Work pertinent to this study was supported in part by National Institutes of Health (NIH) National Heart, Lung, and Blood Institute grants HL082808, HL123984 (R.L.), and F31HL134241 (M.E.Q.). Funding also provided by NIH National Institute of General Medical Sciences grant T32GM008367 (M.E.Q.); and pilot grant funds from Children&#8217;s Healthcare of Atlanta and Emory University Pediatric Flow Cytometry Core. The authors would like to thank Dr. Hans Deckmyn for sharing the 6B4 antibody, and the Emory Children&#39;s Pediatric Research Center Flow Cytometry Core for technical support.

All methods using donor-derived human platelets described herein were approved by the Institutional Review Board of Emory University/Children&#39;s Healthcare of Atlanta.
1. Blood Draw and Platelet Isolation
<ol>
	<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 2...

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The protocol described in this manuscript allows quick and versatile assessment of the effect of laminar shear on platelet and cell surface receptors. The specific representative results presented here underscore how the effects of multimeric or bivalent ligands can be affected by shear flow. In addition to this application, a uniform shear assay has broad applications in observing shear-dependent effects. In the absence of a known ligand-receptor pair, a uniform shear assay can also detect the effects of shear on factor...

Mechanoreceptors are proteins that respond to mechanical stimuli, such as pressure or mechanical perturbation/deformation. For some mechanoreceptors, sensing these mechanical perturbations is explicit to the function of the cell types in which they are expressed. Take, for example, the stretch receptors in baroreceptor neurons; these mechanosensitive ion channels regulate blood pressure by sensing vascular &#34;stretch&#34;1,2. In the inner ear, ion channels on hair cells detect mechanical deformations caused by sound waves3, and cutaneous low threshold mechanoreceptors (LTMRs) facilitate the transmission of tactile information4. In other cases, mechanoreceptors provide important information to the cell for the establishment of adhesion or growth. Cells can sense the rigidity of their local environment, and may rely on contractile forces via the actin cytoskeleton and integrins to dictate growth or spreading5,6.
When studying receptor-ligand interactions in cell or tissue-based models, common assays exist which can quickly and accurately report the effects of altering temperature, pH, ligand concentration, tonicity, membrane potential, and many other parameters which can vary in vivo. However, these same assays may fall short when it comes to detecting the contribution of mechanical force to receptor activation. Whether cells are sensing their microenvironment, detecting sound waves, or responding to stretch, one thing the aforementioned mechanoreceptors have in common is that they are participating in interactions where the ligand, receptor, or both, are anchored to a surface. Assays developed to test the effects of mechanical forces on receptor interactions often reflect this paradigm. Microfluidics and flow chambers are used to study the effects of shear flow on cells and receptors7,8. These types of experiments have the advantage of allowing fine-tuning of shear rates via established flow speeds. Other techniques employ fluorescent molecular probes to detect forces applied by cells on ligand-rich surfaces, yielding an accurate readout of the magnitude and orientations of forces involved in the interaction9,10.
In addition to mechanosensation occurring where one or both partners are anchored to a surface, shear stress may affect proteins and cells in solution. This is often observed in blood cells/proteins which are constantly in the circulation, and may manifest via activation of mechanoreceptors that are normally surface-anchored11, or through exposure of target sequences which would be occluded under static conditions12. However, relatively fewer techniques assay the effects of shear force on particles in solution. Some in-solution approaches introduce shear via vortexing cells in fluid suspension with varying speeds and durations, although these approaches may not allow a very precise determination of the shear stress generated. Rotational viscometers measure viscosity by applying a specific shear force to fluids. Herein we describe an applied method for determining the effect of specific laminar shear rates on cells or cell fragments in solution.
One of the most highly expressed proteins on the platelet surface is the glycoprotein (GP) Ib-IX complex. GPIb-IX is the primary receptor for the plasma protein von Willebrand Factor (VWF). Together, this receptor-ligand pair has long been recognized as the foundation of the platelet response to shear stress13. In the event of vascular damage, VWF binds to exposed collagen in the sub-endothelial matrix14, thus recruiting platelets to the site of injury via the VWF-GPIb-IX interaction. VWF engagement to its binding site in the GPIb&#945; subunit if GPIb-IX under physiological shear stress induces unfolding of a membrane-proximal mechanosensory domain (MSD) which in turn activates GPIb-IX15. In a recent study, we have shown that antibodies against GPIb&#945;, like those generated in many immune thrombocytopenia (ITP) patients, are also capable of inducing platelet signaling via MSD unfolding under shear stress11. However, unlike VWF, which facilitates shear-induced GPIb-IX activation by immobilizing the complex under normal circulation, the bivalent antibodies are able to crosslink platelets via GPIb-IX and unfold the MSD in circulation. In this way, a mechanoreceptor which is normally activated by surface immobilization under shear can be activated in solution. In the present report, we will demonstrate how a viscometer-based uniform shear assay was leveraged to detect the effects of specific levels of shear stress on receptor activation in solution.

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			<td height="34" style="height:35px;width:413px;">APC anti-human CD62P (P-Selectin)</td>
			<td style="width:128px;">BioLegend</td>
			<td style="width:135px;">304910</td>
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			<td height="22" style="height:22px;width:413px;">Brookfield Cap 2000+ Viscometer</td>
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			<td height="21" style="height:21px;width:413px;">FITC-conjugated Erythrina cristagalli lectin (ECL)</td>
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			<td height="21" style="height:21px;">Pooled Normal Human Plasma</td>
			<td>Precision Biologic</td>
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			<td height="52" style="height:52px;width:413px;">Vacutainer Light Blue Blood Collection Tube (Sodium Citrate)</td>
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			<td height="53" style="height:53px;width:413px;">Vacutainer Blood Collection Set, 21G x &#190;&#34; Needle</td>
			<td style="width:128px;">BD</td>
			<td style="width:135px;">367287</td>
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a uniform shear assay for human platelet and cell surface receptors via cone-plate viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

Figure 1 outlines how the trigger model of GPIb-IX activation, initially introduced to explain shear-dependent receptor activation when anchored to the vessel wall, may also support activation of platelets crosslinked by a multivalent ligand. Figure 2 shows readouts of human platelet activation treated by two antibodies targeting the N-terminal domain of GPIb-IX (6B4 and 11A8), and one control antibody (normal IgG) under sheared and static conditions. In <strong...

Watch this Scientific Journal Video about A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry at JoVE.com

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

This protocol allows us to answer a variety of questions about the way that shear acts on cells in solution and provides significant control over shear duration and magnitude. Unlike other techniques, which detect the effect of shear on cells that are immobilized, this technique allows us to apply shear to cells in solution and lends itself to a spectrum of downstream signal detection methods. After obtaining peripheral blood from a consenting, healthy adult donor in a 4.5-milliliter tube of 3.8%trisodium citrate, centrifuge the blood to obtain platelet-rich plasma, and use a pipette tip cut at a 45-degree angle to carefully remove the resulting top, cloudy, yellow platelet-rich plasma layer.Obtain the platelet count via complete blood count, and adjust the platelet count to approximately 2.5 times 10 to the fifth platelets per microliter with pooled human platelet-poor plasma. Then, place the suspension at 22 degrees Celsius with gentle rotation. For ligand treatment, slowly add the antibody or ligand of interest to the platelet-rich plasma, and mix the platelets gently.During the incubation, turn on the cone-plate viscometer, and set the plate temperature to 22 degrees Celsius. After five to 10 minutes, transfer the treated platelet-rich plasma onto the temperature-controlled cone-plate viscometer directly at the center of the plate, taking care that all of the sample is deposited between the cone and plate at the point of contact. To apply shear to the sample, calculate the shear according to the viscometer manual, and apply shear to the sample at the appropriate rate and duration.At the end of the application, lift the cone about two millimeters off the plate, so that the sample remains in contact with both the plate and the cone, and use a gel-loading pipette tip to collect five to 10 microliters from the center of the sample volume. Then incubate the sheared sample with antibodies against the surface markers of interest for 20 minutes at room temperature, and fix the samples in 2%paraformaldehyde for 20 minutes at room temperature. To analyze the samples by flow cytometry, collect at least 20, 000 events for each condition, and quantify the signal strength of each fluorescent marker, using the height value for the intensity of each fluorophore.Then use a negative control with bovine serum albumin or vehicle to draw a gate, excluding negative background fluorescent events, and quantify the percentage of events inside the gate for all experimental conditions. Platelet activation marker expression in response to shear force application is unregulated in a time-dependent manner. Here, representative readouts of human platelet activation in the presence of two antibodies targeting the N-terminal domain of glycoprotein 1b-IX and one control antibody under sheared and static conditions are shown.The expression of all three activation markers was significantly increased in the presence of shear forces and targeting antibodies, compared to either static conditions or in the presence of control antibodies. Notably, treatment with antibody with an unbinding force too low to trigger glycoprotein 1b-IX activation does not trigger platelet activation marker upregulation under static or shear force conditions. Further, plasma from immune thrombocytopenia patients containing antibodies against glycoprotein 1b-IX trigger shear dependent responses in contrast with plasma from patients without glycoprotein 1b-IX targeting antibodies.In addition of flowcytometric analyses, cells that have been exposed to shear can also be lysed for western blot, ELISA, and mass-spec analysis to determine any changes in protein level. As always, when using blood or human samples, be sure to wear proper protective equipment and follow the lab safety and blood born pathogen guidelines of your institution.

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7.5K 조회수.  Children's Healthcare of Atlanta. 이 프로토콜을 사용하면 전단이 용액의 세포에서 작동하는 방식에 대한 다양한 질문에 답할 수 있으며 전단 지속 시간과 크기에 대한 상당한 제어를 제공합니다. 고정된 세포에 대한 전단의 효과를 감지하는 다른 기술과 달리 이 기술은 용액의 세포에 전단을 적용할 수 있게 해주며 다운스트림 신호 검출 방법의 스펙트럼에 빌려줍니다. 동의에서 말초 혈액을 얻은 후, 3.8%의 ...