This work was supported by grants from the Swiss National Science Foundation (310030_162579/1 to Brenda Renata Kwak and 320030_144150 to Pierre Fontana) as well as by a grant from the Swiss Heart Foundation.

Five unrelated healthy volunteers were recruited for blood sampling for platelet isolation and aggregation tests. Written informed consent was obtained and the protocol was approved by the Central Ethics Committee of the Geneva University Hospitals. All volunteers certified to be healthy and to have not taken any platelet-interfering drugs during at least the 10 days preceding the experiments.
1. Buffer Preparation for Human Blood Collection and Washed Platelet Isolation
<ol>
	<li>Prepare a 100 mL aqueous solution of acid-citrate-dextrose (ACD) by dissolving 1.4 g citric acid monohydrate (C6H8O7&#8226;H2O, 66.6 mM), 2.5 g trisodium citrate dihydrate (Na3C6H5O7&#8226;2 H2O, 85 mM) and 2 g of anhydrous D(+)-glucose. The pH of the solution is about 4.5.</li>
	<li>Prepare stock solutions for Tyrode&#39;s buffer as follows
	<ol>
		<li>Prepare stock solution 1 by dissolving 80 g NaCl, 2 g KCl, 10 g NaHCO3 and 0.58 g NaH2PO4*H2O in 500 mL of distilled H2O. The respective final concentrations are 2.73 M, 53.6 mM, 238 mM and 8.4 mM. Keep the solution at 4 &#176;C.</li>
		<li>Prepare stock solution 2 by dissolving 10.15 g MgCl2*6 H2O (100 mM) in 500 mL distilled H2O. Keep solution at 4 &#176;C.</li>
		<li>Prepare stock solution 3 by dissolving 10.95 g (100 mM) CaCl2*6 H2O in 500 mL distilled H2O. Keep solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare Tyrode&#39;s buffer by diluting 2.5 mL of stock solution 1 in a final volume of 50 mL with distilled H2O. This corresponds to final concentrations of 136.5 mM NaCl, 2.68 mM KCl, 11.9 mM NaHCO3 and 0.42 mM NaH2PO4*H2O. Adjust the pH to 7.35 and sterilize by filtering with 0.22-&#956;m filters.</li>
	<li>Prepare Tyrode&#39;s albumin 0.35% buffer (TA buffer7) by diluting 5 mL of stock solution 1, 1 mL of stock solution 2, 2 mL of stock solution 3, 0.5 mL 1M HEPES, 1.8 mL of 200 g/L human serum albumin and 0.1 g of anhydrous D(+)-glucose in a final volume of 100 mL distilled H2O.
	<ol>
		<li>Adjust the pH to 7.35 with 1N HCl and set the osmolarity to 295 mOsm/L by adding distilled H2O (10% of total volume). Final concentrations in this solution are: 124 mM NaCl, 2.44 mM KCl, 10.82 mM NaHCO3, 0.38 mM NaH2PO4*H2O, 0.91 mM MgCl2*6 H2O, 1.82 mM CaCl2*6 H2O. Keep TA buffer at 37 &#176;C during the whole experiment.</li>
	</ol>
	</li>
</ol>
2. Blood Collection
<ol>
	<li>Collect 45-50 mL of venous blood, from the antecubital vein using a 19 G needle and no or low tourniquet, into 50 mL tubes containing ACD anticoagulant (1 volume ACD for 6 volumes of blood). Discard the first 1-2 mL of blood to avoid the presence of thrombin and tissue factor.
	<ol>
		<li>After collection, mix the blood with the ACD by gently inverting the tube. Incubate the sample for 10 min at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Preparation of Human Washed Platelets
<ol>
	<li>Pre-heat the centrifuge to 37 &#176;C. All centrifugation steps below are performed at this temperature.</li>
	<li>Dispatch the collected blood into 15 mL tubes (5 mL per tube) and centrifuge at 250 x g for 13 min to obtain PRP. 
	NOTE: This centrifugation step results in the production of three layers in the sample: 1) The upper layer, composed of plasma, platelets, and a small fraction of white blood cells. 2) The intermediate layer, a portion rich in white blood cells. 3) The bottom layer, which is essentially composed of red blood cells.</li>
	<li>Collect the PRP by pipetting the upper layer carefully into a new 15 mL tube to maximally prevent contamination with red and white blood cells, and incubate for 10 min at 37 &#176;C.</li>
	<li>Centrifuge the PRP at 2,200 x g for 12 min (for 5 mL PRP). 
	NOTE: This centrifugation step should be performed with low brake or without brake.</li>
	<li>Remove the supernatant (platelet-poor plasma), and carefully resuspend the pellet with 10 mL of TA buffer containing 2 &#181;L/mL of heparin (5,000 U/mL) and 2.5 &#181;L/mL of 25 &#956;M PGI2 using a plastic Pasteur pipet. Incubate for 10 min at 37 &#176;C.</li>
	<li>Add 2.5 &#181;L/mL of 25 &#956;M PGI2 and centrifuge for 8 min at 1,900 x g (with low brake or without brake).</li>
	<li>Remove the supernatant and resuspend the pellet with 5 mL TA buffer containing 2.5 &#181;L/mL of 25 &#956;M PGI2 using a plastic Pasteur pipet. Incubate for 10 min at 37 &#176;C.</li>
	<li>During the incubation period, pipet 150 &#181;L of the platelet suspension into a 1.5 mL tube and count platelets, using an automatized cell counter (that detects the size of blood cells by measuring the changes in direct-current resistance).</li>
	<li>After the 10 min incubation, add 2.5 &#181;L/mL of 25 &#956;M PGI2 to the platelet suspension and immediately centrifuge at 1,900 x g for 8 min.</li>
	<li>Remove the supernatant and resuspend the pellet to a concentration of 250,000 platelets/&#181;L with an adequate volume of TA buffer (i.e. if the cell count is 500,000 per &#181;L, resuspend in 10 mL TA buffer) containing 32 &#181;L/mL of apyrase at 0.01 U/mL (final concentration 0.32 U/mL). 
	NOTE: High concentration of apyrase is used to avoid the desensitization of P2X1 receptors induced by spontaneous secretion of ATP10,11 in absence of agonists. This is important because collagen-induced responses are induced by fast paracrine/autocrine activation of P2X1 by ATP released from activated platelets. If the platelet signaling pathway does not critically require preservation of P2X1 function, use 0.02 U/mL apyrase. Several studies (reviewed in Mahaut-Smith et al.10) demonstrated that 0.02 U/mL apyrase avoids ADP receptor P2Y1 desensitization with negligible P2X1 responses.</li>
	<li>Incubate the cell suspension for at least 30 min at 37 &#176;C before performing the aggregometric measurements. The preparation is stable for 5 to 8 h.</li>
</ol>
4. Aggregometry
<ol>
	<li>Prepare fibrinogen (56 mg/mL) in Tyrode&#39;s buffer.</li>
	<li>Pipet 260 &#181;L of platelet suspension into glass cuvettes (Figure 1A; left cuvette) containing 10 &#181;L of fibrinogen (56 mg/mL) and a magnetic stirring rod, then incubate the suspension for 2 - 3 min at 37 &#176;C in incubation wells present in the aggregometer (Figure 1B and 1C).</li>
	<li>Pre-incubate with the Panx1 inhibitor Brilliant Blue FCF by adding 10 &#181;L of a 2.8 mM or 28 mM stock solution (final concentration 100 &#956;M and 1 mM, respectively) for 7 min at 37 &#176;C.</li>
	<li>Calibrate the aggregometer to an assumptive 100% aggregation value by measuring the OD of a cuvette containing 10 &#181;L fibrinogen (56 mg/mL), 10 &#181;L Brilliant Blue FCF (2.8 mM or 28 mM) and TA buffer without platelets.
	<ol>
		<li>Place the cuvette in an aggregation well under automatic stirring and press the corresponding button on the keyboard of the computer linked to the aggregometer (i.e. press F1 if aggregation well 1 is used). 
		NOTE: This experiment described below includes Brilliant Blue FCF. The compound used for calibration has to be adjusted to the experimental condition.</li>
	</ol>
	</li>
	<li>Calibrate the aggregometer to an assumptive 0% aggregation value by using the same platelet sample that will be used for the experiment under automatic stirring.
	<ol>
		<li>Place the cuvette in the aggregation well and press the corresponding button on the keyboard of the computer linked to the aggregometer. Wait for about 20-30 s before proceeding. This delay serves to assure that no aggregation happens before adding the agonists. 
		NOTE: As any difference in platelet number may have an effect on the measured OD, the 0% calibration step needs to be repeated for each individual measurement.</li>
	</ol>
	</li>
	<li>Add 20 &#181;L of desired agonist, such as 15 &#181;g/mL collagen (1 &#181;g/mL final) or 1.125 mM arachidonic acid (75 &#181;M final), into the cuvette. Immediately start the recording under continuous automatic stirring by pressing the corresponding button on the keyboard of the computer linked to the aggregometer. 
	NOTE: The addition of the agonist induces platelet activation. Platelet aggregates can clearly be distinguished in the glass cuvette at the end of the experiment (Figure 1A; right cuvette).</li>
	<li>The recording automatically stops after 6 min. At this point, save the data by clicking on the save icon of the computer. 
	NOTE: The calculation of the rate of aggregation is performed by the computer, which expresses the end results of the aggregation process as a percentage.</li>
	<li>Analyze the data.
	<ol>
		<li>For additional extensive information on protocols for the preparation of washed platelets suspensions and turbidimetric measurement of platelet aggregation, refer to other papers authored by experts in the field12,13.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

There is great interest in finding new drugs capable of modulating platelet function in order to prevent thrombosis without enhancing the risk of bleeding. For this purpose, in vitro laboratory tests which can reliably and reproducibly monitor aggregation responses in human platelets are absolutely necessary. Turbidimetric aggregometry is an easy technique to perform. However, some precautions need to be kept in mind. The measurements need to be performed under continuous stirring as the aggregation process is l...

Platelets are crucial components of blood and their main function-together with coagulation factors-is to stop bleeding after blood vessel injury. Platelets are small (2-3 &#181;m) anuclear fragments derived from megakaryocytes of the bone marrow1. Platelets circulate in non-activated state, during which they appear as lens-shaped structures. Upon interruption of the endothelium, platelets gather to the site of blood vessel injury to plug the hole, a process called primary hemostasis. Initially, platelets attach to sub-endothelial molecules, such as collagen and von Willebrand factor, that are exposed as a result of the injury-adhesion step2. Then, they change shape and secrete chemical messengers-activation step. Finally, they connect to each other by bridging receptors-aggregation step. Primary hemostasis is followed by a secondary process involving activation of the coagulation cascade with fibrin deposition, which stabilizes the initial thrombus2.
Acute ischemic events such as myocardial infarction3 often result from thrombi that form because of physical disruption (rupture) of an atherosclerotic plaque. Current anti-platelet drugs are the cornerstone of the treatment of this widespread disease but their clinical benefit is limited by an increased risk for bleeding. The most prescribed drugs in cardiovascular patients, aspirin and anti-P2Y12 compounds, target the thromboxane A2 and the ADP pathways4, respectively, which are the major pathways leading to platelet activation. However, innovative research towards new targets that would optimally balance antithrombotic effects and haemorragic risk is still necessary.
From the 1960s5 to today, turbidimetric aggregometry has played a crucial role in research, enhancing our knowledge of platelet reactivity and in the monitoring of the potency of anti-thrombotic reagents in humans. Turbidimetry was initially applied to PRP extracted from blood samples. Indeed, blood collection performed in tubes containing citrate allows fast and large production of PRP without having any effect on platelet integrity and function. However, the short-term stability (about 3 h) of PRP and the remaining plasmatic enzymes, such as thrombin, and the low calcium concentration associated with potentially artefactual aggregation profiles are of major inconvenience for the use of PRP. An important step forward has been the development of a method for platelet isolation with additional centrifugation and washing steps6. In short, PRP is isolated from whole blood collected on acid-citrate-dextrose (ACD) and platelets are isolated after serial centrifugation steps before being resuspended in an iso-osmotic phosphate buffer (Tyrode&#39;s buffer) containing glucose, human serum albumin and divalent cations (Ca2+ and Mg2+). To avoid changes in platelet reactivity, the pH of Tyrode&#39;s buffer is carefully kept at 7.35-7.4. Moreover, undesired activation of platelets is prevented by adding prostacyclin (PGI2) before some centrifugation steps. Finally, addition of apyrase prevents washed platelets from becoming resistant against the action of ATP/ADP. The resulting platelet suspension lacks coagulant factors and the stability of platelets is increased by at least two-fold as compared to PRP solutions. In addition, the fact that platelets are inactive but intact warrants the reproducibility of turbidimetric measurements and provides the ability to study the action of agonists or antagonists of platelet aggregation in an optimal way.
Using this method, we have shown in a recent study that inhibiting the formation of Panx1 channels by a genetic approach (knock-out mice) or decreasing Panx1 channel activity by pharmacological approaches reduced collagen-induced platelet aggregation7. Panx1 forms ATP-release channels, which are ubiquitously expressed in many cell types including human platelets7,8. In fact, we demonstrated by turbidimetry on human washed platelets that a 7 min preincubation with a panel of more-or-less specific chemical blockers (probenecid, mefloquine and 10Panx1 peptides) prior to the addition of various agonists, inhibited specifically collagen-induced platelet aggregation while platelet responses to AA and ADP were not affected. We demonstrated that ATP release through Panx1 channels specifically interferes in the GPVI signaling pathway leading to collagen-induced aggregation. Interestingly, multiple FDA-approved compounds with applications in other diseases (probenecid, mefloquine) affect the activity of Panx1 channels in platelets. On one hand, this opens new therapeutic perspectives to selectively modify platelet reactivity. On the other hand, one should consider potential secondary effects of these compounds. In this context, the safe food dye Brilliant Blue FCF used in multiple candies and energy drinks has been described as a selective inhibitor of Panx19. We describe here a protocol for the isolation of human washed platelets and turbidimetric measurements of platelet aggregation adapted to investigate the effect of the Brilliant Blue FCF dye as an antagonist of platelet aggregation.

<table><tbody><tr><td>citric acid monohydrate (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;*H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Roth</td><td>5949-29-1</td><td>danger of eye damage/irritation</td></tr><tr><td>trisodium citrate dihydrate (Na&lt;sub&gt;3&lt;/sub&gt;C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>S1804</td><td>-</td></tr><tr><td>D(+)-glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>-</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888</td><td>-</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td>-</td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S6014</td><td>-</td></tr><tr><td>Sodium dihydrogenophosphate monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;*H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>S9638</td><td>-</td></tr><tr><td>Magnesium chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;*6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>M9272</td><td>-</td></tr><tr><td>Calcium chloride hexahydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;*6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>442909</td><td>danger of eye damage/irritation</td></tr><tr><td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (Hepes)</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>15630</td><td>-</td></tr><tr><td>human serum albumin</td><td>CSL Behring</td><td>00257/374</td><td>-</td></tr><tr><td>hydrochloric acid</td><td>Sigma-Aldrich</td><td>320331</td><td>Corrosive and irritative for the respiratory system. Can cause severe skin and eye damages.</td></tr><tr><td>Eppendorf 5810 R</td><td>Fisher Scientific</td><td>-</td><td>-</td></tr><tr><td>heparin</td><td>Drosspharm AG/SA</td><td>20810</td><td></td></tr><tr><td>prostacyclin I2 (PGI2)</td><td>Cayman</td><td>18220</td><td>-</td></tr><tr><td>apyrase from potatoes</td><td>Sigma-Aldrich</td><td>A6535</td><td>-</td></tr><tr><td>fibrinogen (Haemocomplettan)</td><td>CSL Behring</td><td>HS 73466011</td><td>-</td></tr><tr><td>thrombo-aggragometer SD-Medical</td><td>SD-Innovation</td><td>TA8V</td><td>-</td></tr><tr><td>Brilliant blue FCF (Erioglaucine disodium salt)</td><td>Sigma-Aldrich</td><td>80717</td><td>Harmful to aquatic life with long lasting effects (Avoid release to the environnement)</td></tr><tr><td>collagen</td><td>Horm, Nycomed</td><td></td><td>-</td></tr><tr><td>arachidonic acid</td><td>Bio/Data corporation</td><td>C/N 101297</td><td>-</td></tr><tr><td>cell counter Sysmex KX-21N</td><td>Sysmex Digitana&amp;nbsp;</td><td>-</td><td>-</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-056</td><td>-</td></tr><tr><td>glass cuvettes</td><td>SD-Innovation</td><td>THCV1000</td><td>-</td></tr><tr><td>magnetic stirrers</td><td>SD-Innovation</td><td>THA100</td><td>-</td></tr></tbody></table>

turbidimetry on human washed platelets: the effect of the pannexin1-inhibitor brilliant blue fcf on collagen-induced aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

The aggregometer software automatically produces the aggregation curves and gives the values for maximal aggregation in percentage. The values can be copied to a data analysis software in order to perform statistical analysis and visualize maximal aggregation values in form of bar charts. Optionally, each individual point of the aggregation curves can be exported successively into a spreadsheet software and then to statistical software (e.g. GraphPad) in order to visualize the cu...

Watch this Scientific Journal Video about Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation at JoVE.com

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

turbidimetry-on-human-washed-platelets-effect-pannexin1-inhibitor

Research

JoVE Journal

Medicine

11.9K Views.  University of Geneva. We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Video: Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Biology

Kinetx: A Combined Flow Cytometry Assay and Analysis Software Framework to Quantitatively Measure and Categorize Platelet Activation in Real-time.

Platelets react rapidly to vascular injury and undergo activation in response to a range of stimuli to limit blood loss. Many platelet function tests measure endpoint responses after a defined time period and not the rate of platelet activation. However, the rate at which platelets convert extracellular stimuli into a functional response is an essential factor in determining how efficiently they can respond to injury, bind to a forming thrombus, and signal to recruit other platelets. This paper describes a flow cytometry-based platelet function assay that enables simultaneous data acquisition and sample stimulation and utilizes newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux. Kinetix was developed in R so that users can alter parameters such as degree of smoothing, identification of outlying data points, or time scales. To aid users unfamiliar with the R environment, Kinetix analysis of data can be performed by a single command. Together, this allows real-time platelet activation metrics, such as rate, acceleration, time to peak-rate, time to peak-calcium, and qualitative shape changes, to be accurately and reproducibly measured and categorized. Kinetic measurements of platelet activation give a unique insight into platelets' behavior during the first stages of activation and may provide a method of predicting the recruitment of platelets into a forming thrombus.

Platelets react rapidly to a range of stimuli. This paper describes a real-time flow cytometry-based platelet function assay and a newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux.

Platelets react rapidly to vascular injury and undergo activation in response to a range of stimuli to limit blood loss. Many platelet function tests ...

Biochemistry

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Bioengineering

Platform for Assessing Platelet Function in Trauma and Transfusion

Microfluidics in Assessing Platelet Function

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying ...

Imaging Single Platelet Migration Using a Three-Layer Coating System

An In Vitro Assay to Study Platelet Migration Using RGD-Functionalized Avidin-Biotin Tethers

Despite being anucleated cell fragments, platelets are now widely recognized for their multifaceted abilities. Not only do they form blood clots to prevent bleeding after injury, but they also fight infections and maintain vascular integrity during inflammatory diseases. While hemostatic plugs require the collective activation and aggregation of platelets, their role in protecting inflamed blood vessels is performed at the single-cell level. In this context, recent data have shown that platelets can migrate autonomously, a process dependent on the mechanosensing of their adhesive environment. Here, a detailed protocol for imaging single platelet migration is presented, utilizing a three-layer coating system consisting of a poly-L-lysine graft poly(ethylene glycol) (PLL-PEG)-biotin backbone (1), a fluorescent avidin linker (2), and biotin-cyclic Arg-Gly-Asp (cRGD) (3) as the platelet integrin-binding motif. This reductionist approach allows precise control of substrate adhesion properties and serves as a simple, standardized in vitro assay to study the mechanisms underlying platelet migration. The results indicate that migrating platelets binding to cRGD exert forces capable of disrupting the avidin-biotin bond. Furthermore, the density of biotin-cRGD significantly influences both platelet spreading and migration.

A detailed protocol for imaging single migrating platelets using RGD-functionalized avidin-biotin tethers with tunable density is provided, revealing that platelets generate enough force to rupture the avidin-biotin bond.

Despite being anucleated cell fragments, platelets are now widely recognized for their multifaceted abilities. Not only do they form blood clots to ...

Analyzing Platelet Phosphatidylserine and Microvesicle Release

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

Isolating Quiescent Platelets for Metabolic Research

Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%&#8211;40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.

Platelet metabolism is of interest, particularly as it relates to the role of platelet hyper- and hypoactivity in bleeding and thrombotic disorders. Isolating platelets from plasma is necessary for some metabolic assays; presented here is a method for isolating of intracellular metabolites from washed platelets.

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated ...

An In Vitro Method for the Differentiation of Megakaryocytes and Platelet Formation

Source: Perdomo, J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56420">Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells.</a> J. Vis. Exp. (2017)
This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells &#8212; isolated from human umbilical cord blood &#8212; differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

Source: Perdomo, J., et al. Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells. J. Vis. Exp. (2017)
This video ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Differentiation Techniques

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

Source: Quach, M. E. et al., <a href="https://appjove.unicartagenaproxy.elogim.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.

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

Immunodiagnostics

Cellular Immunodiagnostics

A Turbidimetric Analysis to Monitor Collagen-Induced Aggregation of Pretreated Human Platelets

Source: Molica, F. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55525">Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates turbidimetric analysis to assess collagen-induced platelet aggregation in pre-treated, washed human platelets. Control wells show collagen-induced aggregation, whereas a high concentration of inhibitors blocks Pannexin-1 channels, thereby inhibiting collagen-induced platelet aggregation,&#160; evident from unchanged turbidity.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...