Engineering an Endothelialized Vascular Graft: A Rational Approach to Study Design in a Non-Human Primate Model

After many years of research, small diameter, synthetic vascular grafts still lack the necessary biologic integration to perform ideally in clinical settings. Endothelialization of vascular grafts has the potential to improve synthetic graft function, and endothelial outgrowth cells (EOCs) are a promising autologous cell source. Yet no work has established the link between endothelial cell functions and outcomes of implanted endothelialized grafts. This work utilized steady flow, oscillatory flow, and tumor necrosis factor stimulation to alter EOC phenotype and enable the formulation of a model to predict endothelialized graft performance. To accomplish this, EOC in vitro expression of coagulation and inflammatory markers was quantified. In parallel, in non-human primate (baboon) models, the platelet and fibrinogen accumulation on endothelialized grafts were quantified in an ex vivo shunt, or the tissue ingrowth on implanted grafts were characterized after 1mth. Oscillatory flow stimulation of EOCs increased in vitro coagulation markers and ex vivo platelet accumulation. Steady flow preconditioning did not affect platelet accumulation or intimal hyperplasia relative to static samples. To determine whether in vitro markers predict implant performance, a linear regression model of the in vitro data was fit to platelet accumulation data—correlating the markers with the thromboprotective performance of the EOCs. The model was tested against implant intimal hyperplasia data and found to correlate strongly with the parallel in vitro analyses. This research defines the effects of flow preconditioning on EOC regulation of coagulation in clinical vascular grafts through parallel in vitro, ex vivo, and in vivo analyses, and contributes to the translatability of in vitro tests to in vivo clinical graft performance.     

Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry

IntroductionThe paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry.ResultsThe injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung.ConclusionsInjection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates.     

Cells in 3D matrices under interstitial flow: Effects of extracellular matrix alignment on cell shear stress and drag forces

Interstitial flow is an important regulator of various cell behaviors both in vitro and in vivo, yet the forces that fluid flow imposes on cells embedded in a 3D extracellular matrix (ECM), and the effects of matrix architecture on those forces, are not well understood. Here, we demonstrate how fiber alignment can affect the shear and pressure forces on the cell and ECM. Using computational fluid dynamics simulations, we show that while the solutions of the Brinkman equation accurately estimate the average fluid shear stress and the drag forces on a cell within a 3D fibrous medium, the distribution of shear stress on the cellular surface as well as the peak shear stresses remain intimately related to the pericellular fiber architecture and cannot be estimated using bulk-averaged properties. We demonstrate that perpendicular fiber alignment of the ECM yields lower shear stress and pressure forces on the cells and higher stresses on the ECM, leading to decreased permeability, while parallel fiber alignment leads to higher stresses on cells and increased permeability, as compared to a cubic lattice arrangement. The Spielman–Goren permeability relationships for fibrous media agreed well with CFD simulations of flow with explicitly considered fibers. These results suggest that the experimentally observed active remodeling of ECM fibers by fibroblasts under interstitial flow to a perpendicular alignment could serve to decrease the shear and drag forces on the cell.     

On the choice of outlet boundary conditions for patient-specific analysis of aortic flow using computational fluid dynamics

Boundary conditions (BCs) are an essential part in computational fluid dynamics (CFD) simulations of blood flow in large arteries. Although several studies have investigated the influence of BCs on predicted flow patterns and hemodynamic wall parameters in various arterial models, there is a lack of comprehensive assessment of outlet BCs for patient-specific analysis of aortic flow. In this study, five different sets of outlet BCs were tested and compared using a subject-specific model of a normal aorta. Phase-contrast magnetic resonance imaging (PC-MRI) was performed on the same subject and velocity profiles extracted from the in vivo measurements were used as the inlet boundary condition. Computational results obtained with different outlet BCs were assessed in terms of their agreement with the PC-MRI velocity data and key hemodynamic parameters, such as pressure and flow waveforms and wall shear stress related indices. Our results showed that the best overall performance was achieved by using a well-tuned three-element Windkessel model at all model outlets, which not only gave a good agreement with in vivo flow data, but also produced physiological pressure waveforms and values. On the other hand, opening outlet BCs with zero pressure at multiple outlets failed to reproduce any physiologically relevant flow and pressure features.     

Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow

Background

The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function.

Objectives

Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo.

Methods and Results

We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa.

Conclusions

Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function may prove beneficial in the search for new antithrombotic therapies.     

Platelet Activation in Human Immunodeficiency Virus Type-1 Patients Is Not Altered with Cocaine Abuse

Recent work has indicated that platelets, which are anucleate blood cells, significantly contribute to inflammatory disorders. Importantly, platelets also likely contribute to various inflammatory secondary disorders that are increasingly associated with Human Immunodeficiency Virus Type-1 (HIV) infection including neurological impairments and cardiovascular complications. Indeed, HIV infection is often associated with increased levels of platelet activators. Additionally, cocaine, a drug commonly abused by HIV-infected individuals, leads to increased platelet activation in humans. Considering that orchestrated signaling mechanisms are essential for platelet activation, and that nuclear factor-kappa B (NF-κB) inhibitors can alter platelet function, the role of NF-κB signaling in platelet activation during HIV infection warrants further investigation. Here we tested the hypothesis that inhibitory kappa B kinase complex (IKK) activation would be central for platelet activation induced by HIV and cocaine. Whole blood from HIV-positive and HIV-negative individuals, with or without cocaine abuse was used to assess platelet activation via flow cytometry whereas IKK activation was analyzed by performing immunoblotting and in vitro kinase assays. We demonstrate that increased platelet activation in HIV patients, as measured by CD62P expression, is not altered with reported cocaine use. Furthermore, cocaine and HIV do not activate platelets in whole blood when treated ex vivo. Finally, HIV-induced platelet activation does not involve the NF-κB signaling intermediate, IKKβ. Platelet activation in HIV patients is not altered with cocaine abuse. These results support the notion that non-IKK targeting approaches will be better suited for the treatment of HIV-associated inflammatory disorders.     

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system¹. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury^2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes⁴. This method focuses on neutrophil transmigration across human endothelial cells.One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)⁵. Neutrophil isolation has been described visually elsewhere⁶; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression⁷. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature^7,8.The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro^9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi μslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.     

Platelet Activation Determines Angiopoietin-1 and VEGF Levels in Malaria: Implications for Their Use as Biomarkers

Introduction

The angiogenic proteins angiopoietin (Ang)-1, Ang-2 and vascular endothelial growth factor (VEGF) are regulators of endothelial inflammation and integrity. Since platelets store large amounts of Ang-1 and VEGF, measurement of circulation levels of these proteins is sensitive to platelet number, in vivo platelet activation and inadvertent platelet activation during blood processing. We studied plasma Ang-1, Ang-2 and VEGF levels in malaria patients, taking the necessary precautions to avoid ex vivo platelet activation, and related plasma levels to platelet count and the soluble platelet activation markers P-selectin and CXCL7.

Methods

Plasma levels of Ang-1, Ang-2, VEGF, P-selectin and CXCL7 were measured in CTAD plasma, minimizing ex vivo platelet activation, in 27 patients with febrile Plasmodium falciparum malaria at presentation and day 2 and 5 of treatment and in 25 healthy controls.

Results

Levels of Ang-1, Ang-2 and VEGF were higher at day 0 in malaria patients compared to healthy controls. Ang-2 levels, which is a marker of endothelial activation, decreased after start of antimalarial treatment. In contrast, Ang-1 and VEGF plasma levels increased and this corresponded with the increase in platelet number. Soluble P-selectin and CXCL7 levels followed the same trend as Ang-1 and VEGF levels. Plasma levels of these four proteins correlated strongly in malaria patients, but only moderately in controls.

Conclusion

In contrast to previous studies, we found elevated plasma levels of Ang-1 and VEGF in patients with malaria resulting from in vivo platelet activation. Ang-1 release from platelets may be important to dampen the disturbing effects of Ang-2 on the endothelium. Evaluation of plasma levels of these angiogenic proteins requires close adherence to a stringent protocol to minimize ex vivo platelet activation.     

Gastric prostacyclin (PGI2) prevents stress-induced gastric mucosal injury in rats primarily by inhibiting leukocyte activation.

We investigated whether, in rats, gastric prostacyclin (PGI2) prevented gastric mucosal injury that was induced by water-immersion restraint stress by inhibiting leukocyte activation. Gastric levels of 6-keto-PGF1alpha, a stable metabolite of PGI2, increased transiently 30 min after stress, followed by a decrease to below the baseline 6-8 h after stress. Gastric mucosal blood flow decreased to approximately 40% of the baseline level 8 h after stress. Myeloperoxidase activity was significantly increased 8 h after stress. Treatment with indomethacin before stress inhibited the increase in 6-keto-PGF1alpha levels and markedly reduced mucosal blood flow. It also markedly increased leukocyte accumulation and mucosal lesion formation. Iloprost, a stable PGI2 analog, inhibited the indomethacin-induced decrease in mucosal blood flow, mucosal lesion exacerbation, and increase in leukocyte accumulation. Nitrogen mustard-induced leukocytopenia inhibited the indomethacin-associated lesion exacerbation and the increase in leukocyte accumulation, but not the decreases in mucosal blood flow. These observations indicate that gastric PGI2 decreases gastric mucosal lesion formation primarily by inhibiting leukocyte accumulation.     

Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis

Background

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.

Objective

The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.

Methods and Results

We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84^−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84^−/− mice. In vivo, Cd84^−/− mice exhibited unaltered hemostatic function and arterial thrombus formation.

Conclusion

These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.     

Platelet activation of mechanical versus bioprosthetic heart valves during systole

Thrombus formation is a major concern for recipients of mechanical heart valves (MHVs), which requires them to take anticoagulant drugs for the rest of their lives. Bioprosthetic heart valves (BHVs) do not require life-long anticoagulant therapy but deteriorate after 10–15 years. The thrombus formation is initiated by the platelet activation which is thought to be mainly generated in MHVs by the flow through the hinge and the leakage flow during the diastole. However, our results show that the activation in the bulk flow during the systole phase might play an essential role as well. This is based on our results obtained by comparing the thrombogenic performance of a MHV and a BHV (as control) in terms of shear induced platelet activation under exactly the same conditions. Three different mathematical activation models including linear level of activation, damage accumulation, and Soares model are tested to quantify the platelet activation during systole using the previous simulations of the flow through MHV and BHV in a straight aorta under the same physiologic flow conditions. Results indicate that the platelet activation in the MHV at the beginning of the systole phase is slightly less than the BHV. However, at the end of the systole phase the platelet activation by the bulk flow for the MHV is several folds (1.41, 5.12, and 2.81 for linear level of activation, damage accumulation, and Soares model, respectively) higher than the BHV for all tested platelet activation models.     

Characterization of flow conditions in 2 L and 20 L wave bioreactors® using computational fluid dynamics

Characterization of flow conditions is of great importance to control cell growth and cell damage in animal cell culture because cell viability is influenced by the flow properties in bioreactors. Alternative reactor types like Wave Bioreactors® have been proposed in recent years, leading to markedly different results in cell growth and product formation. An advantage of Wave Bioreactors® is the disposability of the Polyethylenterephthalet‐bags after one single use (fast setup of new production facilities). Another expected advantage is a lower shear stress compared to classical stirred‐tank reactors, due to the gentle liquid motion in the rocking cellbag. This property would considerably reduce possible cell damage. The purpose of the present study is to investigate in a quantitative manner the key flow properties in Wave Bioreactors®, both numerically and experimentally. To describe accurately flow conditions and shear stress in Wave Bioreactors® using numerical simulations, it is necessary to compute the unsteady flow applying Computational Fluid Dynamics (CFD). Corresponding computations for two reactor scales (2 L and 20 L cellbags) are presented using the CFD code ANSYS‐FLUENT®. To describe correctly the free liquid surface, the present simulations employ the Volume of Fluid (VOF) method. Additionally, experimental measurements have been carried out to determine liquid level, flow velocity and liquid shear stress, which are used as a validation of the present CFD simulations. It is shown that the obtained flows stay in the laminar regime. Furthermore, the obtained shear stress levels are well below known threshold values leading to damage of animal cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Realistic non-Newtonian viscosity modelling highlights hemodynamic differences between intracranial aneurysms with and without surface blebs

Most computational fluid dynamic (CFD) simulations of aneurysm hemodynamics assume constant (Newtonian) viscosity, even though blood demonstrates shear-thinning (non-Newtonian) behavior. We sought to evaluate the effect of this simplifying assumption on hemodynamic forces within cerebral aneurysms, especially in regions of low wall shear stress, which are associated with rupture. CFD analysis was performed for both viscosity models using 3D rotational angiography volumes obtained for 26 sidewall aneurysms (12 with blebs, 12 ruptured), and parametric models incorporating blebs at different locations (inflow/outflow zone). Mean and lowest 5% values of time averaged wall shear stress (TAWSS) computed over the dome were compared using Wilcoxon rank-sum test. Newtonian modeling not only resulted in higher aneurysmal TAWSS, specifically in areas of low flow and blebs, but also showed no difference between aneurysms with or without blebs. In contrast, for non-Newtonian analysis, bleb-bearing aneurysms showed significantly lower 5% TAWSS compared to those without (p=0.005), despite no significant difference in mean dome TAWSS (p=0.32). Non-Newtonian modeling also accentuated the differences in dome TAWSS between ruptured and unruptured aneurysms (p<0.001). Parametric models further confirmed that realistic non-Newtonian viscosity resulted in lower bleb TAWSS and higher focal viscosity, especially when located in the outflow zone. The results show that adopting shear-thinning non-Newtonian blood viscosity in CFD simulations of intracranial aneurysms uncovered hemodynamic differences induced by bleb presence on aneurysmal surfaces, and significantly improved discriminant statistics used in risk stratification. These findings underline the possible implications of using a realistic model of blood viscosity in predictive computational hemodynamics.     

Leukocyte- and platelet-derived microvesicle interactions following in vitro and in vivo activation of toll-like receptor 4 by lipopolysaccharide

Background

Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS).

Methodology/Principal Findings

Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens.

Conclusions/Significance

Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood.     

Epinephrine and shear stress synergistically induce platelet aggregation via a mechanism that partially bypasses vWF-Gp Ib interactions

Elevated shear stress levels in pathologically stenosed vessels induce platelet activation and aggregation, and may play a role in the pathogenesis of arterial disease. Increased plasma catecholamine concentrations have also been implicated in the onset of acute coronary ischemic syndromes. This study was designed to examine the synergistic interaction of shear stress and epinephrine in the activation of platelets. Platelets (in PRP) sheared at 60 dyn/cm² showed little or no aggregation unless pretreated with epinephrine. Pretreatment with 250 nM epinephrine followed by shear at 60 dyn/cm² induced >60% platelet aggregation. The specific α₂-adrenergic receptor antagonist yohimbine inhibited the synergistic aggregation, as did the ADP scavenging system phosphocreatine/creatine phosphokinase, indicating a three-way synergism with ADP. Chemical or monoclonal antibody blockade of von Willebrand factor (vWF) interactions with either platelet glycoprotein (Gp) Ib or Gp IIb/IIIa completely inhibited platelet aggregation induced by activating levels of shear stress alone. However, the combination of epinephrine and shear stress induced platelet aggregation that was blocked by 10E5, a monoclonal antibody that inhibits vWF binding to Gp IIb/IIIa, but not by aurin tricarboxylic acid or the monoclonal antibody 6D1, both of which inhibit vWF binding to Gp Ib. Synergistic platelet aggregation in response to epinephrine and shear stress was observed in washed platelets, platelet-rich plasma and whole blood in vitro, and also ex vivo following exercise to elevate endogenous levels of catecholamines. These results indicate that epinephrine synergizes with shear stress to induce platelet aggregation. This synergistic response requires functional Gp IIb/IIIa complexes, but is at least partially independent of vWF-Gp Ib interactions.     

Dependence of leukocyte capture on instantaneous pulsatile flow

Atherosclerosis, an artery disease, is currently the leading cause of death in the United States in both men and women. The first step in the development of atherosclerosis involves leukocyte adhesion to the arterial endothelium. It is broadly accepted that blood flow, more specifically wall shear stress (WSS), plays an important role in leukocyte capture and subsequent development of an atherosclerotic plaque. What is less known is how instantaneous WSS, which can vary by up to 5 Pa over one cardiac cycle, influences leukocyte capture. In this paper we use direct numerical simulations (DNS), performed using an in-house code, to illustrate that leukocyte capture is different whether as a function of instantaneous or time-averaged blood flow. Specifically, a stenotic plaque is modeled using a computational fluid dynamics (CFD) solver through fully three-dimensional Navier-Stokes equations and the immersed boundary method. Pulsatile triphasic inflow is used to simulate the cardiac cycle. The CFD is coupled with an agent-based leukocyte capture model to assess the impact of instantaneous hemodynamics on stenosis growth. The computed wall shear stress agrees well with the results obtained with a commercial software, as well as with theoretical results in the healthy region of the artery. The analysis emphasizes the importance of the instantaneous flow conditions in evaluating the leukocyte rate of capture. That is, the capture rate computed from mean flow field is generally underpredicted compared to the actual rate of capture. Thus, in order to obtain a reliable estimate, the flow unsteadiness during a cardiac cycle should be taken into account.     

GPVI and GPIbα mediate staphylococcal superantigen-like protein 5 (SSL5) induced platelet activation and direct toward glycans as potential inhibitors

Background

Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects.

Methodology/Principal Findings

In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro.

Conclusions/Significance

These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo.     

Pharmacological Modulation of Hemodynamics in Adult Zebrafish In Vivo

Introduction

Hemodynamic parameters in zebrafish receive increasing attention because of their important role in cardiovascular processes such as atherosclerosis, hematopoiesis, sprouting and intussusceptive angiogenesis. To study underlying mechanisms, the precise modulation of parameters like blood flow velocity or shear stress is centrally important. Questions related to blood flow have been addressed in the past in either embryonic or ex vivo-zebrafish models but little information is available for adult animals. Here we describe a pharmacological approach to modulate cardiac and hemodynamic parameters in adult zebrafish in vivo.

Materials and Methods

Adult zebrafish were paralyzed and orally perfused with salt water. The drugs isoprenaline and sodium nitroprusside were directly applied with the perfusate, thus closely resembling the preferred method for drug delivery in zebrafish, namely within the water. Drug effects on the heart and on blood flow in the submental vein were studied using electrocardiograms, in vivo-microscopy and mathematical flow simulations.

Results

Under control conditions, heart rate, blood flow velocity and shear stress varied less than ± 5%. Maximal chronotropic effects of isoprenaline were achieved at a concentration of 50 μmol/L, where it increased the heart rate by 22.6 ± 1.3% (n = 4; p < 0.0001). Blood flow velocity and shear stress in the submental vein were not significantly increased. Sodium nitroprusside at 1 mmol/L did not alter the heart rate but increased blood flow velocity by 110.46 ± 19.64% (p = 0.01) and shear stress by 117.96 ± 23.65% (n = 9; p = 0.03).

Discussion

In this study, we demonstrate that cardiac and hemodynamic parameters in adult zebrafish can be efficiently modulated by isoprenaline and sodium nitroprusside. Together with the suitability of the zebrafish for in vivo-microscopy and genetic modifications, the methodology described permits studying biological processes that are dependent on hemodynamic alterations.     

Impact of Tissue Factor Localization on Blood Clot Structure and Resistance under Venous Shear

The structure and growth of a blood clot depend on the localization of tissue factor (TF), which can trigger clotting during the hemostatic process or promote thrombosis when exposed to blood under pathological conditions. We sought to understand how the growth, structure, and mechanical properties of clots under flow are shaped by the simultaneously varying TF surface density and its exposure area. We used an eight-channel microfluidic device equipped with a 20- or 100-μm-long collagen surface patterned with lipidated TF of surface densities ～0.1 and ～2 molecules/μm². Human whole blood was perfused at venous shear, and clot growth was continually measured. Using our recently developed computational model of clot formation, we performed simulations to gain insights into the clot’s structure and its resistance to blood flow. An increase in TF exposure area resulted not only in accelerated bulk platelet, thrombin, and fibrin accumulation, but also in increased height of the platelet mass and increased clot resistance to flow. Moreover, increasing the TF surface density or exposure area enhanced platelet deposition by approximately twofold, and thrombin and fibrin generation by greater than threefold, thereby increasing both clot size and its viscous resistance. Finally, TF effects on blood flow occlusion were more pronounced for the longer thrombogenic surface than for the shorter one. Our results suggest that TF surface density and its exposure area can independently enhance both the clot’s occlusivity and its resistance to blood flow. These findings provide, to our knowledge, new insights into how TF affects thrombus growth in time and space under flow.     

Flow-induced structural transition in the beta-switch region of glycoprotein Ib

The impact of fluid flow on structure and dynamics of biomolecules has recently gained much attention. In this article, we present a molecular-dynamics algorithm that serves to generate stable water flow under constant temperature, for the study of flow-induced protein behavior. Flow simulations were performed on the 16-residue β-switch region of platelet glycoprotein Ibα, for which crystal structures of its N-terminal domain alone and in complex with the A1 domain of von Willebrand factor have been solved. Comparison of the two structures reveals a conformational change in this region, which, upon complex formation, switches from an unstructured loop to a β-hairpin. Interaction between glycoprotein Ibα and von Willebrand factor initiates platelet adhesion to injured vessel walls, and the adhesion is enhanced by blood flow. It has been hypothesized that the loop to β-hairpin transition in glycoprotein Ibα is induced by flow before binding to von Willebrand factor. The simulations revealed clearly a flow-induced loop→β-hairpin transition. The transition is dominated by the entropy of the protein, and is seen to occur in two steps, namely a dihedral rotation step followed by a side-group packing step.