Kinetics and regulation of coagulation factor X activation by intrinsic tenase on phospholipid membranes.

BACKGROUND: Factor X activation by the phospholipid-bound intrinsic tenase complex is a critical membrane-dependent reaction of blood coagulation. Its regulation mechanisms are unclear, and a number of questions regarding diffusional limitation, pathways of assembly and substrate delivery remain open. METHODS: We develop and analyze here a detailed mechanism-driven computer model of intrinsic tenase on phospholipid surfaces. Three-dimensional reaction-diffusion-advection and stochastic simulations were used where appropriate. RESULTS: Dynamics of the system was predominantly non-stationary under physiological conditions. In order to describe experimental data, we had to assume both membrane-dependent and solution-dependent delivery of the substrate. The former pathway dominated at low cofactor concentration, while the latter became important at low phospholipid concentration. Factor VIIIa-factor X complex formation was the major pathway of the complex assembly, and the model predicted high affinity for their lipid-dependent interaction. Although the model predicted formation of the diffusion-limited layer of substrate for some conditions, the effects of this limitation on the fXa production were small. Flow accelerated fXa production in a flow reactor model by bringing in fIXa and fVIIIa rather than fX. CONCLUSIONS: This analysis suggests a concept of intrinsic tenase that is non-stationary, employs several pathways of substrate delivery depending on the conditions, and is not particularly limited by diffusion of the substrate.

Different modeling approaches in the simulation of extrinsic coagulation factor X activation: Limitations and areas of applicability.

Proteolytic reactions on the phospholipid membrane surface, so-called "membrane-dependent" reactions, play central role in the process of blood clotting. One particularly important example is FX activation by the extrinsic tenase (VIIa/TF). Here we constructed three mathematical models of FX activation by VIIa/TF: (A) a homogeneous "well-mixed" model, (B) a two-compartment "well-mixed" model, (C) a heterogeneous model with diffusion, to investigate the impact and importance of inclusion of each complexity level. All models provided good description of the reported experimental data and were equivalently applicable for <40 µM of phospholipids. Model C provided better predictions than A, B in the presence of TF-negative phospholipid microparticles. Models predicted that for high TF surface density (STF ) and FX deficiency the FX activation rate was limited by the rate of FX binding to the membrane. For low STF and excess of FX the reaction rate was limited by the tenase formation rate. The analysis of the substrate delivery pathways revealed that FX bound to VIIa/TF predominantly from solution for STF >2.8 × 10-3  nmol/cm2 and from the membrane for lower STF . We proposed the experimental setting to distinguish between the collision-limited and non-collision-limited binding. The analysis of models in flow and non-flow conditions revealed that the model of a vesicle in flow might be substituted by model C in the absence of the substrate depletion. Together, this study was the first which provided the direct comparison of more simple and more complex models. The reaction mechanisms were studied in a wide range of conditions.

Asymmetrical Forces Dictate the Distribution and Morphology of Platelets in Blood Clots.

Primary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet-dependent clot retraction reduces the clot volume by extruding the serum. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completely understood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery of retracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots.

Substrate delivery mechanism and the role of membrane curvature in factor X activation by extrinsic tenase.

Membrane-bound enzyme complex of extrinsic tenase (VIIa/TF) is believed to be the primary activator of blood clotting in vivo. This complex (where factor VIIa (FVIIa) is a catalytically active part and tissue factor (TF) is its essential cofactor) activates its primary substrate factor X (FX) leading to factor Xa (FXa) ('a' stands for 'activated'). Both FX and FXa are able to bind to phospholipid membrane and, therefore, are distributed between solution and membrane surface. As a result, two possible mechanisms of substrate delivery to the extrinsic tenase exist: via lateral diffusion on the membrane surface or directly from the solution. Determination of the predominant pathway of substrate delivery is an important key to understanding the precise reaction mechanism. Here we construct a mechanism-driven computational model of FX activation by extrinsic tenase on the surface of phospholipid vesicles of different size. We show that experimentally observed dependence of the tenase activity on the phospholipid concentration could be obtained only if the substrate (FX) is membrane-bound. For correct experimental data description it is also necessary to take into account the dependence of FX/FXa membrane binding parameters (equilibrium dissociation constant and the number of phospholipid molecules per bound FX/FXa) on the membrane curvature. The model predicts that small vesicles promote activation of FX by the extrinsic tenase significantly better than large vesicles (with the same overall phospholipid, factors VIIa, X and TF concentrations in the solution).