Thrombosis (intravascular blood clots) is a major cause of morbidity and mortality in most areas of the world. Thrombus formation occurs in both arteries and veins and is a dynamic process. Both the development (blood coagulation) and dissolution (fibrinolysis) of thrombi must be exquisitely controlled to prevent either severe bleeding or thrombosis. Elevated levels of an important fibrinolysis inhibitor, plasminogen activator inhibitor-1 (PAI-1, gene name: Serpine1) in plasma have been associated with both venous thrombosis and myocardial infarction. Intra-individual variations in platelet PAI-1 levels could have a major impact on thrombotic disease. We have previously found that an occlusive arterial thrombus could not be formed in a mouse model with complete PAI-1 deficiency. Over 90% of circulating PAI-1 is located in the platelet alpha granules. We measured platelet PAI-1 antigen levels among 12 inbred mouse strains. The C57BL/6J mouse strain has essentially no platelet PAI-1, while the LEWES/EiJ strain has ~100 fold higher platelet PAI-1 than the C57BL/6J strain. We used crosses of the LEWES/EiJ and C57BL/6J strains to generate 24 genetically informative F2 progeny and through QTL analysis identified a major locus for the regulation of platelet PAI-1 levels. This 14.4 megabase region on chromosome 5 contains the PAI-1 gene. We hypothesize that a cis-regulatory expression variant is responsible for controlling platelet PAI-1 expression (pPAI1High). In the studies below, we propose to identify this expression variant through the analysis of additional F2 mice and the production of genome- edited mice for validation of the identified candidate variant(s). We will also determine whether this expression variant affects platelet specific genes other than PAI-1 by analyzing the platelet transcriptomes of LEWES/EiJ and C57BL/6J mice. We have also determined that there are differences in PAI-1 expression and antigen levels in liver, heart and adipose tissue in LEWES/EiJ compared to C57BL/6J. These differences could be due to the same or different expression variants as those controlling platelet PAI-1. Our hypothesis is that endothelial PAI-1 expression is the major source of PAI- 1 from these tissues. The heterogeneity of cell types and relative fraction of endothelial cells in each tissue could have a major impact on PAI-1 levels in these tissues, compromising our ability to draw conclusions regarding the cellular origin of PAI-1. In order to dissect the cell types responsible for these expression differences, we will use a Ribotagged mouse model to specifically pull out endothelial messages from LEWES/EiJ and C57BL/6J and perform a comparative transcriptomic analyses. The proposed investigations of PAI-1 regulation will provide insights into platelet and endothelial specific gene expression as well as providing novel therapeutic targets for modulating thrombosis and fibrinolysis.
High plasma levels of plasminogen activator inhibitor-1 (PAI-1), an important regulator of blood clotting, have been consistently associated with cardiovascular disease. We have identified a chromosome 5 locus as a major regulator of PAI-1 levels in the platelets. We are working to identify the responsible genetic variant within this locus and then study how it affects PAI-1 transcription.
| Westrick, Randal J; Tomberg, Kärt; Siebert, Amy E et al. (2017) Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci. Proc Natl Acad Sci U S A 114:9659-9664 |
