Actylise treatment does not influence nitric oxide metabolites serum level.

BACKGROUND: Nitric oxide (NO) is synthesized by Nitric Oxide Synthases (NOS), the family of enzymes capable to conduct the conversion of Arginine (Arg) into the NO and Citrulline (Cit). Currently, only the administration of recombinant tissue plasminogen activator (rtPA) is recommended for acute ischemic stroke (AIS) treatment. To allow solubility of rtPA, Arg is added as a constituent of the drug. Our purpose was to check the effect of alteplase administration on NO metabolites concentration in the blood. METHODS: Eighteen AIS patients were selected into the study. Nine of them received thrombolytic therapy (rtPA group). The serum samples were obtained at 3 time-points for rtPA group (time-point 0: 1st-4th hour of stroke; time-point 1: immediately after rtPA administration; time-point 3: on day 5-7 from stroke onset). Remaining patients (non-rtPA group) had blood collection at two time-points: time-point 1: 1st-10th hour of stroke and time-point 2: on day 5-7 of stroke. Arg and Cit were determined by the automated ion-exchange chromatography using Amino Acids Analyzer. NO serum level was indirectly evaluated with the usage of commercially available kits that measuring the nitrate/nitrite level. RESULTS: Significant increase of Arg serum level was noticed at time-point 1, directly after the iv thrombolysis in comparison to non-rtPA group. However, the products of the reaction catalyzed by NOS (NO and Cit) did not rise after the thrombolysis. CONCLUSIONS: Current study showed that Arg administration simultaneously with rtPA, as a constituent of Actylise, does not affect serum NO metabolites level.

Thrombolytic treatment decreases glutamate/GABA ratio in serum during acute ischaemic stroke: a pilot study.

There is no information about possible effect of recombinant tissue plasminogen activator (rtPA) therapy on excitotoxic/neuroprotective amino acids during acute phase of ischaemic stroke (IS). Our purpose was to evaluate iv thrombolytic treatment on glutamate (Glu) and gamma-aminobutyric acid (GABA) serum levels during acute IS. Eleven thrombolytic (rtPA group) and 12 non-thrombolytic (non-rtPA group) patients with acute IS were enrolled. The serum samples were obtained at three time points for rtPA group (time point 0: first to fourth hour of stroke; time point 1: immediately after rtPA administration; time point 2: on days 5-7 from stroke onset). The remaining patients had blood collection at two time points: time point 1: 5(th)-10(th) hour of stroke and time point 2: on days 5-7 of stroke. Glutamate and GABA were determined by the automated ion-exchange chromatography using Amino Acids Analyser (AAA 400) by INGOS Corp., Praha, Czech Republic. The statistically significant elevation of GABA serum level was noticed directly after thrombolysis (time point 1) in comparison to the corresponding time point in non-rtPA group [0.016 (0.002-0.032) µM/ml vs 0.001 (0.001-0.004) µM/ml for rtPA vs non-rtPA groups, respectively, median (first to third quartile), P < 0.05]. At the same time point, the Glu/GABA ratio was significantly decreased in rtPA group (P < 0.05) suggesting the decrease of excitotoxicity biomarkers in the blood after thrombolysis. Considering the beneficial effect of GABA receptor agonists, the elevation of GABA by rtPA should bring an additional positive features of thrombolytic treatment.

The effect of recombinant tissue plasminogen activator on MMP-2 and MMP-9 activities in vitro.

One of the most significant side effects during recombinant tissue plasminogen activator (rtPA) for acute stroke treatment is intracranial bleeding. Gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] are one of the agents involved in the blood-brain barrier destruction resulting in secondary bleeding into the ischemic area during stroke. Previous papers revealed that patients with high baseline MMP-9 serum level have higher risk of intracranial bleeding after thrombolytic therapy. Our objective was to evaluate rtPA influence on serum MMP-2 and MMP-9 activities in vitro. Nine sera obtained from healthy donors were applied for experiment. The commercially available rtPA (Actylise) were diluted with included solvent and additionally with phosphate-buffered saline (PBS) to get concentrations: 2, 4, 8, and 16 µg/ml. Next, 100 µl of serum was mixed with equal proportion with different concentrations of rtPA to obtain final rtPA concentrations: 1, 2, 4, and 8 µg/ml. The sera together with rtPA were incubated for 1 or 2 hours at 37 °C. The activity of gelatinases was estimated with zymography. The activities of MMP-9 (92 kDa) and MMP-2 (72 kDa) were increased by incubation with rtPA in a dose-dependent manner. Simultaneously, the activity of band at 200 kDa (MMP-9/MMP-9 homodimer) was decreased. The activity of gelatinases incubated for 2 hours was elevated in comparison with 1-hour incubation; however, the increase was observed even for sample without rtPA. In conclusion, this study showed that rtPA can increase the biological activity of MMP-2 and MMP-9 on posttranslational level.

The rtPA increases MMP-9 activity in serum during ischaemic stroke.

BACKGROUND AND PURPOSE: To find the relationship between rtPA treatment vs. MMP-9 activity, MMP-3, and TIMP-1 serum levels related to patients' neurological status during acute ischaemic stroke (IS). MATERIAL AND METHODS: 35 IS patients were enrolled. 14 of them underwent thrombolytic therapy with Actylise (rtPA group). The serum samples were obtained at 3 time-points for rtPA group (time-point 0: 1st-4th hour of stroke; time-point 1 - immediately after rtPA administration; time-point 2 - on day 5-7 from stroke onset). Remaining patients had venous blood collection at two time-points: time-point 1 - 5th-10th hour of stroke and time-point 2 - on day 5-7 of stroke. MMP-9 was analyzed with gelatin zymography, MMP-3 and TIMP-1 serum levels were analyzed with ELISA method. NIHSS improvement ratio (IR) was calculated as a difference between NIHSS score at the admission and discharge of patient. RESULTS: The active form of MMP-9 (86kDa) was not observed in any analyzed samples. Total MMP-9 activity was significantly elevated at time-point 1 in rtPA group in comparison with non-rtPA group. MMP-3 serum level significantly decreased during rtPA administration in comparison with non-rtPA group and it was restored at time-point 2. MMP-3 negatively correlated with IR values (p=0.06). CONCLUSIONS: Thrombolysis applied for IS treatment increases MMP-9 activity in serum, however, rtPA does not facilitate the conversion of pro-MMP-9 into the active form. Our results also suggest the involvement of MMP-3 to the biochemical processes occurring during acute phase of IS.

The significance of matrix metalloproteinase (MMP)-2 and MMP-9 in the ischemic stroke.

There is a continuous urgent need to explore the pathogenesis and biochemical changes within the infarcted area during acute ischemic stroke (IS). Matrix metalloproteinases (MMPs), prevailing extracellular endopeptideses, can digest proteins located extracellulary, e.g. collagen, proteoglycans, elastin or fibronectin. Among MMPs, gelatinases (MMP-2 and MMP-9) are the most investigated enzymes. Gelatinases possess the ability to active numerous pro-inflammatory agents as chemokine CXCL-8, interleukin 1ß or tumor necrosis factor α. Moreover, due to digestion of collagen type IV (the component of basal membranes) and tight junction proteins (TJPs) they facilitate to cross the endothelium by leukocytes. Due to the significant role of gelatinases during brain ischemia, their selective inhibition seems to be an interesting kind of treatment of acute stroke. The synthetic inhibitors of gelatineses decrease the infarct volume in animal models of IS. In clinical practice statins, the lipid-lowering drugs possess the ability to inhibit the activity of MMP-9 during acute IS. This review briefly provides the most important information about the involvement of MMP-2 and MMP-9 in the pathogenesis of brain ischemia.