Co-ordinated spatial propagation of blood plasma clotting and fibrinolytic fronts

Abstract

Fibrinolysis is a cascade of proteolytic reactions occurring in blood and soft tissues, which functions to disintegrate fibrin clots when they are no more needed. In order to elucidate its regulation in space and time, fibrinolysis was investigated using an in vitro reaction-diffusion experimental model of blood clot formation and dissolution. Clotting was activated by a surface with immobilized tissue factor in a thin layer of recalcified blood plasma supplemented with tissue plasminogen activator (TPA), urokinase plasminogen activator or streptokinase. Formation and dissolution of fibrin clot was monitored by videomicroscopy. Computer systems biology model of clot formation and lysis was developed for data analysis and experimental planning. Fibrin clot front propagated in space from tissue factor, followed by a front of clot dissolution propagating from the same source. Velocity of lysis front propagation linearly depended on the velocity clotting front propagation (correlation r2 = 0.91). Computer model revealed that fibrin formation was indeed the rate-limiting step in the fibrinolysis front propagation. The phenomenon of two fronts which switched the state of blood plasma from liquid to solid and then back to liquid did not depend on the fibrinolysis activator. Interestingly, TPA at high concentrations began to increase lysis onset time and to decrease lysis propagation velocity, presumably due to plasminogen depletion. Spatially non-uniform lysis occurred simultaneously with clot formation and detached the clot from the procoagulant surface. These patterns of spatial fibrinolysis provide insights into its regulation and might explain clinical phenomena associated with thrombolytic therapy.

Fig 1

Pictures of fibrin clot growth in the absence of plasminogen activators (2 (A1), 10 (A2) and 20 (A3) minutes after the clotting onset) and clot growth and lysis in the presence of 30 nmol/L of TPA (2 (B1), 10 (B2) and 20 (B3) minutes after the clotting onset). Yellow rectangle on the panel A1 shows the region of the data collection for processing. The scale bar is 1 mm long. Spatial distribution of fibrin in the absence of plasminogen activators (A4) or in the presence of 30 nmol/L of TPA (B4) shows clot propagation and simultaneous clot growth and dissolution, respectively. Black, red, and blue lines show spatial distribution of light scattering signal (proportional to fibrin concentration) at 2^nd, 10^th,and 20^th minute after initiation of coagulation, respectively. When the signal exceeded the threshold level in any area, we considered that the clot appeared there, and when the level of signal decreased below this threshold, the clot dissolved. Coordinates of these events were designated as fronts of clot growth and lysis. Time course of clot growth front (A5) or clot growth and lysis fronts (B5) allows to calculate the velocities of clot growth or lysis as the average velocity of growth or lysis front propagation within the first 5 minutes after the onset of the process. In order to do that we used its linear approximation within the first 5 minutes after the clotting (lysis) lag time. Clotting (lysis) lag time was calculated as the time when clotting (lysis) front coordinate started to increase.

Fig 2

Spatial kinetics of fibrin generation in the absence (A) or in the presence (B) of 50 nmol/L TPA. Spatial fibrin distribution is shown for 10^th (black line), 30^th (blue line) and 60^th (red line) minute of simulation. (C) Time course of clot growth/lysis front during simulation. (D) Scheme of blood coagulation cascade main reactions. Zymogens are shown as blue circles, activated proteins are shown as yellow circles. Inactive cofactors are shown as blue rectangles, activated cofactors are shown as cyan rectangles. Red arrows show activation, black arrows show transition from inactive to active form, and formation of complexes. Green arrows show inhibition. Double arc shows phospholipid surface that is required for complex formation or activation. PgA stands for plasminogen activator; FDP stands for fibrin degradation products.

Fig 3. Pictures of fibrin clot growth and lysis in blood plasma 5, 15 and 30 minutes after the start of experiment.

Grey rectangle on the top of each image is an inset with immobilized TF. Fibrin clot is white, while liquid plasma is black/dark grey. (A) PFP from a patient under 0.03 mg/kg/h TPA therapy. (B) Pooled PFP from healthy volunteers supplemented with 14 nmol/L TPA in vitro. (C) PRP from healthy volunteer supplemented with 14 nmol/L TPA in vitro. (D) Pooled PFP from healthy volunteers supplemented with 14 nmol/L TPA in vitro, clotting was initiated with a TF-bearing fibroblasts confluent monolayer. Clot growth and lysis were monitored in plasma, treated as described in Methods section. Individual experiments are shown.

Fig 4. Three regimes of spatial clot lysis.

Pooled PFP from healthy volunteers supplemented with increasing concentration of TPA. Clot lysis started 200 μm away from the clotting activating surface in the presence of 6 nmol/L TPA (A); complete clot dissolution was observed in the presence of 100 nmol/L TPA (B); spatial clot lysis stopped 300 μm away from clotting activating surface in the presence of 800 nmol/L TPA (C). Clot growth and lysis were monitored in fresh frozen normal pooled plasma, treated as described in Methods section. Individual experiments are shown.

Fig 5

Kinetics of clot growth and lysis fronts coordinate in the presence of 30 nmol/L UPA (A) or 1200 nmol/L SK(B). Spatial clot lysis lag time and velocity depended on the type of plasminogen activator, while spatial clot growth did not change compared to control. The lag times of lysis (C) were significantly different for all plasminogen activators (2.4±0.3 min for SK, 3.4±0.2 min for TPA and 11±1 min for UPA). The velocity of lysis (D) was the same for UPA (48±4 μm/min) and SK (44±3 μm/min), but it was about 1.5 times higher for TPA (78±5 μm/min). Clot growth velocity and lag time were not significantly different from control (74±2 μm/min and 0.77±0.14 min, respectively). Clot growth and lysis were monitored in fresh frozen normal pooled plasma, treated as described in Methods section and supplemented with vehicle (control, n = 7), 50 nmol/L TPA (n = 9), 30 nmol/L UPA (n = 7) or 1200 nmol/L SK (n = 7).

Fig 6. Dependence of clot growth and lysis lag time and velocity on the TPA concentration.

(A) Simulation lag time (solid lines) was approximately 4 times higher than experimental lag time (symbols), both for clot growth (red) and clot lysis (black). Lag time of clot growth did not depend on TPA concentration, but lysis lag time decreased with the increase of TPA concentration up to 200 nmol/L, and further increase of TPA caused increase of lag time. (B) Clot growth velocity did not depend on TPA concentration, while lysis velocity was constant (75–78 μm/min) within the TPA concentration range 30–200 nmol/L, and decreased at TPA concentrations >200 nmol/L and <15 nmol/L. In mathematical simulations clot growth velocity was the same as the experimental one; clot lysis velocity was lower than the experimental for TPA concentrations 15–400 nmol/L. Prohibition of free plasminogen activation by TPA (dotted line) removed the increase of lag time and the drop of lysis velocity at high TPA concentrations. (C) Tenfold increase (orange dash line) of the rate of plasminogen association with fibrin made clot lysis velocity insensitive to TPA concentration. Tenfold decrease of the rate of plasminogen association with fibrin cancelled lysis. (D) Changes in plasma procoagulant state, like supplementation with antithrombin III (fivefold increase compared to the baseline, green dash line) or with phospholipids (fivefold increase compared to the baseline, black dash line) decreased or increased respectively the velocity of clot lysis. Clot growth and lysis were monitored in fresh frozen normal pooled plasma (panels A&B), treated as described in Methods section and supplemented with vehicle (control) or TPA at different concentrations (N = 4–11).

Fig 7. Clot lysis velocity correlated with the clot growth velocity in mathematical simulation (r² = 0.96, opened symbols) and in vitro experiments (r² = 0.91, closed symbols, N = 2–6).

In simulations plasma was supplemented with 100 nmol/L TPA; 1, 10 or 20 nmol/L FXIa (pentagons) or 45, 90 or 180 x 10⁻⁵ nmol/L PL (final concentration, circles); 7 or 17 μmol/L ATIII (final concentration, triangles). In vitro normal pooled plasma was supplemented with 30 (red), 50 (green) or 100 nmol/L (black) of TPA; 0.5 or 4 μmol/L PL (circles); 5, 10 or 50 pmol/L FXIa (pentagons); 2.5 mU/ml of unfractionated heparin (triangle). Spatial clot lysis in PPP, supplemented with 50 nmol/L TPA had a very high clot lysis velocity and was accompanied by a high clot growth velocity (star). Clot growth and lysis were monitored in plasma, treated as described in Methods section.