Thrombosis prophylaxis using plasmin and its combination with alpha-adrenoreceptor antagonists]

Using two models of experimental thrombosis (arterio-venous shunt and Wessler's model) the effect of plasmin and its combination with alpha-adrenoreceptor antagonists on the formation of thrombus was studied on white rats. It was established that the efficacy of prophylactic of thrombosis by plasmin only was low: middle ball of thrombosis was 2.5-3.0. The combination of plasmin with alpha-adrenoblockers dihydroergotoxine or prazosin under these conditions is most efficient against the formation of thrombus (middle ball of thrombus in this case was 0.8-1.1). Prazosin under certain conditions have some advantages.     

Thrombin mediates the extraintestinal thrombosis associated with experimental colitis

Recent evidence implicating tissue factor and the protein C pathway in the hypercoagulable state associated with intestinal inflammation suggests that thrombin is likely to contribute to this response. The objective of this study was to assess the role of thrombin in the extraintestinal thrombosis associated with experimental colitis. Thrombus formation was quantified in microvessels of the cremaster muscle in mice with dextran sodium sulfate (DSS)-induced colonic inflammation. The light/dye endothelial injury model was used to elicit thrombus formation in DSS colitic mice treated with either hirudin, heparin, or antithrombin III. The initiation and propagation/stabilization phases of thrombus formation were quantified using the time of onset of the thrombus and time to blood flow cessation, respectively. Thrombus formation was accelerated in arterioles of DSS colitic mice, as exhibited by significant reductions in the time of thrombus initiation and propagation/stabilization. Colitic mice treated with hirudin, heparin, or antithrombin III did not exhibit a significant change in the time of onset of the thrombus compared with untreated colitic mice. However, all three antithrombin agents largely prevented the DSS-induced reduction in the time to flow cessation following light/dye injury, with hirudin offering complete protection. These findings indicate that thrombin plays a major role in the extraintestinal thrombus formation associated with experimental colitis. Thrombin appears to contribute to the propagation/stabilization, rather than initiation, phase of the colitis-associated thrombogenesis at the distant vascular site. The results support the therapeutic use of antithrombin agents for reducing the risk of thromboembolism in patients with inflammatory bowel disease.     

Strenuous but not moderate exercise increases the thrombotic tendency in healthy sedentary male volunteers.

We have investigated the effect of moderate and strenuous exercise on experimental arterial thrombus formation in men. Thrombogenesis was measured in 15 sedentary healthy male volunteers at rest or immediately after two standardized exercise tests performed for 30 min on a bicycle ergometer. The exercises were performed at a constant load corresponding to either 50 or 70% maximal oxygen uptake. Thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel plate perfusion chamber to native nonanticoagulated blood for 3 min. The shear rate at the collagen surface was 2,600 s(-1). Platelet and fibrin deposition was quantified by immunoenzymatic methods. The results show that moderate exercise did not affect arterial thrombus formation. In contrast, platelet thrombus formation on collagen was increased on the average by 20% after 30 min at 70% maximal oxygen uptake (P = 0.03). Fibrin deposition on collagen remained unchanged with exercise, regardless of its intensity. Thus, with the use of a clinically relevant human experimental model of thrombosis, the present study suggests that exercise of heavy intensity may increase the risk for arterial thrombogenesis in sedentary young healthy male volunteers.     

Methods to Determine the Lagrangian Shear Experienced by Platelets during Thrombus Growth

Platelets can become activated in response to changes in flow-induced shear; however, the underlying molecular mechanisms are not clearly understood. Here we present new techniques for experimentally measuring the flow-induced shear rate experienced by platelets prior to adhering to a thrombus. We examined the dynamics of blood flow around experimentally grown thrombus geometries using a novel combination of experimental (ex vivo) and numerical (in silico) methodologies. Using a microcapillary system, platelet aggregate formation was analysed at elevated shear rates in the presence of coagulation inhibitors, where thrombus formation is predominantly platelet-dependent. These approaches permit the resolution and quantification of thrombus parameters at the scale of individual platelets (2 μm) in order to quantify real time thrombus development. Using our new techniques we can correlate the shear rate experienced by platelets with the extent of platelet adhesion and aggregation. The techniques presented offer the unique capacity to determine the flow properties for a temporally evolving thrombus field in real time.     

Platelet Transport Rates and Binding Kinetics at High Shear over a?Thrombus

Thrombus formation over a ruptured atherosclerotic plaque cap can occlude an artery with fatal consequences. We describe a computational model of platelet transport and binding to interpret rate-limiting steps seen in experimental thrombus formation over a collagen-coated stenosis. The model is used to compute shear rates in stenoses with growing boundaries. In the model, moving erythrocytes influence platelet transport based on shear-dependent enhanced diffusivity and a nonuniform platelet distribution. Adhesion is modeled as platelet-platelet binding kinetics. The results indicate that observed thrombus growth rates are limited by platelet transport to the wall for shear rates up to 6000 s⁻¹. Above 7000 s⁻¹, the thrombus growth rate is likely limited by binding kinetics (10⁻⁴ m/s). Thrombus growth computed from these rate-limiting steps match the thrombus location and occlusion times for experimental conditions if a lag time for platelet activation is included. Using fitted parameters, the model is then used to predict thrombus size and shape at a higher Reynolds number flow consistent with coronary artery disease.     

Modeling Thrombus Shell: Linking Adhesion Receptor Properties and Macroscopic Dynamics

Damage to arterial vessel walls leads to the formation of platelet aggregate, which acts as a physical obstacle for bleeding. An arterial thrombus is heterogeneous; it has a dense inner part (core) and an unstable outer part (shell). The thrombus shell is very dynamic, being composed of loosely connected discoid platelets. The mechanisms underlying the observed mobility of the shell and its (patho)physiological implications are unclear. To investigate arterial thrombus mechanics, we developed a novel, to our knowledge, two-dimensional particle-based computational model of microvessel thrombosis. The model considers two types of interplatelet interactions: primary reversible (glycoprotein Ib (GPIb)-mediated) and stronger integrin-mediated interaction, which intensifies with platelet activation. At high shear rates, the former interaction leads to adhesion, and the latter is primarily responsible for stable platelet aggregation. Using a stochastic model of GPIb-mediated interaction, we initially reproduced experimental curves that characterize individual platelet interactions with a von Willebrand factor-coated surface. The addition of the second stabilizing interaction results in thrombus formation. The comparison of thrombus dynamics with experimental data allowed us to estimate the magnitude of critical interplatelet forces in the thrombus shell and the characteristic time of platelet activation. The model predicts moderate dependence of maximal thrombus height on the injury size in the absence of thrombin activity. We demonstrate that the developed stochastic model reproduces the observed highly dynamic behavior of the thrombus shell. The presence of primary stochastic interaction between platelets leads to the properties of thrombus consistent with in vivo findings; it does not grow upstream of the injury site and covers the whole injury from the first seconds of the formation. А simplified model, in which GPIb-mediated interaction is deterministic, does not reproduce these features. Thus, the stochasticity of platelet interactions is critical for thrombus plasticity, suggesting that interaction via a small number of bonds drives the dynamics of arterial thrombus shell.     

Clot Permeability,Agonist Transport,and Platelet Binding Kinetics in Arterial Thrombosis

The formation of wall-adherent platelet aggregates is a critical process in arterial thrombosis. A growing aggregate experiences frictional drag forces exerted on it by fluid moving over or through the aggregate. The magnitude of these forces is strongly influenced by the permeability of the developing aggregate; the permeability depends on the aggregate’s porosity. Aggregation is mediated by formation of ensembles of molecular bonds; each bond involves a plasma protein bridging the gap between specific receptors on the surfaces of two different platelets. The ability of the bonds existing at any time to sustain the drag forces on the aggregate determines whether it remains intact or sheds individual platelets or larger fragments (emboli). We investigate platelet aggregation in coronary-sized arteries using both computational simulations and in vitro experiments. The computational model tracks the formation and breaking of bonds between platelets and treats the thrombus as an evolving porous, viscoelastic material, which moves differently from the background fluid. This relative motion generates drag forces which the fluid and thrombus exert on one another. These forces are computed from a permeability-porosity relation parameterized from experimental measurements. Basing this relation on measurements from occlusive thrombi formed in our flow chamber experiments, along with other physiological parameter values, the model produced stable dense thrombi on a similar timescale to the experiments. When we parameterized the permeability-porosity relation using lower permeabilities reported by others, bond formation was insufficient to balance drag forces on an early thrombus and keep it intact. Under high shear flow, soluble agonist released by platelets was limited to the thrombus and a boundary layer downstream, thus restricting thrombus growth into the vessel lumen. Adding to the model binding and activation of unactivated platelets through von Willebrand-factor-mediated processes allowed greater growth and made agonist-induced activation more effective.     

Rheological factors in platelet - vessel wall interactions

Rheological aspects of platelet-vessel wall interactions involve cell-cell encounters, platelet - vessel wall encounters and platelet-thrombus interactions. The cell-cell encounters are usually caused by convection of cells in shear flows rather than by Brownian motion; this is important in aggregation and in the enhancement of the diffusion of platelets by red cell motion. Platelet - vessel wall interactions can involve transient adhesion (lasting from a fraction of a second to a few minutes) as well as more permanent adhesion. Reaction rates between platelets and walls are generally very small except on damaged vessels and some artificial surfaces. Ultra-filtration through the vessel wall affects cell-wall interactions. Rheological analyses of thrombus formation have been made and shown interesting relations to experimental observations. Some experimental results have indicated that platelets are capable of reacting within a small fraction of a second. Red cells may act as mechanoreceptors for increases in shear rate and facilitate the speed of response of platelets. Surface geometrical forms such as bumps and cavities tend to prolong residence times and facilitate thrombus formation.     

Prostaglandin endoperoxides and thromboxane A2 in thrombus formation in the hamster cheek pouch in vivo

Electrical stimulation in the presence of ADP of arterioles of the hamster cheek pouch caused endothelial damage and white thrombus formation. The thrombus formation was inhibited by cyclo-oxygenase inhibitors aspirin and sulphinpyrazone and its thioether derivative G 35671. Thromboxane synthetase inhibitors N-(7-carboxyheptyl) imidazole and butylimidazole failed to inhibit thrombus formation, although in the same doses both compounds inhibited serum levels of thromboxane. These results indicate that thromboxane is not important in thrombus formation in the hamster, but that prostaglandin endoperoxides are more significant. However, it is possible that the inhibition of white thrombus formation by aspirin, sulphinpyrazone and G 25671 may be mediated by a different mechanism altogether.     

Introducing the pro-coagulant contact system in the numerical assessment of device-related thrombosis

Thrombosis is a major concern in blood-coated medical devices. Contact activation, which is the initial part of the coagulation cascade in device-related thrombosis, is not considered in current thrombus formation models. In the present study, pro-coagulant reactions including the contact activation system are coupled with a fluid solver in order to evaluate the potential of the contact system to initiate thrombin production. The biochemical/fluid model is applied to a backward-facing step configuration, a flow configuration that frequently appears in medical devices. In contrast to the in vivo thrombosis models in which a specific thrombotic zone (injury region) is set a priori by the user to initiate the coagulation reaction, a reactive surface boundary condition is applied to the whole device wall. Simulation results show large thrombin concentration in regions related to recirculation zones without the need of an a priori knowledge of the thrombus location. The numerical results align well with the regions prone to thrombosis observed in experimental results reported in the literature. This approach could complement thrombus formation models that take into account platelet activity and thrombus growth to optimize a wide range of medical devices.     

Involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase 1 in thrombus formation

The involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase-1 (JNK1) has never been investigated in hemostasis and thrombosis. Using two JNK inhibitors (SP600125 and 6o), we have demonstrated that JNK1 is involved in collagen-induced platelet aggregation dependent on ADP. In these conditions, JNK1 activation requires the coordinated signaling pathways of collagen receptors (alpha2beta1 and glycoprotein (GP)VI) and ADP. In contrast, JNK1 is not required for platelet adhesion on a collagen matrix in static or blood flow conditions (300-1500 s(-1)) involving collagen receptors (alpha2beta1 and GPVI). Importantly, at 1500 s(-1), JNK1 acts on thrombus formation on a collagen matrix dependent on GPIb-von Willebrand factor (vWF) interaction but not ADP receptor activation. This is confirmed by the involvement of JNK1 in shear-induced platelet aggregation at 4000 s(-1). We also provide evidence during rolling and adhesion of platelets to vWF that platelet GPIb-vWF interaction triggers alphaIIbbeta3 activation in a JNK1-dependent manner. This was confirmed with a Glanzmann thrombastenic patient lacking alphaIIbbeta3. Finally, in vivo, JNK1 is involved in arterial but not in venular thrombosis in mice. Overall, our in vitro studies define a new role of JNK1 in thrombus formation in flowing blood that is relevant to thrombus development in vivo.     

Inhibitory effect of hirudin on thrombosis induced by prothrombin complex concentrates

The influence of hirudin on thrombus formation induced by prothrombin complex concentrate (PCC) was studied in two different test series in rats. Within the first test series hirudin was i.v. administered to the animals 10 min before they received PCC. Complete prevention of thrombus formation required a hirudin dose of 0.2 mg/kg. Within the second test series hirudin was added to the transfusion unit of PCC before application of PCC was started. In this case complete prevention of thrombus formation was yielded by addition of 140 micrograms hirudin to the PCC transfusion unit. In comparison with heparin and the synthetic thrombin inhibitor N alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine piperidide, hirudin was most potent.     

Recent advances in understanding endogenous fibrinolysis: implications for molecular-based treatment of vascular disorders

The fate of a forming thrombus is determined through the delicate balance between the coagulation cascade, favouring clot formation, and the fibrinolytic system, favouring clot lysis. These processes occur simultaneously, and enhancement of fibrinolysis has been shown to reduce occlusive thrombus formation in animal models. This review examines the roles of the major fibrinolytic factors involved in clot lysis. The regulation of plasmin activity by plasminogen activators, alpha-2-antiplasmin, plasminogen activator inhibitor 1, and thrombin-activatable fibrinolysis inhibitor, and their effects on thrombus formation in vivo are discussed. Since alterations in fibrinolytic capacity appear to affect thrombus formation in animal models, there is considerable interest in the pharmacological manipulation of fibrinolysis.     

Mathematical analysis of mural thrombogenesis. Concentration profiles of platelet-activating agents and effects of viscous shear flow.

The concentration profiles of adenosine diphosphate (ADP), thromboxane A2 (TxA2), thrombin, and von Willebrand factor (vWF) released extracellularly from the platelet granules or produced metabolically on the platelet membrane during thrombus growth, were estimated using finite element simulation of blood flow over model thrombi of various shapes and dimensions. The wall fluxes of these platelet-activating agents were estimated for each model thrombus at three different wall shear rates (100 s-1, 800 s-1, and 1,500 s-1), employing experimental data on thrombus growth rates and sizes. For that purpose, whole human blood was perfused in a parallel-plate flow chamber coated with type l fibrillar human collagen, and the kinetic data collected and analyzed by an EPl-fluorescence video microscopy system and a digital image processor. It was found that thrombin concentrations were large enough to cause irreversible platelet aggregation. Although heparin significantly accelerated thrombin inhibition by antithrombin lll, the remaining thrombin levels were still significantly above the minimum threshold required for irreversible platelet aggregation. While ADP concentrations were large enough to cause irreversible platelet aggregation at low shear rates and for small aggregate sizes, TxA2 concentrations were only sufficient to induce platelet shape change over the entire range of wall shear rates and thrombi dimensions studied. Our results also indicated that the local concentration of vWF multimers released from the platelet alpha-granules could be sufficient to modulate platelet aggregation at low and intermediate wall shear rates (less than 1,000 s-1). The sizes of standing vortices formed adjacent to a growing aggregate and the embolizing stresses and the torque, acting at the aggregate surface, were also estimated in this simulation. It was found that standing vortices developed on both sides of the thrombus even at low wall shear rates. Their sizes increased with thrombus size and wall shear rate, and were largely dependent upon thrombus geometry. The experimental observation that platelet aggregation occurred predominantly in the spaces between adjacent thrombi, confirmed the numerical prediction that those standing vortices are regions of reduced fluid velocities and high concentrations of platelet-activating substances, capable of trapping and stimulating platelets for aggregation. The average shear stress and normal stress, as well as the torque, acting to detach the thrombus, increased with increasing wall shear rate. Both stresses were found to be nearly independent of thrombus size and only weekly dependent upon thrombus geometry. Although both stresses had similar values at low wall shear rates, the average shear stress became the predominant embolizing stress at high wall shear rates.     

Luteolysis and thrombus formation in ovaries of immature superstimulated golden hamsters

Thrombus appearance during luteolysis with and without exogenous prostaglandin (PGF2 alpha) was studied in immature golden hamsters between days 4 and 7 after stimulation with pregnant mares' serum gonadotrophin (PMSG) followed by human chorionic gonadotrophin. Both ovaries were weighed and cut in series for light-microscopic evaluation. The fibrinolytic activity was determined by the fibrin slide method after treatment with PGF2 alpha on day 4 after PMSG stimulation and compared with controls of days 3 and 4 after PMSG stimulation. There was a marked decrease in ovarian weights in the experimental and the control group between days 4 and 7 after PMSG. Few necrotic cells were seen in corpora lutea on day 5, but many on day 6. All of them had disappeared on day 7. The number of ovaries with thrombi was 80-100% in both groups on day 4 and declined to approximately zero levels on day 7. The amount of thrombus formation appeared to be higher in the PGF2 alpha-treated groups than in controls. Fibrinolytic activity was high in controls on day 3 and low in controls and in PGF2 alpha-treated animals on day 4 after PMSG. It is concluded that thrombus formation occurs in superstimulated ovaries during luteolysis; and thrombus formation is related to a decrease in fibrinolytic activity.     

Mathematical modeling of thrombus growth in mesenteric vessels

Richardson’s phenomenological mathematical model of the thrombi growth in microvessels is extended to describe the realistic features of the phenomenon. The main directions of the generalization of Richardson’s model are: (1) the dependence of platelet activation time on the distance from the injured vessel wall; (2) the non-homogeneity of the platelet distribution in blood flow in the vicinity of the vessel wall; (3) the adequate choice of the phenomenological function describing the dependence of blood velocity on the thrombus size. The generalization of the model corresponds to the main experimental results and theoretical considerations concerning thrombus formation obtained in recent years. The extended model permits to achieve qualitative agreement between model and experimental data.     

Biomechanical behaviour of cerebral aneurysm and its relation with the formation of intraluminal thrombus: a patient-specific modelling study

Cerebral aneurysm is an irreversible dilatation causing intracranial haemorrhage with severe complications. It is assumed that the biomechanical factor plays a significant role in the development of cerebral aneurysm. However, reports on the correlations between the formation of intraluminal thrombus and the flow pattern, wall shear stress (WSS) distribution of the cerebral aneurysm as well as wall compliance are still limited. In this research, patient-specific numerical simulation was carried out for three cerebral aneurysms based on magnetic resonance imaging (MRI) data-sets. The interaction between pulsatile blood and aneurysm wall was taken into account. The biomechanical behaviour of cerebral aneurysm and its relation with the formation of intraluminal thrombus was studied systematically. The results of the numerical simulation indicated that the region of low blood flow velocity and the region of swirling recirculation were nearly coincident with each other. Besides, there was a significant correlation between the slow swirling flow and the location of thrombus deposition. Excessively low WSS was also found to have strong association with the regions of thrombus formation. Moreover, the relationship between cerebral aneurysm compliance and thrombus deposition was discovered. The patient-specific modelling study based on fluid–structure interaction) may provide a basis for future investigation on the prediction of thrombus formation in cerebral aneurysm.     

Continuous Modeling of Arterial Platelet Thrombus Formation Using a Spatial Adsorption Equation

In this study, we considered a continuous model of platelet thrombus growth in an arteriole. A special model describing the adhesion of platelets in terms of their concentration was derived. The applications of the derived model are not restricted to only describing arterial platelet thrombus formation; the model can also be applied to other similar adhesion processes. The model reproduces an auto-wave solution in the one-dimensional case; in the two-dimensional case, in which the surrounding flow is taken into account, the typical torch-like thrombus is reproduced. The thrombus shape and the growth velocity are determined by the model parameters. We demonstrate that the model captures the main properties of the thrombus growth behavior and provides us a better understanding of which mechanisms are important in the mechanical nature of the arterial thrombus growth.     

Thrombosis is reduced by inhibition of COX-1, but unaffected by inhibition of COX-2, in an acute model of platelet activation in the mouse

Background

Clinical use of selective inhibitors of cyclooxygenase (COX)-2 appears associated with increased risk of thrombotic events. This is often hypothesised to reflect reduction in anti-thrombotic prostanoids, notably PGI₂, formed by COX-2 present within endothelial cells. However, whether COX-2 is actually expressed to any significant extent within endothelial cells is controversial. Here we have tested the effects of acute inhibition of COX on platelet reactivity using a functional in vivo approach in mice.

Methodology/Principal Findings

A non-lethal model of platelet-driven thromboembolism in the mouse was used to assess the effects of aspirin (7 days orally as control) diclofenac (1 mg.kg⁻¹, i.v.) and parecoxib (0.5 mg.kg⁻¹, i.v.) on thrombus formation induced by collagen or the thromboxane (TX) A₂-mimetic, U46619. The COX inhibitory profiles of the drugs were confirmed in mouse tissues ex vivo. Collagen and U46619 caused in vivo thrombus formation with the former, but not latter, sensitive to oral dosing with aspirin. Diclofenac inhibited COX-1 and COX-2 ex vivo and reduced thrombus formation in response to collagen, but not U46619. Parecoxib inhibited only COX-2 and had no effect upon thrombus formation caused by either agonist.

Conclusions/Significance

Inhibition of COX-1 by diclofenac or aspirin reduced thrombus formation induced by collagen, which is partly dependent upon platelet-derived TXA₂, but not that induced by U46619, which is independent of platelet TXA₂. These results are consistent with the model demonstrating the effects of COX-1 inhibition in platelets, but provide no support for the hypothesis that acute inhibition of COX-2 in the circulation increases thrombosis.     

Modeling the effect of blood vessel bifurcation ratio on occlusive thrombus formation

Vascular geometry is a major determinant of the hemodynamics that promote or prevent unnecessary vessel occlusion from thrombus formation. Bifurcations in the vascular geometry are repeating structures that introduce flow separation between parent and daughter vessels. We modelled the blood flow and shear rate in a bifurcation during thrombus formation and show that blood vessel bifurcation ratios determine the maximum shear rate on the surface of a growing thrombus. We built an analytical model that may aid in predicting microvascular bifurcation ratios that are prone to occlusive thrombus formation. We also observed that bifurcation ratios that adhere to Murray’s law of bifurcations may be protected from occlusive thrombus formation. These results may be useful in the rational design of diagnostic microfluidic devices and microfluidic blood oxygenators.