Catheter-directed thrombolysis with microplasmin for acute peripheral arterial occlusion (PAO): an exploratory study.

AIM: We performed an open-label, dose-ascending, single-centre, Phase IIa study to explore the safety and efficacy of catheter-directed thrombolysis (CDT) with microplasmin for infrainguinal arterial or bypass occlusions. METHODS: Patients who presented with acute occlusions were subsequently treated with an intrathrombus infusion of five ascending doses of microplasmin: 0.3 mg/kg/h for 4 hours; 0.45 mg/kg/h for 4 hours; 0.6 mg/kg/h for 4 hours; 0.9 mg/kg/h for 4 hours or 0.6 mg/kg/h for 6 hours. Repeat angiograms were obtained to assess the degree of clot lysis. The primary outcome was complete thrombolysis defined as >95% thrombus volume reduction at the end of the microplasmin infusion. Safety evaluation included bleedings, adverse events and coagulation biomarkers. RESULTS: Complete thrombolysis was obtained in 3 of the 19 treated patients at the end of microplasmin infusion. Thrombus volume reduction between 50% and 95% was achieved with all dosing regimens. Clinically significant distal embolization occurred in 8 patients. One major and two non-major bleedings occurred. Microplasmin depleted α2-anti-plasmin and decreased fibrinogen. CONCLUSION: Intrathrombus infusion of microplasmin for 4 or 6 hours resulted in significant clot lysis. Distal embolization appeared the most important limitation.

Lipid peroxidation as the mechanism of modification of brain 5'-nucleotidase activity in vitro.

The effect of lipid peroxidation on the Mg2(+)-independent and Mg2(+)-dependent activity of brain cell membrane 5'-nucleotidase was determined and the affinity of the active sites of Mg2(+)-dependent enzyme for 5'-AMP (substrate) and Mg2+ (activator) was examined. Brain cell membranes were peroxidized at 37 degrees C in the presence of 100 microM ascorbate and 25 microM FeCl2 (resultant) for 10 min. The activity of 5'-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20 +/- 0.10 to 17.5 +/- 1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg2(+)-independent 5'-nucleotidase increased from 0.201 +/- 0.020 in controls to 0.305 +/- 0.028 mumol Pi/mg protein/hr in peroxidized membranes. In the presence of 10 mM Mg2+, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control. In peroxidized preparation, the affinity of active site of Mg2(+)-dependent 5'-nucleotidase for 5'-AMP tripled, as indicated by a significant decrease in Km (Km = 95 +/- 2 microM AMP for control; Km = 32 +/- 2 microM AMP for peroxidized). Vmax was significantly reduced from 3.35 +/- 0.16 in control to 1.70 +/- 0.9 mumoles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg2+ significantly increased (Km = 6.17 +/- 0.37 mM Mg2+ for control; Km = 4.0 +/- 0.31 peroxidized).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP, K+, Na+, and strophanthidin in vitro.

The effect of lipid peroxidation on the affinity of specific active sites of Na+,K+-ATPase for ATP (substrate), K+ and Na+ (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100 microM ascorbate and 25 microM FeCl2 at 37 degrees C for time intervals from 0-20 min. The level of thiobarbituric acid reactive substances and the activity of Na+, K+-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0 +/- 3.8 to 8.8 +/- 0.9 mumol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2 +/- 0.1 to 16.5 +/- 1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K+ and Na+ was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na+, K+-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K+ will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na+, K+-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process.