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Tissue plasminogen activator deficiency: A molecular analysis
J Mol Cell Cardiol 19 (Supplement IV) (1987) 4OGENERATION AND CHARACTERIZATION OF CLEAVAGE-MINUS MUTANTS OF HUMAN TISSUE-PLASMINOGEN ACTIVATOR. Boose, J.A., Kulsmanen, E., Samhrook, J. and Gething, M.-J. University of Texas Health Science Center, Dallas, Texas. The serine protease, tissue-plasminogen activator (t-PA), is believed to be responsible for catalyzing the production of plasmin from plasminogen at the site of blood clots, t-PA is synthesized as a single polypeptide chain which is converted to a two-chaln form upon exposure to proteases. Proteolytic cleavage of the 1-chain polypeptide occurs on the C-termlnal side of arginine-275. The subonlts of two-chaln t-PA are held together by a single disulfide bond. There has been controversy as to the enzymatic activity of the one-chain form of t-PA. To address this issue, we have used oligonucleotide directed mutagenesis to alter arginine-275 to a glycine residue. This amino acid change prevents conversion of one-chain t-PA to the two-chain form. The cDNAs encoding the wild-type and mutant forms of the t-PA have been expressed in monkey cells using an SV40-based vector. We have analyzed wild-type and cleavage-mlnus t-PA for enzymatic activity by direct and indirect chromogenlc assays and by zymography. The results show that cleavage-mlnus t-PA is enzymatically active. However, this activity is depressed compared to that of wild-type due to an increase in the Km and a decrease in the Vmax of cleavage-mlnus t-PA. Using purified inhibitor to t-PA, we have shown that the activity of cleavage minus t-PA is inhibited to a lesser extent than is the activity of wild-type t-PA.
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TISSUE P L A S M I N O G E N A C T I V A T O R D E F I C I E N C Y : A M O L E C U L A R ANALYSIS. G. Phillips, R. K a u f m a n , and S. P i z z o . D e p a r t m e n t s of Medicine, P a t h o l o g y and B i o c h e m i s t r y , Duke Univ. Medical C e n t e r , D u r h a m , N.C. 27710 H y p e r c o a g u l a b l e , f i b r i n o l y t i c d i s o r d e r s m a y r e s u l t f r o m d e c r e a s e d r e l e a s e of v a s c u l a r p l a s m i n o g e n a c t i v a t o r (t-PA) 3) o r i n c r e a s e d p l a s m a l e v e l s of fast a c t i n g t - P A inhibitor. We now r e p o r t a f a m i l y with r e c u r r e n t s t r o k e s w h e r e g e n e t i c p r o b e s h a v e been used to p r o v e t h a t the t - P A g e n e is a b n o r m a l . We o b t a i n e d c D N A p r o b e s f r o m Biogen as follows: 1) BG 328, 5' p r o b e c o n t a i n i n g cDNA s e q u e n c e s f r o m -Z00 to 1; 2) BG 19Z, c o n t a i n i n g a 47Z base p a i r (bp) Eco RI f r a g m e n t f o r m t h e middle of t h e t - P A cDNA s e q u e n c e ; 3) t - P A 480, a 3' p r o b e c o n t a i n i n g t h e 800 bp Eco RI to Bgl II f r a g m e n t of t h e t - P A cDNA. The p r o b e s w e r e e m p l o y e d for S o u t h e r n b l o t t i n g analysis of t h e p r o p o s i t u s and a second u n r e l a t e d p a t i e n t who also p r e s e n t e d with a t h r o m b o t i c s t r o k e . The following r e s u l t s w e r e obtained: Releasable t-PA t - P A Inhibitcr G e n e t i c Structm-e (IU/mL Plasma) 5' End Middle 3' End Pt 1 0.0 Z.11 Normal Normal Absent Pt Z 0.01 Z3.0 Normal Normal Normal Control 0.70+_ 0.10 Z.56;t. 0.Z9 (n:ll8) (n 56) These r e s u l t s suggest t h a t Pt 1 has a d e l e t i o n f r o m t h e 3' end o f t h e t - P A gene w h i l e P t g has an i n t a c t gene at this l e v e l o f e x a m i n a t i o n . It is suggested t h a t Pt I does not m a k e n o r m a l m e s s e n g e r RNA and h e n c e p r o d u c e s no f u n c t i o n a l p r o t e i n .
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EFFECT OF BINDING OF UROKINASE TO ITS RECEPTORS IN A H U M A N E P I D E R M A L T U M O R CELL LINE, CCL 20.2. J.C. Kirchheimer, G. Christ, J. Wojta, B.R. Binder. Lab. Clin.-Exp. Physiology, University of Vienna, Austria. It is gener'cklly known that plasminogen activators convert the extracellular z ~ o g e n plasminogen into plasmin, an active protease that can either directly or indirectly promote degradation of components of the extracellular matrix. Several types of cells including monocytes, fibroblasts and ep,~ermal tumor cells have been shown to possess specific high 'affinity (Kd 10-ruM) receptors for urokinase-type p l a s m i n o g e n activator (u-PA). Binding of u-PA to its membrane receptor with its a m i n o t e r m i ~ l 17kD f r a ~ e n t results in focusirhg of p l a s m i n o g e n activation to the close e n v i r o n m e n t for the cell surface. In addition to this finding it could be shown in the human epidermal tumor cell line, COL 20.2, tb~t binding of the intact active u-PA molecule elicts stimulation of g r o w t h of the cell. The discovery of u-PA receptors and a biological function of bound urokinase su~gest possible importance for this hormone-receptor system in malignant growth as well as angiogenesis.
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TISSUE P L A S M I N O G E N A C T I V A T O R D E F I C I E N C Y : A M O L E C U L A R ANALYSIS. G. Phillips, R. K a u f m a n , and S. P i z z o . D e p a r t m e n t s of Medicine, P a t h o l o g y and B i o c h e m i s t r y , Duke Univ. Medical C e n t e r , D u r h a m , N.C. 27710 H y p e r c o a g u l a b l e , f i b r i n o l y t i c d i s o r d e r s m a y r e s u l t f r o m d e c r e a s e d r e l e a s e of v a s c u l a r p l a s m i n o g e n a c t i v a t o r (t-PA) 3) o r i n c r e a s e d p l a s m a l e v e l s of fast a c t i n g t - P A inhibitor. We now r e p o r t a f a m i l y with r e c u r r e n t s t r o k e s w h e r e g e n e t i c p r o b e s h a v e been used to p r o v e t h a t the t - P A g e n e is a b n o r m a l . We o b t a i n e d c D N A p r o b e s f r o m Biogen as follows: 1) BG 328, 5' p r o b e c o n t a i n i n g cDNA s e q u e n c e s f r o m -Z00 to 1; 2) BG 19Z, c o n t a i n i n g a 47Z base p a i r (bp) Eco RI f r a g m e n t f o r m t h e middle of t h e t - P A cDNA s e q u e n c e ; 3) t - P A 480, a 3' p r o b e c o n t a i n i n g t h e 800 bp Eco RI to Bgl II f r a g m e n t of t h e t - P A cDNA. The p r o b e s w e r e e m p l o y e d for S o u t h e r n b l o t t i n g analysis of t h e p r o p o s i t u s and a second u n r e l a t e d p a t i e n t who also p r e s e n t e d with a t h r o m b o t i c s t r o k e . The following r e s u l t s w e r e obtained: Releasable t-PA t - P A Inhibitcr G e n e t i c Structm-e (IU/mL Plasma) 5' End Middle 3' End Pt 1 0.0 Z.11 Normal Normal Absent Pt Z 0.01 Z3.0 Normal Normal Normal Control 0.70+_ 0.10 Z.56;t. 0.Z9 (n:ll8) (n 56) These r e s u l t s suggest t h a t Pt 1 has a d e l e t i o n f r o m t h e 3' end o f t h e t - P A gene w h i l e P t g has an i n t a c t gene at this l e v e l o f e x a m i n a t i o n . It is suggested t h a t Pt I does not m a k e n o r m a l m e s s e n g e r RNA and h e n c e p r o d u c e s no f u n c t i o n a l p r o t e i n .
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EFFECT OF BINDING OF UROKINASE TO ITS RECEPTORS IN A H U M A N E P I D E R M A L T U M O R CELL LINE, CCL 20.2. J.C. Kirchheimer, G. Christ, J. Wojta, B.R. Binder. Lab. Clin.-Exp. Physiology, University of Vienna, Austria. It is gener'cklly known that plasminogen activators convert the extracellular z ~ o g e n plasminogen into plasmin, an active protease that can either directly or indirectly promote degradation of components of the extracellular matrix. Several types of cells including monocytes, fibroblasts and ep,~ermal tumor cells have been shown to possess specific high 'affinity (Kd 10-ruM) receptors for urokinase-type p l a s m i n o g e n activator (u-PA). Binding of u-PA to its membrane receptor with its a m i n o t e r m i ~ l 17kD f r a ~ e n t results in focusirhg of p l a s m i n o g e n activation to the close e n v i r o n m e n t for the cell surface. In addition to this finding it could be shown in the human epidermal tumor cell line, COL 20.2, tb~t binding of the intact active u-PA molecule elicts stimulation of g r o w t h of the cell. The discovery of u-PA receptors and a biological function of bound urokinase su~gest possible importance for this hormone-receptor system in malignant growth as well as angiogenesis.
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