- Email: [email protected]
34 Structural domains of tissue plasminogen activator (tPA) involved in cellular stimulation of plasmin generation
ORAL COMMUNICATIONS III-2 Cellular Receptors
1
3t
32
DECREASED EXPRESSION OF LOW DENSITY LIPOPROTEIN RELATED PROTEIN (LRP) BY THE NEOINTIMAL SMOOTH MUSCLE CELLS IN RABBIT BALLOON-INJURED ABDOMINAL AORTA. Briggs IM, Kanthou C, Moradoghli-Haftvani A, Esmail A, Kakkar VV. and Lupu F Thrombosis Research Institute, Chelsea, London SW3 6LR, U.K.
CLEARANCE STUDIES OF PAI-I:PROTEASE COMPLEXES SUGGEST THAT PAl- 1CONTAINSA CRYPTIC LRP BINDING SITE. ~ e f a n s s o n , S_Mnhammad & D.A. Lawrealf~Biochemist~ Department J. H. Holland Laboratoc¢ American Red Cross 15601 Crabbs Branch Way Rockville, MD 20855.
Low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor that is responsible for the clearance of lipoprotein remnants, of proteases or cytokines/growth factors in complex with alpha2maeroglobulin as well as of plasminogen activators complexed with inhibitors. We investigated the time course of LRP expression of in healthy and balloon catheter injured abdominal aorta in rabbits. Samples were taken 1, 3, 9 and 15 days after balloon catheter injury, frozen, cryosectioned, immunostained by an indirect fluorescence technique using a monoelonal antibody anti LRP apha-chain and examined by laser scanning confocal microscopy. The cellular components of the vessel wall were identified using antibodies to cell-specific markers. The quantitation of the fluorescence intensity was carried out by computer-assisted image analysis. In the normal vessel wall, LRP was found heterogeneously distributed in all SMC of the media, being in higher amount in the layers toward the adventitia than in the ones located close to the luminal side. Within the first three days following balloon deendothelialization, the pattern of LRP staining was changed: some SMC scattered within the luminal side of the media showed an intense staining while the fluorescence intensity in the external layers displayed a slight decrease. Interestingly, the neointimal SMCs which become visible after 9 and 14 days do show just a faint LRP staining. Our results indicate that balloon induced SMC intimal hyperplasia is accompanied by a strong downregulation of LRP expression. The low expression of LRP by the SMC of the restenotic lesions may be important for the pathogenesis of restenosis after angioplasty by increasing the life-time of growth faetors/cytokines, extracelhilar proteases and other proteins that are normally cleared from the extracellular space by LRP (Supported by British Heart Foundation grant PG/94188)
The low density tipoprotein receptor-related protein (LRP) is a cell surface receptor thought to function as a general clearance receptor for a diverse set of ligands including protease inhibitor complexes. Previously we showed that P A l - l , but not antithrombin III, heparin cofactor II or oq-antitrypsin greatly enhanced the clearance of 1251thrombin, and that the enhancement was dependant upon the presence of native vitronectin (Stefansson, S., Lawrence, D.A and Argraves, W.S. 1996. J. Biol. Chem. In press). Extending these studies we have now compared the clearance of neutrophil elastase in complex with either a mutant form of PAl-1 or cq-antitrypsin. As with PAI-1 :thrombin complexes, the mutant PAl-1 :elastase complexes are cleared much more efficiently than elastase in complex with cq-antitrypsin. Together, these data suggest that the PAI-1 has a significantly higher affinity for L R P than other serpins. To further characterize the relative contribution of PAI-1 in LRP mediated clearance, we have developed a system to enzymatically label active PAL1 with 32p under physiological conditions. This eliminates the well characterized problem o f PAI-1 inactivation associated with iodination that has until now hindered the development of direct binding assays for PAl-1. This mutant (HMK-PAi-1) was constructed by PCR and introduces a six residue peptide tag at the Nterminus of PAl- 1 containing the recognition sequence for the specific protein kinase Heart Muscle Kinase (HMK). The activity of HMK-PAI1 toward uPA and tPA is comparable to wtPAl-1 both before and after labeling with 32p. Finally, cellular clearance studies indicate that only PAI-1 in complex with an enzyme and not free active PAl- 1 is efficiently cleared by LRP and suggests that upon complex formation a neoepitope is exposed on PAI-1 that is responsible for the clearance by L R P
33
34
Vascular smooth muscle cells potentiate cell surface plasminogen activation by both uPA and tPA-dependent mechanisms Vincent Ellis and Simon Whawell Thrombosis Research Institute, London, UK
S T R U C T U R A L D O M A I N S OF T I S S U E P L A S M I N O G E N A C T I V A T O R (tPA) I N V O L V E D IN C E L L U L A R S T I M U L A T I O N OF P L A S M I N GENERATION iMerton RE, ~ S i n n i g e r V, ~Fabregas P, 2Felez J, ~ L o n g s t a f f C. D e p t of H a e m a t o l o g y , NIBSC, H e f t s , UK, EN6 3QG. Dept Receptors Cellulars, IRO, B a r c e l o n a , Spain.
Experiments in both normal and transgenic animals indicate that plasminogen activators play a role in the response of the vessel wall to injury, presumably by mediating the degradation of extracellular matrix (ECM) by vascular smooth muscle cells (VSMC) which is necessary to facilitate their migration and proliferation. We have therefore investigated the ability of human VSMC in culture to assemble specific plasminogen activating systems on their cell surface. uPA bound to a single class of binding sites on VSMC with high affinity (Kd = 2 riM), and this binding could be competed by monoclonal antibodies to the uPA receptor (uPAR). Binding of pro-uPA to these cells resulted in a large potentiation of plasmin generation which was dependent on the cellular binding of plasminogen, consistent with our previous observations on leukocytes and various cells of neoplastic origin. Disruption of caveolae on these cells with cholesterol binding agents had no effect on uPAR-mediated plasminogen activation. tPA was also found to bind to VSMC, using a functional assay to ensure that binding to PAI-1 was not being measured. Analysis of the binding isotherms obtained revealed 2 classes of binding site with Kds of 20 and 600 riM. Neither inactivated uPA or plasminogen could compete this binding. Enzyme kinetic analysis demonstrated that although both of these sites mediated plasminogen activation, the large enhancement of tPA activity observed (greater than 50-fold) was solely due to tPA binding to the higher affinity site and required the cellular binding of plasminogen. The tPA domain deletion mutant K2P was found not to interact with the higher affinity binding site. Comparison of the two specific plasminogen activator binding sites revealed that at saturation the binding of tPA led to an approx. 10-fold greater plasmin generation than uPA. This rose to approx. 25-fold after thrombin stimulation of the cells, which increased tPA but not uPA binding. These data demonstrate the occurrence of a novel specific tPA receptor on human VSMC which may be important for the regulation of plasminogen activation and ECM degradation by these cells in various vascular pathologies.
We h a v e i n v e s t i g a t e d cultured c e l l lines, U937, THPI, K562, Molt4, a n d N a l m 6 a n d s h o w n t h a t t h e y bind tPA and plasminogen (Pgn) a n d are a b l e to act as promoters of lys-pgn activation using p h y s i o l o g i c a l t P A c o n c e n t r a t i o n s . To u n d e r s t a n d the structural features of tPA involved we have performed kinetic studies with tPA domain deletion e n z y m e s c o n s i s t i n g of f u l l l e n g t h g l y c o s y l a t e d a n d n o n - g l y c o s y l a t e d t P A (F-G-K1 K2 P), AF t P A (G K1 K2P), K 2 - P t P A (BM 0 6 . 0 2 2 f r o m B o e h r i n g e r M a n n h e i m ) and protease domain (P) . Deletion variants were expressed and refolded from E.coli. Stimulation, c a l c u l a t e d as r a t i o of p g n a c t i v a t i o n rates with c e l l s / r a t e s w i t h o u t cells, was d e t e r m i n e d at o p t i m u m cell densities. Maximum stimulation w a s u p to 79 fold with full length, glycosylated tPA but stimulation for n o n - g l y c o s y l a t e d full length tPA dropped by 40-50%. L o s s of N t e r m i n a l d o m a i n s as in AF t P A a n d K 2 - P t P A r e s u l t e d in a f u r t h e r loss of stimulation to 20-30% of the full length glycosylated value. The p r o t e a s e d o m a i n a l o n e w a s s t i m u l a t e d p o o r l y , u p to 2 - f o l d . Direct binding via carbohydrate is n o t l i k e l y as s t i m u l a t i o n w a s n o t inhibited by the presence of a selection of monosaccharides u p to 20mM. T h e r a n k i n g o r d e r of s t i m u l a t i o n for t P A v a r i a n t s w a s m a i n t a i n e d o v e r p g n c o n c e n t r a t i o n s f r o m 1-520 nM, a n d a s i m p l e m o d e l h a s been developed to show how tPA cell affinity r e g u l a t e s k i n e t i c s . Thus, we c o n c l u d e t h a t m u l t i p l e s i t e s are i n v o l v e d in t P A - c e l l i n t e r a c t i o n s l e a d i n g to e n h a n c e d k i n e t i c s , a s i m i l a r s i t u a t i o n to the r e g u l a t i o n b y f i b r i n of t P A a c t v i t y .
1
3t
32
DECREASED EXPRESSION OF LOW DENSITY LIPOPROTEIN RELATED PROTEIN (LRP) BY THE NEOINTIMAL SMOOTH MUSCLE CELLS IN RABBIT BALLOON-INJURED ABDOMINAL AORTA. Briggs IM, Kanthou C, Moradoghli-Haftvani A, Esmail A, Kakkar VV. and Lupu F Thrombosis Research Institute, Chelsea, London SW3 6LR, U.K.
CLEARANCE STUDIES OF PAI-I:PROTEASE COMPLEXES SUGGEST THAT PAl- 1CONTAINSA CRYPTIC LRP BINDING SITE. ~ e f a n s s o n , S_Mnhammad & D.A. Lawrealf~Biochemist~ Department J. H. Holland Laboratoc¢ American Red Cross 15601 Crabbs Branch Way Rockville, MD 20855.
Low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor that is responsible for the clearance of lipoprotein remnants, of proteases or cytokines/growth factors in complex with alpha2maeroglobulin as well as of plasminogen activators complexed with inhibitors. We investigated the time course of LRP expression of in healthy and balloon catheter injured abdominal aorta in rabbits. Samples were taken 1, 3, 9 and 15 days after balloon catheter injury, frozen, cryosectioned, immunostained by an indirect fluorescence technique using a monoelonal antibody anti LRP apha-chain and examined by laser scanning confocal microscopy. The cellular components of the vessel wall were identified using antibodies to cell-specific markers. The quantitation of the fluorescence intensity was carried out by computer-assisted image analysis. In the normal vessel wall, LRP was found heterogeneously distributed in all SMC of the media, being in higher amount in the layers toward the adventitia than in the ones located close to the luminal side. Within the first three days following balloon deendothelialization, the pattern of LRP staining was changed: some SMC scattered within the luminal side of the media showed an intense staining while the fluorescence intensity in the external layers displayed a slight decrease. Interestingly, the neointimal SMCs which become visible after 9 and 14 days do show just a faint LRP staining. Our results indicate that balloon induced SMC intimal hyperplasia is accompanied by a strong downregulation of LRP expression. The low expression of LRP by the SMC of the restenotic lesions may be important for the pathogenesis of restenosis after angioplasty by increasing the life-time of growth faetors/cytokines, extracelhilar proteases and other proteins that are normally cleared from the extracellular space by LRP (Supported by British Heart Foundation grant PG/94188)
The low density tipoprotein receptor-related protein (LRP) is a cell surface receptor thought to function as a general clearance receptor for a diverse set of ligands including protease inhibitor complexes. Previously we showed that P A l - l , but not antithrombin III, heparin cofactor II or oq-antitrypsin greatly enhanced the clearance of 1251thrombin, and that the enhancement was dependant upon the presence of native vitronectin (Stefansson, S., Lawrence, D.A and Argraves, W.S. 1996. J. Biol. Chem. In press). Extending these studies we have now compared the clearance of neutrophil elastase in complex with either a mutant form of PAl-1 or cq-antitrypsin. As with PAI-1 :thrombin complexes, the mutant PAl-1 :elastase complexes are cleared much more efficiently than elastase in complex with cq-antitrypsin. Together, these data suggest that the PAI-1 has a significantly higher affinity for L R P than other serpins. To further characterize the relative contribution of PAI-1 in LRP mediated clearance, we have developed a system to enzymatically label active PAL1 with 32p under physiological conditions. This eliminates the well characterized problem o f PAI-1 inactivation associated with iodination that has until now hindered the development of direct binding assays for PAl-1. This mutant (HMK-PAi-1) was constructed by PCR and introduces a six residue peptide tag at the Nterminus of PAl- 1 containing the recognition sequence for the specific protein kinase Heart Muscle Kinase (HMK). The activity of HMK-PAI1 toward uPA and tPA is comparable to wtPAl-1 both before and after labeling with 32p. Finally, cellular clearance studies indicate that only PAI-1 in complex with an enzyme and not free active PAl- 1 is efficiently cleared by LRP and suggests that upon complex formation a neoepitope is exposed on PAI-1 that is responsible for the clearance by L R P
33
34
Vascular smooth muscle cells potentiate cell surface plasminogen activation by both uPA and tPA-dependent mechanisms Vincent Ellis and Simon Whawell Thrombosis Research Institute, London, UK
S T R U C T U R A L D O M A I N S OF T I S S U E P L A S M I N O G E N A C T I V A T O R (tPA) I N V O L V E D IN C E L L U L A R S T I M U L A T I O N OF P L A S M I N GENERATION iMerton RE, ~ S i n n i g e r V, ~Fabregas P, 2Felez J, ~ L o n g s t a f f C. D e p t of H a e m a t o l o g y , NIBSC, H e f t s , UK, EN6 3QG. Dept Receptors Cellulars, IRO, B a r c e l o n a , Spain.
Experiments in both normal and transgenic animals indicate that plasminogen activators play a role in the response of the vessel wall to injury, presumably by mediating the degradation of extracellular matrix (ECM) by vascular smooth muscle cells (VSMC) which is necessary to facilitate their migration and proliferation. We have therefore investigated the ability of human VSMC in culture to assemble specific plasminogen activating systems on their cell surface. uPA bound to a single class of binding sites on VSMC with high affinity (Kd = 2 riM), and this binding could be competed by monoclonal antibodies to the uPA receptor (uPAR). Binding of pro-uPA to these cells resulted in a large potentiation of plasmin generation which was dependent on the cellular binding of plasminogen, consistent with our previous observations on leukocytes and various cells of neoplastic origin. Disruption of caveolae on these cells with cholesterol binding agents had no effect on uPAR-mediated plasminogen activation. tPA was also found to bind to VSMC, using a functional assay to ensure that binding to PAI-1 was not being measured. Analysis of the binding isotherms obtained revealed 2 classes of binding site with Kds of 20 and 600 riM. Neither inactivated uPA or plasminogen could compete this binding. Enzyme kinetic analysis demonstrated that although both of these sites mediated plasminogen activation, the large enhancement of tPA activity observed (greater than 50-fold) was solely due to tPA binding to the higher affinity site and required the cellular binding of plasminogen. The tPA domain deletion mutant K2P was found not to interact with the higher affinity binding site. Comparison of the two specific plasminogen activator binding sites revealed that at saturation the binding of tPA led to an approx. 10-fold greater plasmin generation than uPA. This rose to approx. 25-fold after thrombin stimulation of the cells, which increased tPA but not uPA binding. These data demonstrate the occurrence of a novel specific tPA receptor on human VSMC which may be important for the regulation of plasminogen activation and ECM degradation by these cells in various vascular pathologies.
We h a v e i n v e s t i g a t e d cultured c e l l lines, U937, THPI, K562, Molt4, a n d N a l m 6 a n d s h o w n t h a t t h e y bind tPA and plasminogen (Pgn) a n d are a b l e to act as promoters of lys-pgn activation using p h y s i o l o g i c a l t P A c o n c e n t r a t i o n s . To u n d e r s t a n d the structural features of tPA involved we have performed kinetic studies with tPA domain deletion e n z y m e s c o n s i s t i n g of f u l l l e n g t h g l y c o s y l a t e d a n d n o n - g l y c o s y l a t e d t P A (F-G-K1 K2 P), AF t P A (G K1 K2P), K 2 - P t P A (BM 0 6 . 0 2 2 f r o m B o e h r i n g e r M a n n h e i m ) and protease domain (P) . Deletion variants were expressed and refolded from E.coli. Stimulation, c a l c u l a t e d as r a t i o of p g n a c t i v a t i o n rates with c e l l s / r a t e s w i t h o u t cells, was d e t e r m i n e d at o p t i m u m cell densities. Maximum stimulation w a s u p to 79 fold with full length, glycosylated tPA but stimulation for n o n - g l y c o s y l a t e d full length tPA dropped by 40-50%. L o s s of N t e r m i n a l d o m a i n s as in AF t P A a n d K 2 - P t P A r e s u l t e d in a f u r t h e r loss of stimulation to 20-30% of the full length glycosylated value. The p r o t e a s e d o m a i n a l o n e w a s s t i m u l a t e d p o o r l y , u p to 2 - f o l d . Direct binding via carbohydrate is n o t l i k e l y as s t i m u l a t i o n w a s n o t inhibited by the presence of a selection of monosaccharides u p to 20mM. T h e r a n k i n g o r d e r of s t i m u l a t i o n for t P A v a r i a n t s w a s m a i n t a i n e d o v e r p g n c o n c e n t r a t i o n s f r o m 1-520 nM, a n d a s i m p l e m o d e l h a s been developed to show how tPA cell affinity r e g u l a t e s k i n e t i c s . Thus, we c o n c l u d e t h a t m u l t i p l e s i t e s are i n v o l v e d in t P A - c e l l i n t e r a c t i o n s l e a d i n g to e n h a n c e d k i n e t i c s , a s i m i l a r s i t u a t i o n to the r e g u l a t i o n b y f i b r i n of t P A a c t v i t y .