Platelet GPVI is a pivotal receptor that initiates adhesion to collagen and propagates thrombus formation in vivo in both mice and men. A significant variability in GPVI expression and/or activity has been noted in both species. Elimination of the mouse gene Gp6 causes a uniform and expected defect in platelet responses to collagen in vitro, but on a mixed background, the in vivo phenotype is dichotomous, with either normal thrombus formation or severely impaired hemostasis. Using these """"""""sensitized"""""""" mice, I have identified a single dominant locus, Modifier of hemostasis (MH), which is in linkage with the bleeding phenotype and maps to an 8 Mb region of chromosome 4. I propose that one or more modifier genes in MH regulate the in vivo phenotypes. To identify the modifier genes in this locus, I propose in Specific Aim 1.1, to positionally clone modifier gene(s) contributing to the in vivo phenotypes, through recombination cloning. Having obtained preliminary data that one of the candidate genes may be Klf4, the gene for Kruppel-like Factor 4, I have designed a strategy to evaluate in vivo the relevance of endothelial expression of Klf4 and any additional candidate genes that are identified. For this reason, Specific Aim 1.2 is to develop transgenic mice in which the relevance of the candidate modifier gene(s) can be assessed in vivo. These in vivo models will enable inducible (tetON), conditional (endothelial cell) knockdown or over expression of Klf4, as the prototype target. The models are adaptable to other relevant vascular cell types, such as smooth muscle cells or fibroblasts.
In Specific Aim 2, I propose to use these transgenic mice to assess the relevance of the modifier gene products using in vivo thrombosis models. Emphasis will be placed on the results of the carotid artery injury model, with which I have the most experience, but alternative models can also be employed. The characterization of MH and the composite genes responsible for the bleeding/thrombotic phenotype will make a significant contribution to our understanding of the in vivo regulation of hemostasis. GPVI and other platelet receptors are implicated in many cases of human thrombosis. However, the severity of the disease phenotype is variable, suggesting the existence of modifying gene traits. Identification of genes that can modify the incidence and severity of thrombotic disease is of significant value to general hematology and cardiovascular disease.
Blood platelets are the smallest cellular component of the blood and are responsible for the formation of clots in humans and most animals, including mice. When working on mice for research purposes, we have the ability to add or remove genes and to thus find out how these genes influence the ability of platelets to form clots. This information is potentially valuable to our understanding of risk for and prevention of coronary artery disease and stroke in humans. I will take advantage of the mouse to remove one of the key genes that we believe controls platelet stickiness, and then I will add back into the same mice either the same human gene or a modified mouse gene. Then, when the appropriate system is in place, I can chemically induce an increase or a decrease in either the mouse or the human gene in a specific cell in the whole animal and measure the effect that this gene manipulation has on the formation of clots in that live mouse. The severity of coronary artery disease and stroke in man is variable, suggesting that there are differences between individuals in these modifying genes. It is my intention to discover gene differences in the mouse that are shared by humans and will help us predict the risk for or even prevent coronary artery disease or stroke in man.
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|Kunicki, Thomas J; Williams, Shirley A; Nugent, Diane J (2012) Genetic variants that affect platelet function. Curr Opin Hematol 19:371-9|
|Kunicki, Thomas J; Williams, Shirley A; Diaz, Daniel et al. (2012) Platelet adhesion to decorin but not collagen I correlates with the integrin ?2 dimorphism E534K, the basis of the human platelet alloantigen (HPA)-5 system. Haematologica 97:692-5|
|Cheli, Yann; Williams, Shirley A; Ballotti, Robert et al. (2010) Enhanced binding of poly(ADP-ribose)polymerase-1 and Ku80/70 to the ITGA2 promoter via an extended cytosine-adenosine repeat. PLoS One 5:e8743|
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|Trifiro, Elisabetta; Williams, Shirley A; Cheli, Yann et al. (2009) The low-frequency isoform of platelet glycoprotein VIb attenuates ligand-mediated signal transduction but not receptor expression or ligand binding. Blood 114:1893-9|
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