E. Van Houtte

Protective effect of parvalbumin on excitotoxic motor neuron death.

The mechanism responsible for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is poorly understood. Several lines of evidence indicate that susceptibility of motor neurons to Ca(2+) overload induced by excitotoxic stimuli is involved. In this study, we investigated whether the high density of Ca(2+)-permeable AMPA receptors on motor neurons gives rise to higher Ca(2+) transients in motor neurons compared to dorsal horn neurons. Dorsal horn neurons were chosen as controls as these cells do not degenerate in ALS. In cultured spinal motor neurons, the rise of the cytosolic Ca(2+) concentration induced by kainic acid (KA) and mediated by the AMPA receptor was almost twice as high as in spinal neurons from the dorsal horn. Furthermore, we investigated whether increasing the motor neurons cytosolic Ca(2+)-buffering capacity protects them from excitotoxic death. To obtain motor neurons with increased Ca(2+) buffering capacity, we generated transgenic mice overexpressing parvalbumin (PV). These mice have no apparent phenotype. PV overexpression was present in the central nervous system, kidney, thymus, and spleen. Motor neurons from these transgenic mice expressed PV in culture and were partially protected from KA-induced death as compared to those isolated from nontransgenic littermates. PV overexpression also attenuated KA-induced Ca(2+) transients, but not those induced by depolarization. We conclude that the high density of Ca(2+)-permeable AMPA receptors on the motor neurons surface results in high Ca(2+) transients upon stimulation and that the low cytosolic Ca(2+)-buffering capacity of motor neurons may contribute to the selective vulnerability of these cells in ALS. Overexpression of a high-affinity Ca(2+) buffer such as PV protects the motor neuron from excitotoxicity and this protective effect depends upon the mode of Ca(2+) entry into the cell.

An α-mercaptoacrylic acid derivative (PD150606) inhibits selective motor neuron death via inhibition of kainate-induced Ca2+ influx and not via calpain inhibition

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron death. The exact mechanism responsible for this selectivity is not clear, although it is known that motor neurons are very sensitive to excitotoxicity. This high sensitivity is due to a high density of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors on their surface and to a limited Ca(2+) buffering capacity. Ca(2+) can enter the cell upon stimulation through voltage-operated Ca(2+) channels and through the Ca(2+)-permeable portion of AMPA receptors. How this Ca(2+) kills motor neurons is incompletely understood. In the present study, we report that kainate (KA)-induced motor neuron death is purely mediated through Ca(2+) entering motor neurons through Ca(2+)-permeable AMPA receptors and that voltage-operated Ca(2+) channels play no significant role. In contrast to what has been observed in other neuronal models or after N-methyl-D-aspartate stimulation, NO synthase inhibition and a number of antioxidants did not protect motor neurons from KA-induced death. Only PD150606, derived from alpha-mercaptoacrylic acid and considered as a selective calpain antagonist, inhibited dose-dependently the KA-induced motor neuron death. However, other calmodulin and calpain inhibitors were not effective. At least part of the inhibitory effect of PD150606 is due to an irreversible inhibition of the Ca(2+) influx through the Ca(2+)-permeable AMPA receptor. These results demonstrate the interesting property of PD150606 to interfere with excitotoxicity-dependent motor neuron death and show that PD150606 is not an exclusive calpain/calmodulin antagonist.

Streptokinase-induced platelet aggregation. Prevalence and mechanism.

BackgroundStreptokinase (SK) is a bacteria-derived protein and one of the plasminogen activators that is currently available for therapeutic use. Exposure to SK induces synthesis of specific antibodies that may initiate platelet aggregation and paradoxical clot propagation during treatment. Methods and ResultsUsing platelet-rich plasma (PRP), we found that SK (5,000 units/ml) but not urokinase (2,500 units/ml) or recombinant tissue-type plasminogen activator (2,500 units/ml) caused platelet aggregation in PRP from 14 of 100 normal volunteers. In 13 consecutive patients teated with SK for acute myocardial infarction, SK-mediated platelet aggregation was induced in five patients within 1 week after treatment. SK-mediated platelet aggregation was associated with significantly increased titers of both anti-SK antibodies and SK-neutralizing activity in plasma; it was partially inhibited by aspirin (1 mM) and by aprotinin (500 kallikrein inhibitor units/ml) and completely inhibited by tranexamic acid (1 mM) and by prostaglandin E1 (9, M). Addition of SK (1,000 or 5,000 units/ml) induce a statistically significant dose-dependent thromboxane B2 release in mixtures of PRP with plasma from subjects with SK-induced aggregation but not in samples of PRP mixed with plasma from nonresponders; addition of recombinant tissue-type plasminogen (1 50 jug/ml) did induce thromboxane B2 release. Mixing experiments with PRP and immunoglobulin G from reactive and nonreactive donors revealed that SK-induced aggregation rquires the presence of anti-SK antibodies. When 1251-SK (50 nM) was used, platelets preincubated with plasminogen (0.5 μM) bound 9,500 ± 600 (mean ± SEM, n = 6) molecules SK/platelet, which increased to 25,000 ± 3,100 molecules/platelet after thrombin stimulation. Tranexamic acid (1 mM) blocked specific binding of SK to resting platelets. ConcluionsThese data demonstrate that SK-induced platelet aggregation is initiated by the binding of anti-SK antibodies to the SK-plasminogen complex located on the platelet surface. SK-induced platelet activation may limit the therapeutic effectiveness of the drug, and in view of the high prevalence of aggregation in a normal population, prospective evaluation of the effects of platelet aggregation during treatment with SK is warranted. (Circulation 1991;84:84–91)

Studies of recombinant plasminogen activator inhibitor-1 in rabbits. Pharmacokinetics and evidence for reactivation of latent plasminogen activator inhibitor-1 in vivo.

The pharmacokinetics of human recombinant plasminogen activator inhibitor-1 (rPAI-1) was studied in rabbits. Latent rPAI-1 (0-2 units of tissue-type plasminogen activator neutralizing activity per microgram protein); reactivated rPAI-1 (approximately 150 units/micrograms); and chloramine T-oxidized, nonreactivatable rPAI-1 (approximately 0.7 units/microgram) were studied. The pharmacokinetic parameters for the disposition of rPAI-1 antigen after an intravenous bolus injection of 1.0 or 2.5 mg/kg rPAI-1 were very similar for all three forms: the initial volume of distribution was approximately 60 ml/kg, the initial half-life in plasma was 6 minutes, and the plasma clearance was approximately 4 ml/kg/min. The disposition of PAI activity after injection of reactivated rPAI-1 was similar to that of rPAI-1 antigen. Injection of latent rPAI-1 was associated with a nearly threefold increase in the specific activity of circulating PAI-1 from 2 units/micrograms to 5.0 +/- 1.1 units/micrograms (p less than 0.01) within 1 minute, followed by a cumulative 25-fold increase in specific activity over 1 hour (p = 0.01). In contrast, the specific activity of oxidized or reactivated preparations of rPAI-1 did not increase in the first several minutes after injection. These findings support the existence of a fast-acting but low-capacity mechanism for the reactivation of rPAI-1 in vivo.

Urokinase binds to platelets through a specific saturable, low affinity mechanism

Summary Specific receptors for urokinase have previously been identified on a number of cell types, including peripheral blood monocytes and endothelial cells. In the present study, the existence of specific receptors for single chain urokinase-type plasminogen activator (scu-PA) on gel-filtered human platelets was investigated. Platelets were found to bind 125 I-labelled recombinant scu-PA ( 125 I-rscu-PA) rapidly, specifically, saturably and reversibly, with a binding capacity of 92000±10000 molecules (mean±SEM) per platelet, and an apparent K d of 130±20nM. Unlabelled rscu-PA and high molecular weight urokinase efficiently completed with binding, whereas low molecular weight urokinase (lacking the epidermal growth factor and kringle domains) did not. Other molecules known to bind to platelets, including fibrinogen, tissue-type plasminogen activator and plasminogen, had little effect on the binding of 125 I-rscu-PA. Binding was not significantly altered by the addition of 1.5 mM Ca ++ or by 5mM EDTA. Thus, platelets have a high density, specific mechanism that binds scu-PA and potentially localises fibrinolytic activity to their surface.

Pharmacokinetic properties of mutants of recombinant single chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys158, Ile159 and Ile160

Abstract Single chain urokinase-type plasminogen activator (scu-PA, prourokinase) is converted to two chain urokinase-type plasminogen activator (tcu-PA) by hydrolysis of the Lys158-Ile159 peptide bond with plasmin. In this study we have evaluated the pharmacokinetic properties in rabbits of rscu-PAs with specific amino acid substitutions at the cleavage site, including rscu-PA-Arg158 (rscu-PA with Lys158 substituted with Arg), rscu-PA-Gly158 (Lys158 substituted with Gly), rscu-PA-Glu158 (Lys158 substituted with Glu), rscu-PAG1y159 (Ile159 substituted with Gly), rscu-PA-Pro159 (Ile159 substituted with Pro) and rscu-PA-Gly158,Lys160 (Lys158 substituted with Gly and Ile160 with Lys). The mutant or wild type rscu-PAs (at doses between 1.6 and 4.0 mg/kg) were infused intravenously over 4 h in groups of 2 to 5 rabbits, and the initial disposition rate of rscu-PA related antigen from plasma was monitored over a 30 min period after the end of the infusion. The disappearance rate of antigen from plasma was biphasic for rscu-PA and for the mutants and could be described by a sum of two exponential terms with an a half-life of 2.0 to 3.0 min (2.6 min for rscu-PA) and a β half-life of 11 to 14 min (11 min for rscu-PA). The plasma clearance rate ranged between 21 and 31 ml per min (30 ml per min for rscu-PA). These results indicate that the amino acid sequence around the plasmin cleavage site in scu-PA is not a significant determinant for its in vivo pharmacokinetic properties and that scu-PA is not cleared via activation to tcu-PA and neutralisation by protease inhibitors.