A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity--a staphylokinase variant. I. In vitro study.

We attempted to construct a new recombinant protein characterized by fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. To the C-terminal part of recombinant staphylokinase (r-SAK), which is a promising profibrinolytic agent, we assembled: (i) the Kringle 2 domain (K2) of tissue-type plasminogen activator (t-PA), containing a fibrin-specific binding site, (ii) the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation and (iii) the antithrombotic agent - hirudin. The cDNA for hybrid protein SAK-RGD-K2-Hir was cloned into pESP-3 yeast protein expression vector. The introduction of K2 t-PA, RGD sequence and hirudin into r-SAK molecule did not alter the SAK activity. The plasminogen activation rate (determined by K(M) and K(cat)) of SAK-RGD-K2-Hir was not significantly different from that of r-SAK. Affinity and binding strength of the recombinant protein to fibrin immobilized on the biosensor were higher than to r-SAK. We observed a higher clot lysis potency of SAK-RGD-K2-Hir as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirudin part of the recombinant fusion protein SAK-RGD-K2-Hir was the same as that of r-Hir alone. In conclusion, the results of the in vitro study suggest that the SAK-RGD-K2-Hir construct can be a more potent and faster-acting thrombolytic agent with antithrombin and antiplatelet properties compared with standard r-SAK.

Differential effects of somatostatin and its analog on protein tyrosine kinases activity in the rat pituitary and the murine colonic tumors.

The effects of the native somatostatin-14 (SST-14) and of its analog octreotide (OCT) on the activity of protein tyrosine kinases (PTK) in the normal rat anterior pituitary gland, diethylstilbestrol (DES)-induced rat pituitary tumor and murine colonic cancer Colon 38 were studied in vitro. PTK activity was estimated in tissue homogenates using gamma-[32P]ATP and poly (Glu80, Tyr20) as a substrate. It was found that both SST-14 and OCT suppressed the PTK activity in all examined tissues. The suppressive effect was more pronounced in DES-induced pituitary tumor than in normal anterior pituitary gland, and in the former, OCT was more effective than SST-14. In contrast, SST-14 stronger suppressed PTK activity in colonic cancer than OCT. We hypothesize that SST-14 acts on PTK activity in colonic cancer mainly via SSTR-1 subtype of somatostatin receptors.

Vanadium inhibition of human parietal lobe ATPases.

In vitro influence of vanadate and vanadyl ions on the activities of Na,K- and Ca,Mg-ATPase from synaptosomal membranes of the parietal lobe of the human brain were compared. 1. Vanadate and vanadyl inhibit the enzymes activities in the investigated fraction. 2. Vanadate is a more effective inhibitor of both ATPases in the concentrations above 50 microM and vanadyl is an effective inhibitor in a very low concentration (10 nM). 3. Vanadate seems to be an uncompetitive inhibitor of Na,K-ATPase (K1 = 880 nM).

A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain.

The influence in vitro of SP and C-terminal fragments of analogues SP(5-11) (pyroGlu5, Tyr8); SP(6-11) (pyroGlu6, Tyr8); SP(6-11) (pyroGlu6, D-Phe7); SP(6-11) (pyroGlu6, D-Phe8) on the (Ca, Mg) and (Na, K) ATPases activities from synaptosomal membranes of cerebral cortex and hippocampus of rat brain were compared. The data obtained in this study indicate the following: 1. Substance P stimulates the activities of (Na, K) and (Ca, Mg) ATPases more effectively in synaptosomal membranes from hippocampus than cerebral cortex. 2. Heptapeptide SP(5-11) (pyroGlu5, Tyr8) causes a more distinct increase of (Ca, Mg) ATPase activity in cortical synaptosomal membranes than SP does. 3. The change of L-Phe conformation to D in position 7 in hexapeptide induces reduction of enzymes activities in hippocampus. 4. Especially important for the maintenance of biological activity of drugs is the replacement of Gln5 with pyroGlu6 and conformation of Phe residues. 5. SP and shorter analogues of fragments SP C-terminal SP regulate the active cation transport in synaptosomal membranes of cerebral cortex and hippocampus.

Role of theophylline during the action of SP in vitro on the activity of synaptosomal membrane ATPase (EC 3.6.1.3) from different areas of rat brain.

The in vitro action of SP and theophylline on the activities of (Na K) and (Ca Mg) dependent adenosine-5'-triphosphates (ATPase) of crude synaptosomal membranes (SM) of four different areas of rat brain was investigated. The highest activity of (Ca Mg)ATPase was observed in SM from hippocampus. In thalamus with hypothalamus region the highest activity of (Na K)ATPase and the lowest activity of (Ca Mg)ATPase occurred. It was found, that 10 mumol of SP stimulating "alternatively" both (Na K) and (Ca Mg)ATPase activities from different regions may constitute a regulating agent for ionic transport in CNS of rat. Theophylline on concentration of 5 mumol modulates the action of SP on the activities of the investigated enzymes.

Effect of certain agents on subcellular cAMP level in different areas of rat brain.

The influence was studied in vitro of certain agents (adenosine, ADP, ATP, theophylline, together with F- ions) on the cAMP concentrations in the nuclear (N) and mitochondrial (M) fractions from different areas of rat brain. F- ions caused a slight decrease of the cAMP concentrations in nuclear fractions of the thalamus with hypothalamus and a marked decrease of this cyclic nucleotide in M fractions from the cerebral cortex. After incubation with adenosine and F- ions a distinct decrease of cAMP level was observed in N fractions from the midbrain and thalamus with hypothalamus and in mitochondrial fractions obtained from all the investigated regions. The incubation with ATP and F- ions resulted in a distinct decrease of cAMP values in the nuclear fractions from all regions. The concentrations of cAMP in the mitochondria of the midbrain and thalamus with hypothalamus incubated with ATP and F- ions increased 2-3 times. The incubation of the nuclear fraction with theophylline and F- ions caused an increase of cAMP concentration in the cortex and a decrease of cAMP values in the midbrain. The level of cAMP after the incubation with theophylline and fluoride on the mitochondrial fraction is increased in the cortex and decreased in the thalamus with hypothalamus.

Influence of Ca2+ and substance P on the (Ca Mg) ATPase (EC 3.6.1.3) activity of synaptosomes from different areas of rat brain.

The influence of SP in vitro on the (Ca Mg) ATPase activity of synaptosomes from cerebral cortex, hippocampus, midbrain and thalamus with hypothalamus of rats was studied. It was confirmed that SP increases the activity of the enzyme from hippocampus, midbrain and thalamus with hypothalamus. A condition of this stimulation is the maintenance of Ca2+ concentration between 1 and 2 X 10(-4) M. Ca2+ concentration above and below the optimal (0.2 mM), reduced the SP stimulation of (Ca Mg) ATPase activity.

Ca2+ Mg2+ -ATPase activity of synaptosome fraction and synaptosomal membranes from different areas of rat brain.

1. Crude synaptosomal fractions obtained from four areas of rat brain were studied; cerebral cortex, hippocampus, midbrain, thalamus with hypothalamus, using the Cotman & Matthews method (1971) Biochim, biophys. Acta 249, 350-394. 2. The purity of synaptosomal fractions was controlled by electron microscopy, and by determination of some marker enzymes such as: LDH, MAO, AChE and cytochrome-c oxidase. 3. Synaptosomes were disrupted by hypoosmotical shock. 4. Crude synaptosomal membrane preparations indicated on increased Ca2+ Mg2+ -ATPase activity in comparison to the activity of this enzyme in synaptosomal fractions. 5. The incubation of crude synaptosomal membranes with cAMP and theophylline caused the subsequent increase of Ca2+ Mg2+ -ATPase activity, but mainly, in hippocampal region.

Cyclic AMP, DNA and proteins in various areas of rat brain.

Values of cyclic AMP, DNA and protein content were determined in six regions of the rat brain: cerebral cortex, hippocampus, midbrain, thalamus with hypothalamus, cerebellum, and medulla. The obtained results indicated significant differences in the levels of protein in several areas of rat brain. There is some relationship between the changes in the content of DNA and cyclic AMP in these regions of the brain. These observations support the view that adenosine 3': 5'-monophosphate can play a positive role in regulating DNA synthesis in rat brain tissue.