Blood flow controls coagulation onset via the positive feedback of factor VII activation by factor Xa

<p>Abstract</p> <p>Background</p> <p>Blood coagulation is a complex network of biochemical reactions, which is peculiar in that it is time- and space-dependent, and has to function in the presence of rapid flow. Recent experimental reports suggest that flow plays a significant role in its regulation. The objective of this study was to use systems biology techniques to investigate this regulation and to identify mechanisms creating a flow-dependent switch in the coagulation onset.</p> <p>Results</p> <p>Using a detailed mechanism-driven model of tissue factor (TF)-initiated thrombus formation in a two-dimensional channel we demonstrate that blood flow can regulate clotting onset in the model in a threshold-like manner, in agreement with existing experimental evidence. Sensitivity analysis reveals that this is achieved due to a combination of the positive feedback of TF-bound factor VII activation by activated factor X (Xa) and effective removal of factor Xa by flow from the activating patch depriving the feedback of "ignition". The level of this trigger (i.e. coagulation sensitivity to flow) is controlled by the activity of tissue factor pathway inhibitor.</p> <p>Conclusions</p> <p>This mechanism explains the difference between red and white thrombi observed <it>in vivo </it>at different shear rates. It can be speculated that this is a special switch protecting vascular system from uncontrolled formation and spreading of active coagulation factors in vessels with rapidly flowing blood.</p

Systems Biology Approach for Personalized Hemostasis Correction

The correction of blood coagulation impairments of a bleeding or thrombotic nature employs standard protocols where the type of drug, its dose and the administration regime are stated. However, for a group of patients, such an approach may be ineffective, and personalized therapy adjustment is needed. Laboratory hemostasis tests are used to control the efficacy of therapy, which is expensive and time-consuming. Computer simulations may become an inexpensive and fast alternative to real blood tests. In this work, we propose a procedure to numerically define the individual hemostasis profile of a patient and estimate the anticoagulant efficacy of low-molecular-weight heparin (LMWH) based on the computer simulation of global hemostasis assays. We enrolled a group of 12 patients receiving LMWH therapy and performed routine coagulation assays (activated partial thromboplastin time and prothrombin time) and global hemostasis assays (thrombodynamics and thrombodynamics-4d) and measured anti-Xa activity, fibrinogen, prothrombin and antithrombin levels, creatinine clearance, lipid profiles and clinical blood counts. Blood samples were acquired 3, 6 and 12 h after LMWH administration. We developed a personalized pharmacokinetic model of LMWH and coupled it with the mechanism-driven blood coagulation model, which described the spatial dynamics of fibrin and thrombin propagation. We found that LMWH clearance was significantly lower in the group with high total cholesterol levels. We generated an individual patient&rsquo;s hemostasis profile based on the results of routine coagulation assays. We propose a method to simulate the results of global hemostasis assays in the case of an individual response to LMWH therapy, which can potentially help with hemostasis corrections based on the output of global tests

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

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

Parameters of clot growth and lysis in the absence and presence of thrombolytics.

<p>Parameters of clot growth and lysis in the absence and presence of thrombolytics.</p

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

<p>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.</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

Three regimes of spatial clot lysis.

<p>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.</p