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

<div><p>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.</p></div

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

<p>(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).</p

Co-ordinated spatial propagation of blood plasma clotting and fibrinolytic fronts - Fig 2

<p>Spatial kinetics of fibrin generation in the absence <b>(A)</b> or in the presence <b>(B)</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. <b>(C)</b> Time course of clot growth/lysis front during simulation. <b>(D)</b> 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.</p

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

<p>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). <i>In vitro</i> 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.</p

Co-ordinated spatial propagation of blood plasma clotting and fibrinolytic fronts - Fig 1

<p>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.</p

Thrombodynamics—A new global hemostasis assay for heparin monitoring in patients under the anticoagulant treatment

<div><p>Background</p><p>Heparin therapy and prophylaxis may be accompanied by bleeding and thrombotic complications due to individual responses to treatment. Dosage control based on standard laboratory assays poorly reflects the effect of the therapy. The aim of our work was to compare the heparin sensitivity of new thrombodynamics (TD) assay with sensitivity of other standard and global coagulation tests available to date.</p><p>Study population and methods</p><p>A total of 296 patients with high risk of venous thromboembolism (deep vein thrombosis (DVT), early postoperative period, hemoblastosis) were enrolled in the study. We used a case-crossover design to evaluate the sensitivity of new thrombodynamics assay (TD) to the hemostatic state before and after unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) therapy/prophylaxis and to compare it with the activated partial thromboplastin time (APTT), anti-Xa activity test, thrombin generation test (TGT) and thromboelastography (TEG). A receiver operating characteristic (ROC) curve analysis was used to evaluate changes before and after heparin prophylaxis and therapy. Blood was sampled before heparin injection, at the time of maximal blood heparin concentration and before the next injection.</p><p>Results</p><p>Hypercoagulation before the start of heparin treatment was detected by TD, TGT and TEG but not by APTT. The area under the ROC curve (AUC) was maximal for TD and anti-Xa, intermediate for TGT and TEG and minimal for APTT.</p><p>Conclusions</p><p>These results indicate that TD has a high sensitivity to the effects of UFH and LMWH after both prophylactic and therapeutic regimes and may be used for heparin monitoring.</p></div

Characteristics of patients.

<p>Characteristics of patients.</p

APTT vs TD parameters before heparin treatment.

<p>(A) APTT and (B) V in TD before heparin treatment in groups: healthy volunteers (control group), group 1, group 2 and group 3. The box plots indicate the following parameters: the mean value (the dot inside the box), the median (the horizontal line inside the box), the 25th and 75th percentiles (the bottom and top of the box, respectively) and the 5th and 95th percentiles (the ends of the whiskers). * indicates a significant difference from healthy volunteers group (p<0.01, Mann-Whitney test).</p

Heparin effect on TEG vs TD.

<p>ROC curves for alpha in TEG (gray lines) and V in TD (black lines) for prophylaxis with LMWH (group 2) at Point 1 (A) and Point 2 (B) or UFH (group 3) at Point 1 (C). Data of the same patients before the first heparin injection were used as controls.</p