Model systems of genetically modified platelets

Abstract

Although platelets are the smallest cells in the blood, they are implied in various processes ranging from immunology and oncology to thrombosis and hemostasis. Many large-scale screening programs, genome-wide association, and "omics" studies have generated lists of genes and loci that are probably involved in the formation or physiology of platelets under normal and pathologic conditions. This creates an increasing demand for new and improved model systems that allow functional assessment of the corresponding gene products in vivo. Such animal models not only render invaluable insight in the platelet biology, but in addition, provide improved test systems for the validation of newly developed antithrombotics. This review summarizes the most important models to generate transgenic platelets and to study their influence on platelet physiology in vivo. Here we focus on the zebrafish morpholino oligonucleotide technology, the (platelet- specific IntroductionBlood platelets play part in a myriad of processes, such as inflammation, tumor growth and metastasis, immunology and, of course, thrombosis and blood clotting where they provide a first and crucial line of defense against vascular injury, thus maintaining normal hemostasis. 1,2 Primary hemostasis starts when platelets recognize a site of vascular injury where the subendothelial matrix is exposed, bind to collagen, and become activated. 3 The subsequent rise in intracellular calcium triggers conformational changes in integrin receptors, degranulation, exposition of a procoagulant surface, and generation and release of secondary agonists resulting in a thrombus that will cover the site of injury and prevent further blood loss. 4 Platelets are furthermore an important factor in thrombotic events, such as stroke and myocardial infarction. 5 To identify more proteins regulating platelet function that may serve as new targets for the development of anti-thrombotics or in the prevention of bleeding, the platelet research community has seen the completion of several large-scale screening programs and the spectacular rise in the "platelet-omics" field. Several genome-wide association studies and subsequent meta-analysis in patients with coronary artery disease and healthy volunteers identified numerous genetic loci that are possibly involved in regulating platelet formation, count, volume, and function and might confer a risk for coronary artery disease. [6][7][8][9][10][11] On the other hand, gene expression profiling of healthy volunteer platelets, in combination with comparative microarray analysis between in vitro differentiated megakaryocytes (MKs) and closely related cell types, established a comprehensive platelet transcriptome. 6,[12][13][14][15][16][17][18][19] Finally, advanced proteomics studies identified proteins of the platelet sheddome, secretome, interactome, kinome, and phosphoproteome potentially involved in platelet function. 20 The overall result is a large number of newly identified gene products for which we are only beginning to understand their ...

FAK regulates platelet extravasation and tumor growth after antiangiogenic therapy withdrawal

“…Pertuy et al found that 0.3% of CD45 + leukocytes as well as a small percentage of intestinal epithelial cells showed expression of Pf4. Despite these results, the authors confirmed that the Pf4-Cre system is extremely valuable and a powerful instrument to study the function of megakaryocytes and platelets in diverse physiological and pathological settings, as also shown by others (58 with regard to tumor growth and tumor rebound after withdrawal of antiangiogenic therapy were found using the FAK inhibitor GSK2256098, which combined well when given together with the antiangiogenic agents pazopanib or bevacizumab. GSK2256098 is a highly specific small-molecule inhibitor for FAK, both for humans and mice, and has been designed to block FAK Y397 phosphorylation which is necessary for kinase activation (59).…”

Simple and Rapid Quantification of Thrombocytes in Zebrafish Larvae

“…1 Defects in platelet number or function often result in primary hemostatic disorders or thrombotic events such as myocardial infarction and stroke. 2,3 A significant body of research has been dedicated to the identification of proteins that regulate platelet number and function with the goal of developing therapies for affected individuals. Through genome-wide association and proteomic studies, a number of genes have been recognized to play a role in platelet activity.…”

“…3,8,9 ). Unlike mammals, zebrafish thrombocytes are nucleated, but they share structural and functional features with platelets.…”

“…This strain expresses green fluorescent protein (GFP) under regulation of the cd41 promoter, and it has been used for multiple studies that have led to novel insights into platelet function. 3,8,9 Quantitative analysis of thrombocytes has relied on in vivo or ex vivo flow cytometry 14,15 or peripheral blood smears. 10 However, these methods usually require euthanasia for blood collection and/or timeconsuming and expensive analyses.…”

Animal Models of Thrombosis From Zebrafish to Nonhuman Primates

“…[138][139][140][141][142] Not only are these zebrafish genes syntenic to human genes, functional assays have also shown that extrinsic and intrinsic coagulation factors and several platelet surface receptors are also present in zebrafish blood or thrombocytes. 143,144 Zebrafish have nucleated thrombocytes which are functionally equivalent to enucleated platelets in mammals. 145 Similarly, the vascular endothelium in zebrafish possesses several factors found in human endothelial cells.…”