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 t...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>CaCl2, 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&#160;</td>
			<td>Stago</td>
			<td>86924</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionophore&#160;</td>
			<td>Calbiochem/VWR</td>
			<td>100105</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl&#160;</td>
			<td>Merck</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M0250</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>VWR</td>
			<td>27810.295</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Merck</td>
			<td>1.06498</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Merck</td>
			<td>6329</td>
			<td>NaHCO3</td>
		</tr>
		<tr>
			<td>Thrombin&#160;</td>
			<td>Hyphen Biomed</td>
			<td>EZ 006 A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Na3C6H5O7*2H2O</td>
		</tr>
		<tr>
			<td>&#945;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

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

Platelet procoagulant formation is crucial to maintain hemostasis. This process involves expression of phospholipids and microvesicle release at the platelet surface, which is essential for the coagulation steps. Assessments of this process have provide new data on how platelet procoagulant responses are in diverse human disease.Flow cytometry has been a cornerstone and has gained widespread acceptance in the evaluation of phosphatidylserine expression on platelets and microvesicle release. The isolation and analysis of purified platelets from blood are time-consuming and necessitates specialized laboratory equipment, and are thus not currently available for routine clinical diagnosis. Analyzing phosphatidylserine exposure and microvesicle release is also challenging due to their small size and the low number of positive events for markers of interest.To begin, collect peripheral venous blood in collection tubes containing the active anticoagulant trisodium citrate with citric acid and dextrose. Gently invert the tube to mix the blood with the anticoagulant and let the mixture rest at room temperature for 15 minutes. Count platelets using an automated cell counter to determine the correct speed for centrifugation.To prepare the platelet-rich plasma, centrifuge the blood at 250 g for 10 minutes at room temperature without applying the break. Then add 1 milliliter of ACDA and 6.25 microliters of apyrase to 9 milliliters of the platelet-rich plasma. Centrifuge the mixture at 1, 100 g for 10 minutes at room temperature.Using a Pasture pipette, remove the supernatant containing platelet-poor plasma and re-suspend the platelet pellet gently in 1 milliliter of washing buffer. Then add 3 to 4 milliliters of washing buffer to the tube and centrifuge it again. After removing the supernatant, re-suspend the pellet in 1 milliliter of washing buffer.Transfer 150 microliters of the platelet suspension to a 1.5 milliliter tube for counting the cells in an automated cell counter. Add 3 to 4 milliliters of washing buffer to the remaining suspension to perform an additional wash as demonstrated earlier. Next, re-suspend the platelet pellet in the reaction buffer to achieve a final concentration of 5 x 10 to the power of 11 platelets per liter.To begin, obtain washed platelets in the desired buffer for flow cytometry. Prepare all agonists and fluorochromes in the reaction buffer. For internal assays, add 100 microliters of washed platelets at a concentration of 50 x 10 to the power of 9 platelets per liter to different treatment tubes.Gently shake each aliquot and incubate at 37 degrees Celsius for 5 minutes. Then add 5 microliters of non-diluted Annexin V-FITC to each microtube and incubate. Afterward, add 500 microliters of reaction buffer to each tube to stop the process.Gate the platelet population using forward scatter or FSC and use density plots to identify each population independently. Set the negative cutoff at the far right of the resting state platelet population. After activation, classify events to the right of this cutoff as platelets exposing phosphatidylserine.For differentiating microvesicles, set the cutoff at the lowest FSC value observed in the resting platelet population. After activation, classify phosphatidylserine positive events below this threshold as microvesicles. Baseline measurements indicated that less than 2.9%of non-activated platelets exhibited phosphatidylserine exposure.Upon activation, 26.7%of platelets became phosphatidylserine positive with collagen, 9.1%with thrombin, and 36.2%with both collagen and thrombin. Ionophore treatment resulted in 49.6%phosphatidylserine positive platelets. The baseline proportion of microvesicles was 0.3%Significant increases were observed only with collagen and thrombin together at about 11.4%and more so with ionophores at 44%

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...

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

Blood Collection and Preparation of Washed Platelets for Coagulation Studies

Characterization of Procoagulant Platelets and Microvesicles by Flow Cytometry

295 Views.  University Hospital of Bordeaux.  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.

Analyzing Platelet Phosphatidylserine and Microvesicle Release

Summary