Genetic analysis of thrombosis and haemostasis in zebrafish

Platelets are a central cell in the process of haemostasis, that mechanism that the body employs to stop you bleeding when you cut yourself. The role platelets play in this process is to stick to the cut blood vessel and then to each other, to form a plug to stop the bleeding as rapidly as possible. In order to do this platelets have very active mechanisms to increase their stickiness (adhesion) and to spread rapidly over a wide surface area, to effectively form a living elastoplast over the wound site. These events involve the activation of multiple signals in the cells and switching on of adhesion receptors and massive regulated rearrangements of the cellular architecture (cytoskeleton). Our laboratyr is trying to determine the molecules that underlie and mediate these dramatic functional events. One approach that has yielded a tremendous amount of data over the past 5 years or so is an approach called proteomics. For platelets this has revealed the identity of a huge number of proteins in the cell, but leaves the question: what do they all do? This question of function of proteins, and the genes that encode them, is a real problem for biology at present because of the massive amounts of data that may be derived from both proteomics and genomics. As a laboratory interested in platelet signalling and cell biology we use a variety of technical approaches to address these questions, but each have their limitations. The zebrafish represents an excellent, very powerful and rapid way to analyse the functioning of novel genes in regulating haemostasis and platelet function in general. The reason for this is that the zebrafish is much easier to modify genetically than mammalian organisms, and it is therefore possible rapidly to modulate genes and assess the effect that has on various cellular functions in the animal, including haemostasis. As a laboratory we currently have no zebrafish model established, but can see the enormous potential this system would have for rapid functional screening of genes that may regulate haemostasis. In this proposal we intend to establish the various protocols necessary to use this approach in zebrafish, and address the hypothesis that 4 different genes we have identified from previous studies may or may not play a significant role in regulating thrombocyte activation and haemostasis in vivo. The work will therefore establish the approach here in Bristol and form the basis of a larger and more ambitious grant proposal in 18 months time.

This Fellowship application is to establish a zebrafish model system within my laboratory for analysis of genes that regulate haemostasis and thrombus formation in vivo and thrombocyte function in vitro. This is a new approach for our laboratory, and indeed is set up for haemostatic analysis in only a handful of laboratories worldwide, although the numbers are increasing rapidly. The value and power of the zebrafish model centres upon its great genetic tractability and the ease with which cellular processes, such as haemostasis, may be visualised in vivo due to the translucency of its tissues. It therefore adds an excellent new dimension of functional analysis of genes to our laboratory, and in particular feeds directly from data derived from proteomic studies of platelets. In this Fellowship I propose taking 18 months to develop the techniques to generate zebrafish by in vitro fertilisation, microinject embryos with morpholino antisense oligonucleotides, analyse haemostatic function in vivo in young larvae by a vascular occlusion approach, take blood from adult zebrafish and establish several assays of thrombocyte functionality in vitro. The proposal will address the function of 4 proteins found in a previous proteomic study to interact with the tyrosine phosphatase SHP-1: coronin 1C, b-parvin, Rab39b and WASp. This will provide sufficient data to submit a full grant application in 18 months, to follow up function of related and other proteins found by our ongoing proteomic analyses to be substrates either of SHP-1 phsophatase or of protein kinase C isoforms in vitro, which we are presently performing by a chemical genetic approach. I envisage therefore that the zebrafish model will form an invaluable part of the phenotypic analysis of genes identified in human platelet proteomic studies, and will complement the mouse model systems human platelet cell biological analyses that we already undertake.