Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke

AbstractBackgroundTissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment. A novel direct-acting thrombolytic agent, Δ(K2-K5) plasmin, promising an improved safety profile was examined for safety in the snare ligature model of stroke in the rat.MethodsMale spontaneously hypertensive rats were subjected to 6 hours middle cerebral artery occlusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosed with saline, vehicle, Δ(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissue-type plasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60 minutes. The rats were assessed for bleeding score, infarct volume, modified Bederson score and general behavioral score. In a parallel study, temporal progression of infarct volume was determined. In an in vitro study, whole blood clots from humans, canines and rats were exposed to Δ(K2-K5). Clot lysis was monitored by absorbance at 280 nm.ResultsThe main focus of this study was intracranial hemorrhage safety. Δ(K2-K5) plasmin treatment at the highest dose caused no more intracranial hemorrhage than the lowest dose of recombinant tissue type plasminogen activator, but showed at least a 5-fold superior safety margin. Secondary results include: temporal infarct volume progression shows that the greatest expansion of infarct volume occurs within 2–3 hours of middle cerebral artery occlusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6 hours ischemia with reflow. Δ(K2-K5) plasmin tended to reduce infarct volume and improve behavior compared to controls. In vitro data suggests that Δ(K2-K5) plasmin is equally effective at lysing clots from humans, canines and rats.ConclusionsThe superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Δ(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agent a good candidate for clinical evaluation in the treatment of acute ischemic stroke

Dynamic Protein Domains: Identification, Interdependence, and Stability

Existing methods of domain identification in proteins usually provide no information about the degree of domain independence and stability. However, this information is vital for many areas of protein research. The recently developed hierarchical clustering of correlation patterns (HCCP) technique provides machine-based domain identification in a computationally simple and physically consistent way. Here we present the modification of this technique, which not only allows determination of the most plausible number of dynamic domains but also makes it possible to estimate the degree of their independence (the extent of correlated motion) and stability (the range of environmental conditions, where domains remain intact). With this technique we provided domain assignments and calculated intra- and interdomain correlations and interdomain energies for >2500 test proteins. It is shown that mean intradomain correlation of motions can serve as a quantitative criterion of domain independence, and the HCCP stability gap is a measure of their stability. Our data show that the motions of domains with high stability are usually independent. In contrast, the domains with moderate stability usually exhibit a substantial degree of correlated motions. It is shown that in multidomain proteins the domains are most stable if they are of similar size, and this correlates with the observed abundance of such proteins