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227 Fibrinolytic activity of nerve growth factor (NGF)
66 P O S T E R P R E S E N T A T I O N S
P-XI P e r i c e l l u l a r P r o t e o l y s i s : G r o w t h F a c t o r s a n d C y t o k i n e s
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IMPAIRMENT OF IN VITRO FIBRINOLYSIS BY PAI-I RELEASED FROM STIMULATED HUMAN ENDOTHELIAL CELLS.
FIBRINOLYTIC ACTIVITY OF NERVE GROWTH FACTOR
Monies R, Chord~ C, Orbe J, Zabalegui N, Hermida ,1, P6ramo JA, Rocha E. Laboratory of Vascular Biology and Thrombosis. School of Medicine. University of Navarra. Pamplona. Spain.
Isanbaev Ch., Kazantseva D., Vypova N., Sagdiev N. Laboratory of Pharmacology, Institute of Bioorg~nic Chemistry, Tashkent, Uzbeklstan
(NGF)
PAl-1 can be found in three different conformations: active, latent and forming
complexes with tissue plasminogen activator (t-PA). The stimulation of endothelial cells with endotoxin and cytokines induces the release of active PAI-1, which spontaneously converts into its latent (inactive form). To study the effect of endothelial PAI-I on in vitro fibrinolysis, cultured human umbilical vein endothelial cells (HUVEC) were stimulated with endotoxin, TNF-ct and IL-lc~ (100 ng/ml). Samples from conditioned media, recovered 2 and 24 hours after stimulation were incorporated (34% v/v) to a reaction mixture containing Glu-plasminogen (25 gtg/ml), CaCt2 (0.1 mol/l), fibrinogen (5gmol/I) and t-PA (10 IU/ml). Clotting was initiated by addition of thrombin (10 NIH/ml). The formation and lysis of the resulting clots was monitored in a spectrophotometer at 350 nm and 37°C. Active PAI-I levels were also measured in the conditioned media by an amidolytic assay. We found a marked delay in the lysis times when conditioned media recovered 24 hours after stimulation were incorporated into forming clots (mean 58 rain vs 39 min in control cultures, representing a 49% delay) whereas no differences in the lysis times were observed between control and cultures recovered 2 hours after stimulation. A significant increase in active PAI-1 was observed in the samples taken at 24 hours as compared to both controls and samples taken 2 hours after stimulation (p<0.01). Interestingly, a strong correlation (r=0.85) was observed between active PAI-1 and lysis times in the whole data series. To determine the contribution of PAl-1 on the lysis times, a molar excess (50 gtg/ml) of a neutralizing anti-PAl-1 monoclonal antibody was added to the reaction mixture containing PAl-1 rich conditioned media. The antibody virtually reversed the delayed lysis time. We conclude that cultured HUVEC secrete significant amounts of active PAl-1, which effectively inhibits in vitro fibrinolysis.
228 LOCAL GOMOLOGIES TO PATTERN 3 8 - 6 3 OF THROMBIN RECEPTOR. D u g i n a T . , Rakhmanov S . , Strukova S., B r o d s k i y L. Moscow S t a t e University, Moscow, R u s s i a .
Thrombin cleaves its 7 - d o m a i n r e c e p t o r on cells to reveal a tethered l i g a n d whose a c tivity is m i m i c k e d by p e p t i d e a n a l o g u e o f the newly exposed N-terminus. There are evid e n c e s t h a t t h e a c t i o n o f t h r o m b i n on c e l l s is not limited t o t h a t m e d i a t e d by i t s c l o ned t ~ r o m b i n r e c e p t o r and t h e r e may be more t h e n ~ne t h r o m b i n receptor. In order to find probable candidates mediating action o f t h r o m b i n on c e l l s among t h e c l o n e d prot e i n s we u s e d t h e c o m p u t e r s e a r c h f o r gomologies to functionally important pattern
known to interact with thrombin. Using programm set GeneBee the search of gomologies to site 38-63 thrombin, binding to the active center of enzyme and the hirudin-like domain,docked in the anion-binding exocite of thrombin, was undertaken. The high degree of gomology of this pattern to fragments of Ras-related protein, light chain B of plasminogen (662-886) and extracellular domain of FGF 2 receptor was revealed. The synthetic peptide agonist of thrombin receptor SFLLRN (unlike to thrombin) has no reactions considered as the mitogenic or able to maintain prolonged mitogen-activated protein(MAP)kinase stimulation. So we suggest that the full growth responce of thrombin is supported not only by cooperation of growth factors with thrombin receptor peptide agonists but also the interaction of thrombin with another receptor.
Fibrinolytic activity of highly-molecular 7S NGF is conditioned by its gamma-subunit possessing esteropeptidase activity. Shown is a possibility of gamma NGF to induce fibrinolysls by transformation of plasminogen into plasmin bosh in salivary gland and in the blood of mice (Isanbaev q99@). Besides, 7S NGF can induce fibrinolysis not only through gamma subunit, but also through beta subunit which does not obtain enzymatic property. Such an effect of NGF on the lysis of fibrin, as our study showed, may take place through the histamine of the mast cells, eosinophils and basophils. Histamin, by itself, notably activizes the process of fibrinolysis, and the population of the mast cells, which produce it in an organism, has specific receptors to NGF. Interaction of NGF with them causes an intensive degranulation of the mast cells with the release of histamine into blood, which indicates the potential role of NGF as a natural liberator of histamine, whose role in biochemical processes of initiation of inflammation and regeneration is well known. Thus, our study assumes an important role of NGF in the processes of fibrino- and thrombolysis, and each component may in different ways (gamma subunit directly and beta subunit indirectly) perform its effect on the fibrinolytic system.
P-XI P e r i c e l l u l a r P r o t e o l y s i s : G r o w t h F a c t o r s a n d C y t o k i n e s
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227
IMPAIRMENT OF IN VITRO FIBRINOLYSIS BY PAI-I RELEASED FROM STIMULATED HUMAN ENDOTHELIAL CELLS.
FIBRINOLYTIC ACTIVITY OF NERVE GROWTH FACTOR
Monies R, Chord~ C, Orbe J, Zabalegui N, Hermida ,1, P6ramo JA, Rocha E. Laboratory of Vascular Biology and Thrombosis. School of Medicine. University of Navarra. Pamplona. Spain.
Isanbaev Ch., Kazantseva D., Vypova N., Sagdiev N. Laboratory of Pharmacology, Institute of Bioorg~nic Chemistry, Tashkent, Uzbeklstan
(NGF)
PAl-1 can be found in three different conformations: active, latent and forming
complexes with tissue plasminogen activator (t-PA). The stimulation of endothelial cells with endotoxin and cytokines induces the release of active PAI-1, which spontaneously converts into its latent (inactive form). To study the effect of endothelial PAI-I on in vitro fibrinolysis, cultured human umbilical vein endothelial cells (HUVEC) were stimulated with endotoxin, TNF-ct and IL-lc~ (100 ng/ml). Samples from conditioned media, recovered 2 and 24 hours after stimulation were incorporated (34% v/v) to a reaction mixture containing Glu-plasminogen (25 gtg/ml), CaCt2 (0.1 mol/l), fibrinogen (5gmol/I) and t-PA (10 IU/ml). Clotting was initiated by addition of thrombin (10 NIH/ml). The formation and lysis of the resulting clots was monitored in a spectrophotometer at 350 nm and 37°C. Active PAI-I levels were also measured in the conditioned media by an amidolytic assay. We found a marked delay in the lysis times when conditioned media recovered 24 hours after stimulation were incorporated into forming clots (mean 58 rain vs 39 min in control cultures, representing a 49% delay) whereas no differences in the lysis times were observed between control and cultures recovered 2 hours after stimulation. A significant increase in active PAI-1 was observed in the samples taken at 24 hours as compared to both controls and samples taken 2 hours after stimulation (p<0.01). Interestingly, a strong correlation (r=0.85) was observed between active PAI-1 and lysis times in the whole data series. To determine the contribution of PAl-1 on the lysis times, a molar excess (50 gtg/ml) of a neutralizing anti-PAl-1 monoclonal antibody was added to the reaction mixture containing PAl-1 rich conditioned media. The antibody virtually reversed the delayed lysis time. We conclude that cultured HUVEC secrete significant amounts of active PAl-1, which effectively inhibits in vitro fibrinolysis.
228 LOCAL GOMOLOGIES TO PATTERN 3 8 - 6 3 OF THROMBIN RECEPTOR. D u g i n a T . , Rakhmanov S . , Strukova S., B r o d s k i y L. Moscow S t a t e University, Moscow, R u s s i a .
Thrombin cleaves its 7 - d o m a i n r e c e p t o r on cells to reveal a tethered l i g a n d whose a c tivity is m i m i c k e d by p e p t i d e a n a l o g u e o f the newly exposed N-terminus. There are evid e n c e s t h a t t h e a c t i o n o f t h r o m b i n on c e l l s is not limited t o t h a t m e d i a t e d by i t s c l o ned t ~ r o m b i n r e c e p t o r and t h e r e may be more t h e n ~ne t h r o m b i n receptor. In order to find probable candidates mediating action o f t h r o m b i n on c e l l s among t h e c l o n e d prot e i n s we u s e d t h e c o m p u t e r s e a r c h f o r gomologies to functionally important pattern
known to interact with thrombin. Using programm set GeneBee the search of gomologies to site 38-63 thrombin, binding to the active center of enzyme and the hirudin-like domain,docked in the anion-binding exocite of thrombin, was undertaken. The high degree of gomology of this pattern to fragments of Ras-related protein, light chain B of plasminogen (662-886) and extracellular domain of FGF 2 receptor was revealed. The synthetic peptide agonist of thrombin receptor SFLLRN (unlike to thrombin) has no reactions considered as the mitogenic or able to maintain prolonged mitogen-activated protein(MAP)kinase stimulation. So we suggest that the full growth responce of thrombin is supported not only by cooperation of growth factors with thrombin receptor peptide agonists but also the interaction of thrombin with another receptor.
Fibrinolytic activity of highly-molecular 7S NGF is conditioned by its gamma-subunit possessing esteropeptidase activity. Shown is a possibility of gamma NGF to induce fibrinolysls by transformation of plasminogen into plasmin bosh in salivary gland and in the blood of mice (Isanbaev q99@). Besides, 7S NGF can induce fibrinolysis not only through gamma subunit, but also through beta subunit which does not obtain enzymatic property. Such an effect of NGF on the lysis of fibrin, as our study showed, may take place through the histamine of the mast cells, eosinophils and basophils. Histamin, by itself, notably activizes the process of fibrinolysis, and the population of the mast cells, which produce it in an organism, has specific receptors to NGF. Interaction of NGF with them causes an intensive degranulation of the mast cells with the release of histamine into blood, which indicates the potential role of NGF as a natural liberator of histamine, whose role in biochemical processes of initiation of inflammation and regeneration is well known. Thus, our study assumes an important role of NGF in the processes of fibrino- and thrombolysis, and each component may in different ways (gamma subunit directly and beta subunit indirectly) perform its effect on the fibrinolytic system.