Annotation of the results obtained in 2023
During the reported period, according to the research plan, all-atomic molecular dynamics modeling of the platelet adhesive protein GPIb in complex with its ligand, the A1 domain of the von Willebrand factor (VWF) protein, was carried out. Equilibrium and steered molecular dynamics approaches were used. The alpha-subunit of the GPIb protein, which is directly involved in the formation of adhesive protein bridges during the primary attachment of platelets to VWF, was considered. Using controlled molecular dynamics, we plot the dependence of the adhesion force of the GPIb-A1VWF protein complex on time and the adhesion force of the GPIb-A1VWF protein complex on deformation at different stretching rates. The average force potential for deaggregation of the GPIb-A1VWF protein complex was measured using the umbrella sampling method. Modeling was carried out for both wild-type proteins (corresponding to conditionally healthy platelets) and mutant proteins (leading to various types of von Willebrand disease). A comparative analysis of the molecular mechanical characteristics of these proteins was carried out. The conformations of the GPIb and A1VWF proteins were analyzed during the process of stretching the complex of these proteins for both wild type and mutant forms. Structural changes in the GPIb and A1VWF proteins were identified, which are responsible for the effect of the trapping bond during stretching of the complex of these proteins. The work used the GROMACS software package to perform all-atomic molecular dynamics simulations, as well as the VMD (Visual Molecular Dynamics) program to visualize and analyze the results obtained. The calculations were carried out on the Lomonosov-2 supercomputer at the Research Computing Center of Moscow State University. The conformations of the GPIb and A1VWF proteins were analyzed during the elongation process. Three stages of deformation of the A1VWF-GPIb protein complex were observed during the deaggregation process. At high stretching rates, partial unfolding of the GPIb protein was also observed until complete deaggregation of the proteins. Modeling was also carried out for some known point mutations in the A1 domain of the VWF protein, which can lead to von Willebrand disease types 2B (increased platelet aggregation) and 2M (decreased platelet aggregation). Using the modeling of mutant structures and comparative analysis, the structural factors responsible for the effect of the trapping bond (that is, strengthened by tension) in the GPIb-A1VWF protein complex were identified. Local electrostatic interactions of these amino acid residues according to the principle of salt bridges contribute to the fact that at low and high tensile forces the complex deaggregates relatively quickly, and at medium ones it lives as long as possible. Thanks to this nanomechanics of proteins, determined by their geometry, it appears that “tuning” of the optimal adhesion interaction between platelets and von Willebrand factor is provided in the operating range of hydrodynamic conditions. Understanding these mechanisms may facilitate the development of new therapies for type 2B von Willebrand disease, which is associated with point mutations in the A1 domain. Mutations of the A1 domain of VWF, leading to von Willebrand disease, affect the kinetics of interaction of the molecules in question, in particular, they change the type of time dependence of the force applied to GPIb. Some of these mutations are located close to A1VWF binding sites and may directly affect protein interactions. At the same time, other dangerous mutations may be located in the center of the globule and affect its rigidity or the formation of intramolecular bonds. Based on molecular dynamics calculations, more accurate parameters of the interaction potentials between platelets and von Willebrand factor were selected for the cellular scale model. During the second year of the project, an updated stochastic model of ligand-receptor connections between platelets and von Willebrand factor was formulated mathematically, implemented in software code, and added to a cell-scale computer model to account for the trapping effect and study the effect of this effect on adhesion dynamics platelets to von Willebrand factor during thrombosis in blood microvessels. The new model allows us to take into account mutations in the GPIb and A1VWF proteins by changing the rate constants of bond cleavage, which in turn can be calculated from the results of the molecular dynamics part of the work, namely from the potentials of average force. Using cell-scale modeling, the dynamics of platelet motion in a shear fluid flow near a flat wall with an immobilized layer of von Willebrand factor multimers attached to one of the ends was studied. A comparison with experiments was carried out and based on this comparison the numerical values of the free parameters of the model were refined for “healthy” platelets and VWF multimers, as well as for mutant proteins that cause von Willebrand disease types 2B and 2M. A new modification of the coarse-grained model of the GPIb-A1VWF protein complex has also been developed, taking into account changes in its conformation of these proteins under the influence of mechanical tension and the effect of mechanoreception. Based on the modeling, it was concluded that during the primary adhesion of platelets to von Willebrand factor in arteries and arterioles, sufficient tensile forces act on platelet receptors for mechanical activation of platelets at shear stresses > 1 Pa. The updated cellular model of platelet hemostasis, developed during the reporting period, now explicitly takes into account the dynamics and elastic properties of red blood cells at a realistic hematocrit. We improved and verified the classical mechanical model of the spectrin network of the erythrocyte by comparing modeling and experimental data from literature sources. A new technique has been proposed to implicitly take into account the difference in viscosity inside and outside the erythrocyte based on rescaling the coefficient of interaction of erythrocyte membrane particles with liquid. Calculations were carried out for a model of a section of a cylindrical microvessel with a section of inflamed endothelium, which secreted von Willebrand factor protein multimers into the lumen of the vessel. In the model, collisions with red blood cells lead to platelets being “pressed” into the VWF protein layer on the walls of the inflamed endothelium and forming a greater number of adhesive bonds with it. At the same time, red blood cells also push platelets already attached to VWF, which leads to an increase in stretched VWF subunits, and also increases the stretch of mechanosensitive receptors (GPIb). These mechanical interactions, as follows from the model, can contribute to the mechanical activation of platelets in narrow microvessels. All planned tasks were completed in full. Based on the results of the work, 3 papers were published in peer-reviewed scientific journals (two in Q1 journals).

 

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Annotation of the results obtained in 2022
The computational approach developed in the project is based on computer simulation using the MPI multiprocessor technology. To solve the tasks set, a specially developed software package for parallel computing will be used. It is based on a combination of the Lattice Boltzmann (LB) method for calculating hydrodynamic velocities and the Immersed Boundary (IB) method for describing the dynamics of cell membranes. The model consists of three main components: a viscous fluid (blood plasma), blood cells (platelets), and von Willebrand factor (VWF) multimers represented as linear polymer chains. The fluid was represented by a continuum model, and the fluid mechanics was calculated based on the Boltzmann lattice equation method. During the reporting period, the software package was finalized to solve the tasks set in the project. The mechanism of regulation of the length of the von Willebrand factor by the ADAMTS13 metalloproteinase enzyme has been added to the computer model. The modified model suggests that the A2 domain of von Willebrand factor undergoes a conformational change (unfolding) under the action of a tensile mechanical force above a threshold value (approximately 20 pN, according to experimental data) and becomes open for cutting by the enzyme. Proteolysis of the open domain A2 of the von Willebrand factor in the model occurs in a probabilistic manner (Monte Carlo algorithm), the value of the reaction rate constant was found in the literature. The next important modification of the model, carried out in the reporting period, was to take into account the activation and deactivation of the A1 domain of the von Willebrand factor under the action of a tensile force. The model makes it possible to take into account this mechanochemical effect both deterministically and probabilistically, similarly to the unfolding of the A2 domain. The model of adhesion receptors on the platelet membrane has also been improved. Now, the GPIb receptors and GPIIb/IIIa integrins are explicitly presented as particles associated with platelet membrane (cytoskeleton) particles by elastic bonds. This method makes it possible to measure the tension force of the receptor, to study the mechanical signal transmission between the receptor and the platelet cytoskeleton, to test one of the hypotheses about the mechanical activation of platelets in a wide range of shear stresses in the near-wall layer of the liquid (blood). In order to test the hypothesis about the regulatory role of changes in the conformation of the von Willebrand factor (compact-stretched) and mechanical activation of the A1 domain under the action of a shear flow of a viscous fluid, a series of calculations was carried out using the model we created on the Lomonosov-2 supercomputer of Moscow State University. During the first stage of the work, computer modeling of platelet adhesion to von Willebrand factor (VWF) multimers attached to a solid wall was carried out, taking into account the realistic shape of platelets and their mechanical movement in a shear flow of a viscous fluid, a quantitative assessment of the force acting on VWF and GPIb receptors, and studies possibilities of biomechanical activation of VWF. The cases of Couette flow and Poiseuille flow were considered. The next task was to study the mechanisms of thrombosis initiation during inflammation of the endothelium of the vessel walls and the secretion of ultra-long von Willebrand factor multimers (ULVWF). To solve this problem, several series of model calculations were carried out in various configurations and with varying model parameters. The sensitivity analysis of the model to the parameters was carried out. Also, using the model developed by our research team, we tested the hypothesis about the regulatory action of ADAMTS13 metalloproteinase in the process of primary platelet adhesion to von Willebrand factor multimers of various lengths. The dynamics of platelet accumulation at the site of damage or inflammation of the endothelium was studied depending on the activity of the ADAMTS13 enzyme. By using computer simulation, the dependences of the number of attached platelets on the size, quantity, and mechanical properties of von Willebrand factor multimers secreted by the endothelium were identified and numerically characterized under various hydrodynamic conditions in the model system. The results of the first year of work confirmed the hypothesis about the regulatory effect of hemodynamic forces on the process of primary platelet hemostasis: an increase in the number of active monomers with an increase in shear stress also correlates with an increase in the number of adherent platelets during the simulation time. The results of numerical calculations revealed a competition between the processes of activation of the A1 domain and unfolding of the A2 domain of the von Willebrand factor by mechanical forces acting on the VWF multimer attached to the wall in a shear flow of a viscous fluid. The model confirmed the relevance of the biophysical mechanism of hydrodynamic activation of thrombus formation, according to which successive changes in the conformation of the multimeric VWF protein molecule of various scales, controlled by viscous friction forces, lead to increased platelet adhesion and trigger the process of thrombus formation at the site of inflammation of the endothelium of microvessels. Based on the results of the work in the reporting period, 2 papers were prepared and sent to the editors of peer-reviewed scientific journals indexed by WoS and Scopus. Both articles are currently undergoing peer review. The results of the work were presented in the form of oral presentations at 5 scientific conferences in the reporting period. All tasks set for the first year of the project implementation were completed in full.

 

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