Cofactor and substrate-assisted activation of coagulation factor IXa

The blood coagulation cascade is an interdependent system of serine proteases, their cofactors and inhibitors. This complex protease machinery rapidly stops life-threatening blood loss caused by vasculature lesions, while avoiding uncontrolled clot formation. To this end, the involved proteases and cofactors are produced on stock as inactive precursors, which are converted to the active form by proteolytic cleavage. Among the coagulation factors the serine protease factor IX and its associated protein cofactor VIII have attracted particular attention, because defects in either of these X-chromosome encoded gene products cause haemophilia, affecting 1 in about 5,000 males. Intriguingly, proteolytically activated factor IXa (fIXa) is almost inactive and exhibits substantial proteolytic activity only in the presence of its protein cofactor fVIIIa, which similarly requires proteolytic activation. Furthermore, the activation of fIXa by fVIIIa in the so called intrinsic Xase complex is strictly specific to its physiologic substrate coagulation factor X (fX), whereas the Xase complex shows no activity enhancement towards peptidic substrates. Physiologically, the assembly of this Xase complex as well as the catalysed fX activation take place on the membranes of blood platelets which also contribute to reach a total reaction acceleration of up to 1,000,000-fold. The molecular mechanisms underlying this concerted catalysis remain largely unclear and represent the centre of the present research proposal. Extending on our previous structural and functional studies on super-active fIXa mutants, we propose to study a simplified model system of the Xase where we focus on the protein components (fVIIIa, fIXa, fX), eliminating (and ignoring) the contribution of the membrane. Consequently, and to further simplify the system, we will work with engineered protein factors that lack the membrane-binding domains. The resulting "soluble Xase" system is of unmatched chemical and conformational homogeneity, making its crystal structure analysis feasible. In our first aim (Aim A) we propose to crystallise an engineered fIXa-fVIIIa complex. In Aim B we will enzymatically and thermodynamically characterise the proposed engineered Xase system and calibrate it towards the classical full-length Xase system. Finally, in Aim C we will investigate the mechanisms how substrates assist their catalytic turnover by the Xase. To this end we will map the substrate requirements by using proteomically derived substrate libraries. The observed substrate preferences will guide the development of substrate-like inhibitors, including their complex crystal structure analysis. By using stabilised, non-cleavable protein variants, we will also attempt to crystallise the ternary fVIIIa-fIXa-fX complex.

Upon vessel injury, the life-threatening blood loss must be stopped in short time, a task accomplished by several physiological processes, including the blood coagulation system. Importantly, the haemostasis must be kept in balance to avoid thrombotic complications. This delicate balance of haemostasis and thrombosis is maintained self-regulating networks of biochemical reactions. This can be exemplified by the blood coagulation cascade where proteases are present in the blood circulation in an inactive form, which sequentially activate each other upon an external stimulus, i.e. the vessel injury. Of particular interest, coagulation factor IX requires not only the classical activation to factor IXa but additionally the stimulation by the protein cofactor VIIIa; otherwise the coagulation activity will be choked,as witnessed clinically as haemophilia, affecting approximately 1 in 10,000 men. Consequently, the aim of the current project was to decipher the mechanism of factor IXa activity enhancement by its protein co-factor as well as its substrate factor X, as such understanding opens therapeutic opportunities for haemophiliacs, particularly a cofactor-independent activity stimulation of factor IXa. We pursued parallel approaches to tackle this challenge.By using mutagenesis studies we could identify cues to the unusually low enzymatic activity of factor IXa: Different from other related enzymes, there is network of molecular brakes which cooperatively downregulate factor IXa's proteolytic activity. These regulatory elements are distributed on several loops. We could further show how substrate specificity and affinity of factor IXa and and also IX were influenced by co-factor VIIIa-derived peptides. These findings could be qualitatively confirmed by the protein co-factor VIIIa. Finally, we were also attempting to evaluate these results with the recombinantly produced factor VIII domain A2, which we managed to produce in an eukaryotic expression system. However, the production criteria turned out to be rather variable, making the reproducibility of the A2 preparation difficult. Nonetheless, also these results represent an important milestone for further mechanistic studies in this field.