On this blog we have discussed the rates of activation among hospital blood banks and platelet suppliers, we have given an overview on the sources of platelet activation, and the impact transfusions of activated platelets have on platelet utilization. However, the validity of all these posts hinges on a foundational understanding that microparticle content is a reliable indicator of platelet activation status. For this blog post, we will explore this question: How do we know that microparticle content is a good marker of platelet activation status?
The first step in answering this post’s question is to clarify what we mean when we talk about platelet activation. Platelet activation is the natural process during which platelets undergo a drastic change and transition from non-activated, discoid platelets, to an irregular shape with numerous protrusions often called pseudopods (from Greek pseudo “like or fake” and pod “foot”). This shape change is accompanied by numerous other changes including the budding of surface membrane vesicles, the activation of GPIIb/IIIa sites which prime the platelet surface to bind with fibrinogen, and the secretion of many substances from the three types of platelet granules: dense granules, alpha granules, and lysosomal granules.1,2 Platelets are very sensitive, and activation can occur when platelets adhere to exposed collagen or artificial surfaces, shear stress while in circulation, or are exposed to one of a wide range of stimulators including thrombin, ADP, complement, high shear, or low temperature.3-5
The images below show non-activated (left) and activated (right) platelets imaged via transmission electron microscopy (top) and scanning electron microscopy(bottom).
Platelets on right were activated by cold; micrographs provided by E. Maurer-Spurej
Platelet microparticles (MPs) can be either the result of budded off surface membrane vesicles or intracellular vesicles released during platelet activation. The platelet microparticles that have budded off from the platelet surface maintain a similar surface marker profile as an activated platelet. Platelet MPs have significant pro-coagulant and hemostatic properties3,6,7
In samples of non-activated platelets, very few MPs are present.8,9 Furthermore, 70-90% of MPs in plasma are generated by platelets.10,11 These two findings suggest that the vast majority of circulating MPs are from activated platelets. In addition to MPs formed during activation, in vivo platelet MPs are known to form during platelet storage over 7 days.12 This MP generation during storage is at least partially due to contact with artificial surfaces.13 The additional formation of MP’s during storage is especially prevalent when the platelets are already activated from their donors and are therefore more stress-sensitive.14-17
The special vulnerability of activated platelets to further MP generation helps to explain why we did not find a difference between the activation rates in day 2 platelets versus day 5 platelets. The platelet units that begin storage in a non-activated state typically stay non-activated while the platelet units that begin activated have significant increases in microparticle content but no change in platelet activation status.18
Determining Platelet Activation Status
Due to the very prominent shape change associated with platelet activation, morphological assessment under sufficient magnification and contrast enhancement has served as one of the earliest methods for determining platelet activation status. However, there are significant limitations for the routine use of morphological assessments including the technical difficulty of stabilizing platelets to prevent further activation, and inter-operator variability.
The most common method to determine platelet activation is by flow cytometry. Flow cytometry allows for the determination of platelet activation using labeling with specific antibodies including PAC-1 for detecting the activation of GPIIb/IIIa, CD62P (P-selectin) from alpha granule membranes and CD63 from dense granules.7,19-25 Flow cytometry also gives the ability to determine MP levels of the sample which have been correlated with the expression of many of the above antigens.23-26 There are some limitations to flow cytometry however as it is generally a very technical process that is not adaptable to routine use. Furthermore, the size calibration and gating methods used in flow cytometry mean that often only the largest microparticles are detected 3 Other methods to measure MPs and their association with platelet activation have also been developed,27-30 including dynamic light scattering-based ThromboLUX.14,31-34
Platelet Activation in Diseases
Platelets function both as innate immune cells35-37 and hemostatic cells. Because of this dual role, platelet activation is a defining phenomenon of many pathological conditions. Microparticles are elevated under these pathological conditions25,38-47 which often culminate in thrombocytopenia.48,49 For example, increased platelet activation and elevated platelet-derived microparticle (PMP) levels have been found during acute malaria infection,50 antiphospholipid syndrome,51 gastrointestinal diseases,52-54 psoriasis,55 scleroderma,56 arthritis,57 diabetes,58,59 spontaneous abortion,60 cancer,61,62 thalassemia,63 and cardiovascular pathologies.64-74 Studies have also investigated the diagnostic value of MP changes in response to treatment.75-81
Adenovirus and other viruses induce PMP release during platelet activation82 and promote the formation of platelet-leukocyte aggregates accompanied by cellular microparticle release both in vitro and in vivo.83-85 Increased P-selectin expression by activated platelets in patients with sepsis is associated with increased formation of platelet microparticles, which express surface receptors that enable them to interact with leukocytes.35
Based on published literature, it is well established that platelet activation leads to microparticle generation. The use of MP content as a marker of platelet activation status is supported by the fact that hardly any MPs are present in samples of non-activated platelets and the vast majority of MPs are generated by platelets. The connection between MPs and various disease states further supports the conclusion that MPs are generated during all forms of platelet activation, not just during clotting. The scientific evidence connecting platelet activation and microparticle generation led platelet expert Dr. Alan Michelson to say, “There is no doubt that platelet activation results in microparticles.”
We’d love to hear your input on this subject. If you have any questions or comments about the connection between platelet activation and microparticles please leave a comment in the area below the references.
- Abrams CS. Platelet Biology. In: Timauer JS, ed. UpToDate. UpToDate (Accessed on July 10, 2018).
- Heijnen HFG, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. Activated platelets release two types of membrane vesicles: Microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood 1999;94:3791-9.
- Nieuwland R, Van der Pol E, Gardiner C, Sturk A. Platelet-Derived Microparticles. In: Michelson AD, ed. Platelets. 3rd ed: Elsevier; 2013:453-67.
- Maurer-Spurej E, Pfeiler G, Maurer N, Lindner H, Glatter O, Devine DV. Room temperature activates human blood platelets. Laboratory Investigation 2001;81.
- Spurej E, Glatter O, Pfeiler G. Shape change of human blood platelets: reliable and fast detection by quasi-elastic light scattering. Experientia 1992;48:71-9.
- Matijevic N, Wang YW, Holcomb JB, Kozar R, Cardenas JC, Wade CE. Microvesicle phenotypes are associated with transfusion requirements and mortality in subjects with severe injuries. J Extracell Vesicles 2015;4:29338.
- Michelson AD, Furman MI. Laboratory markers of platelet activation and their clinical significance. Curr Opin Hematol 1999;6:342-8.
- Flaumenhaft R, Dilks JR, Richardson J, et al. Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles. Blood 2009;113:1112-21.
- van der Zee PM, Biro E, Ko Y, et al. P-selectin- and CD63-exposing platelet microparticles reflect platelet activation in peripheral arterial disease and myocardial infarction. Clin Chem 2006;52:657-64.
- Flaumenhaft R. Formation and fate of platelet microparticles. Blood Cells Molecules and Diseases 2006;36:182-7.
- Keuren JF, Magdeleyns EJ, Govers-Riemslag JW, Lindhout T, Curvers J. Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation. Br J Haematol 2006;134:307-13.
- Bode AP, Orton SM, Frye MJ, Udis BJ. Vesiculation of platelets during in vitro aging. Blood 1991;77:887-95.
- Gemmell CH. Activation of platelets by in vitro whole blood contact with materials: increases in microparticle, procoagulant activity, and soluble P-selectin blood levels. J Biomater Sci Polym Ed 2001;12:933-43.
- Kanzler P, Mahoney A, Leitner G, Witt V, Maurer-Spurej E. Microparticle detection to guide platelet management for the reduction of platelet refractoriness in children – A study proposal. Transfus Apher Sci 2017;56:39-44.
- Gustafson CM, Shepherd AJ, Miller VM, Jayachandran M. Age- and sex-specific differences in blood-borne microvesicles from apparently healthy humans. Biol Sex Differ 2015;6:10.
- Rank A, Nieuwland R, Liebhardt S, et al. Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles. Vox Sang 2011;100:179-86.
- Ueba T, Nomura S, Inami N, et al. Correlation and association of plasma interleukin-6 and plasma platelet-derived microparticles, markers of activated platelets, in healthy individuals. Thromb Res 2010;125:e329-34.
- Maurer-Spurej E, Larsen R, Labrie A, Heaton A, Chipperfield K. Microparticle content of platelet concentrates is predicted by donor microparticles and is altered by production methods and stress. Transfus Apher Sci 2016;55:35-43.
- Michelson AD, Barnard MR, Krueger LA, Frelinger AL, 3rd, Furman MI. Evaluation of platelet function by flow cytometry. Methods 2000;21:259-70.
- Connor DE, Ma DD, Joseph JE. Flow cytometry demonstrates differences in platelet reactivity and microparticle formation in subjects with thrombocytopenia or thrombocytosis due to primary haematological disorders. Thromb Res 2013;132:572-7.
- Frelinger AL, 3rd, Torres AS, Caiafa A, et al. Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation. Platelets 2016;27:128-35.
- Perez-Pujol S, Marker PH, Key NS. Platelet microparticles are heterogeneous and highly dependent on the activation mechanism: studies using a new digital flow cytometer. Cytometry A 2007;71:38-45.
- Nolan JP, Jones JC. Detection of platelet vesicles by flow cytometry. Platelets 2017;28:256-62.
- Burdess A, Michelsen AE, Brosstad F, Fox KA, Newby DE, Nimmo AF. Platelet activation in patients with peripheral vascular disease: reproducibility and comparability of platelet markers. Thromb Res 2012;129:50-5.
- Moritz A, Walcheck BK, Weiss DJ. Flow cytometric detection of activated platelets in the dog. Vet Clin Pathol 2003;32:6-12.
- Tan KT, Tayebjee MH, Lynd C, Blann AD, Lip GY. Platelet microparticles and soluble P selectin in peripheral artery disease: relationship to extent of disease and platelet activation markers. Ann Med 2005;37:61-6.
- Xiong G, Aras O, Shet A, Key NS, Arriaga EA. Analysis of individual platelet-derived microparticles, comparing flow cytometry and capillary electrophoresis with laser-induced fluorescence detection. Analyst 2003;128:581-8.
- Taube J, McWilliam N, Luddington R, Byrne CD, Baglin T. Activated protein C resistance: Effect of platelet activation, platelet-derived microparticles, and atherogenic lipoproteins. Blood 1999;93:3792-7.
- Ponomareva AA, Nevzorova TA, Mordakhanova ER, et al. Intracellular origin and ultrastructure of platelet-derived microparticles. J Thromb Haemost 2017;15:1655-67.
- Osumi K, Ozeki Y, Saito S, et al. Development and assessment of enzyme immunoassay for platelet-derived microparticles. Thromb Haemost 2001;85:326-30.
- Maurer-Spurej E, Chipperfield K. Could Microparticles Be the Universal Quality Indicator for Platelet Viability and Function? J Blood Transfus 2016;2016:6140239.
- Maurer-Spurej E, Labrie A, Pittendreigh C, et al. Platelet quality measured with dynamic light scattering correlates with transfusion outcome in hematologic malignancies. Transfusion 2009;49:2276-84.
- Xu Y, Nakane N, Maurer-Spurej E. Novel test for microparticles in platelet-rich plasma and platelet concentrates using dynamic light scattering. Transfusion 2011;51:363-70.
- Millar D, Murphy L, Labrie A, Maurer-Spurej E. Routine Screening Method for Microparticles in Platelet Transfusions. J Vis Exp 2018.
- Semple JW, Italiano JE, Jr., Freedman J. Platelets and the immune continuum. Nature Reviews Immunology 2011;11:264-74.
- Peerschke EI, Yin W, Ghebrehiwet B. Platelet mediated complement activation. Adv Exp Med Biol 2008;632:81-91.
- Hamad OA, Mitroulis I, Fromell K, et al. Contact activation of C3 enables tethering between activated platelets and polymorphonuclear leukocytes via CD11b/CD18. Thromb Haemost 2015;114:1207-17.
- Nomura S, Miyazaki Y, Miyake T, et al. Detection of platelet-derived microparticles in patients with diabetes. Am J Hematol 1993;44:213.
- Holme PA, Solum NO, Brosstad F, Roger M, Abdelnoor M. Demonstration of Platelet-Derived Microvesicles in Blood from Patients with Activated Coagulation and Fibrinolysis using a Filtration Technique and Western Blotting. Thrombosis and haemostasis 1994;72:666-71.
- Nomura S, Suzuki M, Katsura K, et al. Platelet-derived microparticles may influence the development of atherosclerosis in diabetes mellitus. Atherosclerosis 1995;116:235-40.
- Nomura S. Extracellular vesicles and blood diseases. Int J Hematol 2017;105:392-405.
- Nomura S, Shouzu A, Omoto S, et al. Effect of cilostazol on soluble adhesion molecules and platelet-derived microparticles in patients with diabetes. Thromb Haemost 1998;80:388-92.
- Jacoby RC, Owings JT, Holmes J, Battistella FD, Gosselin RC, Paglieroni TG. Platelet activation and function after trauma. J Trauma 2001;51:639-47.
- Hughes M, Hayward CP, Warkentin TE, Horsewood P, Chorneyko KA, Kelton JG. Morphological analysis of microparticle generation in heparin-induced thrombocytopenia. Blood 2000;96:188-94.
- Wang Y, Luo L, Morgelin M, Thorlacius H. Rac1 regulates sepsis-induced formation of platelet-derived microparticles and thrombin generation. Biochem Biophys Res Commun 2017;487:887-91.
- Tahmasbi L, Karimi M, Kafiabadi SA, et al. Evaluation of Plasma Platelet Microparticles in Thrombotic Thrombocytopenic Purpura. Ann Clin Lab Sci 2017;47:62-7.
- Duarte D, Taveira-Gomes T, Sokhatska O, et al. Increased circulating platelet microparticles as a potential biomarker in asthma. Allergy 2013;68:1073-5.
- Wang J, Zhang W, Nardi MA, Li Z. HIV-1 Tat-induced platelet activation and release of CD154 contribute to HIV-1-associated autoimmune thrombocytopenia. J Thromb Haemost 2011;9:562-73.
- Stravitz RT, Ellerbe C, Durkalski V, et al. Thrombocytopenia Is Associated With Multi-organ System Failure in Patients With Acute Liver Failure. Clin Gastroenterol Hepatol 2016;14:613-20 e4.
- Campos FM, Franklin BS, Teixeira-Carvalho A, et al. Augmented plasma microparticles during acute Plasmodium vivax infection. Malar J 2010;9:327.
- Chaturvedi S, Cockrell E, Espinola R, et al. Circulating microparticles in patients with antiphospholipid antibodies: characterization and associations. Thromb Res 2015;135:102-8.
- Andoh A, Yoshida T, Yagi Y, et al. Increased aggregation response of platelets in patients with inflammatory bowel disease. J Gastroenterol 2006;41:47-54.
- Tziatzios G, Polymeros D, Spathis A, et al. Increased levels of circulating platelet derived microparticles in Crohn’s disease patients. Scand J Gastroenterol 2016;51:1184-92.
- Wilhelmsen P, Kjaer J, Thomsen KL, et al. Elevated platelet expression of CD36 may contribute to increased risk of thrombo-embolism in active inflammatory bowel disease. Arch Physiol Biochem 2013;119:202-8.
- Tamagawa-Mineoka R, Katoh N, Kishimoto S. Platelet activation in patients with psoriasis: increased plasma levels of platelet-derived microparticles and soluble P-selectin. J Am Acad Dermatol 2010;62:621-6.
- Nomura S, Inami N, Ozaki Y, Kagawa H, Fukuhara S. Significance of microparticles in progressive systemic sclerosis with interstitial pneumonia. Platelets 2008;19:192-8.
- Boilard E, Blanco P, Nigrovic PA. Platelets: active players in the pathogenesis of arthritis and SLE. Nat Rev Rheumatol 2012;8:534-42.
- Zhang X, McGeoch SC, Johnstone AM, et al. Platelet-derived microparticle count and surface molecule expression differ between subjects with and without type 2 diabetes, independently of obesity status. J Thromb Thrombolysis 2014;37:455-63.
- Omoto S, Nomura S, Shouzu A, et al. Significance of platelet-derived microparticles and activated platelets in diabetic nephropathy. Nephron 1999;81:271-7.
- Kaptan K, Beyan C, Ifran A, Pekel A. Platelet-derived microparticle levels in women with recurrent spontaneous abortion. Int J Gynaecol Obstet 2008;102:271-4.
- Toth B, Liebhardt S, Steinig K, et al. Platelet-derived microparticles and coagulation activation in breast cancer patients. Thromb Haemost 2008;100:663-9.
- Liang H, Yan X, Pan Y, et al. MicroRNA-223 delivered by platelet-derived microvesicles promotes lung cancer cell invasion via targeting tumor suppressor EPB41L3. Mol Cancer 2015;14:58.
- Pattanapanyasat K, Gonwong S, Chaichompoo P, et al. Activated platelet-derived microparticles in thalassaemia. Br J Haematol 2007;136:462-71.
- Zeiger F, Stephan S, Hoheisel G, Pfeiffer D, Ruehlmann C, Koksch M. P-Selectin expression, platelet aggregates, and platelet-derived microparticle formation are increased in peripheral arterial disease. Blood Coagul Fibrinolysis 2000;11:723-8.
- Inoue T, Komoda H, Kotooka N, et al. Increased circulating platelet-derived microparticles are associated with stent-induced vascular inflammation. Atherosclerosis 2008;196:469-76.
- Ismail EA, Youssef OI. Platelet-derived microparticles and platelet function profile in children with congenital heart disease. Clin Appl Thromb Hemost 2013;19:424-32.
- Wang ZT, Wang Z, Hu YW. Possible roles of platelet-derived microparticles in atherosclerosis. Atherosclerosis 2016;248:10-6.
- Vasina E, Heemskerk JW, Weber C, Koenen RR. Platelets and platelet-derived microparticles in vascular inflammatory disease. Inflamm Allergy Drug Targets 2010;9:346-54.
- Vajen T, Mause SF, Koenen RR. Microvesicles from platelets: novel drivers of vascular inflammation. Thromb Haemost 2015;114:228-36.
- Suades R, Padro T, Alonso R, Mata P, Badimon L. High levels of TSP1+/CD142+ platelet-derived microparticles characterise young patients with high cardiovascular risk and subclinical atherosclerosis. Thromb Haemost 2015;114:1310-21.
- Lukasik M, Rozalski M, Luzak B, et al. Enhanced platelet-derived microparticle formation is associated with carotid atherosclerosis in convalescent stroke patients. Platelets 2013;24:63-70.
- Skeppholm M, Mobarrez F, Malmqvist K, Wallen H. Platelet-derived microparticles during and after acute coronary syndrome. Thrombosis and haemostasis 2012;107:1122-9.
- Ohuchi M, Fujino K, Kishimoto T, et al. Association of the Plasma Platelet-Derived Microparticles to Platelet Count Ratio with Hospital Mortality and Disseminated Intravascular Coagulopathy in Critically Ill Patients. J Atheroscler Thromb 2015;22:773-82.
- Nagy B, Jr., Szuk T, Debreceni IB, Kappelmayer J. Platelet-derived microparticle levels are significantly elevated in patients treated by elective stenting compared to subjects with diagnostic catheterization alone. Platelets 2010;21:147-51.
- Nomura S, Inami N, Iwasaka T, Liu Y. Platelet activation markers, microparticles and soluble adhesion molecules are elevated in patients with arteriosclerosis obliterans: therapeutic effects by cilostazol and potentiation by dipyridamole. Platelets 2004;15:167-72.
- Yahata T, Suzuki C, Yoshioka A, Hamaoka A, Ikeda K. Platelet activation dynamics evaluated using platelet-derived microparticles in Kawasaki disease. Circ J 2014;78:188-93.
- Kagawa H, Nomura S, Nagahama M, Ozaki Y, Fukuhara S. Effect of ticlopidine on platelet-derived microparticles in patients with connective tissue diseases. Haemostasis 1999;29:255-61.
- Kereiakes DJ, Michelson AD. Platelet activation and progression to complications. Rev Cardiovasc Med 2006;7:75-81.
- Krajewski S, Kurz J, Geisler T, Peter K, Wendel HP, Straub A. Combined blockade of ADP receptors and PI3-kinase p110beta fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation. PLoS One 2012;7:e38455.
- Shouzu A, Nomura S, Hayakawa T, et al. Effect of sarpogrelate hydrochloride on platelet-derived microparticles and various soluble adhesion molecules in diabetes mellitus. Clin Appl Thromb Hemost 2000;6:139-43.
- Nomura S, Shouzu A, Omoto S, Nishikawa M, Fukuhara S, Iwasaka T. Losartan and simvastatin inhibit platelet activation in hypertensive patients. J Thromb Thrombolysis 2004;18:177-85.
- Hottz ED, Lopes JF, Freitas C, et al. Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation. Blood 2013;122:3405-14.
- Othman M, Labelle A, Mazzetti I, Elbatarny HS, Lillicrap D. Adenovirus-induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediating accelerated platelet clearance. Blood 2007;109:2832-9.
- Punyadee N, Mairiang D, Thiemmeca S, et al. Microparticles provide a novel biomarker to predict severe clinical outcomes of dengue virus infection. J Virol 2015;89:1587-607.
- El-Menshawy N, Eissa M, Farag R, Aboalyazed A. CD235a (Glycophorin-A) Is the Most Predictive Value Among Different Circulating Cellular Microparticles in Thrombocytopenic Human Immunodeficiency Virus Type 1. J Clin Lab Anal 2016;30:235-43.