| The coagulation system is a potent mechanism that prevents blood loss after vascular injury and that consists of a number of linked enzymatic reactions resulting in thrombin generation. Thrombin converts soluble fibrinogen into a fibrin clot. The clot is subsequently removed by the fibrinolytic system upon wound healing. Thrombin-activatable fibrinolysis inhibitor (TAFI) provides an important link between the coagulation and fibrinolytic systems. Activated TAFI, TAFIa, down-regulates fibrinolysis. TAFIa is a labile enzyme with a half-life of less than ten minutes at 37°C. It has been shown in vitro that a more stable form of TAFIa results in enhanced clot protection. However, the exact nature of the instability is still unclear. It is our hypothesis that TAFIa (in)stability has an important effect on its function. The main objective of this proposal is to identify the molecular determinants that regulate (in)stability of TAFIa. We will try to crystallize TAFI, TAFIa and the inactivated form TAFIai. After establishing optimal crystallization conditions three-dimensional structures will be determined using established methods. Mutagenesis of TAFI will be used to prepare mutants with an altered stability, which should lead to a better understanding of the determinants in TAFI that are responsible for instability. Elucidation of the nature and relevance of TAFIa instability may have important clinical benefits, since decreasing the stability of TAFIa may be beneficial for patients with thrombosis, whereas increasing the stability of TAFIa may be useful in the treatment of patients with bleeding disorders. |
