Structural studies of the human chimerical plasminogen activator Amediplase® (K2tu-PA)
Doriano Lamba, Alberto Cassetta, Cecilia Bartolucci

Plasminogen activators (PA) are serine proteases that convert the inactive proenzyme plasminogen into the active enzyme plasmin, thereby functioning as important initiators of fibrinolysis. Two types of plasminogen activators can be distinguished, the urinary-type (u-PA) and the tissue type (t-PA).
u-PA and t-PA are of clinical interest for the treatment of acute vascular diseases. u-PA is also involved in physiological and pathological tissue destruction and cell migration processes. To obtain artery reperfusion, high blood levels of PA are required. Such pharmaceutical use is associated with some side effects. Therefore, more effective and safe fibrinolytic agents are of great interest. Both u-PA and t-PA are mosaic glycoproteins containing individual protein domains with autonomous functions: signal peptide (SP), pro-sequence (PS), a finger (F), epidermal growth factor (EGF), kringle (K) and serine protease (P) domains.
In the presence of fibrin, t-PA hydrolyses the R560-V561 peptide bond of plasminogen more efficiently. Although the exact mechanism of this enhancement is not well understood the presence of the K2 domain seems to be of essential importance. Efforts have been made to obtain improved PA variants. A hybrid variant K2tu-PA (Amediplase®), constructed from the sequences coding for the kringle-2 domain of t-PA and for the protease domain of u-PA combined at the gene level [1], is being researched by Menarini Biotech S.r.l, Pomezia (Roma), Italy. The recombinant protein was produced in a CHO cell line using a mouse cytomegalovirus-promoter/enhancer driven expression cassette.
In animal models, Amediplase® is a more potent and longer-lasting thrombolytic agent than Alteplase® (rt-PA), the standard thrombolytic drug for the treatment of acute myocardial infarction. Amediplase® is indicated for administration as a single bolus, which constitutes an advantage over the standard thrombolytic treatments [2]. Amediplase® has recently completed phase II clinical trials with excellent results both in terms of efficacy and safety.
The chimerical plasminogen activator has been crystallised, structure has been solved by Patterson search methods and refined at 2.5Å.The kringle 2 domain is covalently bound to the catalytic module via the two residues Thr and Ser linker. However no intimate extended contacts are observed between the two domains and their mutual orientation differs in the two molecules present in the asymmetric unit. This observation suggests that the two domains may function autonomously. The two molecules present in the asymmetric unit interact one each other in a head-to-tail fashion. A lysine residue belonging to the catalytic domain of either of the two molecules occupies the kringle-2 lysine-binding site of either of the correspondent molecules. This observation may represent a rationale for the recently reported clot penetration and fibrin binding properties of Amediplase® [3].

[1]Colucci, M., et al., Thromb. Haemost. (1993) 69, 466-472; [2] Doggrell, S.A., Curr. Opin. Investig. Drugs (2004) 5, 344-347; [3]Rijken, D.C., et al., Thromb. Haemost. (2004) 71, 52-60.