fibrin network. Furthermore, the spatial modification of thrombin activity significantly affects TAFI activation, as indicated by the reduced accumulation of plasminogen. Dotted blue lines represent the threshold of TAFI activation…