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Method Article

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

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DOI:

10.3791/67042

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November 29th, 2024

 ,  , 
1French Reference Centre for Inherited Platelet Disorders, University Hospital of Bordeaux, 2Department of Haematology, University Hospital of Bordeaux, 3Inserm U1034, Biology of Cardiovascular Disease

In This Article

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 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 µm 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' 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'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.

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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.

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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...

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Discussion

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...

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Disclosures

The authors declare having no conflict of interest.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
(CD42b, GPIX) APCBeckman CoulterB13980
ACD-A blood collection tubesBD Vacutainer366645
Annexin-V FITCBD Pharmingen560931
Apyrase Grade VIISigma-AldrichA6410
Bovine Serum Albumin 30%Sigma-AldrichA9576
CaCl2, 0.25MSigma-AldrichC3881
Citric acid monohydrateMerck5949-29-1
Collagen Stago86924
Glucose Sigma-AldrichG8270
Hepes Sigma-AldrichH3375
Ionophore Calbiochem/VWR100105
KCl Merck7447-40-7
MgCl2Sigma-AldrichM0250
NaCl VWR27810.295
NaOH Merck1.06498
Sodium hydrogen carbonateMerck6329NaHCO3
Thrombin Hyphen BiomedEZ 006 A
Trisodium citrate dihydrate Sigma-AldrichG8270Na3C6H5O7*2H2O
αIIb integrin (CD41, GPIIb) PC7Beckman Coulter6607115

References

  1. Chu, Y., Guo, H., Zhang, Y., Qiao, R. Procoagulant platelets: Generation, characteristics, and therapeutic target. J Clin Lab Anal. 35 (5), e23750(2021).
  2. Agbani, E. O., Hers, I., Poole, A. W. Platelet procoagulant membrane dynamics: A key distinction between thrombosis and hemostasis. Blood Adv. 7 (8), 1615-1619 (2022).
  3. Veuthey, L., Aliotta, A., Bertaggia Calderara, D., Pereira Portela, C., Alberio, L. Mechanisms underlying dichotomous procoagulant COAT platelet generation-A conceptual review summarizing current knowledge. Int J Mol Sci. 23 (5), 2536(2022).
  4. Bourguignon, A., Tasneem, S., Hayward, C. P. M. Update on platelet procoagulant mechanisms in health and in bleeding disorders. Int J Lab Hematol. 44 Suppl 1, 89-100 (2022).
  5. Suades, R., Padró, T., Vilahur, G., Badimon, L. Platelet-released extracellular vesicles: The effects of thrombin activation. Cel lMo lLife Sci. 79 (3), 190(2022).
  6. Dai, Z., et al. Platelets and platelet extracellular vesicles in drug delivery therapy: A review of the current status and future prospects. Front Pharmacol. 13, 1026386(2022).
  7. Kalluri, R., LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Science (New York, N.Y.). 367 (6478), eaau6977(2020).
  8. Doyle, L. M., Wang, M. Z. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells. 8 (7), 727(2019).
  9. Berumen Sánchez, G., Bunn, K. E., Pua, H. H., Rafat, M. Extracellular vesicles: Mediators of intercellular communication in tissue injury and disease. Cell communsignal. 19 (1), 104(2021).
  10. Arraud, N., et al. Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration. J Thromb Haemost. 12 (5), 614-627 (2014).
  11. Eustes, A. S., Dayal, S. The role of platelet-derived extracellular vesicles in immune-mediated thrombosis. Int J Mol Sci. 23 (14), 7837(2022).
  12. Chaudhary, P. K., Kim, S., Kim, S. Shedding light on the cell biology of platelet-derived extracellular vesicles and their biomedical applications. Life. 13 (6), 1403(2023).
  13. Sinauridze, E. I., et al. Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets. Thromb Haemost. 97 (3), 425-434 (2007).
  14. Cai, Z., et al. Platelet-derived extracellular vesicles play an important role in platelet transfusion therapy. Platelets. 34 (1), 2242708(2023).
  15. Chaudhary, P. K., Kim, S., Kim, S. An insight into recent advances on platelet function in health and disease. Int J Mol Sci. 23 (11), 6022(2022).
  16. Tyagi, T., et al. A guide to molecular and functional investigations of platelets to bridge basic and clinical sciences. Nat Cardiovasc Res. 1 (3), 223-237 (2022).
  17. Hechler, B., Dupuis, A., Mangin, P. H., Gachet, C. Platelet preparation for function testing in the laboratory and clinic: Historical and practical aspects. Res Pract Thromb Haemost. 3 (4), 615-625 (2019).
  18. Perez, G. I., et al. Phosphatidylserine-exposing Annexin A1-positive extracellular vesicles: Potential cancer biomarkers. Vaccines. 11 (3), 639(2023).
  19. Inglis, H., Norris, P., Danesh, A. Techniques for the analysis of extracellular vesicles using flow cytometry. J Vis Exp. (97), e52484(2015).
  20. Dachary-Prigent, J., Freyssinet, J. M., Pasquet, J. M., Carron, J. C., Nurden, A. T. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: A flow cytometry study showing a role for free sulfhydryl groups. Blood. 81 (10), 2554-2565 (1993).
  21. Abaeva, A. A., et al. Procoagulant platelets form an α-granule protein-covered "cap" on their surface that promotes their attachment to aggregates. J Biol Chem. 288 (41), 29621-29632 (2013).
  22. Fager, A. M., Wood, J. P., Bouchard, B. A., Feng, P., Tracy, P. B. Properties of procoagulant platelets: defining and characterizing the subpopulation binding a functional prothrombinase. Arterioscler Thromb Vasc Biol. 30 (12), 2400-2407 (2010).
  23. Cazenave, J. P., Hemmendinger, S., Beretz, A., Sutter-Bay, A., Launay, J. Platelet aggregation: A tool for clinical investigation and pharmacological study. Methodology. Ann Biol Clin. 41 (3), 167-179 (1983).
  24. Han, P., Ardlie, N. G. The influence of pH, temperature, and calcium on platelet aggregation: Maintenance of environmental pH and platelet function for in vitro studies in plasma stored at 37 degrees C. Br J Haematol. 26 (3), 373-389 (1974).
  25. Ardlie, N. G., Perry, D. W., Packham, M. A., Mustard, J. F. Influence of apyrase on stability of suspensions of washed rabbit platelets. Proc Soc Exp Biol Med. 136 (4), 1021-1023 (1971).
  26. Kang, J. K., et al. Increased intracellular Ca2+ concentrations prevent membrane localization of PH domains through the formation of Ca2+-phosphoinositides. Proc Natl Acad Sci USA. 114 (45), 11926-11931 (2017).
  27. Frelinger, A. L., et al. Consensus recommendations on flow cytometry for the assessment of inherited and acquired disorders of platelet number and function: Communication from the ISTH SSC Subcommittee on Platelet Physiology. J Thromb Haemost. 19 (12), 3193-3202 (2021).
  28. Khattab, M. H., et al. Increased procoagulant platelet levels are predictive of death in COVID-19. GeroScience. 43 (4), 2055-2065 (2021).
  29. Denorme, F., Campbell, R. A. Procoagulant platelets: Novel players in thromboinflammation. Am J Physiol Cell Physiol. 323 (4), C951-C958 (2022).
  30. Gianazza, E., et al. Platelets in healthy and disease states: From biomarkers discovery to drug targets identification by proteomics. Int J Mol Sci. 21 (12), 4541(2020).
  31. Menck, K., Bleckmann, A., Schulz, M., Ries, L., Binder, C. Isolation and characterization of microvesicles from peripheral blood. J VisExp. (119), (2017).
  32. Alberca, R. W., et al. Platelet-based biomarkers for diagnosis and prognosis in COVID-19 Patients. Life. 11 (10), 1005(2021).
  33. Suzuki, J., Umeda, M., Sims, P. J., Nagata, S. Calcium-dependent phospholipid scrambling by TMEM16F. Nature. 468 (7325), 834-838 (2010).
  34. Boisseau, P., et al. A new mutation of ANO6 in two familial cases of Scott syndrome. Br J Haematol. 180 (5), 750-752 (2018).
  35. Castoldi, E., Collins, P. W., Williamson, P. L., Bevers, E. M. Compound heterozygosity for 2 novel TMEM16F mutations in a patient with Scott syndrome. Blood. 117 (16), 4399-4400 (2011).
  36. Zwaal, R. F. A., Comfurius, P., Bevers, E. M. Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids. Biochim Biophys Acta Mol Cell Biol Lipids. 1636 (2), 119-128 (2004).
  37. Agbani, E. O., Poole, A. W. Procoagulant platelets: Generation, function, and therapeutic targeting in thrombosis. Blood. 130 (20), 2171-2179 (2017).
  38. Reddy, E. C., Rand, M. L. Procoagulant phosphatidylserine-exposing platelets in vitro and in vivo. Front Cardiovasc Med. 7, 15(2020).
  39. Reddy, E. C., et al. Analysis of procoagulant phosphatidylserine-exposing platelets by imaging flow cytometry. Res Pract Thromb Haemost. 2 (4), 736-750 (2018).
  40. Magnette, A., Chatelain, M., Chatelain, B., Ten Cate, H., Mullier, F. Pre-analytical issues in the haemostasis laboratory: Guidance for the clinical laboratories. Thromb J. 14, 49(2016).
  41. Schoenfeld, H., Muhm, M., Doepfmer, U., Exadaktylos, A., Radtke, H. Platelet activity in washed platelet concentrates. Anesth Analg. 99 (1), 17-20 (2004).
  42. Koessler, J., et al. Role of purinergic receptor expression and function for reduced responsiveness to adenosine diphosphate in washed human platelets. PLoS ONE. 11 (1), e0147370(2016).
  43. Baurand, A., et al. Desensitization of the platelet aggregation response to ADP: Differential down-regulation of the P2Y1 and P2cyc receptors. Thromb Haemost. 84 (3), 484-491 (2000).
  44. Alberio, L., et al. Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT platelet formation. Thromb Haemost. 117 (06), 1101-1114 (2017).
  45. van Kruchten, R., et al. Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation. Blood. 121 (10), 1850-1857 (2013).
  46. Rochat, S., Alberio, L. Formaldehyde-fixation of platelets for flow cytometric measurement of phosphatidylserine exposure is feasible. Cytometry A. 87 (1), 32-36 (2015).
  47. Josefsson, E. C., Ramström, S., Thaler, J., Lordkipanidzé, M. COAGAPO study group Consensus report on markers to distinguish procoagulant platelets from apoptotic platelets: communication from the Scientific and Standardization Committee of the ISTH. J Thromb Haemost. 21 (8), 2291-2299 (2023).
  48. Choo, H. J., Kholmukhamedov, A., ChengZing, Z., Shawn, J. Inner mitochondrial membrane disruption links apoptotic and agonist-initiated phosphatidylserine externalization in platelets. Arterioscler Thromb Vasc Biol. 37 (8), 1503-1512 (2017).
  49. Johnson, L., Pearl Lei, P., Waters, L., Padula, M. P., Marks, D. C. Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry. Sci Rep. 13 (1), 1221(2023).
  50. Montague, S. J. Comprehensive functional characterization of a novel ANO6 variant in a new patient with Scott syndrome. J Thromb Haemost. S1538 - 7836 (24), 00127-00132 (2024).

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