Blood Flow Controls Coagulation Onset Via the Positive Feedback of Factor

Shibeko et al. BMC Systems Biology 2010, 4:5 http://www.biomedcentral.com/1752-0509/4/5 RESEARCH ARTICLE Open Access Blood flow controls coagulation onset via the positive feedback of factor VII activation by factor Xa Alexey M Shibeko1, Ekaterina S Lobanova1,2, Mikhail A Panteleev1,3, Fazoil I Ataullakhanov1,3,4* Abstract Background: 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. Results: 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. Conclusions: This mechanism explains the difference between red and white thrombi observed in vivo 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. Background have been discovered over the last fifteen years [1], the Blood coagulation is a complex reaction network that present understanding of the functioning of this system functions to form a fibrin clot that covers damaged ves- is limited. The incredible biochemical complexity of coa- sel wall and prevents blood loss [1]. The clotting process gulation additionally complicated by protein diffusion is initiated by tissue factor (TF), a transmembrane pro- and blood flow makes it extremely difficult to establish tein exposed in the damaged parts of the wall. This pro- a correlation between the roles of individual reactions tein forms a complex called extrinsic tenase with plasma and the functioning of the clotting system in vivo as a protein activated factor VII (VIIa). Extrinsic tenase acti- whole. For diffusion, a number of recent studies brought vates factor X, which activates thrombin, the main pro- attention to the essential role of spatial non-uniformity tein of blood coagulation. Activated factor X (factor Xa) and rate-limiting diffusion of specific coagulation factors activates factor VII in complex VII-TF (inactive extrinsic thus proposing new concepts of clotting regulation, tenase), thus forming a positive feedback. Extrinsic alternative to the classic “cascade” paradigm [1,3-6]. tenase is inhibited by tissue factor pathway inhibitor This is not the case for blood flow. (TFPI) in a complex factor Xa-dependent manner [2]. A major role of flow in hemostasis and thrombosis Thrombin forms fibrin, which polymerizes to create a was recognized as early as 19th century, flow being one clot. of the components of the famous Virchow’striad[7]. Although reactions of the coagulation cascade are well Two primary hemostatic mechanisms, platelet plug for- known, and no new essential components of this system mation and blood coagulation, are known to differently depend on the flow conditions: platelet adhesion and * Correspondence: [email protected] aggregation require high blood flow velocities, while 1 National Research Center for Hematology, 4a Novyi Zykovskii pr., Moscow fibrin deposition occurs better in slowly flowing blood 125167, Russia © 2010 Shibeko et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Shibeko et al. BMC Systems Biology 2010, 4:5 Page 2 of 12 http://www.biomedcentral.com/1752-0509/4/5 [8,9]. Moreover, a recent report suggests that fibrin clot near the activator (dark grey in Fig. 1), while the large formation is inhibited by flow in a threshold-like man- thrombus downstream primarily consisted of low den- ner [10]. This can be illustrated in vivo by formation of sity clot (dark blue in Fig. 1). fibrin-rich red thrombi containing erythrocytes in veins This following study was concentrated on the most (where shear rate is low) and of platelet-rich white obvious and immediate effect of the flow, the increase thrombi in arteries [11]. However, the phenomenon of of time between TF exposure and the clot appearance. blood clotting inhibition by flow has not been studied in In order to characterize this quantitatively, we intro- detail and was assumed to be a self-evident consequence duced the parameter of lagtime, i.e. time required to of the removal of active coagulation factors from the cover half of the activator with high density fibrin clot site of vascular damage by flow. (3,800 nM). In order to gain insight into this phenomenon, a mod- A series of computational experiments on fibrin throm- ular decomposition strategy was used. We created a bus formation at different flow rates was performed. Fig. detailed quantitative mechanism-driven mathematical 2a (black line) shows the above-defined lagtime as a func- model of thrombus formation in flowing plasma. In tion of wall shear rate. It can be approximated with a func- c·x agreement with experimental reports, clot formation in tion of exponential growth y = a+be ,a= 1.54 ± 0.73, the model depended on blood flow shear rate in a b = 2.86 ± 0.32, c = 0.129 ± 0.004, where y is lagtime and threshold-like manner. Sensitivity analysis of this model x is wall shear rate. From a practical point of view, this was performed to identify reactions forming a module means that, at small shear rates (0-13 s-1), the lagtime (or subsystem) within this biochemical network that was increased slowly with the increase of shear rate from 2.5 responsible for this effect. We demonstrated that a spe- min without flow up to 10-20 min. However, this increase cially designed positive feedback of factor VII activation became very rapid at higher shear rates, and by 28 s-1 the combined with chemical inhibition of extrinsic tenase lag time reached 100-120 min. The results in Fig. 2a can and flow-induced removal of factor Xa becomes a be summarized as follows: 1) clotting is inhibited by flow; “switcher” that can “decide": whether to start clotting or 2) this inhibition is strongly non-linear, i.e. the system can to abstain from it. It can be speculated that this switch- resist inhibition caused by flow within a certain range of ing can serve to prevent uncontrolled spreading of coa- flow rate, but it is efficiently “turned off” at higher shear gulation factor by flow throughout the vasculature. rates. Dependence of the phenomena on model assumptions Results While the vast majority of model parameters are based The effect of flow on fibrin thrombus formation on experimental data and have been thoroughly vali- Functioning of blood coagulation network was simulated dated by comparison with experiments without flow [4], in a two-dimensional region with constant pressure flow two assumptions were made in this study: specific values as described under Methods. Fig. 1 (see also Additional for fibrin gelation concentration and the fibrin level to files 1 and 2) illustrates the design of computational calculate lagtime. In order to estimate the effect of these experiments and the general effect of flow on coagula- assumptions, we performed calculations at different tion. The clot initially appeared downstream from the values. For this, we introduced two numerical para- S activator and slowly propagated upstream; the clot size meters characterizing control of clotting by flow: Cij downstream from the activator was much greater than (overall inhibition of clotting by flow in the interval of the activator size. This clotting downstream from the shear rates i-j) and R’ (non-linearity of this inhibition) as activator, which may seem not physiological, was most defined under Methods. likely caused by lack of thrombomodulin and heparane Theincreaseofthegelationpointconcentration sulfate on the chamber walls in our simulations. Throm- increased the influence of flow on the system and vice bomodulin present on the endothelium of vessels, is versa quantitatively, but not qualitatively. The principal able to inhibit spatial clot propagation [4] and is prob- effect (that coagulation system can resist flow within ably responsible for the lack of uncontrolled fibrin for- some range of flow velocities and is suppressed by more mation downstream from the damage site in vivo. rapid flow) remained (Fig. 2a, Table 1). Furthermore, However, this study was focused on the initial stages of the exact value of fibrin concentration at which clot clot formation, which are not influenced by thrombo- became solid did not influence the observed flow effect modulin [4]; therefore, we chose not include this addi- at all (Fig. 2b, Table 1). The most likely explanation is tional system into the model. Further, it is interesting to that, when a portion of the activator became protected note that the clot is heterogeneous: the high density from the flow by the clot, coagulation reactions within region (Fn concentration higher than 3,800 nM, i.e. the clot could proceed rapidly, and complete fibrinogen more than 1/2 of fibrinogen concentration) is localized conversion could occur with minimal delays.

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