The forward rate of binding of surface-tethered reactants: effect of relative motion between two surfaces

The reaction of molecules confined to two dimensions is of interest in cell adhesion, specifically for the reaction between cell surface receptors and substrate-bound ligand. We have developed a model to describe the overall rate of reaction of species that are bound to surfaces under relative motion, such that the Peclet number is order one or greater. The encounter rate between reactive species is calculated from solution of the two-dimensional convection-diffusion equation. The probability that each encounter will lead to binding depends on the intrinsic rate of reaction and the encounter duration. The encounter duration is obtained from the theory of first passage times. We find that the binding rate increases with relative velocity between the two surfaces, then reaches a plateau. This plateau indicates that the increase in the encounter rate is counterbalanced by the decrease in the encounter duration as the relative velocity increases. The binding rate is fully described by two dimensionless parameters, the Peclet number and the Damk?hler number. We use this model to explain data from the cell adhesion literature by incorporating these rate laws into "adhesive dynamics" simulations to model the binding of a cell to a surface under flow. Leukocytes are known to display a "shear threshold effect" when binding selectin-coated surfaces under shear flow, defined as an increase in bind rate with shear; this effect, as calculated here, is due to an increase in collisions between receptor and ligand with increasing shear. The model can be used to explain other published data on the effect of wall shear rate on the binding of cells to surfaces, specifically the mild decrease in binding within a fixed area with increasing shear rate.     

Tortuosity triggers platelet activation and thrombus formation in microvessels

Tortuous blood vessels are often seen in humans in association with thrombosis, atherosclerosis, hypertension, and aging. Vessel tortuosity can cause high fluid shear stress, likely promoting thrombosis. However, the underlying physical mechanisms and microscale processes are poorly understood. Accordingly, the objectives of this study were to develop and use a new computational approach to determine the effects of venule tortuosity and fluid velocity on thrombus initiation. The transport, collision, shear-induced activation, and receptor-ligand adhesion of individual platelets in thrombus formation were simulated using discrete element method. The shear-induced activation model assumed that a platelet became activated if it experienced a shear stress above a relative critical shear stress or if it contacted an activated platelet. Venules of various levels of tortuosity were simulated for a mean flow velocity of 0.10?cm s(-1), and a tortuous arteriole was simulated for a mean velocity of 0.47?cm s(-1). Our results showed that thrombus was initiated at inner walls in curved regions due to platelet activation in agreement with experimental studies. Increased venule tortuosity modified fluid flow to hasten thrombus initiation. Compared to the same sized venule, flow in the arteriole generated a higher amount of mural thrombi and platelet activation rate. The results suggest that the extent of tortuosity is an important factor in thrombus initiation in microvessels.     

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.     

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.     

Platelet adhesion receptors and (patho)physiological thrombus formation

In thrombus formation associated with hemostasis or thrombotic disease, blood platelets first undergo a rapid transition from a circulating state to an adherent state, followed by activation and aggregation. Under flow conditions in the bloodstream, this process potentially involves platelet-platelet, platelet-endothelium, platelet-subendothelial matrix, and platelet-leukocyte interactions. Specific adhesion receptors on platelets mediate these interactions, by engaging counter-receptors on other cells, or noncellular ligands in the plasma or matrix. The glycoprotein (GP) Ib-IX-V complex on platelets initiates adhesion at high shear stress by binding the adhesive ligand, von Willebrand Factor (vWF). GP Ib-IX-V may also mediate platelet-endothelium or platelet-leukocyte adhesion, by recognition of P-selectin or Mac-1, respectively. Other membrane glycoproteins, such as the collagen receptor GP VI, may trigger platelet activation at low shear rates. Engagement of GP Ib-IX-V or GP VI leads ultimately to platelet aggregation mediated by the integrin, alphaIIbbeta3 (GP IIb-IIIa). This review will focus on recent advances in understanding structure-activity relationships of GP Ib-IX-V, its role in initiating thrombus formation, and its emerging relationships with other vascular cell adhesion receptors.     

Primary platelet adhesion receptors

Thrombotic diseases such as heart attack and stroke remain a major health concern in the Western world despite existing anti-thrombotic drugs. Current studies are revealing structure-function relationships of primary platelet adhesion receptors mediating adhesion, activation and aggregation, and the molecular mechanisms underlying platelet thrombus formation. Platelet adhesion is relevant not only to thrombotic disease, but there is increasing evidence of a specific role for platelets in vascular processes such as inflammation and atherogenesis. This review focuses on recent advances in understanding the molecular basis for platelet thrombus formation, in particular the receptors, glycoprotein (GP)Ib-IX-V and GPVI, that initiate platelet adhesion and activation at high shear stress.     

Fluid shear regulates the kinetics and receptor specificity of Staphylococcus aureus binding to activated platelets

The interaction between surface components on the invading pathogen and host cells such as platelets plays a key role in the regulation of endovascular infections. However, the mechanisms mediating Staphylococcus aureus binding to platelets under shear remain largely unknown. This study was designed to investigate the kinetics and molecular requirements of platelet-S. aureus interactions in bulk suspensions subjected to a uniform shear field. Hydrodynamic shear-induced collisions augment platelet-S. aureus binding, which is further potentiated by platelet activation with stromal derived factor-1beta. Peak adhesion efficiency occurs at low shear (100 s(-1)) and decreases with increasing shear. The molecular interaction of platelet alpha(IIb)beta(3) with bacterial clumping factor A through fibrinogen bridging is necessary for stable bacterial binding to activated platelets under shear. Although this pathway is sufficient at low shear (相似文献   

Efficiency of platelet adhesion to fibrinogen depends on both cell activation and flow

The kinetics of adhesion of platelets to fibrinogen (Fg) immobilized on polystyrene latex beads (Fg-beads) was determined in suspensions undergoing Couette flow at well-defined homogeneous shear rates. The efficiency of platelet adhesion to Fg-beads was compared for ADP-activated versus "resting" platelets. The effects of the shear rate (100-2000 s(-1)), Fg density on the beads (24-2882 Fg/microm(2)), the concentration of ADP used to activate the platelets, and the presence of soluble fibrinogen were assessed. "Resting" platelets did not specifically adhere to Fg-beads at levels detectable with our methodology. The apparent efficiency of platelet adhesion to Fg-beads readily correlated with the proportion of platelets "quantally" activated by doses of ADP, i.e., only ADP-activated platelets appeared to adhere to Fg-beads, with a maximal adhesion efficiency of 6-10% at shear rates of 100-300 s(-1), decreasing with increasing shear rates up to 2000 s(-1). The adhesion efficiency was found to decrease by only threefold when decreasing the density of Fg at the surface of the beads by 100-fold, with only moderate decreases in the presence of physiologic concentrations of soluble Fg. These adhesive interactions were also compared using activated GPIIbIIIa-coated beads. Our studies provide novel model particles for studying platelet adhesion relevant to hemostasis and thrombosis, and show how the state of activation of the platelet and the local flow conditions regulate Fg-dependent adhesion.     

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.     

Two independent light signals cooperate in the activation of the plastid psbD blue light-responsive promoter in Arabidopsis

Though phospholipase C PLCgamma2 is known to play an important role in platelet activation by collagen and fibrinogen, its importance in GPIb-mediated platelet activation is less well understood. To better understand the role of PLCgamma2 in GPIb-mediated adhesion and thrombus formation, we examined the ability of wild-type and PLCgamma2- deficient murine platelets to spread on immobilized von Willebrand factor (VWF) under static conditions, and to attach to and form thrombi on VWF under conditions of arterial shear. While absence of PLCgamma2 had only a minimal effect on platelet adhesion to immobilized VWF, its absence impaired spreading and profoundly affected thrombus growth and stability on VWF.     

Preferential binding of platelets to monocytes over neutrophils under flow

This study was undertaken to systematically investigate the binding kinetics of platelet recruitment by monocytes relative to neutrophils in bulk suspensions subjected to shear as well as the molecular requirements of leukocyte-platelet binding. Hydrodynamic shear-induced collisions augment the proportion of monocytes with adherent platelets more drastically than that of neutrophils with bound platelets. These heterotypic interactions are further potentiated by platelet activation with thrombin or to a lesser extent by monocyte stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Monocyte-platelet heteroaggregation increases with increasing shear rate and shear exposure time. Platelet P-selectin binding to monocyte P-selectin-glycoprotein-ligand-1 is solely responsible for maximal platelet adhesion to unstimulated monocytes in shear flow. However, the enhanced platelet binding to fMLP-treated monocytes involves a sequential two-step process, wherein P-selectin-PSGL-1 interactions are stabilized by CD18-integrin involvement. Blocking platelet alpha(IIb)beta(3) or monocyte beta(1)-integrin function had no effect. This study underscores the preferential recruitment of platelets by monocytes relative to neutrophils in shear flow, and demonstrates that the shear environment of the vasculature coupled to the state of cell activation modulates the dynamics and molecular constituents mediating monocyte-platelet adhesion.     

PI 3-kinase p110beta: a new target for antithrombotic therapy

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.     

The effect of the active component of Dendrostellera lessertii on the adhesive property of human platelets and HL-60 cells

To evaluate the mediation of cell adhesion by Dendrostellera lessertii, an anti-cancer medicinal plant in Iranian folk medicine, the adhesion of thrombin activated human platelets to the cultured monocytes or HL-60 cells was investigated under the effect of the plant extract (0.4 mg/ml) and one of its purified components, 3-hydrgenkwadaphnin (0.2 nM). Treatment of the platelets with the plant extract or/3-hydrgenkwadaphnin, for various time intervals, followed by their activation by thrombin resulted in 80-90% reduction in the number of monocytes with more than 10 attached platelets. Similarly, under almost all identical conditions, the adhesion of the activated platelets to HL-60 cells was decreased by 90%. The adhesion of thrombin activated platelets to the plant extract treated HL-60 cells was also reduced significantly (95%). These data clearly indicates that Dendrostellera lessertii is capable of mediating tumor metastasis through affecting the adhesion properties of the cells.     

An integrated fluid–structure interaction and thrombosis model for type B aortic dissection

False lumen thrombosis (FLT) in type B aortic dissection has been associated with the progression of dissection and treatment outcome. Existing computational models mostly assume rigid wall behavior which ignores the effect of flap motion on flow and thrombus formation within the FL. In this study, we have combined a fully coupled fluid–structure interaction (FSI) approach with a shear-driven thrombosis model described by a series of convection–diffusion reaction equations. The integrated FSI-thrombosis model has been applied to an idealized dissection geometry to investigate the interaction between vessel wall motion and growing thrombus. Our simulation results show that wall compliance and flap motion can influence the progression of FLT. The main difference between the rigid and FSI models is the continuous development of vortices near the tears caused by drastic flap motion up to 4.45 mm. Flap-induced high shear stress and shear rates around tears help to transport activated platelets further to the neighboring region, thus speeding up thrombus formation during the accelerated phase in the FSI models. Reducing flap mobility by increasing the Young’s modulus of the flap slows down the thrombus growth. Compared to the rigid model, the predicted thrombus volume is 25% larger using the FSI-thrombosis model with a relatively mobile flap. Furthermore, our FSI-thrombosis model can capture the gradual effect of thrombus growth on the flow field, leading to flow obstruction in the FL, increased blood viscosity and reduced flap motion. This model is a step closer toward simulating realistic thrombus growth in aortic dissection, by taking into account the effect of intimal flap and vessel wall motion.

     

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.     

Platelet thrombi produced on cultured endothelial cells by the dye/light method.

Platelet adhesion and aggregation were induced on cultured endothelial cells using the fluorescent dye/light method. A cone-and-plate apparatus was newly developed to observe interactions between platelets and cultured endothelial cells under a shear flow condition. The platelet deposition grew on the light-irradiated area of the cells. Degree of endothelial cell injury induced by the dye/light reaction seemed to depend on the dye concentration. Application of either aspirin or indomethacin significantly inhibited the growth of platelet aggregation, but was not effective for the platelet adhesion to endothelial cells. The platelet thrombi were formed on endothelial cells without their denudation. It was found by transmission electron microscopy that platelets directly adhered to endothelial cells which were not seriously damaged. This thrombus model is expected to be applicable to some physiological and pharmacological studies investigating platelet-endothelial cell interaction and mechanism of platelet thrombus formation in blood vessels.     

Severe streptococcal infection is associated with M protein-induced platelet activation and thrombus formation

Disturbed haemostasis is a central finding in severe Streptococcus pyogenes infection. In particular, microthrombi are found both at the local site of infection and at distant sites. Platelets are responsible for maintaining vascular function and haemostasis. We report here that M1 protein of S. pyogenes triggers immune-mediated platelet activation and thrombus formation. M1 protein is released from the bacterial surface and forms complexes with plasma fibrinogen. These complexes bind to the fibrinogen receptor on resting platelets. When these complexes also contain immunoglobulin G (IgG) against M1 protein, this will engage the Fc receptor on the platelets and activation will occur. Activation of the platelets leads to platelet aggregation and the generation of platelet-rich thrombi. Neutrophils and monocytes are in turn activated by the platelets. Platelet thrombi are deposited in the microvasculature, and aggregated platelets, IgG and M1 protein colocalize in biopsies from patients diagnosed with S. pyogenes toxic shock syndrome. This chain of events results in a pro-coagulant and pro-inflammatory state typical of severe S. pyogenes infection.     

Spatial Distribution of Factor Xa,Thrombin, and Fibrin(ogen) on Thrombi at Venous Shear

Background

The generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro)thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.

Methodology/Principal Findings

Fluorescence microscopy was used to study the accumulation of platelets, FXa, (pro)thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca²⁺ signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro)thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl₃. Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro)thrombin and fibrin(ogen).

Conclusions/Significance

FXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).