Dmitry Yu. Nechipurenko

The mechanisms driving spatial heterogeneity of arterial thrombus and its three-stage dynamics are poorly understood. To investigate the potential principles regulating the size of the thrombus core and shell we developed a 3D continuum computational model that describes thrombus heterogeneity, thrombin-induced platelet dense granule secretion and clot propagation through thrombin and ADP-induced platelet activation. The continuum model predicted that spatial confinement of the thrombus core was a result of thrombin transport and a threshold-like dependence of platelet activation on thrombin concentration. This new model recapitulated three-stage dynamics observed in vivo and explained it with a burst-like ADP concentration dynamics due to the confinement of thrombus core propagation and rapid dense granule pool depletion within the core. The maximal shell size in silico was regulated by the transport of ADP and the kinetics of thrombin-dependent dense granules secretion. Simulations also predicted that partial propagation of thrombin inside the thrombus shell caused irreversible platelet activation by the low-dose thrombin and defined the residual shell size. Moreover, our results provided an explanation for the reduced size of a thrombus core observed in the mouse models of Hermansky-Pudlak syndrome. The continuum model was then applied to describe a FeCl ₃ -induced thrombosis in macrocirculation, and described the thrombin-flux-depending switch between occlusive and non-occlusive thrombosis scenarios in mouse carotid artery. Finally, our simulations reinforced the hypothesis suggesting the importance of the large ADP-dependent thrombus shell for sealing the breach in case of a penetrating injury. Taken together, our results suggest a novel mechanism that may regulate arterial thrombus dynamics and offer several insights and сlarification to the core-and-shell model of arterial thrombus organization, as well as a possible role of the large thrombus shell in hemostasis.

Gelation of blood plasma is key to normal hemostasis. The regulation of coagulation in blood flow has been studied for several decades, but the key problems in the field remain unresolved. Understanding the major mechanisms governing coagulation under flow conditions is important for the development of new diagnostic tools, therapeutic agents, and novel strategies addressing pathological states associated with both hypo- and hypercoagulation. In this review, we highlight the general features of plasma coagulation under flow conditions, including its spatiotemporal dynamics and threshold dependence on the key parameters. A short overview of in vitro and in silico models and important breakthroughs that became possible with their combination is followed by a brief introduction into the analysis of the balance between convection, diffusion, and reactions using Peclet and Damköhler numbers. We highlight the major unresolved challenges in the field and share an overall perspective of the future directions that might help to clarify the important questions that emerged during the last decade of intensive research.

Microfluidic assays offer a promising solution for accessing the state of the hemostasis system, testing drugs, and adjusting anticoagulant dosages. However, standardization of such assays is still missing. We aimed to design a robust and readily accessible microfluidic assay, which follows recent scientific and standardization committee (SSC) guidelines and is sensitive to hemostatic abnormalities. We optimized key parameters of the whole blood perfusion system to produce a reliable assay suitable for rapid evaluation of primary hemostasis in patients. The optimized protocol includes anticoagulation with hirudin, 5 minutes of perfusion at a shear rate of 1,000 s−1 over the millimeter-wide fibrillar collagen patch at room temperature, and evaluation of thrombus formation using the upright fluorescent microscope. Percentage of the activator area covered by thrombi was a reliable parameter demonstrating reproducible results for a given donor over a time course of months. Analysis of the whole blood from 7 patients with Wiscott–Aldrich syndrome, 34 patients with immune thrombocytopenia (ITP), and 8 patients with X-linked agammaglobulinemia showed a significant decrease in thrombus surface coverage compared to that of healthy individuals. Importantly, the microfluidic assay was able to differentiate between ITP patients with distinct clinical bleeding scores better than platelet counts and cytofluorometric parameters. The developed robust microfluidic assay represents an accessible tool for the assessment of primary haemostasis in patients and is promising for clinical use.

The formation of a hemostatic thrombus is a key response of the hemostasis system to a wide range of possible vessel injuries. The basic mechanism of thrombus formation at high shear rate is platelet adhesion and aggregation. It is known that arterial thrombi are spatially heterogeneous. Such heterogeneity is thought to be due to the heterogeneous distribution of the platelet activators inside the thrombus. Spatiotemporal dynamics of molecules, which are involved in thrombus formation, depends on rates at which the substrates are transported. To explore the dynamics of arterial thrombus formation, continuum models that represent the thrombus as a porous media are currently widely used. Still, choosing parameters for these models is complicated due to a high level of uncertainty in the published experimental data. This review is focused on the analyses of the literature data on physical parameters of the arterial thrombus as a porous medium. Special attention is paid to the parameters of the thrombus shell, which is generally characterized by the higher values of porosity and permeability.

Objective: Subendothelial matrix exhibits a distinctive organization, with collagen fibers beneath the endothelium oriented primarily parallel to the flow and comprising mainly types III and I collagen in arteries and arterioles. However, the significance of such organization in initiating rapid thrombus formation remains unclear. Approach and Results: To investigate the role of collagen fibers orientation in triggering thrombus formation we utilized the in vitro microfluidic model of thrombosis. Human whole blood was perfused through a system with collagen fibers oriented either parallel or perpendicular to the flow, and the primary stages of platelet adhesion and thrombi growth were analyzed using a high-speed fluorescence microscopy. At the shear rates of 200 and 1000 s ^-1 no significant difference in thrombogenicity was observed between the collagen fibers orientations. However, at a high shear rate of 2000 s ^-1 , thrombi on the parallel fibers were higher and covered a much larger area than those on the perpendicular ones. High-speed microscopy revealed that platelets were able to achieve stable adhesion only after translocating for over several seconds under all studied conditions. Analysis of single platelets dynamics revealed that platelets interacted longer and translocated farther during their interaction with parallel fibers at high shear, suggesting that flow-aligned fibers facilitate stable adhesion by enabling platelets to translocate along them and stay in contact with collagen. Importantly, only a small fraction of collagen fibers belonging to type III collagen admixture - a common component of a standard type I collagen preparation - accumulated plasma vWF and supported platelet adhesion at high shear rate. Conclusion: Type III, but not type I collagen fibers oriented parallel to the flow facilitate stable platelet adhesion at high shear rates. Thus, axial orientation of subendothelial collagen fibers observed in vivo may have physiological relevance for triggering rapid thrombus formation at high shear rates. Graphical

Thrombin is a key enzyme of the blood coagulation system, which has been actively studied since the beginning of the last century. The formation of thrombin from prothrombin in the area of vessel injury leads not only to the formation of fibrin – an important structural component of the hemostatic clot – but also to the activation of platelets, endothelium and immune system cells. The binding of thrombin to the platelet surface is thought to play a critical role in the process of platelet activation and may also ensure the maintenance of a high concentration of thrombin within the thrombus due to the concentration of protease on the platelet surface. To date, all major thrombin receptors on platelets have been thoroughly characterized: through various experimental methods, the physicochemical parameters of the corresponding intermolecular interactions have been established. Since the interaction of thrombin with platelets leads to their activation, which includes changes in the number of receptors as a result of granule secretion, the interpretation of the observed kinetic binding curves faces a number of difficulties. It is known that some receptors as a result of platelet activation are able to redistribute on the membrane and form dimers and clusters, which makes the kinetics of thrombin binding to platelets an extremely complex process depending on many factors, such as activator concentrations, platelet state, and other local parameters of the system. This review aims to describe the current understanding of the interaction of thrombin with the platelet membrane and to outline important unresolved issues in this area of research. The survey provides not only information on structural and kinetic features of thrombin binding to individual platelet membrane proteins, but also analyzes the relationship between the relevant interaction parameters and previously obtained data on the integral kinetics of protease binding to the platelet surface.

Thrombin is a key enzyme of the blood coagulation system, which has been actively studied since the beginning of the last century. The formation of thrombin from prothrombin in the vicinity of vessel injury leads not only to the formation of fibrin—an important structural component of the hemostatic clot—but also to the activation of platelets, endothelium and immune system cells. The binding of thrombin to the platelet surface is thought to play a critical role in the process of platelet activation and may also ensure the maintenance of a high concentration of thrombin within the thrombus due to the concentration of protease on the platelet surface. Nowadays, all major thrombin receptors on platelets have been thoroughly characterized: through various experimental methods, the physicochemical parameters of the corresponding intermolecular interactions have been established. Since the interaction of thrombin with platelets leads to their activation, which includes changes in the number of receptors as a result of granule secretion, the interpretation of the observed kinetic binding curves faces a number of difficulties. It is known that as a result of platelet activation some receptors are able to redistribute on the membrane and form dimers and clusters, which makes the kinetics of thrombin binding to platelets an extremely complex process dependent on many factors, such as activator concentrations, platelet state, and other local parameters of the system. This review aims to describe the current understanding of the interaction of thrombin with the platelet membrane and to outline important unresolved issues in this area of research. The review provides not only information on structural and kinetic features of thrombin binding to individual platelet membrane proteins, but also analyzes the relationship between the relevant interaction parameters and previously obtained data on the integral kinetics of protease binding to the platelet surface.

The formation of a hemostatic thrombus is a key response of the hemostatic system to a wide range of possible vascular damage. The main mechanism of thrombus growth in conditions of high shear rates is platelet adhesion and aggregation. It is known that arterial thrombi have significant spatial heterogeneity, which is associated with the heterogeneity of the distribution of platelet activators in the thrombus structure. The spatiotemporal dynamics of the molecules involved in thrombosis depends on the parameters of the transfer of these substances in the thrombus. To study the dynamics of arterial blood thrombi formation, continuum models representing a blood thrombus as a porous medium are actively used today. However, when choosing the parameters of such models, researchers face significant uncertainty caused by conflicting experimental data. This review analyzes the published data on the physical parameters of an arterial thrombus as a porous medium. Special attention is paid to the analysis of parameters for the external part of the thrombus, the so-called shell, which is generally characterized by higher values of porosity and permeability.