SECRET LIVES OF BLOOD CELLS IN DISEASE
Platelets: more than a sack of glue Andrew S. Weyrich1 1Molecular
Medicine Program and the Department of Internal Medicine, University of Utah, Salt Lake City, UT
Platelets are primary effector cells in hemostasis. Emerging evidence over the last decade, however, demonstrates that platelets also have critical roles in immunity and inflammation. These nontraditional functions of platelets influence the development, progression, and evolution of numerous diseases, including arthritis, cancer, cardiovascular disease, and infectious syndromes. This chapters reviews recently discovered attributes of platelets that contribute to human disease, paying particular attention to the inflammatory activities of this anucleate cytoplast.
Learning Objective ●
To provide a brief understanding of recently discovered inflammatory functions of platelets that contribute to human disease
Background Since their discovery, platelets have been known for their roles in hemostasis, in which they rapidly bind to damaged blood vessels and prevent excessive bleeding. The hemostatic functions of platelets have been and continue to be the focus of intense investigation, especially in disease situations in which platelets inadvertently occlude vessels that should remain patent. Indeed, drugs that dampen the adhesive functions of platelets are commonplace in the clinical setting because they have proven benefits in the treatment of cardiovascular disease. Because of their primary role in hemostasis, many view platelets as “sacks of glue.” However, emerging evidence demonstrates that platelets are far more complex than previously considered and their intracellular machinery is not used solely for hemostatic activities. The newly recognized inflammatory functions of platelets are the focus of this review.
Phylogeny of platelets The phylogeny of platelets suggests that they are destined to have prominent inflammatory functions. In-depth discussion of how platelets evolved from primitive, multifunctional cells can be found in previous reviews.1-2 In brief, platelets likely evolved from invertebrate cells that have ancient origins in host defense.2 By the vertebrate stage, blood cells adapted to perform more distinct functions. For example, fish, reptiles, and birds have circulating thrombocytes that are phenotypically and functionally similar to platelets.2 Unlike platelets, however, thrombocytes are nucleated cells that regulate hemostatic and inflammatory processes.3 Some of the activities of thrombocytes are driven and controlled by their nucleus. Therefore, one might expect that platelets do not express nuclear components or perform “nuclear-like functions” because they circulate as anucleate cytoplasts. New evidence, however, suggests otherwise. Platelets possess numerous transcription factors that have nongenomic functions,4-6 an active spliceosome,7-8 and an extensive repertoire of miRNA and mRNAs.9-12 Platelets also have diverse inflammatory functions, including a recently described inflammasome function.13 Therefore, platelets are complex cells with highly evolved, nonhemostatic functions.
400
Platelets induce inflammatory responses in leukocytes Platelets have a diverse array of surface receptors that allow them to interact with leukocytes, pathogens, pathogen-released products, and inflamed endothelium.1,14 These interactions contribute to microvascular occlusion, thrombosis, and propagation of inflammatory damage elicited at the vascular wall.1 The reader is referred to recent reviews that detail the types and functions of these receptors in platelet-mediated responses, especially in relation to interactions with bacteria, bacterial-derived toxins, and endothelial cells.1,14-16 Under homeostatic conditions, platelets generally do not bind to leukocytes. However, upon activation, platelets adhere to neutrophils and monocytes and interactions with lymphocytes have also been reported.1,17-18 These heterotypic aggregates have several inflammatory consequences, which may be good or bad depending on the pathologic situation (Figure 1). Although less studied, reciprocal activation of platelets also occurs.19 Interactions of platelets with neutrophils and monocytes have received the most attention in regard to inflammatory responses. As described in previous reviews, binding between the cell types is primarily mediated by P-selectin, which translocates to the surface of activated platelets, and P-selectin glycoprotein-1 (PSGL-1) on target leukocytes, but P-selectin/PSGL-1 interactions tend to be more transient in neutrophils than in monocytes.20 Plateletneutrophil and platelet-monocyte aggregates (PMAs) have been detected in the blood of humans with a variety of diseases1 and are considered one of the most sensitive markers of platelet activation in human whole blood.21 When platelets bind neutrophils, they trigger the release of chemokines and the formation of neutrophil extracellular traps (NETs),17,22 which are lattices of chromatin, histones, and granule enzymes that are intended to capture and kill microbes.23 NETs have known roles in bacterial sepsis, but they are also generated in transfusion-related acute lung injury (TRALI), venous thrombosis, and a variety of other infectious syndromes.24-26 Untimely generation of NETs can also induce vascular injury and trap platelets, which may lead to adverse responses in many disease settings. Lipopolysaccharidestimulated platelets trigger NET formation by mechanisms that remain unresolved and, in TRALI, NETs accumulate in lung microvessels in a platelet-dependent fashion.22,24 Human platelets also trigger NET formation via the release of -defensin 1,27 a potent antimicrobial and signaling molecule.
American Society of Hematology
Figure 1. Platelets are effectors of thrombotic and inflammatory injury. Platelet signaling of leukocytes and/or prolonged newly recognized functions contribute to the pathophysiology of arthritis, cancer, cardiovascular (CV) disease, sepsis, and other clinical syndromes.
Platelets also induce an array of inflammatory responses in monocytes, and targeted disruption of PMAs may have therapeutic value in mitigating thromboinflammation at the vascular wall. Circulating PMAs are elevated in a variety of diseases, and cigarette smoke acutely increases the number of PMAs in the bloodstream.1,28 Several studies have shown that as platelets bind to monocytes, they induce monocyte-derived synthesis of critical cytokines including IL-8, IL-1, cyclooxygenase-2 (COX-2), and monocyte chemotactic protein-1 (MCP-1).18,29 Recent studies also demonstrate that PMAs synthesize soluble Flt-1,30 which has critical functions in preeclampsia, and dengue infection induces the formation of PMAs and synthesis of both proinflammatory and antiinflammatory products.31 Platelets also release a variety of factors that signal leukocytes, including angiogenic factors, chemokines, and CD40L, which is a pivotal immune signaling molecule in the adaptive immune response.32 Taken together, these findings indicate that platelets regulate and amplify responses in leukocytes through a variety of mechanisms that involve direct binding and release of soluble products. These activities, and others, are now widely recognized in the field and have led to the growing appreciation that platelets are critically involved in thrombosis and inflammation.
Platelet RNAs: effectors of cellular function and predictors of disease Although it is not hard to imagine that platelets can induce inflammatory gene expression in other cells, one might not predict that platelets themselves are capable of synthesizing proteins. This was our premise in 1998 when we examined the expression of B cell lymphoma 3 (Bcl-3) in PMAs and unexpectedly found that platelets, not monocytes, synthesize Bcl-3 protein upon activation.4 We subsequently demonstrated that Bcl-3, a presumed transcription factor, regulates cytoskeletal processes including platelet-dependent clot retraction.4,33 Perhaps more importantly, we realized that platelets function for extended periods of time and at least one of their prolonged functions is de novo synthesis of proteins. We have since shown that platelets also synthesize IL-1, a proinflammatory cytokine.34 Synthesis of IL-1 is controlled at many steps, starting first with splicing of IL-1 pre-mRNA into mature message.7 Demonstration that platelets splice pre-mRNAs provided the first example that an active spliceosome, one of the most complex eukaryotic signaling systems, resided outside of the nuclear milieu. It also demonstrated that platelets are far more sophisticated than previously thought despite their anucleate stature.
Hematology 2014
In addition to splicing pre-mRNA, platelets process newly synthesized pro-IL-1 protein into its mature, active form.7,34 Processing of IL-1 protein occurs via the inflammasome, another complex intracellular system recently described in platelets.13 Ultimately, platelets package IL-1 into microparticles that induce endothelial cell permeability.13,34 Recent evidence has also shown that IL-1 is produced at higher levels when platelets are exposed to endotoxin compared with classic platelet agonists, and dengue induces significant amounts of IL-1 synthesis by platelets.8,13 These findings demonstrate that platelets sense infectious stimuli and respond in a discrete fashion to produce active, inflammatory products. In addition to expressing transcripts for Bcl-3 and IL-1, it is now known that platelets possess thousands of mRNAs.12 The advent of next-generation RNA sequencing has shown that mRNAs in platelets have classic features, including capped and polyadenylated tails that control stability and translation.35 The repertoire of mRNAs in platelets is similar among healthy subjects, but levels of expression often differ between individuals. Perhaps the best example of this is a recent publication by Edelstein et al showing that several platelet mRNAs are differently expressed between white and black subjects, including phosphatidylcholine transfer protein (PCTP).36 This group demonstrated that PCTP contributes to racial differences in PAR4-mediated platelet activation, which indicates that the effects of race need to be considered when developing future antiplatelet drugs. It also opens the door for personalized therapeutic regimens in cohorts of subjects with defined risk factors. Platelet mRNA expression analysis has also been used to predict whether genes are conserved between mouse and humans,12 which has proven particularly useful as one considers whether the expense, time, and effort to manipulate a gene in mice will yield interpretable results. Platelet mRNA expression profiling also provides key insights into the types of genes that are being regulated in megakaryocytes,37 especially genes that are actively transcribed in response to environmental cues such as toxins, cytokines, and lipid mediators. In addition to mRNAs, platelets have a very diverse and abundant repertoire of miRNAs.9 Platelet miRNA expression has been particularly useful in predicting how mRNAs are regulated in megakaryocytes.38 It is likely, however, that miRNAs also function in platelets. In support of this, human platelets contain functional pre-miRNA processing machinery, including Dicer and Ago2.38 Platelet miRNAs are also transferred to other cells, as are platelet mRNAs, and both types of RNA continue to function in target cells.39-40 Paracrine delivery of miRNAs and mRNAs to target cells
401
represents yet another fascinating function of platelets that was not previously considered until the last few years.
Summary This brief review highlights several nontraditional functions of platelets, but it is not intended to be a comprehensive review of the literature. Instead, its sole intent is to introduce readers to previously unrecognized functions of platelets that contribute to human disease. These unexpected functions of platelets are reviewed in more detail elsewhere1,14-15 and will continue to evolve and be refined over the coming years. With each discovery, we should expect the unexpected. One thing we can say for sure, however, is that platelets are far more complex than a “sack of glue.”
Disclosures Conflict-of-interest disclosure: The author declares no competing financial interests. Off-label drug use: None disclosed.
Correspondence Andrew S. Weyrich, PhD, Professor of Internal Medicine, Eccles Institute of Human Genetics, Bldg 533, Rm 4220, University of Utah, Salt Lake City, UT 84112; Phone: (801)585-0727; Fax: (801)585-0701; e-mail: [email protected].
References 1. Rondina MT, Weyrich AS, Zimmerman GA. Platelets as cellular effectors of inflammation in vascular diseases. Circ Res. 2013;112(11): 1506-1519. 2. Weyrich AS, Lindemann S, Zimmerman GA. The evolving role of platelets in inflammation. J Thromb Haemost. 2003;1(9):1897-1905. 3. Khandekar G, Kim S, Jagadeeswaran P. Zebrafish thrombocytes: functions and origins. Adv Hematol. 2012;2012:857058. 4. Weyrich AS, Dixon DA, Pabla R, et al. Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets. Proc Natl Acad Sci U S A. 1998;95(10):5556-5561. 5. Spinelli SL, Casey AE, Pollock SJ, et al. Platelets and megakaryocytes contain functional nuclear factor-kappaB. Arterioscler Thromb Vasc Biol. 2010;30(3):591-598. 6. Moraes LA, Spyridon M, Kaiser WJ, et al. Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation. J Thromb Haemost. 2010;8(3):577-587. 7. Denis MM, Tolley ND, Bunting M, et al. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell. 2005; 122(3):379-391. 8. Shashkin PN, Brown GT, Ghosh A, Marathe GK, McIntyre TM. Lipopolysaccharide is a direct agonist for platelet RNA splicing. J Immunol. 2008;181(5):3495-3502. 9. Ple H, Landry P, Benham A, Coarfa C, Gunaratne PH, Provost P. The repertoire and features of human platelet microRNAs. PLoS One. 2012;7(12):e50746. 10. Nagalla S, Shaw C, Kong X, et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood. 2011;117(19):51895197. 11. Simon LM, Edelstein LC, Nagalla S, et al. Human platelet microRNAmRNA networks associated with age and gender revealed by integrated plateletomics. Blood. 2014;123(16):e37-45. 12. Rowley JW, Oler AJ, Tolley ND, et al. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes. Blood. 2011;118(14):e101e111. 13. Hottz ED, Lopes JF, Freitas C, et al. Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation. Blood. 2013;122(20):3405-3414. 14. Semple JW, Italiano JE Jr, Freedman J. Platelets and the immune continuum. Nat Rev Immunol. 2011;11(4):264-274.
402
15. Semple JW, Freedman J. Platelets and innate immunity. Cell Mol Life Sci. 2010;67(4):499-511. 16. Vieira-de-Abreu A, Campbell RA, Weyrich AS, Zimmerman GA. Platelets: versatile effector cells in hemostasis, inflammation, and the immune continuum. Semin Immunopathol. 2012;34(1):5-30. 17. Hidari KI, Weyrich AS, Zimmerman GA, McEver RP. Engagement of P-selectin glycoprotein ligand-1 enhances tyrosine phosphorylation and activates mitogen-activated protein kinases in human neutrophils. J Biol Chem. 1997;272(45):28750-28756. 18. Weyrich AS, Elstad MR, McEver RP, et al. Activated platelets signal chemokine synthesis by human monocytes. J Clin Invest. 1996;97(6): 1525-1534. 19. Li N, Hu H, Lindqvist M, Wikstrom-Jonsson E, Goodall AH, Hjemdahl P. Platelet-leukocyte cross talk in whole blood. Arterioscler Thromb Vasc Biol. 2000;20(12):2702-2708. 20. Zimmerman GA. Two by two: the pairings of P-selectin and P-selectin glycoprotein ligand 1. Proc Natl Acad Sci U S A. 2001;98(18):1002310024. 21. Michelson AD, Barnard MR, Krueger LA, Valeri CR, Furman MI. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation. 2001;104(13):1533-1537. 22. Clark SR, Ma AC, Tavener SA, et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med. 2007;13(4): 463-469. 23. Brinkmann V, Zychlinsky A. Neutrophil extracellular traps: is immunity the second function of chromatin? J Cell Biol. 2012;198(5):773-783. 24. Caudrillier A, Kessenbrock K, Gilliss BM, et al. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. J Clin Invest. 2012;122(7):2661-2671. 25. Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood. 2014;123(18):2768-2776. 26. Hahn S, Giaglis S, Chowdhury CS, Hosli I, Hasler P. Modulation of neutrophil NETosis: interplay between infectious agents and underlying host physiology. Semin Immunopathol. 2013;35(4):439-453. 27. Kraemer BF, Campbell RA, Schwertz H, et al. Novel anti-bacterial activities of beta-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation. PLoS Pathog. 2011;7(11):e1002355. 28. Lehr HA, Weyrich AS, Saetzler RK, et al. Vitamin C blocks inflammatory platelet-activating factor mimetics created by cigarette smoking [see comments]. J Clin Invest. 1997;99(10):2358-2364. 29. Dixon DA, Tolley ND, Bemis-Standoli K, et al. Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling. J Clin Invest. 2006;116(10): 2727-2738. 30. Major HD, Campbell RA, Silver RM, Branch DW, Weyrich AS. Synthesis of sFlt-1 by platelet-monocyte aggregates contributes to the pathogenesis of preeclampsia. Am J Obstet Gynecol. 2014;210(6): 547.e1-e7. 31. Hottz ED, Medeiros-de-Moraes IM, Vieira-de-Abreu A, et al. Platelet activation and apoptosis modulate inflammatory responses in dengue. J Immunol. 2014;193(4):1864-1872. 32. Habets KL, Huizinga TW, Toes RE. Platelets and autoimmunity. Eur J Clin Invest. 2013;43(7):746-757. 33. Weyrich AS, Denis MM, Schwertz H, et al. mTOR-dependent synthesis of Bcl-3 controls the retraction of fibrin clots by activated human platelets. Blood. 2007;109(5):1975-1983. 34. Lindemann S, Tolley ND, Dixon DA, et al. Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis. J Cell Biol. 2001;154(3):485-490. 35. Schubert S, Weyrich AS, Rowley JW. A tour through the transcriptional landscape of platelets. Blood. 2014;124(4):493-502. 36. Edelstein LC, Simon LM, Montoya RT, et al. Racial differences in human platelet PAR4 reactivity reflect expression of PCTP and miR-376c. Nat Med. 2013;19(12):1609-1616. 37. Cecchetti L, Tolley ND, Michetti N, Bury L, Weyrich AS, Gresele P.
American Society of Hematology
Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events. Blood. 2011;118(7):1903-1911. 38. Landry P, Plante I, Ouellet DL, Perron MP, Rousseau G, Provost P. Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol. 2009;16(9):961-966.
Hematology 2014
39. Hulsmans M, Holvoet P. MicroRNA-containing microvesicles regulating inflammation in association with atherosclerotic disease. Cardiovasc Res. 2013;100(1):7-18. 40. Risitano A, Beaulieu LM, Vitseva O, Freedman JE. Platelets and platelet-like particles mediate intercellular RNA transfer. Blood. 2012; 119(26):6288-6295.
403
Platelets: more than a sack of glue Andrew S. Weyrich1 1Molecular
Medicine Program and the Department of Internal Medicine, University of Utah, Salt Lake City, UT
Platelets are primary effector cells in hemostasis. Emerging evidence over the last decade, however, demonstrates that platelets also have critical roles in immunity and inflammation. These nontraditional functions of platelets influence the development, progression, and evolution of numerous diseases, including arthritis, cancer, cardiovascular disease, and infectious syndromes. This chapters reviews recently discovered attributes of platelets that contribute to human disease, paying particular attention to the inflammatory activities of this anucleate cytoplast.
Learning Objective ●
To provide a brief understanding of recently discovered inflammatory functions of platelets that contribute to human disease
Background Since their discovery, platelets have been known for their roles in hemostasis, in which they rapidly bind to damaged blood vessels and prevent excessive bleeding. The hemostatic functions of platelets have been and continue to be the focus of intense investigation, especially in disease situations in which platelets inadvertently occlude vessels that should remain patent. Indeed, drugs that dampen the adhesive functions of platelets are commonplace in the clinical setting because they have proven benefits in the treatment of cardiovascular disease. Because of their primary role in hemostasis, many view platelets as “sacks of glue.” However, emerging evidence demonstrates that platelets are far more complex than previously considered and their intracellular machinery is not used solely for hemostatic activities. The newly recognized inflammatory functions of platelets are the focus of this review.
Phylogeny of platelets The phylogeny of platelets suggests that they are destined to have prominent inflammatory functions. In-depth discussion of how platelets evolved from primitive, multifunctional cells can be found in previous reviews.1-2 In brief, platelets likely evolved from invertebrate cells that have ancient origins in host defense.2 By the vertebrate stage, blood cells adapted to perform more distinct functions. For example, fish, reptiles, and birds have circulating thrombocytes that are phenotypically and functionally similar to platelets.2 Unlike platelets, however, thrombocytes are nucleated cells that regulate hemostatic and inflammatory processes.3 Some of the activities of thrombocytes are driven and controlled by their nucleus. Therefore, one might expect that platelets do not express nuclear components or perform “nuclear-like functions” because they circulate as anucleate cytoplasts. New evidence, however, suggests otherwise. Platelets possess numerous transcription factors that have nongenomic functions,4-6 an active spliceosome,7-8 and an extensive repertoire of miRNA and mRNAs.9-12 Platelets also have diverse inflammatory functions, including a recently described inflammasome function.13 Therefore, platelets are complex cells with highly evolved, nonhemostatic functions.
400
Platelets induce inflammatory responses in leukocytes Platelets have a diverse array of surface receptors that allow them to interact with leukocytes, pathogens, pathogen-released products, and inflamed endothelium.1,14 These interactions contribute to microvascular occlusion, thrombosis, and propagation of inflammatory damage elicited at the vascular wall.1 The reader is referred to recent reviews that detail the types and functions of these receptors in platelet-mediated responses, especially in relation to interactions with bacteria, bacterial-derived toxins, and endothelial cells.1,14-16 Under homeostatic conditions, platelets generally do not bind to leukocytes. However, upon activation, platelets adhere to neutrophils and monocytes and interactions with lymphocytes have also been reported.1,17-18 These heterotypic aggregates have several inflammatory consequences, which may be good or bad depending on the pathologic situation (Figure 1). Although less studied, reciprocal activation of platelets also occurs.19 Interactions of platelets with neutrophils and monocytes have received the most attention in regard to inflammatory responses. As described in previous reviews, binding between the cell types is primarily mediated by P-selectin, which translocates to the surface of activated platelets, and P-selectin glycoprotein-1 (PSGL-1) on target leukocytes, but P-selectin/PSGL-1 interactions tend to be more transient in neutrophils than in monocytes.20 Plateletneutrophil and platelet-monocyte aggregates (PMAs) have been detected in the blood of humans with a variety of diseases1 and are considered one of the most sensitive markers of platelet activation in human whole blood.21 When platelets bind neutrophils, they trigger the release of chemokines and the formation of neutrophil extracellular traps (NETs),17,22 which are lattices of chromatin, histones, and granule enzymes that are intended to capture and kill microbes.23 NETs have known roles in bacterial sepsis, but they are also generated in transfusion-related acute lung injury (TRALI), venous thrombosis, and a variety of other infectious syndromes.24-26 Untimely generation of NETs can also induce vascular injury and trap platelets, which may lead to adverse responses in many disease settings. Lipopolysaccharidestimulated platelets trigger NET formation by mechanisms that remain unresolved and, in TRALI, NETs accumulate in lung microvessels in a platelet-dependent fashion.22,24 Human platelets also trigger NET formation via the release of -defensin 1,27 a potent antimicrobial and signaling molecule.
American Society of Hematology
Figure 1. Platelets are effectors of thrombotic and inflammatory injury. Platelet signaling of leukocytes and/or prolonged newly recognized functions contribute to the pathophysiology of arthritis, cancer, cardiovascular (CV) disease, sepsis, and other clinical syndromes.
Platelets also induce an array of inflammatory responses in monocytes, and targeted disruption of PMAs may have therapeutic value in mitigating thromboinflammation at the vascular wall. Circulating PMAs are elevated in a variety of diseases, and cigarette smoke acutely increases the number of PMAs in the bloodstream.1,28 Several studies have shown that as platelets bind to monocytes, they induce monocyte-derived synthesis of critical cytokines including IL-8, IL-1, cyclooxygenase-2 (COX-2), and monocyte chemotactic protein-1 (MCP-1).18,29 Recent studies also demonstrate that PMAs synthesize soluble Flt-1,30 which has critical functions in preeclampsia, and dengue infection induces the formation of PMAs and synthesis of both proinflammatory and antiinflammatory products.31 Platelets also release a variety of factors that signal leukocytes, including angiogenic factors, chemokines, and CD40L, which is a pivotal immune signaling molecule in the adaptive immune response.32 Taken together, these findings indicate that platelets regulate and amplify responses in leukocytes through a variety of mechanisms that involve direct binding and release of soluble products. These activities, and others, are now widely recognized in the field and have led to the growing appreciation that platelets are critically involved in thrombosis and inflammation.
Platelet RNAs: effectors of cellular function and predictors of disease Although it is not hard to imagine that platelets can induce inflammatory gene expression in other cells, one might not predict that platelets themselves are capable of synthesizing proteins. This was our premise in 1998 when we examined the expression of B cell lymphoma 3 (Bcl-3) in PMAs and unexpectedly found that platelets, not monocytes, synthesize Bcl-3 protein upon activation.4 We subsequently demonstrated that Bcl-3, a presumed transcription factor, regulates cytoskeletal processes including platelet-dependent clot retraction.4,33 Perhaps more importantly, we realized that platelets function for extended periods of time and at least one of their prolonged functions is de novo synthesis of proteins. We have since shown that platelets also synthesize IL-1, a proinflammatory cytokine.34 Synthesis of IL-1 is controlled at many steps, starting first with splicing of IL-1 pre-mRNA into mature message.7 Demonstration that platelets splice pre-mRNAs provided the first example that an active spliceosome, one of the most complex eukaryotic signaling systems, resided outside of the nuclear milieu. It also demonstrated that platelets are far more sophisticated than previously thought despite their anucleate stature.
Hematology 2014
In addition to splicing pre-mRNA, platelets process newly synthesized pro-IL-1 protein into its mature, active form.7,34 Processing of IL-1 protein occurs via the inflammasome, another complex intracellular system recently described in platelets.13 Ultimately, platelets package IL-1 into microparticles that induce endothelial cell permeability.13,34 Recent evidence has also shown that IL-1 is produced at higher levels when platelets are exposed to endotoxin compared with classic platelet agonists, and dengue induces significant amounts of IL-1 synthesis by platelets.8,13 These findings demonstrate that platelets sense infectious stimuli and respond in a discrete fashion to produce active, inflammatory products. In addition to expressing transcripts for Bcl-3 and IL-1, it is now known that platelets possess thousands of mRNAs.12 The advent of next-generation RNA sequencing has shown that mRNAs in platelets have classic features, including capped and polyadenylated tails that control stability and translation.35 The repertoire of mRNAs in platelets is similar among healthy subjects, but levels of expression often differ between individuals. Perhaps the best example of this is a recent publication by Edelstein et al showing that several platelet mRNAs are differently expressed between white and black subjects, including phosphatidylcholine transfer protein (PCTP).36 This group demonstrated that PCTP contributes to racial differences in PAR4-mediated platelet activation, which indicates that the effects of race need to be considered when developing future antiplatelet drugs. It also opens the door for personalized therapeutic regimens in cohorts of subjects with defined risk factors. Platelet mRNA expression analysis has also been used to predict whether genes are conserved between mouse and humans,12 which has proven particularly useful as one considers whether the expense, time, and effort to manipulate a gene in mice will yield interpretable results. Platelet mRNA expression profiling also provides key insights into the types of genes that are being regulated in megakaryocytes,37 especially genes that are actively transcribed in response to environmental cues such as toxins, cytokines, and lipid mediators. In addition to mRNAs, platelets have a very diverse and abundant repertoire of miRNAs.9 Platelet miRNA expression has been particularly useful in predicting how mRNAs are regulated in megakaryocytes.38 It is likely, however, that miRNAs also function in platelets. In support of this, human platelets contain functional pre-miRNA processing machinery, including Dicer and Ago2.38 Platelet miRNAs are also transferred to other cells, as are platelet mRNAs, and both types of RNA continue to function in target cells.39-40 Paracrine delivery of miRNAs and mRNAs to target cells
401
represents yet another fascinating function of platelets that was not previously considered until the last few years.
Summary This brief review highlights several nontraditional functions of platelets, but it is not intended to be a comprehensive review of the literature. Instead, its sole intent is to introduce readers to previously unrecognized functions of platelets that contribute to human disease. These unexpected functions of platelets are reviewed in more detail elsewhere1,14-15 and will continue to evolve and be refined over the coming years. With each discovery, we should expect the unexpected. One thing we can say for sure, however, is that platelets are far more complex than a “sack of glue.”
Disclosures Conflict-of-interest disclosure: The author declares no competing financial interests. Off-label drug use: None disclosed.
Correspondence Andrew S. Weyrich, PhD, Professor of Internal Medicine, Eccles Institute of Human Genetics, Bldg 533, Rm 4220, University of Utah, Salt Lake City, UT 84112; Phone: (801)585-0727; Fax: (801)585-0701; e-mail: [email protected].
References 1. Rondina MT, Weyrich AS, Zimmerman GA. Platelets as cellular effectors of inflammation in vascular diseases. Circ Res. 2013;112(11): 1506-1519. 2. Weyrich AS, Lindemann S, Zimmerman GA. The evolving role of platelets in inflammation. J Thromb Haemost. 2003;1(9):1897-1905. 3. Khandekar G, Kim S, Jagadeeswaran P. Zebrafish thrombocytes: functions and origins. Adv Hematol. 2012;2012:857058. 4. Weyrich AS, Dixon DA, Pabla R, et al. Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets. Proc Natl Acad Sci U S A. 1998;95(10):5556-5561. 5. Spinelli SL, Casey AE, Pollock SJ, et al. Platelets and megakaryocytes contain functional nuclear factor-kappaB. Arterioscler Thromb Vasc Biol. 2010;30(3):591-598. 6. Moraes LA, Spyridon M, Kaiser WJ, et al. Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation. J Thromb Haemost. 2010;8(3):577-587. 7. Denis MM, Tolley ND, Bunting M, et al. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell. 2005; 122(3):379-391. 8. Shashkin PN, Brown GT, Ghosh A, Marathe GK, McIntyre TM. Lipopolysaccharide is a direct agonist for platelet RNA splicing. J Immunol. 2008;181(5):3495-3502. 9. Ple H, Landry P, Benham A, Coarfa C, Gunaratne PH, Provost P. The repertoire and features of human platelet microRNAs. PLoS One. 2012;7(12):e50746. 10. Nagalla S, Shaw C, Kong X, et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood. 2011;117(19):51895197. 11. Simon LM, Edelstein LC, Nagalla S, et al. Human platelet microRNAmRNA networks associated with age and gender revealed by integrated plateletomics. Blood. 2014;123(16):e37-45. 12. Rowley JW, Oler AJ, Tolley ND, et al. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes. Blood. 2011;118(14):e101e111. 13. Hottz ED, Lopes JF, Freitas C, et al. Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation. Blood. 2013;122(20):3405-3414. 14. Semple JW, Italiano JE Jr, Freedman J. Platelets and the immune continuum. Nat Rev Immunol. 2011;11(4):264-274.
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