- Email: [email protected]
Introduction of a lysine binding kringle in u-PA does not result in lysine binding and fibrin-dependent plasminogen activation
POSTER PRESENTATIONS P-l: Plasminogen and Plasminogen Activators
83 INTRODUCTION OF A LYSINE BINDING KRINGLE IN u-PA DOES NOT RESULT IN LYSINE BINDING AND FIBRIN-DEPENDENT PLASMINOGEN ACTIVATION. Bakker AHF and Verheiien JH Gaubius Laboratory, TNO-PG, Leiden, The Netherlands. Plasminogen activators are composed of domain-like structures, which are throught to be structurally and functionally independent. Tissue-type plasminogen activator (t-PA, FGK,K,P) consists of a finger-like domain (F), a growth factor-like domain (G), two kringle-like domains (K, and K,) and a serine protease-like domain (P). In the urokinase-type plasminogen activator (u-PA, GKP), a G, K and P domain can be distinguished. In the presence of fibrin t-PA hydrolyses the R560-Vs6’ peptide bond of plasminogen more efficiently. Although the exact mechanism of this enhancement is notwell understood the presence of the K, domain seems to be of essential importance. The K, domain is known to bind lysine
84 ACTIVE SITE LABELLING WITH DANSYL-GLUTAMAYL-GLYCYL-ARGINYL CHLOROMETHYL KETONE DEMONSTRATES THE FULL ACTIVITY OF THE REFOLDED AND PURIFIED TISSUE-TYPE PLASMINOGEN ACTIVATOR VARIANT BM 06.022 Ulrich Kohnert, Manfred Wozny, Anita Roos and Stephan Fischer
Manuel Llinas,
Boehringer Mannheim GmbH, Biochemical search Center Penzberg, Nonnenwald 2, D-82377 Penzberg, FRG
Re-
31
analogues like epsilon amino caproic acid (EACA) and it was hypothesized that lysine binding equals fibrin binding and is a prerequisite for fibrin-dependent plasminogen activation. Introduction of a lysine binding site in K, domain of K,P supported this view. Based on the assumption that differences in primary amino acid structure are responsible for differences in function, we introduced three stretches of K, specific amino acid residues 2XMILIGK33,“DGDs9, 69NRRLTW74 into the kringle of u-PA, substituting 28ATVLQQ33, “DNRS9, 69GLKPLV74in this u-PA kringle, respectively. No lysine binding, fibrin binding or fibrin-dependent plasminogen activation was observed. However, when the K domain of u-PA was replaced by the K, domain of t-PA also no lysine binding, fibrin binding and fibrin-dependent plasminogen activation was observed. This observation suggests that domains in plasminogen activators do not always function autonomously.
inclusion bodies in E.coli and transferred into the active enzyme by an in vitro refolding process. Active site labelling with dansyl-glutamyl-glycylarginyl chloromethyl ketone provides evidence that the purified BM 06.022 is fully active and that misfolded species are completely removed by affinity chromatography on ETI-Sepharose. The comparison of the kinetics of the inhibition of BM 06.022 with that of CHO-t-PA indicates that the active center of both enzymes is rather similar. The further evaluation of the site of interaction of BM 06.022 and GGACK by mass spectroscopy and amino acid sequence analysis revealed that the inhibitor is bound only to HisaZZ which is part of the catalytic triad of the enzyme.
BM 06.022 is a tissue-type plasminogen activator deletion variant which is comprised of the kringle 2 and the protease domain of the native molecule. BM 06.022 is expressed as inactive
85 IN VITRO STABILITY OF THE RECOMBINANT PLASMINOGEN ACTIVATOR BM 06.022 IN HUMAN PLASMA. Riiken DC, Groeneveld E and Barrett-Bergshoeff MM Gaubius Laboratory, TNO-PG, Leiden, The Netherlands. BM 06.022 is a nonglycosylated mutant of human tissuetype plasminogen activator (t-PA) comprising only the kringle-2 and protease domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.0:22 in plasma was further investigated. Varying concentrations of BM 06.022 were incubated in plasma for O-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 62 min at 0.3 pg/ml to 38 min at 10 pg/ml. SDS-polyacrylamide gel electrophoresis (SDS-
PAGE) followed by fibrin zymography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, cr,-antiplasmin and a,-antitrypsin. During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a,-antiplasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i.e. within 45 min) converted into its two-chain form at concentrations of 5 pg/ml BM 06.022 and higher. In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, q-antiplasmin and a,-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.
83 INTRODUCTION OF A LYSINE BINDING KRINGLE IN u-PA DOES NOT RESULT IN LYSINE BINDING AND FIBRIN-DEPENDENT PLASMINOGEN ACTIVATION. Bakker AHF and Verheiien JH Gaubius Laboratory, TNO-PG, Leiden, The Netherlands. Plasminogen activators are composed of domain-like structures, which are throught to be structurally and functionally independent. Tissue-type plasminogen activator (t-PA, FGK,K,P) consists of a finger-like domain (F), a growth factor-like domain (G), two kringle-like domains (K, and K,) and a serine protease-like domain (P). In the urokinase-type plasminogen activator (u-PA, GKP), a G, K and P domain can be distinguished. In the presence of fibrin t-PA hydrolyses the R560-Vs6’ peptide bond of plasminogen more efficiently. Although the exact mechanism of this enhancement is notwell understood the presence of the K, domain seems to be of essential importance. The K, domain is known to bind lysine
84 ACTIVE SITE LABELLING WITH DANSYL-GLUTAMAYL-GLYCYL-ARGINYL CHLOROMETHYL KETONE DEMONSTRATES THE FULL ACTIVITY OF THE REFOLDED AND PURIFIED TISSUE-TYPE PLASMINOGEN ACTIVATOR VARIANT BM 06.022 Ulrich Kohnert, Manfred Wozny, Anita Roos and Stephan Fischer
Manuel Llinas,
Boehringer Mannheim GmbH, Biochemical search Center Penzberg, Nonnenwald 2, D-82377 Penzberg, FRG
Re-
31
analogues like epsilon amino caproic acid (EACA) and it was hypothesized that lysine binding equals fibrin binding and is a prerequisite for fibrin-dependent plasminogen activation. Introduction of a lysine binding site in K, domain of K,P supported this view. Based on the assumption that differences in primary amino acid structure are responsible for differences in function, we introduced three stretches of K, specific amino acid residues 2XMILIGK33,“DGDs9, 69NRRLTW74 into the kringle of u-PA, substituting 28ATVLQQ33, “DNRS9, 69GLKPLV74in this u-PA kringle, respectively. No lysine binding, fibrin binding or fibrin-dependent plasminogen activation was observed. However, when the K domain of u-PA was replaced by the K, domain of t-PA also no lysine binding, fibrin binding and fibrin-dependent plasminogen activation was observed. This observation suggests that domains in plasminogen activators do not always function autonomously.
inclusion bodies in E.coli and transferred into the active enzyme by an in vitro refolding process. Active site labelling with dansyl-glutamyl-glycylarginyl chloromethyl ketone provides evidence that the purified BM 06.022 is fully active and that misfolded species are completely removed by affinity chromatography on ETI-Sepharose. The comparison of the kinetics of the inhibition of BM 06.022 with that of CHO-t-PA indicates that the active center of both enzymes is rather similar. The further evaluation of the site of interaction of BM 06.022 and GGACK by mass spectroscopy and amino acid sequence analysis revealed that the inhibitor is bound only to HisaZZ which is part of the catalytic triad of the enzyme.
BM 06.022 is a tissue-type plasminogen activator deletion variant which is comprised of the kringle 2 and the protease domain of the native molecule. BM 06.022 is expressed as inactive
85 IN VITRO STABILITY OF THE RECOMBINANT PLASMINOGEN ACTIVATOR BM 06.022 IN HUMAN PLASMA. Riiken DC, Groeneveld E and Barrett-Bergshoeff MM Gaubius Laboratory, TNO-PG, Leiden, The Netherlands. BM 06.022 is a nonglycosylated mutant of human tissuetype plasminogen activator (t-PA) comprising only the kringle-2 and protease domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.0:22 in plasma was further investigated. Varying concentrations of BM 06.022 were incubated in plasma for O-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 62 min at 0.3 pg/ml to 38 min at 10 pg/ml. SDS-polyacrylamide gel electrophoresis (SDS-
PAGE) followed by fibrin zymography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, cr,-antiplasmin and a,-antitrypsin. During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a,-antiplasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i.e. within 45 min) converted into its two-chain form at concentrations of 5 pg/ml BM 06.022 and higher. In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, q-antiplasmin and a,-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.