Platelets play an important role in mammalian hemostasis. Thrombocytes of early vertebrates are functionally equivalent to mammalian platelets. A substantial amount of research has been done to study platelet function in humans as well as in animal models. However, to date only limited functional genomic studies of platelets have been performed but are low throughput and are not cost-effective. Keeping this in mind we introduced zebrafish, a vertebrate genetic model to study platelet function. We characterized zebrafish thrombocytes and established functional assays study not only their hemostatic function but to also their production. We identified a few genes which play a role in their function and production. Since we introduced the zebrafish model for the study of hemostasis and thrombosis, other groups have adapted this model to study genes that are associated with thrombocyte function and a few novel genes have also been identified. Furthermore, transgenic zebrafish with GFP-tagged thrombocytes have been developed which helped to study the production of thrombocytes and their precursors as well as their functional roles not only in hemostasis but also hematopoiesis. This paper integrates the information available on zebrafish thrombocyte function and its formation. 1. Introduction Hemostasis is a defense mechanism to prevent loss of blood in the event of an injury in an organism that has a vasculature [1]. It consists of the platelet response to injury which results in platelet aggregation and plugging the wound, termed primary hemostasis, followed by the interplay of a complex cascade of coagulation factors on the platelet surface ultimately resulting in a fibrin clot, termed secondary hemostasis. After their primary hemostatic function platelets, also repair the damaged endothelium [2]. In primary hemostasis platelets adhere to collagen in the subendothelial matrix in response to injury and are subsequently activated by a complex signaling cascade resulting in secretion of their granular contents. These contents also result in the amplification of platelet aggregation at the site of injury and formation of a platelet plug which is stabilized further with help of fibrin [1]. This hemostatic plug prevents loss of blood from the site of injury. Thus, platelets that play a role in hemostasis and defects in platelet function have been shown to be involved in bleeding disorders as well as many pathophysiological conditions like thrombosis, inflammation, and even cancer [3]. Platelets have a number of receptors on their membrane surface that help
References
[1]
P. Jagadeeswaran, M. Gregory, K. Day, M. Cykowski, and B. Thattaliyath, “Zebrafish: a genetic model for hemostasis and thrombosis,” Journal of Thrombosis and Haemostasis, vol. 3, no. 1, pp. 46–53, 2005.
[2]
G. Davì and C. Patrono, “Mechanisms of disease: platelet activation and atherothrombosis,” The New England Journal of Medicine, vol. 357, no. 24, pp. 2482–2494, 2007.
[3]
P. Harrison, “Platelet function analysis,” Blood Reviews, vol. 19, no. 2, pp. 111–123, 2005.
[4]
R. J. Westrick, M. E. Winn, and D. T. Eitzman, “Murine models of vascular thrombosis (Eitzman series),” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 27, no. 10, pp. 2079–2093, 2007.
[5]
K. D. Mason, M. R. Carpinelli, J. I. Fletcher et al., “Programmed anuclear cell death delimits platelet life span,” Cell, vol. 128, no. 6, pp. 1173–1186, 2007.
[6]
C. I. Jones, S. Bray, S. F. Garner et al., “A functional genomics approach reveals novel quantitative trait loci associated with platelet signaling pathways,” Blood, vol. 114, no. 7, pp. 1405–1416, 2009.
[7]
T. Thijs, H. Deckmyn, and K. Broos, “Model systems of genetically modified platelets,” Blood, vol. 119, no. 7, pp. 1634–1642, 2012.
[8]
D. Carradice and G. J. Lieschke, “Zebrafish in hematology: sushi or science?” Blood, vol. 111, no. 7, pp. 3331–3342, 2008.
[9]
L. Solnica-Krezel, A. F. Schier, and W. Driever, “Efficient recovery of ENU-induced mutations from the zebrafish germline,” Genetics, vol. 136, no. 4, pp. 1401–1420, 1994.
[10]
G. Streisinger, C. Walker, and N. Dower, “Production of clones of homozygous diploid zebra fish (BrachyDanio rerio),” Nature, vol. 291, no. 5813, pp. 293–296, 1981.
[11]
M. N. O'Connor, I. I. Salles, A. Cvejic et al., “Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins,” Blood, vol. 113, no. 19, pp. 4754–4762, 2009.
[12]
P. Jagadeeswaran, J. P. Sheehan, F. E. Craig, and D. Troyer, “Identification and characterization of zebrafish thrombocytes,” British Journal of Haematology, vol. 107, no. 4, pp. 731–738, 1999.
[13]
T. Grosser, S. Yusuff, E. Cheskis, et al., “Developmental expression of functional cyclooxygenases in zebrafish,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 12, pp. 8418–8423, 2002.
[14]
S. Kim, M. Carrillo, V. Kulkarni, and P. Jagadeeswaran, “Evolution of primary hemostasis in early vertebrates,” PLoS ONE, vol. 4, no. 12, article e8403, 2009.
[15]
M. Carrillo, S. Kim, S. K. Rajpurohit, V. Kulkarni, and P. Jagadeeswaran, “Zebrafish von Willebrand factor,” Blood Cells, Molecules, and Diseases, vol. 45, no. 4, pp. 326–333, 2010.
[16]
M. R. Lang, G. Gihr, M. P. Gawaz, and I. Müller, “Hemostasis in Danio rerio: is the zebrafish a useful model for platelet research?” Journal of Thrombosis and Haemostasis, vol. 8, no. 6, pp. 1159–1169, 2010.
[17]
M. Gregory, R. Hanumanthaiah, and P. Jagadeeswaran, “Genetic analysis of hemostasis and thrombosis using vascular occlusion,” Blood Cells, Molecules & Diseases, vol. 29, no. 3, pp. 286–295, 2002.
[18]
P. Jagadeeswaran, M. Carrillo, U. P. Radhakrishnan, S. K. Rajpurohit, and S. Kim, “Laser-induced thrombosis in Zebrafish,” Methods in Cell Biology, vol. 101, pp. 197–203, 2011.
[19]
P. Jagadeeswaran, M. Cykowski, and B. Thattaliyath, “Vascular occlusion and thrombosis in zebrafish,” Methods in Cell Biology, vol. 76, pp. 489–500, 2004.
[20]
P. Jagadeeswaran, Y. C. Liu, and J. P. Sheehan, “Analysis of hemostasis in the zebrafish,” Methods in Cell Biology, no. 59, pp. 337–357, 1999.
[21]
P. Jagadeeswaran, R. Paris, and P. Rao, “Laser-induced thrombosis in zebrafish larvae: a novel genetic screening method for thrombosis,” Methods in Molecular Medicine, vol. 129, pp. 187–195, 2006.
[22]
M. Gregory and P. Jagadeeswaran, “Selective labeling of zebrafish thrombocytes: quantitation of thrombocyte function and detection during development,” Blood Cells, Molecules, and Diseases, vol. 28, no. 3, pp. 418–427, 2002.
[23]
B. Thattaliyath, M. Cykowski, and P. Jagadeeswaran, “Young thrombocytes initiate the formation of arterial thrombi in zebrafish,” Blood, vol. 106, no. 1, pp. 118–124, 2005.
[24]
P. Jagadeeswaran, S. Lin, B. Weinstein, A. Hutson, and S. Kim, “Loss of GATA1 and gain of FLI1 expression during thrombocyte maturation,” Blood Cells, Molecules, and Diseases, vol. 44, no. 3, pp. 175–180, 2010.
[25]
R. J. Berckmans, R. Nieuwland, A. N. B?ing, F. P. H. T. M. Romijn, C. E. Hack, and A. Sturk, “Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation,” Thrombosis and Haemostasis, vol. 85, no. 4, pp. 639–646, 2001.
[26]
D. E. Connor, T. Exner, D. D. F. Ma, and J. E. Joseph, “The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib,” Thrombosis and Haemostasis, vol. 103, no. 5, pp. 1044–1052, 2010.
[27]
A. Tesse, M. C. Martínez, F. Meziani et al., “Origin and biological significance of shed-membrane microparticles,” Endocrine, Metabolic and Immune Disorders, vol. 6, no. 3, pp. 287–294, 2006.
[28]
M. C. Martínez, A. Tesse, F. Zobairi, and R. Andriantsitohaina, “Shed membrane microparticles from circulating and vascular cells in regulating vascular function,” American Journal of Physiology, vol. 288, no. 3, pp. H1004–H1009, 2005.
[29]
M. Merten, R. Pakala, P. Thiagarajan, and C. R. Benedict, “Platelet microparticles promote platelet interaction with subendothelial matrix in a glycoprotein IIb/IIIa-dependent mechanism,” Circulation, vol. 99, no. 19, pp. 2577–2582, 1999.
[30]
R. Nieuwland, R. J. Berckmans, S. McGregor et al., “Cellular origin and procoagulant properties of microparticles in meningococcal sepsis,” Blood, vol. 95, no. 3, pp. 930–935, 2000.
[31]
P. A. Holme, N. O. Solum, F. Brosstad, M. Roger, and M. Abdelnoor, “Demonstration of platelet-derived microvesicles in blood from patients with activated coagulation and fibrinolysis using a filtration technique and Western blotting,” Thrombosis and Haemostasis, vol. 72, no. 5, pp. 666–671, 1994.
[32]
M. Gawaz, F. J. Neumann, I. Ott, A. Schiessler, and A. Sch?mig, “Platelet function in acute myocardial infarction treated with direct angioplasty,” Circulation, vol. 93, no. 2, pp. 229–237, 1996.
[33]
S. Kim, M. Carrillo, U. P. Radhakrishnan, and P. Jagadeeswaran, “Role of thrombocyte and non-thrombocyte microparticles in hemostasis,” Blood Cells Molecules and Diseases, vol. 48, no. 3, pp. 188–196, 2012.
[34]
R. Bahadori, O. Rinner, H. B. Schonthaler et al., “The zebrafish fade out mutant: a novel genetic model for Hermansky-Pudlak syndrome,” Investigative Ophthalmology and Visual Science, vol. 47, no. 10, pp. 4523–4531, 2006.
[35]
A. Nasevicius and S. C. Ekker, “Effective targeted gene “knockdown” in zebrafish,” Nature Genetics, vol. 26, no. 2, pp. 216–220, 2000.
[36]
K. Day, N. Krishnegowda, and P. Jagadeeswaran, “Knockdown of prothrombin in zebrafish,” Blood Cells, Molecules, and Diseases, vol. 32, no. 1, pp. 191–198, 2004.
[37]
A. Srivastava, et al., “The role of acvr1, ift122, poldip2 and ripk5 in zebrafish hemostasis,” in Proceedings of the International Conference for Zebrafish Development and Genetics, p. 577, Madison, Wis,USA, 2008.
[38]
P. Jagadeeswaran and P. Rao, “Role of KIAA0472, a Novel Ser/Thr Kinase in zebrafish thrombosis,” in Proceedings of the International Conference on Zebrafish Development and Genetics, Madison, Wis, USA, 2006.
[39]
E. Tournoij, G. J. Weber, J. W. N. Akkerman et al., “Mlck1a is expressed in zebrafish thrombocytes and is an essential component of thrombus formation,” Journal of Thrombosis and Haemostasis, vol. 8, no. 3, pp. 588–595, 2010.
[40]
C. M. Williams, et al., “Protein kinase C alpha and beta are positive regulators of thrombus formation in vivo in a zebrafish (Danio rerio) model of thrombosis,” Journal of Thrombosis and Haemostasis, vol. 9, no. 12, pp. 2457–2465, 2011.
[41]
C. M. Williams and A. W. Poole, “Using zebrafish (Danio rerio) to assess gene function in thrombus formation,” Methods in Molecular Biology, vol. 788, pp. 305–319, 2012.
[42]
S. Kim, U. P. Radhakrishnan, S. K. Rajpurohit, V. Kulkarni, and P. Jagadeeswaran, “Vivo-Morpholino knockdown of αIIb: a novel approach to inhibit thrombocyte function in adult zebrafish,” Blood Cells, Molecules, and Diseases, vol. 44, no. 3, pp. 169–174, 2010.
[43]
P. Jagadeeswaran, M. Gregory, S. Johnson, and B. Thankavel, “Haemostatic screening and identification of zebrafish mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes in man,” British Journal of Haematology, vol. 110, no. 4, pp. 946–956, 2000.
[44]
V. Kulkarni, et al., “Separation of young and mature thrombocytes by a novel immuno-selection method,” Blood Cells Molecules and Diseases, vol. 48, no. 3, pp. 183–187, 2012.
[45]
J. F. Amatruda and L. I. Zon, “Dissecting hematopoiesis and disease using the zebrafish,” Developmental Biology, vol. 216, no. 1, pp. 1–15, 1999.
[46]
A. J. Davidson and L. I. Zon, “The “definitive” (and “primitive”) guide to zebrafish hematopoiesis,” Oncogene, vol. 23, no. 43, pp. 7233–7246, 2004.
[47]
H. Lin, et al., “Production and characterization of transgenic zebrafish (Danio rario) with fluorescent thrombocytes and thrombocyte precursors,” Blood, vol. 98, p. 2147, 2001.
[48]
H. F. Lin, D. Traver, H. Zhu et al., “Analysis of thrombocyte development in CD41-GFP transgenic zebrafish,” Blood, vol. 106, no. 12, pp. 3803–3810, 2005.
[49]
J. Y. Bertrand, A. D. Kim, S. Teng, and D. Traver, “CD41+ cmyb+ precursors colonize the zebrafish pronephros by a novel migration route to initiate adult hematopoiesis,” Development, vol. 135, no. 10, pp. 1853–1862, 2008.
[50]
K. Kissa, E. Murayama, A. Zapata et al., “Live imaging of emerging hematopoietic stem cells and early thymus colonization,” Blood, vol. 111, no. 3, pp. 1147–1156, 2008.
[51]
D. Ma, J. Zhang, H. F. Lin, J. Italiano, and R. I. Handin, “The identification and characterization of zebrafish hematopoietic stem cells,” Blood, vol. 118, no. 2, pp. 289–297, 2011.
[52]
M. L. Kalev-Zylinska, J. A. Horsfield, M. V. C. Flores et al., “Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX-1-CBF2T1 transgene advances a model for studies of leukemogenesis,” Development, vol. 129, no. 8, pp. 2015–2030, 2002.
[53]
R. Sood, M. A. English, C. L. Belele et al., “Development of multilineage adult hematopoiesis in the zebrafish with a runx1 truncation mutation,” Blood, vol. 115, no. 14, pp. 2806–2809, 2010.
[54]
Y. Zhang, et al., “cMyb regulates hematopoietic stem/progenitor cell mobilization during zebrafish hematopoiesis,” Blood, vol. 118, no. 15, pp. 4093–4101, 2011.
[55]
C. Grabher, E. M. Payne, A. B. Johnston et al., “Zebrafish microRNA-126 determines hematopoietic cell fate through c-Myb,” Leukemia, vol. 25, no. 3, pp. 506–514, 2011.
[56]
A. G. Muntean and J. D. Crispino, “Differential requirements for the activation domain and FOG-interaction surface of GATA-1 in megakaryocyte gene expression and development,” Blood, vol. 106, no. 4, pp. 1223–1231, 2005.
[57]
J. D. Amigo, G. E. Ackermann, J. J. Cope et al., “The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish,” Blood, vol. 114, no. 21, pp. 4654–4663, 2009.
[58]
H. J. Johnson, M. J. Gandhi, E. Shafizadeh et al., “In vivo inactivation of MASTL kinase results in thrombocytopenia,” Experimental Hematology, vol. 37, no. 8, pp. 901–908, 2009.
[59]
M. R. Tijssen, A. Cvejic, A. Joshi et al., “Genome-wide analysis of simultaneous GATA1/2, RUNX1, FLI1, and SCL binding in megakaryocytes identifies hematopoietic regulators,” Developmental Cell, vol. 20, no. 5, pp. 597–609, 2011.
[60]
C. Gieger, et al., “New gene functions in megakaryopoiesis and platelet formation,” Nature, vol. 480, no. 7376, pp. 201–208, 2011.
[61]
C. A. Albers, A. Cvejic, R. Favier et al., “Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome,” Nature Genetics, vol. 43, no. 8, pp. 735–737, 2011.
[62]
S. Louwette, V. Labarque, C. Wittevrongel et al., “Regulator of G-protein signaling 18 controls megakaryopoiesis and the cilia-mediated vertebrate mechanosensory system,” The FASEB Journal, vol. 26, no. 5, pp. 2125–2136, 2012.
[63]
J. Kulinski, D. Besack, C. A. Oleykowski, A. K. Godwin, and A. T. Yeung, “CEL I enzymatic mutation detection assay,” BioTechniques, vol. 29, no. 1, pp. 44–48, 2000.
[64]
E. Wienholds, F. van Eeden, M. Kosters, J. Mudde, R. H. A. Plasterk, and E. Cuppen, “Efficient target-selected mutagenesis in zebrafish,” Genome Research, vol. 13, no. 12, pp. 2700–2707, 2003.
[65]
Y. Doyon, J. M. McCammon, J. C. Miller et al., “Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases,” Nature Biotechnology, vol. 26, no. 6, pp. 702–708, 2008.
[66]
S. C. Ekker, “Zinc finger-based knockout punches for zebrafish genes,” Zebrafish, vol. 5, no. 2, pp. 121–123, 2008.
[67]
K. J. Clark, D. F. Voytas, and S. C. Ekker, “A TALE of two nucleases: gene targeting for the masses?” Zebrafish, vol. 8, no. 3, pp. 147–149, 2011.
[68]
D. Caroll and B. Zhang, “Primer and interviews: advances in targeted gene modification,” Developmental Dynamics, vol. 240, no. 12, pp. 2688–96, 2011.