Name: Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets (Video) | JoVE Journal
Uploaded: 2024-11-29T13:50:00.000+00:00
Duration: 5 min 49 s
Description: Learn to quantify phosphatidylserine exposure and microvesicle release from human purified platelets using flow cytometry. This method enables precise characterization of procoagulant platelet formation in hemostasis research.

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5 minutes

The protocol follows the guidelines and was approved by the University Hospital of Bordeaux Human Research Ethics Committee. Blood samples were obtained from healthy volunteers who provided informed consent, and these samples were processed according to institutional protocols. Donors who had taken any substances that could affect platelet function were excluded if such substances were taken within 10 days preceding the experiments. Informed consent was obtained from healthy volunteers to collect their blood and publish their data. The details of the reagents and the equipment used are listed in the Table of Materials.
1. Blood collection and preparation of washed platelets
<ol>
	<li>Collect peripheral venous blood in collection tubes containing the active anticoagulant Tri-sodium Citrate with Citric Acid and Dextrose (ACD) after discarding the first 3 mL.</li>
	<li>After collection, gently invert the tube to mix the blood with ACD and allow the mixture to rest at room temperature for 15 min before centrifugation.</li>
	<li>Count platelets using an automated cell counter before centrifugation to determine the correct speed for centrifugation (Table 1). Prepare platelet-rich plasma (PRP) by centrifugation at 250 x g for 10 min at room temperature (RT) without applying the brake to optimize platelet recovery. 
	NOTE: This step will produce three layers in the sample: the upper layer containing plasma, platelets, and a small fraction of white blood cells; a middle layer enriched with white blood cells; and a bottom layer consisting of red blood cells. Carefully transfer the PRP from the upper layer into a new 50 mL conical tube to avoid contamination with red and white blood cells.</li>
	<li>To the PRP, add 1 mL of ACD-A (see Table 2) and 6.25 &#181;L of apyrase per 9 mL. 
	NOTE: Apyrase, at a concentration of 0.02 U/mL, degrades traces of ATP or ADP secreted by the platelets, thereby preventing desensitization of platelet ADP receptors and maintaining platelet shape25. A prostacyclin PGI2 inhibitor (0.5 &#181;M) can also be included with apyrase to prevent platelet activation.</li>
	<li>Prepare the platelet pellet by centrifugation at RT at 1100 x g for 10 min. Aspirate the supernatant containing Platelet Poor Plasma (PPP) using a gentle method, such as a Pasteur pipette, and resuspend the pellet in 1 mL of washing buffer (see Table 3 and Table 4). Homogenize gently with a pipette, then add an additional 3-4 mL of washing buffer. 
	NOTE: The calcium level is reduced to prevent coagulation with clot factors present in the plasma. At this stage, the platelet-poor plasma has been completely removed, preventing clot formation and platelet activation by thrombin production.</li>
	<li>Centrifuge again at RT at 1100 x g for 10 min. Aspirate the supernatant and resuspend the pellet in 1 mL of washing buffer. Transfer 150 &#181;L of the platelet suspension to a 1.5 mL tube for cell counting using an automated cell counter. Then, resuspend the pellet with 3-4 mL of washing buffer.</li>
	<li>Perform a final centrifugation at 1100 x g for 10 min (at RT). Aspirate the supernatant and resuspend the platelet pellet in a volume of reaction buffer (see Table 5 and Table 6) to achieve a final concentration of 5 x 1011 platelets/L.</li>
	<li>Allow the washed platelets to rest for at least 30 min before experiments to allow residual inhibitors to wear off and for the platelets to acclimatize to the buffer.</li>
</ol>
2. Assay preparation
<ol>
	<li>Prepare all agonists and fluorochromes in the reaction buffer. Add 5 &#181;L of thrombin to 45 &#181;L of buffer (dilution 1:10) and 5 &#181;L of ionophore to 495 &#181;L of buffer (dilution 1:500).
	<ol>
		<li>For internal assays, benchmark non-activated platelets against those treated with single and dual agonists. Add, in different aliquots, 100 &#181;L of washed platelets at 50 x 109 platelets/L to: (i) 10 &#181;L of reaction buffer, (ii) 3 &#181;L of non-diluted collagen (final concentration of 30 &#181;g/mL), (iii) 10 &#181;L of pre-diluted thrombin (0.5 IU/mL), (iv) 3 &#181;L of non-diluted collagen + 10 &#181;L of pre-diluted thrombin, and (v) 10 &#181;L of pre-diluted ionophore (final concentration of 2 &#181;M), which is known to directly increase intracellular calcium levels as previously noted26. 
		NOTE: Treatment with a calcium ionophore, such as A23187 or ionomycin, is critical as it induces extensive phospholipid membrane scrambling and enhanced PS externalization.</li>
		<li>Gently shake each aliquot by hand and incubate at 37 &#176;C for 5 min. 
		NOTE: Longer agonist incubation (30 min) may allow better differentiation between platelet populations during the flow cytometry analysis process.</li>
	</ol>
	</li>
	<li>Add 5 &#181;L of non-diluted Annexin-V FITC to each microtube and incubate at room temperature for 10 min in the dark. 
	NOTE: A monoclonal antibody against one of the platelet-specific receptors, GPIX (CD42b) or &#945;IIb integrin (CD41, GPIIb), could also be included to better identify platelet cells.</li>
	<li>Add 500 &#181;L of reaction buffer to stop the process and proceed with the analysis of the sample using a flow cytometer.</li>
</ol>
3. Characterization of procoagulant platelets and microvesicles by Flow Cytometry (FC)
NOTE: For reliable platelet function analysis, utilize a flow cytometry (FC) instrument configured according to established standards27. The instrument must be capable of detecting forward scatter (FSC) and at least one fluorescence signal. Set the light scatter and fluorescence detectors to logarithmic gain. Dilute the samples suitably for FC to ensure only individual platelets are counted at a reduced data acquisition speed.
<ol>
	<li>Set the flow cytometer to a &#39;slow&#39; flow rate with a threshold of at least 10,000 platelet events. Monitor fluorescence emission using a pass filter for FITC.</li>
	<li>Gate the platelet population using forward scatter (FSC). Examine platelets and released microvesicles according to their sizes. Use density plots to identify platelets and released microvesicles for both resting and stimulated platelets; analyze each gated population independently.</li>
	<li>Distinguish procoagulant platelets according to the FL1 axis. Place the negative cut-off at the far right of the platelet population in the resting state (i.e., in the presence of reaction buffer). After platelet activation, classify all events localized to the right of this cut-off as platelets exposing phosphatidylserine.</li>
	<li>Differentiate microvesicles, being smaller than platelets, by their unique light scattering characteristics. Set the cut-off at the lowest FSC value observed in the resting platelet population. After activation, classify any phosphatidylserine-positive events falling below this threshold as microvesicles. 
	NOTE: Account for individual variation in platelet size by adjusting the cut-off if necessary. Ensure that less than 1% of microvesicles are present in the resting state.</li>
</ol>

Analyzing Platelet Phosphatidylserine and Microvesicle Release

The authors declare having no conflict of interest.

Recent studies on procoagulant platelets have highlighted their changes during several diseases1,28,29, underscoring the importance of their detailed analysis and characterization30,31,32. While current clinical tests for assessing platelet procoagulant formation are limited, there has been a significant rise in clinical interest over the past two decades. For example, the calcium-dependent scramblase TMEM16F (ANO6) is involved in procoagulant phosphatidylserine (PS) exposure33. Variants of TMEM16F are associated with Scott syndrome33,34,35, a rare bleeding disorder characterized by impaired phospholipid scrambling and reduced thrombin generation in activated platelets36. Moreover, elevated procoagulant platelet levels are linked with thrombotic strokes and coronary artery disease, sparking interest in therapies to curb platelet procoagulant activity37. Of the methods currently available to evaluate procoagulant platelet formation, assessment of PS exposure by Annexin-V binding and microvesicle release is one of the most commonly used38,39.
The protocol outlined here enables the isolation of human-purified platelets and the characterization of procoagulant platelets by measuring PS exposure and microvesicle release using flow cytometry. It is crucial to advise healthy volunteers to abstain from medications that may impact platelet function for at least 10 days before blood sampling. Blood collection requires care to avoid vein trauma or excessively slow flow, as these can induce thrombin generation and platelet activation. Sodium citrate-anticoagulated blood is not recommended for preparing washed platelets, as residual calcium can enable thrombin generation and platelet activation during centrifugation17. ACD-anticoagulated blood has a pH of 6.5 and a higher citrate concentration of 22 mmol/L compared to citrate-anticoagulated blood&#39;s pH of 7.5 and 13 mmol/L citrate concentration17. The acidic pH and lowered extracellular calcium concentration in ACD-anticoagulated blood prevent platelet aggregation24. Minimizing thrombin generation during the collection process is essential. A non-traumatic venipuncture using at least a 21 G needle is recommended to prevent venous stasis or excessive pressure. Additionally, the first few milliliters of blood should be discarded to avoid contamination with tissue factors and trace amounts of thrombin17. Minimizing the time between venipuncture and sample processing is also essential, as prolonged blood storage may lead to platelet activation, thereby increasing PS exposure and microvesicle release40. Specific attention must be paid during the entire platelet preparation procedure to prevent spontaneous platelet preactivation, which can occur during the preparation process itself41,17. Moreover, isolating purified platelets necessitates careful avoidance of contamination by other blood cells17. This involves thoroughly aspirating the supernatant after centrifuging the platelets to ensure a clean preparation. As the pellet is typically visible and firmly adhered to the tube wall, the supernatant can be readily aspirated using a pipette tip.
During centrifugation wash steps, platelets are resuspended in a buffer that simulates physiological conditions. Using washed platelets in a physiological buffer isolates them from anticoagulants and plasma, thereby preventing thrombin-mediated artifacts. While platelet-rich plasma (PRP) is a rapid method to obtain high platelet concentrations, its use for functional studies is limited by anticoagulant-induced pH changes and the plasma&#39;s extracellular calcium and protein content, which can influence platelet activation17. Furthermore, high levels of PS generated during platelet activation would be sufficient to promote the assembly of plasma coagulation complexes, subsequent thrombin burst, and clot formation in PRP, rendering experimental results uninterpretable. In contrast, resuspension in a buffer containing apyrase is recommended to maintain platelet functionality by preventing desensitization of purinergic receptors through the degradation of ATP and ADP42,43. Although repeated centrifugation and washing steps require more time, suspensions of washed platelets prepared by this method offer the advantage of greater stability (5-8 h) compared with citrated PRP preparations, which are stable for no more than 1-3 h23.
This study demonstrates the feasibility and simplicity of quantifying microvesicles by flow cytometry. However, microvesicle production can get lost in the debris and noise of the machine. Therefore, it is critical to ensure that the flow cytometer has undergone extensive cleaning and that the buffers used do not contain any contaminating particles that might lead to a high background during flow cytometry analysis. Alternatively, specialized flow cytometers with small particle detection capabilities can be used to identify microvesicle formation. The binding of fluorescein-labeled Annexin-V as a function of membrane PS content was also studied, highlighting the critical role of sufficient calcium concentrations in the buffer for Annexin-V binding and procoagulant platelet response44.
Data supporting the stability of platelet samples over time are currently scarce. Several laboratories have addressed this issue by stabilizing samples through the addition of a fixative solution, such as a formaldehyde-containing buffer. Adding a fixative to freshly collected platelet samples has the advantage of preserving samples for later analysis. Nevertheless, formaldehyde fixation can cause artifactually increased Annexin-V binding. Rochat et al. demonstrated that platelet fixation with a low-concentration, calcium-free formaldehyde solution does not alter the proportion of Annexin-V positive platelets45. However, researchers who require fixation should optimize their specific protocol to ensure that it does not cause artifactually altered results. Otherwise, samples should be processed immediately.
Another pathway that may also lead to PS exposure involves intrinsic apoptosis, a process known to regulate platelet lifespan in circulation46. Activating pro-apoptotic BAK and BAX proteins results in caspase activation, which promotes the externalization of PS, leading to the in vivo clearance of platelets. Recently, it has been suggested that another platelet surface marker, P-selectin/CD62P, in addition to Annexin-V, be used to distinguish procoagulants from apoptotic platelets47. Procoagulant platelets are expected to be positive for both PS and P-selectin markers, while apoptotic platelets are positive for Annexin-V but negative for P-selectin. However, time is also a differentiating factor between procoagulant and apoptotic platelets47. Generating procoagulant platelets in vitro is a rapid process, whereas intrinsic apoptosis is a slower process, taking hours48. In this context, the use of additional markers to assess procoagulant platelets is not necessary, simplifying the method. Sustained increases in intracellular Ca2+ and procoagulant platelet formation occur within a few minutes with very strong agonists, such as ionophore or combined activation with collagen and thrombin, whereas a BCL-XL inhibitor (or BH3 mimetic) triggers the formation of apoptotic platelets by activating pro-apoptotic BAK and BAX, causing mitochondrial outer membrane permeabilization, cytochrome C release, and caspase activation49.
In conclusion, the protocol described here facilitates the isolation of purified platelets and their subsequent characterization for procoagulant activity using standard laboratory equipment and flow cytometry. This procedure paves the way for future research on procoagulant platelets in patient blood samples, with potential applications in clinical practice, such as the diagnosis of the rare Scott syndrome50.

Platelet procoagulant formation is crucial to maintaining hemostasis1,2,3. This process involves agonist-induced expression of phospholipids on the platelet membrane, which is essential for the assembly of tenase and prothrombinase complexes1,3,4. After platelet activation, platelet microvesicles are also continuously released5,6. Microvesicles (50 nm to over 1 &#181;m in diameter) encapsulate both the membrane structure and cytoplasmic constituents of the originating cells7,8. Microvesicles are important mediators of intercellular signaling9,10 and also repositories of growth factors that promote wound healing and inflammatory modulation11,12. Microvesicles have also been suggested to be 50- to 100-fold more procoagulant than activated platelets13. Notably, recent evidence suggests that stored platelets become activated, leading to the production of microvesicles-this has potential ramifications for the practice of platelet transfusion therapy. Microvesicles&#39; extended storage duration and heightened procoagulant activity render them a promising substitute for platelets in transfusion applications14.
Methodological innovations have been made to, directly and indirectly, assess platelet procoagulant formation in both health and disease conditions4,14,15. Assessments of phosphatidylserine exposure and microvesicle release using purified platelets have provided new data on how platelet procoagulant responses are perturbed in diverse human diseases1,4. This growing field of research also opens avenues for therapeutic interventions1,4. However, the isolation and analysis of purified platelets from blood are time-consuming, necessitate specialized laboratory equipment, and are, thus, not currently adaptable for routine clinical diagnostics16,17. Analyzing phosphatidylserine exposure and microvesicle release is also challenging due to their small size and the number of positive events for markers of interest18,19.
Flow cytometry, leveraging fluorescent Annexin-V binding, has been a cornerstone in the evaluation of PS expression on platelets and microvesicles since its inception two decades ago20. It has gained widespread acceptance in the examination of procoagulant platelets and microvesicles21,22. Therefore, an optimized protocol is presented here that can be used for the isolation of purified platelets from blood samples using the technique developed by Cazenave&#39;s group23, and the subsequent characterization of phosphatidylserine exposure and microvesicle release by flow cytometry after platelet activation24. This protocol will facilitate further study and in-depth characterization of procoagulant platelets in clinical populations of interest.

<table><tbody><tr><td>(CD42b, GPIX) APC</td><td>Beckman Coulter</td><td>B13980</td><td></td></tr><tr><td>ACD-A blood collection tubes</td><td>BD Vacutainer</td><td>366645</td><td></td></tr><tr><td>Annexin-V FITC</td><td>BD Pharmingen</td><td>560931</td><td></td></tr><tr><td>Apyrase Grade VII</td><td>Sigma-Aldrich</td><td>A6410</td><td></td></tr><tr><td>Bovine Serum Albumin 30%</td><td>Sigma-Aldrich</td><td>A9576</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2, &lt;/sub&gt;0.25M</td><td>Sigma-Aldrich</td><td>C3881</td><td></td></tr><tr><td>Citric acid monohydrate</td><td>Merck</td><td>5949-29-1</td><td></td></tr><tr><td>Collagen&amp;nbsp;</td><td>Stago</td><td>86924</td><td></td></tr><tr><td>Glucose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Hepes&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Ionophore&amp;nbsp;</td><td>Calbiochem/VWR</td><td>100105</td><td></td></tr><tr><td>KCl&amp;nbsp;</td><td>Merck</td><td>7447-40-7</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M0250</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>VWR</td><td>27810.295</td><td></td></tr><tr><td>NaOH&amp;nbsp;</td><td>Merck</td><td>1.06498</td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Merck</td><td>6329</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Thrombin&amp;nbsp;</td><td>Hyphen Biomed</td><td>EZ 006 A</td><td></td></tr><tr><td>Trisodium citrate dihydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G8270</td><td>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></tr><tr><td>&amp;alpha;IIb integrin (CD41, GPIIb) PC7</td><td>Beckman Coulter</td><td>6607115</td><td></td></tr></tbody></table>

procoagulant platelet characterization by measuring phosphatidylserine exposure and microvesicle release from human purified platelets

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.

Quantification of procoagulant platelets and microvesicles is achieved using Annexin-V staining, with at least 50,000 events recorded per sample. As stated in step 2.2, baseline platelet measurements were taken from samples incubated with reaction buffer, as depicted in Figure 1A. Platelet sizes were gauged using the median forward scatter (FSC) value. While FSC is influenced by various factors, it remains a common proxy for cell size estimation, including that of platelets. Density plots of area (Figure 1A) were used to identify and gate resting platelets and released microvesicles.
In the case of non-activated platelets, less than 2.9% displayed phosphatidylserine (PS) as determined by Annexin-V staining. Upon activation of washed platelets with either collagen alone or in conjunction with thrombin or calcium ionophore, there was an observable rise in PS exposure. Collagen alone prompted a 26.7% increase in PS-positive platelets. Thrombin had minimal effects on PS exposure (9.1%), but when associated with collagen, it increased the percentage of PS-exposing platelets to 36.2%. For reference, treatment with 2 µM ionophore resulted in 49.6% of platelets becoming procoagulant.
Figure 1 illustrates that the baseline proportion of microvesicles from washed platelets in a healthy individual was minimal (0.3%). While thrombin and collagen minimally increased microvesicle formation, it was only with the combination of collagen and thrombin that microvesicle formation significantly increased (Figure 1D; 11.4%). Adding 2 µM ionophore resulted in 44.0% of the microvesicles formed by control platelets being PS-exposing.
Figure 2 provides information regarding the variation, repeatability, and reproducibility to be expected from this process.
<img alt="Platelet flow cytometry results, FS Log vs. Annexin-V. Diagrams show PSneg and PSpos platelet distributions." class="xfigimg" src="/files/ftp_upload/67042/67042fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: PS exposure and microvesicle formation in resting and activated control platelets. Platelets were either activated or unactivated (Resting) (A), with 30 µg/mL collagen (B), with 0.5 U/mL thrombin (C), with combinations of collagen and thrombin (D) with 2 µM ionophore. Representative dot plots display platelet size (forward scatter (FSC log)) versus Annexin-V binding. Horizontal and vertical lines (E) delineate the platelet-negative population. <a href="https://www.jove.com/files/ftp_upload/67042/67042fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Phosphatidylserine exposure and microvesicle formation box plots; % positive cells vs. ionomycin levels." class="xfigimg" src="/files/ftp_upload/67042/67042fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Repeatability and reproducibility of PS exposure and microvesicle formation after incubation with ionophore. Analysis of PS exposure and microvesicle formation after incubation with either 2 µM or 5 µM ionophore in washed platelets, treated or untreated with citrate (a calcium chelator): (A) from 4 different healthy controls; (B) from one healthy control repeated 5 times; and (C) from two healthy controls prepared by two different technicians. <a href="https://www.jove.com/files/ftp_upload/67042/67042fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Platelet count in whole blood (109 per mL)</td>
			<td>Relative centrifugal forces (g)</td>
		</tr>
		<tr>
			<td>&lt;30</td>
			<td>70</td>
		</tr>
		<tr>
			<td>30-50</td>
			<td>100</td>
		</tr>
		<tr>
			<td>51-100</td>
			<td>130</td>
		</tr>
		<tr>
			<td>101-150</td>
			<td>200</td>
		</tr>
		<tr>
			<td>151-300</td>
			<td>250</td>
		</tr>
		<tr>
			<td>301-600</td>
			<td>290</td>
		</tr>
		<tr>
			<td>&gt;600</td>
			<td>340</td>
		</tr>
	</tbody>
</table>
Table 1: Relative centrifugal forces for platelet-rich plasma preparation in relation to platelet count in whole blood.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Citric acid monohydrate (C6H8O7.H2O, 38 mM)</td>
			<td>8 g</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate (Na3C6H5O7.2 H2O, 61 mM)</td>
			<td>180 g</td>
		</tr>
		<tr>
			<td>Glucose (C6H12O6, 136 mM)</td>
			<td>24.6 g</td>
		</tr>
		<tr>
			<td colspan="2">The pH of the solution should not be adjusted. Keep the solution at 4 °C.</td>
		</tr>
	</tbody>
</table>
Table 2: Preparation of a 1 L aqueous solution of acid-citrate-dextrose (ACD).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Citric acid monohydrate (C6H8O7.H2O, 36 mM)</td>
			<td>7.56 g</td>
		</tr>
		<tr>
			<td>NaCl (103 mM)</td>
			<td>6 g</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>4 g</td>
		</tr>
		<tr>
			<td> MgCl2.6H2O (1 mM) </td>
			<td>0.203 g</td>
		</tr>
		<tr>
			<td colspan="2">Adjust the pH at 6.5. Keep the solution at 4 °C. </td>
		</tr>
	</tbody>
</table>
Table 3: Preparation of stock solution of Washing Buffer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>30% Bovine Serum Albumin (BSA)</td>
			<td>583 µL</td>
		</tr>
		<tr>
			<td>CaCl2, 0.25 M</td>
			<td>400 µL</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>40 mg</td>
		</tr>
		<tr>
			<td>Apyrase</td>
			<td>31.5 µL </td>
		</tr>
	</tbody>
</table>
Table 4: Preparation of a 50 mL extemporaneous solution of Washing Buffer from the stock solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>NaCl (137 mM)</td>
			<td>8 g</td>
		</tr>
		<tr>
			<td>KCl (2.7 mM)</td>
			<td>0.2 g</td>
		</tr>
		<tr>
			<td>MgCl2.6H2O (1 mM)</td>
			<td>0.203 g</td>
		</tr>
		<tr>
			<td>HEPES (5 mM) </td>
			<td>1.19 g</td>
		</tr>
		<tr>
			<td>NaHCO3 (12 mM) </td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>NaH2PO4.2H2O; 0.3 mM</td>
			<td>0.05 g</td>
		</tr>
		<tr>
			<td colspan="2">Adjust the pH at 7.4. Keep the solution at 4 °C. </td>
		</tr>
	</tbody>
</table>
Table 5: Preparation of a stock solution of Reaction Buffer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>30% Bovine Serum Albumin (BSA)</td>
			<td>167 µL</td>
		</tr>
		<tr>
			<td>CaCl2, 0.25 M</td>
			<td>400 µL </td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>50 mg</td>
		</tr>
	</tbody>
</table>
Table 6: Preparation of a 50 mL extemporaneous solution of Reaction Buffer from the stock solution.

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 ...

procoagulant-platelet-characterization-measuring-phosphatidylserine

Research

JoVE Journal

Biology

This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells — isolated from human umbilical cord blood — differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

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

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

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.

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

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

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.

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Routine Screening Method for Microparticles in Platelet Transfusions

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.

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

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 in Assessing Platelet Function

Multi-color flow cytometry for platelets can identify new platelet subtypes within classical platelet subpopulations, such as resting, aggregatory, procoagulant, and apoptotic platelets. This allows comparison of different subpopulation patterns induced by various platelet agonists. Here, a procedure for establishing a multi-color flow cytometry panel is described in detail.

Analyzing Platelet Subpopulations by Multi-color Flow Cytometry

The present protocol provides a comprehensive set of procedures necessary to analyze procoagulant platelets, which exhibit overlapping features of necrosis, apoptosis, and platelet activation.

Comprehensive Analysis of Procoagulant Platelets Exhibiting Features of Necrosis, Apoptosis and Platelet Activation

This work describes a microfluidic assay that uses shear stress to trigger thrombus formation in blood and characterizes the thrombus by multiple dimensions of readout. The assay can measure the prothrombotic tendency of blood samples, and is thus useful in disease diagnosis, drug discovery, as well as basic mechanistic studies related to thrombosis.

Thrombus Profiling Assay: A Microfluidics-Based Platform for Comprehensively Characterizing Biomechanical Thrombogenesis

Learn to quantify phosphatidylserine exposure and microvesicle release from human purified platelets using flow cytometry. This method enables precise characterization of procoagulant platelet formation in hemostasis research.

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets (Video) | JoVE Journal

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

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