Rearrangements of the Fibrin Network and Spatial Distribution of Fibrinolytic Components during Plasma Clot Lysis

Abstract

Binding of components of the fibrinolytic system to fibrin is important for the regulation of fibrinolysis. In this study, decomposition of the fibrin network and binding of plasminogen and plasminogen activators (PAs) to fibrin during lysis of a plasma clot were investigated with confocal microscopy using fluorescein-labeled preparations of fibrinogen, plasminogen, tissue-type PA (t-PA), and two-chain urokinase-type PA (tcu-PA). Lysis induced by PAs present throughout the plasma clot was accompanied by a gradual loss of fibrin content of fibers and by accumulation of plasminogen onto the fibers. Two sequential phases could be distinguished: a phase of prelysis, during which the fibrin network remained immobile, and a phase of final lysis, during which fibers moved with a tendency to shrink and eventually disappeared. The two phases occurred simultaneously but in different locations when lysis was induced by PAs present in the plasma surrounding the clot. The zone of final lysis was located within a 5-8 microns superficial layer, where fibers were mobile, a surface-associated fibrin agglomerates appeared. Plasminogen accumulated in these agglomerates up to 30-fold as compared with its concentration in the outer plasma. t-PA was also highly concentrated in the agglomerates, and tcu-PA bound to them slightly. The zone of prelysis, where plasminogen was moderately accumulated on the immobile fibers, was located deeper in the clot. This zone was much thinner in the case of t-PA-induced lysis than in the case of tcu-PA-induced lysis, reflecting the difference in penetration of the two PAs into the clot. We conclude that under conditions of diffusional transport of fibrinolytic enzymes from outside a plasma clot, extensive lysis is spatially restricted to a zone not exceeding 5-8 microns from the clot surface. In this zone the structure of the fibrin network undergoes significant changes, and strikingly high accumulation of fibrinolytic components takes place.

Influence of Fibrin Network Conformation and Fibrin Fiber Diameter on Fibrinolysis Speed

“…16,19 This model was based on the characterization of fibrin degradation products released from clots and confirmed later by confocal microscopy showing that plasma fibrin degradation resulted in 2 sequential phases. 7,8 During the prelysis phase, which is characterized by very few structural changes of the fibrin matrix, plasminogen accumulates on the surface as more C-terminal lysine binding sites are exposed. Then, the fibrin network becomes mobile before collapsing and disappearing during the second (end) stage.…”

“…Second, although of unknown molecular mechanism, fibrin fiber retraction phenomena that occur in the prelysis zone of plasma clots (a region of few micrometers away from the lysis front) are another potential explanation for this paradox. 8 Impaired retraction in plasma clots with a tight network conformation could explain the significant difference of the lysis-front thickness between coarse and fine clots ( Figure 3) and may contribute to hindered lysis. 23 Measurements of FITC-rtPA binding-front velocity of native hydrated cross-linked plasma fibrin clots provide strong evidence for the crucial role of the fibrin network architecture rather than fibrin fiber diameter as a limiting factor of fibrinolysis speed.…”

“…8 To a stock solution of 2 mg/mL rtPA in TNE buffer (140 mmol NaCl and Tris-HCl 20 mmol, pH 7.4) was added FITC dissolved in 0.01 mol/L Tris, 0.1 mol/L NaCl, and 1 mmol/L EDTA, pH 8, at a final concentration of 50 g/mL. After 1 hour of incubation, free FITC was removed by gel filtration on a G-25 Sephadex column (3.5 mL) equilibrated with TNE buffer.…”

“…6 Recent structural studies have emphasized that fibrin digestion proceeds locally by transverse cutting across fibers rather than by progressive cleavage uniformly around the fiber and that changes of the fibrin network structure are spatially restricted to a zone in which high accumulation of fibrinolytic components takes place. [7][8][9][10] However, none of these findings provide sufficient conclusions regarding the impact of the fibrin network structure and the fibrin fiber diameter on fibrinolysis speed.…”

Fibrinolytic Activation Promoted by the Cyclopentapeptide Malformin: Involvement of Cytoskeletal Reorganization

“…The importance of local plasminogen accumulation in the fibrinolytic process is well documented. 18) When U937 cells were treated with Alexa 594-plasminogen in plasminogen-deficient plasma, partial focal localization of labeled plasminogen was observed (Fig. 5A, upper).…”