eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Blood Draw and Platelet Isolation
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	<li>Draw human blood from consenting healthy adult donors via venipuncture on the day of the experiment into 3.8% trisodium citrate. One 4.5 mL tube of blood is sufficient to yield enough platelet-rich plasma (PRP) for 20-25 conditions in donors whose platelet counts are close to 250 x 103&#160;per &#956;L...

the effects of shear stress on platelets treated with antibodies against mechanosensory receptors

Source: Quach, M. E. et al., <a href="https://app.jove.com/v/59704">A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry.</a> J. Vis. Exp.&#160;(2019)
This video demonstrates the method to assess the impact of shear stress on platelets. Antibody treatment induces dissociation and conformational changes in mechanoreceptor complexes, activating the platelets and expressing markers on the surface. These markers are observed via immunostaining and flow cytometry.

The Effects of Shear Stress on Platelets Treated with Antibodies Against Mechanosensory Receptors

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Blood Draw and Platelet 
...

Begin with a multi-well plate containing platelet-rich plasma. Platelets express surface mechanoreceptor complexes &#8212; responsive to mechanical stimuli.
The mechanoreceptor complex contains the extracellular ligand-binding domain and macro-glycopeptide region linked to the mechanosensory domain.
Introduce anti-mechanoreceptor antibodies, cross-linking with the ligand-binding domains of the receptors on opposing platelets.
Place a droplet of the antibody-treated plasma suspension between the assemblies of the viscometer.
Apply shear stress, simulating mechanical force, pulling the antibody-bound receptors away from the platelets, leading to macro-glycopeptide region dissociation from the mechanosensory domain.
This triggers a conformational change in the mechanosensory domain, activating platelets and resulting in the expression of surface markers.
Transfer the sheared platelet suspension to a new well within the multi-well plate.
Introduce a cocktail of fluorophore-labeled marker-specific molecules interacting with corresponding platelet surface markers and fix the cells with a fixative solution.
In flow cytometry, distinct fluorescence signals correlate with the effects of shear stress on antibody-treated platelets.

the-effects-shear-stress-on-platelets-treated-with-antibodies-against

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

JoVE Journal

Immunology and Infection

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release. The majority of VWF released by endothelial cells circulates in the plasma; however, a proportion of VWF is anchored to the endothelial cell surface. Under conditions of physiological shear, endothelial-anchored VWF can bind to platelets, forming a VWF-platelet string that may represent the nidus of thrombus formation. A flow chamber system can be used to visually observe the release of VWF from endothelial cells and the subsequent platelet capture in a manner that is reproducible and relevant to the pathophysiology of VWF-mediated thrombus formation. Using this methodology, endothelial cells are cultured in a flow chamber and are subsequently stimulated with secretagogues to induce WPB exocytosis. Washed platelets are then perfused over the activated endothelium. The platelets are activated and subsequently bind to elongated VWF strings in the direction of fluid flow. Using extracellular histones as a procoagulant and proinflammatory stimulus, we observed increased VWF-platelet string formation on histone-treated endothelial cells compared to untreated endothelial cells. This protocol describes a quantitative, visual, and real-time assessment of the activation of VWF-platelet interactions in models of thrombosis and hemostasis.

In this paper, we describe a method to assess endothelial von Willebrand factor release and the subsequent platelet capture under fluid shear stress in response to inflammatory stimuli using an in vitro flow chamber system.

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

Medicine

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

The Effects of Shear Stress on Platelets Treated with Antibodies Against Mechanosensory Receptors

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