Visualization of Domain- and Concentration-Dependent Impact of Thrombomodulin on Differential Regulation of Coagulation and Fibrinolysis

Abstract

Background: Thrombomodulin (TM) functions as a dual modulator-anticoagulant and antifibrinolytic potential-by the thrombin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). Activated TAFI cleaves the C-terminal lysine of partially degraded fibrin and inhibits both plasminogen binding and its activation on the fibrin surface. We have reported previously that activated platelets initiate fibrin network formation and trigger fibrinolysis after the accumulation of tissue-type plasminogen activator and plasminogen.

Objective: To analyze the effects of domain-deletion variants of TM on coagulation and fibrinolysis at different concentrations.

Methods: Domain-deletion variants of TM, such as D123 (all extracellular regions), E3456 (minimum domains for thrombin-dependent activation of protein C and TAFI), and E456 (minimum domains for that of protein C but not TAFI), were used at 0.25 to 125 nM for turbidimetric assay to determine the clotting time and clot lysis time and to visualize fibrin network formation and lysis in platelet-containing plasma.

Results and conclusions: A low concentration of either D123 or E3456, but not of E456, prolonged clot lysis time, and delayed the accumulation of fluorescence-labeled plasminogen at the activated platelets/dense fibrin area due to effective TAFI activation. Conversely, only the highest concentrations of all three TM variants delayed the clotting time, though fibrin network formation in the vicinity of activated platelets was almost intact. TAFI activation might be affected by attenuation in thrombin activity after the clot formation phase. These findings suggest that the spatiotemporal balance between the anticoagulant and antifibrinolytic potential of TM is controlled in domain- and concentration-dependent manners.

Fig. 1

The effects of recombinant thrombomodulin (rTM) variants on the platelet-containing plasma clotting time and clot lysis time determined by the turbidimetric assay. Diluted thromboplastin (tissue factor)-triggered clotting time ( A, C, E ) and tissue-type plasminogen activator-initiated clot lysis time ( B, D, E ) of the platelet-containing plasma (4.0 × 10 ⁴ platelets/µL) in the presence of D123 (A, B), E3456 (C, D), and E456 ( E, F ) are shown. For all conditions, >15 samples in total, unevenly from 6 healthy individuals were used. Data are shown as the median and interquartile range, and the increasing trends were analyzed using the Shirley–Williams multiple comparison tests (* p  < 0.05, ** p  < 0.01).

Fig. 2

Reduction of TM-associated prolongation of clot lysis time by TAFIa inhibitor. Platelet-containing plasma (4.0 × 10 ⁴ platelets/µL) clotting time ( A, C ) and clot lysis time ( B, D ) in the absence (open bars) or presence of 5 µM TAFIa inhibitor (closed bars) are shown. (A) and (B) represent E3456, and (C) and (D) represent E456. For all conditions, >8 samples in total, unevenly from 3 healthy individuals were used. Data are shown as median and interquartile ranges and were analyzed using the Mann–Whitney U -test ( ^## p  < 0.01). TAFIa, activated thrombin-activatable fibrinolysis inhibitor; TM, thrombomodulin.

Fig. 3

Visualization of fibrin network formation and lysis by confocal laser scanning microscopy. A montage of an Alexa Fluor 488-labeled fibrinogen (top) panel, an Alexa Fluor 568-labeled plasminogen (middle) panel, and an overlaid (bottom; green: fibrinogen, red: plasminogen) panel is shown. Coagulation and fibrinolysis were initiated by the addition of tissue factor and 2 nM tPA to recalcified platelet-containing plasma (4.0 × 10 ⁴ platelets/µL) in the absence of rTM. The indicated time refers to the time after the initiation of image capture. Scale: 250 µm. rTM, recombinant thrombomodulin; tPA, tissue-type plasminogen activator.

Fig. 4

rTM-modified fibrin network formation, plasminogen accumulation, and lysis. Representative sequential overlaid images of Alexa Fluor 488-labeled fibrinogen (green) and Alexa Fluor 568-labeled plasminogen (red) are displayed at the time indicated after starting the video capture. All conditions showed similar tissue factor-triggered fibrin formation initiated by activated platelets from the initiation of image capture. In control ( A ) and E456 ( C ), plasminogen also accumulated at the dense fibrin network formed at the site of activated platelets where fibrinolysis was initiated and propagated at the periphery with the addition of 2 nM tPA. In the presence of either D123 ( B ) or E3456 ( D ), plasminogen accumulation and fibrinolysis initiation were significantly delayed. Scale: 250 µm. rTM, recombinant thrombomodulin; tPA, tissue-type plasminogen activator.

Fig. 5

Estimation of TAFI activation by attenuation of labeled plasminogen accumulation. The average fluorescence intensities of labeled fibrinogen (closed circles) and labeled plasminogen (open circles) within a 30-pixel diameter circle of dense fibrin regions under the conditions in Fig. 3 are traced and shown in ( A ) control: without TM, ( B ) 5 nM D123, ( C ) 5 nM E456, and ( D ) 5 nM E3456 in arbitrary unit (a.u.). The double-headed arrow represents the time from the appearance of the fibrin network formation to the maximum intensity of the labeled plasminogen. This time was defined as plasminogen accumulation time. ( E ) Values of plasminogen accumulation time (median and interquartile range) in 3 to 5 regions selected from >3 experiments under each condition are shown, and a significant difference was analyzed using Student's t -test ( ^## p  < 0.01). TAFI, thrombin-activatable fibrinolysis inhibitor; TM, thrombomodulin.

Fig. 6

TM-dependent activation of TAFI was strongly blocked by the TAFIa inhibitor. Representative sequential overlaid images of Alexa Fluor 488-labeled fibrinogen (green) and Alexa Fluor 568-labeled plasminogen (red) at early time points (up to 12 minutes) in the presence of different concentrations (5 nM and 125 nM) of E3456 are shown in ( A ) and their modifications by TAFIa are shown in ( B ). Scale: 250 µm. ( C ) Values of plasminogen accumulation times in 3 to 5 regions selected from >3 experiments under each condition are shown as median and interquartile range and were analyzed using Student's t -test ( ^## p  < 0.01). aTAFI, activated thrombin-activatable fibrinolysis inhibitor; TM, thrombomodulin.

Fig. 7

The difference in anticoagulant effect between low and high concentrations of TMs at an early phase of fibrin network formation. The fluorescence intensity of Alexa Fluor 488-labeled fibrinogen in the same region of interest as in Fig. 5 was measured and represented as a percentage from the initial intensity to the maximum intensity. The median and interquartile ranges from 3 to 5 regions selected from >3 experiments at the time point after triggering the coagulation cascade are shown in ( A ) control, ( B ) E456, and ( C ) E3456. The gray lines and the diamonds of interquartile ranges indicate 5 nM, and the black lines and the minus symbols of interquartile ranges indicate 125 nM of TMs (B, C), respectively. A significant difference in the value between 5 and 125 nM was analyzed using the Mann–Whitney U -test (* p  < 0.05, ** p  < 0.01). TM, thrombomodulin.