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417 Endo- ɛ - ( - Glu-) -Lys isopeptidolysis as a novel hypotetic mechanism of fibrinolysis
POSTER PRESENTATIONS P-XVII Lipoprotein(a) and Regulation of Fibrinolysis
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o~2-ANTIPLASMIN IS A SUBSTRATE OF TISSUE TRANSGLUTAMINASE Hevessy Zs, Hdrsfalvi J and Muszbek L Department of Clinical Chemistry, University School of Medicine, Debrecen, Hungary.
IN VITRO AND IN VIVO STUDIES ON OLIGONUCLEOTIDE PS16 WHICH IS ANTISENSE TO PAl-1 mRNA ICierniewski CS, ZPawlowska Z, IPluskota E, IStasiak M, ZKobylanska A, 2Koziolkiewicz M, 2Maciaszek A, 2Misiura K, 2Stec W J, JWyroba E ~Department of Biophysics, Medical University in Lodz, 2Center for Molecular and Macromolecular Studies, and 3Nencki's Institute in Warsaw, Poland
a2-Antiplasmin (c~2AP) is a highly potent inhibitor of plasmin. The main physiological protective mechanism against the prompt fibrinolytic elimination of fibrin clot involves the covalent attachment of o~2AP to the c~-chain of fibrin by a transglutaminase, activated blood coagulation factor XIII (FXIII). As endothelial cells contain another transglotaminase, tissue tranglutaminase (transghmnninase C; TGC) which, in certain pathological conditions, has also been implicated in fibrin stabilization. The aim of this study was to explore if TGC stabilized fibrin(ogen) clots could also be protected by this mechanism. As detected by SDS PAGE and fluorography, tissue TG catalyzed the incorporation of TGC substrate primary amines (14C glycin ethylester, 3H putrescine and dansylcadaverine) into ~2AP indicating that the protein can 9rovide acyl donor glutamine residue to the crosslinking reaction. The maxhnal extent of incorporation always renmined below 1 tool amine per 1 tool ~2AP. The crosslinking of o~2AP to fibrin(ogen) was investigated by SDS PAGE and imnaunoblotting. TGC crosslinked ot2AP to fibrin and fibrinogen, but not to another c~2AP molecule, in a time and concentration dependent manner. After the fast formation of a heterodimer of 140 kDa consisting of ot2AP and fibrin o>chain the appearance of higher Mr (>300 kDa) products coukl be observed and, eventually, the heterodimers were incorporated into the highly crosslinked ~-chain polymerincapable of penetrating the concentrating gel. It is proposed that TGC released from injured endothelial cell is involved m the production of fibrinolysis-resistant fibrin(ogen) network by incorporating cz2AP into the fibrin (oge n) matrix.
Recently we described a group of phosphorothioate oligodeoxynucleotides antisense to PAI-1 mRNA which selectively inhibited synthesis and release of this protein from endothelial cells. PS 16 (16-residue phosphorothioate oligodeoxynucleotide) was found to be the strongest inhibitor. In present studies, to improve its antisense activity we synthesized a number of derivatives which contained cholesterol or bulky dimethoxytritylphosphonate residues attached to 5' and 3' ends of PS16. In addition, this hexadecanucleotide phosphorothioate was obtained via stereocontrolled oxathiaphospholane methods in [All-Sp]- and [All-Rp]-diastereomerically pure forms. The effect on PAI-1 expression in both endothelial cells and permanent human cell line EA-hy 926 was measured by ELISA and activity assay. PS 16 uptake into cells was monitored by eonfocal microscopy. Independently, the effect of PS 16 on blood PAI-1 activity in rats was measured after single dose injection. In addition, its antithrombotic effect was evaluated in venous model of thrombosis in rats in which thrombus is formed following ligature of the inferior vena cava. Based on these experiments, the following conclusions can be drawn: (a) all derivatives of PS 16 are strong inhibitors of PAI-1 expression, (b) This effect appears to be dependent upon chirality; [All-Sp]diastereoisomers were stronger than [All-Rp]-forms, (c) PS16 in dose 1 mg reduced both PAI-1 activity of rat blood plasma and the mean weight of the thrombi indicating antithrombotic effect.
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ENDO- ~ - ( ~ -GLU-)-LYS I S O P E P T I D O L Y S I S AS A ~OVEL HYBOTET~C M E C H A N I S M OF F~BRINOLYSIS. Baskova IP, -Zavalova LL and B a s a n o v a AV.
FIBRINOLYSIS BY UROKINASE: EFFECT OF CLOT COMPOSITION ON THE LYSIS KINETICS Moukhametova L.I., Aisina R.B., Firsova E. and Varfoiomeyev S.D. Chemical Enzymology Department, Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119899, Russia
IBiologic~l D e p a r t m e n t of H o s c o w State University and -HM Sherayakin and YuA O v c h i n n i k o v Institute of Bioorganic Chemistry, Moscow, Russia. It is known that the medicinal leech saliva which is deprived of pro%eollvtic activity and full of serpins, sti~nuiates tnroinoolysis during the leeching of patient~ ~ith t h o m D o p h l e b i t i s of different ethiology. The novel enzyme destabilase - e n d o - ~ , - ( ~ - G l u ) - L y s - i s o p e p t i d a s e isolated from the secretion of salivary glands and from the water extracts of the medicinal leeches dissolves stabilized fibrin but not n o n - s t a b i l i z e d and stimulates t h r o m o o l y s i s in e x p e r i m e n t a l animals. Highly purified destabilase specifically splits cross-links in stabilized fibrin and in its fragment D-D dimer. The proposed m e c h a n i s m of c r o s s - l i n k e d fibrin dissolving may be explained by the a c c u m u l a t i o n of negative charges of Glu carboxic groups (instead of starting Gln-residues) w h i c h appear as a result of splitting C - ( ~ -Glu)L y s - i s o p e p t i d e bonds. The change<~f charge d i s t r i b u t i o n in fibrin p o l y m e r i z a t i o n sites may be followed by the spontaneous d e p o l y m e r i zation of d e s t a b i l i z e d fibrin. The proving of this m e c h a n i s m of fibrinolysis is in progress.
The purpose of this work was to study the kinetics of fibrinolysis by urokinase (Uk) in a biphase in vitro system of different composition and to evaluate the contribution of plasminogen activation and plasmin (Pm) inhibition by ~t2-antiplasmin (AP) in fibrinolysis by Uk. Fibrin clots containing plasminogen (0.3 p,M) with and without AP or human plasma clots were formed in glass tubes (d=10 mm). Plasma or buffer (pH 7.4) were poured out over the clots. Lysis was initiated by addition of Uk or Pm in the liquid phase. Kinetics of lysis was traced • by the decrease of clot height at 37°C. The kinetic curves of plasma clots lysis by Uk in buffer or in plasma were observed to be S-shaped. To evaluate the effect of plasminogen activation and inhibition of forming Pm by AP on the initial lag time, the series experiments were carried out. Fibrin clots (8.8 [aM) lysis by Uk showed that lag time was also presented at the curves in absence of AP in both phases. When the lysis rate increased with Uk concentration (from 25 IU/ml to 2500 IU/ml), the lag time decreased from 22 rain to 2 min. Incorporation of AP in fibrin clot reduced the lysis rate by Uk, but it had a slight influence on lag time. Curves of Pm-catalyzed lysis of fibrin clots with and without AP, as well as of plasma clots had no lag time. When fibrin clots were substituted for fibrin clots with AP or for plasma clots, the lysis rates by Uk and Pm diminished by a similar way. Substitution of buffer for plasma in liquid phase had a less effect on the |ysis rate by Uk. From the results follows that AP included in clot has a greater inhibitory effect on the lysis rate by Uk than AP being in liquid phase. The lag time on the curves of Uk-induced fibrinolysis is determined primarily by plasminogen activation rate on the clot surface.
12
415
416
o~2-ANTIPLASMIN IS A SUBSTRATE OF TISSUE TRANSGLUTAMINASE Hevessy Zs, Hdrsfalvi J and Muszbek L Department of Clinical Chemistry, University School of Medicine, Debrecen, Hungary.
IN VITRO AND IN VIVO STUDIES ON OLIGONUCLEOTIDE PS16 WHICH IS ANTISENSE TO PAl-1 mRNA ICierniewski CS, ZPawlowska Z, IPluskota E, IStasiak M, ZKobylanska A, 2Koziolkiewicz M, 2Maciaszek A, 2Misiura K, 2Stec W J, JWyroba E ~Department of Biophysics, Medical University in Lodz, 2Center for Molecular and Macromolecular Studies, and 3Nencki's Institute in Warsaw, Poland
a2-Antiplasmin (c~2AP) is a highly potent inhibitor of plasmin. The main physiological protective mechanism against the prompt fibrinolytic elimination of fibrin clot involves the covalent attachment of o~2AP to the c~-chain of fibrin by a transglutaminase, activated blood coagulation factor XIII (FXIII). As endothelial cells contain another transglotaminase, tissue tranglutaminase (transghmnninase C; TGC) which, in certain pathological conditions, has also been implicated in fibrin stabilization. The aim of this study was to explore if TGC stabilized fibrin(ogen) clots could also be protected by this mechanism. As detected by SDS PAGE and fluorography, tissue TG catalyzed the incorporation of TGC substrate primary amines (14C glycin ethylester, 3H putrescine and dansylcadaverine) into ~2AP indicating that the protein can 9rovide acyl donor glutamine residue to the crosslinking reaction. The maxhnal extent of incorporation always renmined below 1 tool amine per 1 tool ~2AP. The crosslinking of o~2AP to fibrin(ogen) was investigated by SDS PAGE and imnaunoblotting. TGC crosslinked ot2AP to fibrin and fibrinogen, but not to another c~2AP molecule, in a time and concentration dependent manner. After the fast formation of a heterodimer of 140 kDa consisting of ot2AP and fibrin o>chain the appearance of higher Mr (>300 kDa) products coukl be observed and, eventually, the heterodimers were incorporated into the highly crosslinked ~-chain polymerincapable of penetrating the concentrating gel. It is proposed that TGC released from injured endothelial cell is involved m the production of fibrinolysis-resistant fibrin(ogen) network by incorporating cz2AP into the fibrin (oge n) matrix.
Recently we described a group of phosphorothioate oligodeoxynucleotides antisense to PAI-1 mRNA which selectively inhibited synthesis and release of this protein from endothelial cells. PS 16 (16-residue phosphorothioate oligodeoxynucleotide) was found to be the strongest inhibitor. In present studies, to improve its antisense activity we synthesized a number of derivatives which contained cholesterol or bulky dimethoxytritylphosphonate residues attached to 5' and 3' ends of PS16. In addition, this hexadecanucleotide phosphorothioate was obtained via stereocontrolled oxathiaphospholane methods in [All-Sp]- and [All-Rp]-diastereomerically pure forms. The effect on PAI-1 expression in both endothelial cells and permanent human cell line EA-hy 926 was measured by ELISA and activity assay. PS 16 uptake into cells was monitored by eonfocal microscopy. Independently, the effect of PS 16 on blood PAI-1 activity in rats was measured after single dose injection. In addition, its antithrombotic effect was evaluated in venous model of thrombosis in rats in which thrombus is formed following ligature of the inferior vena cava. Based on these experiments, the following conclusions can be drawn: (a) all derivatives of PS 16 are strong inhibitors of PAI-1 expression, (b) This effect appears to be dependent upon chirality; [All-Sp]diastereoisomers were stronger than [All-Rp]-forms, (c) PS16 in dose 1 mg reduced both PAI-1 activity of rat blood plasma and the mean weight of the thrombi indicating antithrombotic effect.
417
418
ENDO- ~ - ( ~ -GLU-)-LYS I S O P E P T I D O L Y S I S AS A ~OVEL HYBOTET~C M E C H A N I S M OF F~BRINOLYSIS. Baskova IP, -Zavalova LL and B a s a n o v a AV.
FIBRINOLYSIS BY UROKINASE: EFFECT OF CLOT COMPOSITION ON THE LYSIS KINETICS Moukhametova L.I., Aisina R.B., Firsova E. and Varfoiomeyev S.D. Chemical Enzymology Department, Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119899, Russia
IBiologic~l D e p a r t m e n t of H o s c o w State University and -HM Sherayakin and YuA O v c h i n n i k o v Institute of Bioorganic Chemistry, Moscow, Russia. It is known that the medicinal leech saliva which is deprived of pro%eollvtic activity and full of serpins, sti~nuiates tnroinoolysis during the leeching of patient~ ~ith t h o m D o p h l e b i t i s of different ethiology. The novel enzyme destabilase - e n d o - ~ , - ( ~ - G l u ) - L y s - i s o p e p t i d a s e isolated from the secretion of salivary glands and from the water extracts of the medicinal leeches dissolves stabilized fibrin but not n o n - s t a b i l i z e d and stimulates t h r o m o o l y s i s in e x p e r i m e n t a l animals. Highly purified destabilase specifically splits cross-links in stabilized fibrin and in its fragment D-D dimer. The proposed m e c h a n i s m of c r o s s - l i n k e d fibrin dissolving may be explained by the a c c u m u l a t i o n of negative charges of Glu carboxic groups (instead of starting Gln-residues) w h i c h appear as a result of splitting C - ( ~ -Glu)L y s - i s o p e p t i d e bonds. The change<~f charge d i s t r i b u t i o n in fibrin p o l y m e r i z a t i o n sites may be followed by the spontaneous d e p o l y m e r i zation of d e s t a b i l i z e d fibrin. The proving of this m e c h a n i s m of fibrinolysis is in progress.
The purpose of this work was to study the kinetics of fibrinolysis by urokinase (Uk) in a biphase in vitro system of different composition and to evaluate the contribution of plasminogen activation and plasmin (Pm) inhibition by ~t2-antiplasmin (AP) in fibrinolysis by Uk. Fibrin clots containing plasminogen (0.3 p,M) with and without AP or human plasma clots were formed in glass tubes (d=10 mm). Plasma or buffer (pH 7.4) were poured out over the clots. Lysis was initiated by addition of Uk or Pm in the liquid phase. Kinetics of lysis was traced • by the decrease of clot height at 37°C. The kinetic curves of plasma clots lysis by Uk in buffer or in plasma were observed to be S-shaped. To evaluate the effect of plasminogen activation and inhibition of forming Pm by AP on the initial lag time, the series experiments were carried out. Fibrin clots (8.8 [aM) lysis by Uk showed that lag time was also presented at the curves in absence of AP in both phases. When the lysis rate increased with Uk concentration (from 25 IU/ml to 2500 IU/ml), the lag time decreased from 22 rain to 2 min. Incorporation of AP in fibrin clot reduced the lysis rate by Uk, but it had a slight influence on lag time. Curves of Pm-catalyzed lysis of fibrin clots with and without AP, as well as of plasma clots had no lag time. When fibrin clots were substituted for fibrin clots with AP or for plasma clots, the lysis rates by Uk and Pm diminished by a similar way. Substitution of buffer for plasma in liquid phase had a less effect on the |ysis rate by Uk. From the results follows that AP included in clot has a greater inhibitory effect on the lysis rate by Uk than AP being in liquid phase. The lag time on the curves of Uk-induced fibrinolysis is determined primarily by plasminogen activation rate on the clot surface.