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

Summary

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.

Abstract

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.

Introduction

Platelet procoagulant formation is crucial to maintaining hemostasis^1,2,3. This process involves agonist-induced expression of phospholipids on the platelet membrane, which is essential for the assembly of tenase and prothrombinase complexes^1,3,4. After platelet activation, platelet microvesicles are also continuously released^5,6. Microvesicles (50 nm to over 1 µm in diameter) encapsulate both the membrane structure and cytoplasmic constituents of the originating cells^7,8. Microvesicles are important mediators of intercellular signaling^9,10 and also repositories of growth factors that promote wound healing and inflammatory modulation^11,12. 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' 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 conditions^4,14,15. Assessments of phosphatidylserine exposure and microvesicle release using purified platelets have provided new data on how platelet procoagulant responses are perturbed in diverse human diseases^1,4. This growing field of research also opens avenues for therapeutic interventions^1,4. 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 diagnostics^16,17. Analyzing phosphatidylserine exposure and microvesicle release is also challenging due to their small size and the number of positive events for markers of interest^18,19.

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 microvesicles^21,22. 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'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.

Protocol

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

1. 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.
2. 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.
3. 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.
4. To the PRP, add 1 mL of ACD-A (see Table 2) and 6.25 µL 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 µM) can also be included with apyrase to prevent platelet activation.
5. 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.
6. 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 µL 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.
7. 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.
8. 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.

2. Assay preparation

1. Prepare all agonists and fluorochromes in the reaction buffer. Add 5 µL of thrombin to 45 µL of buffer (dilution 1:10) and 5 µL of ionophore to 495 µL of buffer (dilution 1:500).
   1. For internal assays, benchmark non-activated platelets against those treated with single and dual agonists. Add, in different aliquots, 100 µL of washed platelets at 50 x 10⁹ platelets/L to: (i) 10 µL of reaction buffer, (ii) 3 µL of non-diluted collagen (final concentration of 30 µg/mL), (iii) 10 µL of pre-diluted thrombin (0.5 IU/mL), (iv) 3 µL of non-diluted collagen + 10 µL of pre-diluted thrombin, and (v) 10 µL of pre-diluted ionophore (final concentration of 2 µM), 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.
   2. Gently shake each aliquot by hand and incubate at 37 °C for 5 min.
      NOTE: Longer agonist incubation (30 min) may allow better differentiation between platelet populations during the flow cytometry analysis process.
2. Add 5 µL 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 αIIb integrin (CD41, GPIIb), could also be included to better identify platelet cells.
3. Add 500 µL of reaction buffer to stop the process and proceed with the analysis of the sample using a flow cytometer.

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.

1. Set the flow cytometer to a 'slow' flow rate with a threshold of at least 10,000 platelet events. Monitor fluorescence emission using a pass filter for FITC.
2. 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.
3. 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.
4. 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.

Results

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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Platelet count in whole blood (109 per mL)	Relative centrifugal forces (g)
<30	70
30-50	100
51-100	130
101-150	200
151-300	250
301-600	290
>600	340

Table 1: Relative centrifugal forces for platelet-rich plasma preparation in relation to platelet count in whole blood.

Reagent	Amount
Citric acid monohydrate (C6H8O7.H2O, 38 mM)	8 g
Trisodium citrate dihydrate (Na3C6H5O7.2 H2O, 61 mM)	180 g
Glucose (C6H12O6, 136 mM)	24.6 g
The pH of the solution should not be adjusted. Keep the solution at 4 °C.

Table 2: Preparation of a 1 L aqueous solution of acid-citrate-dextrose (ACD).

Reagent	Amount
Citric acid monohydrate (C6H8O7.H2O, 36 mM)	7.56 g
NaCl (103 mM)	6 g
NaOH	4 g
MgCl2.6H2O (1 mM)	0.203 g
Adjust the pH at 6.5. Keep the solution at 4 °C.

Table 3: Preparation of stock solution of Washing Buffer.

Reagent	Amount
30% Bovine Serum Albumin (BSA)	583 µL
CaCl2, 0.25 M	400 µL
Glucose	40 mg
Apyrase	31.5 µL

Table 4: Preparation of a 50 mL extemporaneous solution of Washing Buffer from the stock solution.

Reagent	Amount
NaCl (137 mM)	8 g
KCl (2.7 mM)	0.2 g
MgCl2.6H2O (1 mM)	0.203 g
HEPES (5 mM)	1.19 g
NaHCO3 (12 mM)	1 g
NaH2PO4.2H2O; 0.3 mM	0.05 g
Adjust the pH at 7.4. Keep the solution at 4 °C.

Table 5: Preparation of a stock solution of Reaction Buffer.

Reagent	Amount
30% Bovine Serum Albumin (BSA)	167 µL
CaCl2, 0.25 M	400 µL
Glucose	50 mg

Table 6: Preparation of a 50 mL extemporaneous solution of Reaction Buffer from the stock solution.

Discussion

Recent studies on procoagulant platelets have highlighted their changes during several diseases^1,28,29, underscoring the importance of their detailed analysis and characterization^30,31,32. 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 syndrome^33,34,35, 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 used^38,39.

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'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 itself^41,17. 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'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 ADP^42,43. 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(CD42b, GPIX) APC	Beckman Coulter	B13980
ACD-A blood collection tubes	BD Vacutainer	366645
Annexin-V FITC	BD Pharmingen	560931
Apyrase Grade VII	Sigma-Aldrich	A6410
Bovine Serum Albumin 30%	Sigma-Aldrich	A9576
CaCl2, 0.25M	Sigma-Aldrich	C3881
Citric acid monohydrate	Merck	5949-29-1
Collagen	Stago	86924
Glucose	Sigma-Aldrich	G8270
Hepes	Sigma-Aldrich	H3375
Ionophore	Calbiochem/VWR	100105
KCl	Merck	7447-40-7
MgCl2	Sigma-Aldrich	M0250
NaCl	VWR	27810.295
NaOH	Merck	1.06498
Sodium hydrogen carbonate	Merck	6329	NaHCO3
Thrombin	Hyphen Biomed	EZ 006 A
Trisodium citrate dihydrate	Sigma-Aldrich	G8270	Na3C6H5O7*2H2O
αIIb integrin (CD41, GPIIb) PC7	Beckman Coulter	6607115