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Adsorption to fibrin of native fragments of known primary structure from human plasminogen
377
Biochimica et Biophysica Acta, 6 6 8 ( 1 9 8 1 ) 3 7 7 - - 3 8 7 E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 38670
ADSORPTION TO FIBRIN OF NATIVE FRAGMENTS OF KNOWN PRIMARY STRUCTURE FROM HUMAN PLASMINOGEN
S I X T U S T H O R S E N a, I N G E C L E M M E N S E N a, L A R S S O T T R U P - J E N S E N b a n d STAFFAN MAGNUSSON b
a Department o f C!inical Chemistry, University o f Copenhagen, Hvidovre Hospital, DK-2650 Hvidovre and b Department o f Molecular Biology and Plant Physiology, University o f Aarhus, DK-8000 Aarhus C (Denmark) (Received October 14th, 1980)
Key words: Plasminogen fragment: Fibrin binding; Lysine-binding site; Aminohexanoic acid; Tranexamic acid; as -Antiplasmin; (Human plasma)
Summary Limited proteolysis o f native Glu-plasminogen with pancreatic elastase produced three major fragments, K1+2+3, K4, K5-1ight chain (miniplasminogen). Fibrin-binding was determined by clotting fibrinogen in the presence of 12sIlabelled fragments and measuring l~sI in the washed fibrin and in the supernatant. Of the fragments miniplasminogen showed the highest fibrin-binding, the strength o f which was intermediate between those of Glu-plasminogen and Lysplasminogen. The fibrin-binding of all three f~agments was decreased by 6-aminohexanoic acid or tranexamic acid. This decrease was most pronounced with K1+2+3. The fibrin-binding o f K1+2+3, but not that of K4 and miniplasminogen was decreased by a2-antiplasmin. The fibrin-binding of K1+2+3 and miniplasminogen was lower in a plasma clot than in a purified fibrin clot. Our results indicate that each of the three fragments can bind to fibrin. They confirm that an a2-antiplasmin-binding site is located on K1+2+3. Furthermore two of the fragments, namely K4 and K1+2+3 contain lysine-binding site(s).
Introduction Native human Glu-plasminogen is a single peptide chain of known primary structure [1--3]. It consists of three parts. (a) The N-terminal part comprising Abbreviations: SDS, sodium dodecyl sulfate; GalNH 2, Galactosamine; GleNH2, Glueosamine; KI unit, kallil~xein inhibitor unit; NIH unit, National Institutes of Health unit; tranexamic acid, trans-4-aminomethylcyelohexane-l-caxboxylic acid; D-Val-Leu-Lys-pNA, D-valyl-L-leucyl-L-lysine 4-nitroanilide. 0 0 0 5 - 2 7 9 5 / 8 1 / 0 0 0 0 - - 0 0 0 0 / $ 0 2 . 5 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
378 residues 1--76 (M~ = 8800). The removal of this part appreciably alters the conformation of plasminogen [4]. (b) The intermediate part comprising residues 77--560 (Mr = 57 000), which contains five kringle structures (K1--K5) that are homologous with the two kringle structures in prothrombin [5 ]. This part of the molecule contains sites that bind to fibrin and a~-antiplasmin and sites, denoted lysine-binding sites, that bind a number of co-aminocarboxylic acids, such as L-lysine, 6-aminohexanoic acid and tranexamic acid [6--14]. These sites are to some extent functionally interdependent but their exact structural bases have not been elucidated. (c) The C-terminal part, residues 561--790 (Mr = 25 700), which upon activation becomes the light chain and is the serine proteinase part of plasmin. It shows extensive homology with other serine proteinases such as thrombin, trypsin, chymotrypsin and elastase [1,3, 15]. Limited proteolysis of Glu-plasminogen with elastase (pancreatic or leucocyte) produces three major fragments with known primary structure [1]. They are K1+2+3, K4 and K5-1ight chain (neoplasminogen-Val-442, miniplasminogen). The present work describes the fibrin-binding of these fragments and the effect of 6-aminohexanoic acid, tranexamic acid or a2-antiplasmin on this binding. Preliminary data on this work have been presented [16]. Materials and Methods
Buffers. Buffer 1, 0.05 M Tris-HC1/0.10 M NaC1, adjusted to pH 7.70 (20°C) with NaOH, ionic strength 0.15. B u f f e r 2 , 0.05 M Tris-HC1/0.25 M NaC1, adjusted to pH 7.70 (20°C) with NaOH, ionic strength 0.30.6-Aminohexanoic acid, tranexamic acid and D-Val-Leu-Lys-pNA (S-2251) were from Kabi Ltd., Stockholm. Bovine thrombin (8 • 104 NIH units/g) was from Leo Pharmaceuticals, Copenhagen. Solutions of these reagents were prepared in Buffer 1. Plasma was obtained from human blood stabilized by 0.1 vol. 0.11 M trisodium citrate. Specific rabbit immunoglobulins against human plasminogen were from Dakopatts, Copenhagen. Specific rabbit immunoglobulins against a2-antiplasmin were isolated from antiserum [17] which was produced as described [18]. SDS-polyacrylamide gel electrophoresis was performed as described [19]. Gel slabs (2.7 X 82 × 82 mm) contained 7.5% or 10% acrylamide with 6 M urea. Concentration of proteins. The concentration (absorbance) of fibrinogen, clottable fibrinogen or purified Glu- or Lys-plasminogen was determined at 280 nm [20,21]. The concentration of K1+2+3, K4 or miniplasminogen was determined by amino acid analysis. The concentration (active site) of Glu- or Lysplasminogen or miniplasminogen was determined by titration with aprotinin (Trasylol) after its conversion to plasmin [20]. The plasmin activity was measured at 25°C by rate assay on D-Val-Leu-Lys-pNA (substrate concentration, 0.36 M in Buffer 1) [6]. The concentrations (active site) were only used when stated. The concentration of purified a~-antiplasmin was determined at 280 nm using nlcrnA!% 6.7 and M~ = 67 000 [22] or by titration with plasmin [18]. The residual plasmin activity was measured on D-Val-Leu-Lys-pNA. Titrated concentrations were used unless otherwise stated. The concentration of Glu-plasminogen or a2-antiplasmin in plasma was determined by electroimmuno assay [23] using purified Glu-plasminogen or a2-antiplasmin as standard. Preparation of proteins. Human fibrinogen from IMCO Corporation, Ltd., =
379 Stockholm. Contaminating plasminogen was removed b y specific binding to lysine-Sepharose 4B (Pharmacia, Uppsala) as previously described [24], except that fibrinogen was dialysed against Buffer 2 and passed through a lysineSepharose column equilibrated and eluted with the same buffer. Prior to use the stock solution of fibrinogen was diluted with I vol. distilled water and further diluted with Buffer 1. The coagulability (mean -+ S.E. (n)), expressed as the ratio, (clottable fibrinogen concentration)/(fibrinogen concentration), was 0.98 + 0.019 (11). SDS-polyacrylamide gel electrophoresis of a reduced fibrinogen sample indicated that the A(a)-chain was intact in a large fraction of the fibrinogen molecules (Fig. 1). Plasminogen and plasminogen fragments. Procedures used were as follows. Glu-plasminogen was purified from outdated Acid-Citrate-Dextrose stabilized plasma b y affinity chromatography on lysine-Sepharose 4B [20]. Spontaneous proteolytic activity was removed by treatment o f the plasminogen eluate from 2000 ml plasma with 20 ml aprotinin-Sepharose 4B (400 nmol aprotinin per ml gel) for 20 h at 4°C [25]. This was followed b y gel filtration on Ultrogel AcA 44 (LKB, Stockholm) and ion exchange chromatography on DEAESephadex A-50 (Pharmacia) [26]. Lys-plasminogen was prepared as described [25]. The Glu-plasminogen eluate from the affinity chromatography step was allowed to spontaneously convert to the modified form and spontaneous proteolytic activity was removed b y aprotinin-Sepharose as above. This was followed b y gel filtration on Ultrogel AcA 44. K1+2+3, K4 and miniplasmi-
D
° 8 2
3
4
5
6
7
'"
Fig. 1. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in u r e a o f r e d u c e d s a m p l e s o f (1) h u m a n f i b r i n o g e n f r o m I M C O ; (2) G l u - p l a s m i n o g e n ; (3) L y s - p l a s m i n o g e n ; (4) K l + 2 + 3 a ; (5) K l + 2 + 3 b ; (6) K 4 ; (7) m i n i p l a s m i n o geM; (8) c~2 - a n t i p l a s m i n . T h e K 1 + 2 + 3 v a r i a n t s axe d e f i n e d in T a b l e I. E l e c t r o p h o r e s i s w a s c a r r i e d o u t in gel slabs (2,7 X 82 X 82 r a m ) c o n t a i n i n g 10% ( r u n 6) or 7.5% a c z y l a m i d e in 6 M u r e a a n d 0.1 M s o d i u m p h o s p h a t e , p H 7.0. T h e a m o u n t o f p r o t e i n a p p l i e d to t h e gel w a s 37 p g i n (1), 72 p g in (6), a n d 1 3 - - 2 0 p g in t h e o t h e r r u n s . (2) to (5) w e r e r u n in t h e s a m e gel slab.
380 nogen (defined in Table I) were prepared b y specific limited proteolysis of Gluplasminogen using porcine elastase (pancreatic) and isolated b y gel filtration on Sephadex G-75 (Pharmacia) followed b y affinity chromatography on lysineSepharose using a linear gradient of 6-aminohexanoic acid [1]. The identities of the Glu- and Lys-plasminogen were verified by N-terminal amino acid analysis [27] and by molecular weight determination b y SDS-polyacrylamide gel electrophoresis. K1+2+3, K4 and miniplasminogen were identified by their primary structures [1]. The ratio, (concentration (active site))/(concentration (absorbance)), ranged between 0.77 and 1.0 with preparations of Glu-plasminogen, Lys-plasminogen and miniplasminogen. The spontaneous catalytic amount of plasmin as fraction of the total amount of plasmin + activatable plasminogen was less than 0.004 in all preparations. Glu-plasminogen and its derivatives are depicted in SDS-polyacrylamide gel electrophoresis in Fig. 1. ~2-Antiplasmin was purified in a five-step procedure from plasma stabilized by AcidCitrate-Dextrose. Step 1. Adsorption of plasminogen to lysine-Sepharose using 0.2 vol. packed gel. Step 2. Fractional (NH4)2SO4-precipitation of the plasminogen-depleted supernatant [18]. Step 3. Affinity chromatography on plasminogen~Sepharose. This was performed as described [22 ], except that ~2-antiplasmin was eluted with 0 . 0 4 M sodium phosphate, pH 7.4 containing I mM tranexamic acid. Setp 4. Ion exchange chromatography. This was performed as described [22], but at pH 7.4. Step 5. Gel filtration on Ultrogel AcA 44, equilibrated and eluted with 0.05 M Tris-HC1/0.55 M NaC1, adjusted to pH 7.70 (20°C) with NaOH. The eluted ~2-antiplasmin was concentrated and dialysed against Buffer 1. Dilutions were made with the same buffer. The ratio, (concentration (titrated))/(concentration (absorbance)), was 0.81. Most of the inhibitor had retained its ability to bind to plasminogen-Sepharose [28]. SDS-polyacrylamide gel electrophoresis of the purified inhibitor is shown in Fig. 1. Radioiodination of proteins. This was performed according to McFarlane [29] with some modifications [30] by using iodine-125 (Na~2SI, carrier free, in NaOH solution, pH 8--11, free from reducing agent) from the Radiochemical Centre, Amersham. U n b o u n d iodine and protein in the iodination solution was separated on Sephadex G-25 (Pharmacia) equilibrated and eluted with Buffer 2 (miniplasminogen) or Buffer 1 (other proteins). Prior to use the labelled miniplasminogen solution was diluted with 1 vol. distilled water and further diluted with Buffer 1. The other labelled protein solutions were diluted with Buffer 1. The iodine substitution level ranged from 0.008 to 0.3 atoms of iodine per mole of protein. The specific radioactivity ranged from 0.04 to 3.3 mCi per ~mol of protein. Lysine-Sepharose affinity chromatography of a mixture of unlabelled and labelled K1+2+3, K4 or miniplasminogen showed that radioactivity and protein (monitored by measuring absorbance at 280 nm) were eluted in the same peak, when a linear gradient of 6~aminohexanoic acid was used [1]. Agarose gel electrophoresis at pH 8.6 [31] of a mixture of unlabelled and labelled Glu- or Lys-plasminogen followed by autoradiography on Kodak X-Omat duplicating film showed that radioactivity and protein had the same mobility (fi- or 3,-mobility, respectively). The ratio, (concentration (active site))/(concentration absorbance)) did not change after iodination of Gluplasminogen, Lys-plasminogen or miniplasminogen. Fibrin-binding of labelled plasminogen fragments. This was determined in
381 fibrin or plasma clots (vol. = 500 pl) by clotting a mixture of fibrinogen or plasma and 125I-labelled fragment with a t h r o m b i n solution (final t h r o m b i n co n c e n tr atio n , 4 • 103 NIH U/l). In assays o f the effect of 6-aminohexanoic acid, tranexamic acid or a2-antiplasmin the substance was present during fibrin f o r m a t io n . The fibrin threads were separated from the liquid phase by winding them up on a glass rod at r o o m t e m p e r a t u r e (20°C) over a period of 30 min [21]. The fibrin was squeezed free of liquid and washed briefly in 5 ml 0.15 M NaC1. Finally th e fibrin was dried by blotting on a piece of filter paper and then dissolved in 1 ml 2.5 M NaOH by incubation for 20 min at 100°C followed by dilution with 9 vols. distilled water. Radioactivity was measured in the resulting 0.25 M NaOH-solution, in the liquid remaining after removal of fibrin, and in a control with the same composition as the clotting mixture, except that it did n o t contain th r om bi n. Radioactivity of the control differed little from the sum o f the radioactivities of the fibrin and the liquid phase. The complete recovery o f fibrin on the glass rod was checked by measuring the absorbance at 280 nm of the 0.25 M NaOH solution. Fibrin-binding was expressed as, (radioactivity o f fibrin)/(radioactivity of control). Fibrin-binding of unlabelled miniplasminogen. This was determined in fibrin clots as previously r e p o r t e d for Glu- or Lys-plasminogen [21], except that miniplasminogen after its conversion to miniplasmin was determined by using D-Val-Leu-Lys-pNA as a substrate. Results
Table I shows that a large fraction o f the labelled Lys-plasminogen (0.71-0.74) or labelled miniplasminogen (0.44--0.45), in the respective clotting mixtures, was b o u n d to fibrin. Separate experiments showed that this also applied to unlabelled miniplasminogen. The fraction b o u n d was 0.55 (mean of two determinations) in a clotting mixture containing the same concentrations of fibrinogen and fragment as in Table I. In the case o f the two labelled variants of K1+2+3 (Table I), the variant with an oligosaccharide group attached to Thr345 (K1+2+3a) was found to bind to fibrin to the same e x t e n t (0.17--0.19) as labelled Glu-plasminogen (0.17). The second variant ( K 1 + 2 + 3 b ) w i t h oligosaccharide groups attached to Asn-288 and to Thr-345, and the labelled K4 fragment exhibited the weakest fibrin-binding (0.033--0.090). The fraction o f each fragment b o u n d to fibrin was essentially independent of its concent rat i on in the clotting mixture. Similar results were obtained with 2 or 3 different preparations of each fragment (results with all preparations are included in Table I). Inclusion o f 6oaminohexanoic acid or tranexamic acid decreased the fibrint)inding o f the labelled plasminogen fragments (Fig. 2, Table II). If az-antiplasmin was included only the binding of Kl+2+3a and Lys-plasminogen was decreased (Fig. 3). There was no effect of a2-antiplasmin on the binding o f K4 or miniplasminogen (Table III). The fibrin-binding of labelled Kl+2+3a, miniplasminogen or Lys-plasminogen in a fibrin clot was 1.6 to 2.6 times higher than that of the same fragment in a plasma clot (Table IV). Inclusion of the same amounts o f Glu-plasminogen and a2-antiplasmin in the fibrin clots as were present in the plasma clots
382 TABLE
I
FIBRIN-BINDING
OF 125I.LABELLED
The clotting mixture
contained
PLASMINOGEN
FRAGMENTS
9.2 ~M clottable fibrinogen.
Fragment (Sequence; Mz)
Concentration in c l o t t i n g mixture (#M)
Fraction of labelled fzagment bound to fibrin Mean
S.E. (n)
K1 + 2 + 3 a (73/79--353;
34 000) *
1.8 0.5 0.2
0.19 0.19 0.17
0.0067 0.013 0.0058
( 6) ( 6) ( 3)
K1 + 2 + 3 b (73/79--353;
39000)
1.9
0.033
0.0029
( 9)
1.2 0.4 0.2
0.065 0.090 0.082
0.0049 0.0054 0.0056
(6) (6) ( 7)
1.2 0.3 0.1
0.45 0.44 0.44
0.014 0.0089 0.0058
(17) (12) (3)
1.2 0.2
0.71 0.74
0.0081 0.012
(14) ( 3)
1.2
0.17
0.0082
( 6)
K4 (354--439;
**
9701)
Miniplasminogen
(442--790;
38 000)
Lys-plasminogen
(77--790;
Glu-plasminogen
(1--790 ; 90 000)
82 000)
* GalNH2-based oligosaccharide gzoup attached to Thr-345 in the K3-K4 connecting strand. * * S a m e as ( * ) e x c e p t t h a t a n a d d i t i o n a l G l c N H 2 - b a s e d o l i g o s a c c h a z i d e g ~ o u p is a t t a c h e d t o A s n - 2 8 8 i n K3.
"~ 0.8 3 R
-~ 0,B
e-
o~0.6
E
® ~0.6
~'gE
oo ._ c
EE
_~ -r -d 0A (3.(3-
"'------o
,.tm
._ m
"5
g
h
~
0.2
<,, 10 °
,
. 10~
.
.
.
, 10 z
10~
,
, 10"
6 - A H A or tr~mexamic acid c o n c . ( p M )
02
u_
I 2 3 4 5 6 7 c(z-Antiplasmin conc. ( p M )
F i g . 2. F i b r i n - b i n d i n g o f 1 2 5 i . l a b e l l e d m i n i p l a s m i n o g e n o r 1 2 S i _ l a b e l l e d L y s - p l a s m i n o g e n as a f u n c t i o n o f the concentration of 6-aminohexanoic acid (6-AHA) or tranexamic acid. Miniplasminogen with 6-AHA ( e ) ; m i n i p l a s m i n o g e n w i t h t r a n e x a m i c a c i d (A); L y s - p l a s m i n o g e n w i t h 6 - A H A (©); L y s - p l a s m i n o g e n w i t h txanexamic acid (a). The clotting mLxture contained 1.2 pM plasminogen derivative and 9.2 #M clottablc fibrinogen. Each point on the cuxves represents the mean of 2--5 determinations with S.E. ~ 0.020. F i g . 3. F i b r i n - b i n d i n g o f 125 I - l a b e l l e d K 1 + 2 + 3 a ( d e f i n e d i n T a b l e I ) o r 125 I - l a b e l l e d L y s - p l a s m i n o g e n as a function of a2-antiplasmin concentration. K1+2+3 a (e); Lys-plasminogen (c). The clotting mixture contained 1.2 pM plasminogen derivative and 9.2 #M clottable fibrinogen. Each point on the curves represents the mean of 2--4 determinations with S.E. ~ 0.0074 with K1+2+3 a and S.E. ~ 0.015 with Lysplasminogen.
383 decreased the fibrin-binding of Kl+2+3a to the same level as was observed in the plasma clot. The fibrin-binding of miniplasminogen decreased insignificantly (P < 0.1) while the fibrin-binding of Lys-plasminogen decreased to a level which was intermediate between that in the fibrin clot and that in the plasma clot. Elimination of Glu-plasminogen from the fibrin clots did not change the results (not shown).
T A B L E II I N F L U E N C E O F 6 - A M I N O H E X A N O I C A C I D OR T R A N E X A M I C A C I D ON F I B R I N - B I N D I N G OF 1 2 5 I . L A B E L L E D K 1 + 2 + 3 a ( D E F I N E D IN T A B L E I) OR 1 2 S I . L A B E L L E D K 4 T h e c l o t t i n g m i x t u r e c o n t a i n e d 9.2 #M c l o t t a b l e f i b r i n o g e n a n d 1.2 pM f r a g m e n t . A m i n o acid concentration (~M)
A m i n o acid
None 6 - A m i n o h e x a n o i c acid 6 - A m i n o h e x a n o i c acid T r a n e x a m i c acid T r a n e x a m i c acid
F r a c t i o n of labelled f r a g m e n t b o u n d to fibrin, m e a n S.E. (n = 4)
0 30 300 10 100
K1+2+3 a
K4
0.17 0,083 0.046 0,065 0,040
0.058 0.041 0.021 0.051 0.020
0.0075 0.0013 0.0047 0.0029 0.0021
0.0023 0.0028 0.0015 0.0024 0.0018
TABLE III I N F L U E N C E O F ~ 2 " A N T I P L A S M I N ON F I B R I N - B I N D I N G O F 1 2 5 1 - L A B E L L E D K 4 OR 125 I - L A B E L LED M1NIPLASMINOGEN T h e c l o t t i n g m i x t u r e c o n t a i n e d 9.2 /sM c l o t t a b l e f i b r i n o g e n a n d 1.2 pM f r a g m e n t . c~2 - A n t i p l a s m i n concentration (#M)
F r a c t i o n o f labelled f r a g m e n t b o u n d to fibrin, m e a n S.E. (n) K4
0 6,8
0.079 0.072
Miniplasminogen 0 . 0 0 5 5 (2) 0 . 0 0 2 0 (47
0.50 0.50
0 . 0 2 3 (47 0 . 0 2 0 (4)
TABLE IV F I B R I N B I N D I N G O F 1 2 5 I _ L A B E L L E D P L A S M I N O G E N F R A G M E N T S IN P L A S M A C L O T S , F I B R I N C L O T S C O N T A I N I N G G l u - P L A S M I N O G E N A N D ~ 2 - A N T I P L A S M I N , A N D IN F I B R I N C L O T S K 1 + 2 + 3 a is d e f i n e d in T a b l e I. T h e c l o t t i n g m i x t u r e c o n t a i n e d 6.1 pM f i b r i n o g e n (all s y s t e m s ) , 1.8 /aM G l u - p l a s m i n o g e n (PC a n d FC 1), 1.0 pM c~2 - a n t i p l a s m i n (PC and FC 1) a n d 0.2 pM labelled f r a g m e n t (all s y s t e m s ) . T h e P l a s m a vol. f r a c t i o n = 0.8 in PC. PC, p l a s m a clots; FC 1, fibrin clots c o n t a i n i n g Glu-plasmin o g e n a n d c~2 - a n t i p l a s m i n ; FC 2, fibrin clots. System
F r a c t i o n of labelled f r a g m e n t b o u n d to fibrin, m e a n S,E. (n) K1+2+3 a
PC FC 1 FC 2
0.038 0.041 0.10
0 . 0 0 1 7 (4) 0 . 0 0 1 5 (4) 0 . 0 0 7 4 (47
Miniplasminogen
Lys-plasminogen
0.24 0.36 0.39
0.27 0.44 0.56
0 . 0 2 7 (6) 0 . 0 1 5 (4) 0 . 0 0 4 2 (6)
0 . 0 2 2 (6) 0 . 0 1 9 (4) 0 . 0 1 6 (6)
384 TABLE V C O R R E L A T I O N B E T W E E N D I S S O C I A T I O N C O N S T A N T S ( K d ) IN p M , A N D C O N C E N T R A T I O N S (c50) I N pM O F 6 - A M I N O H E X A N O I C A C I D , T R A N E X A M I C A C I D A N D c ~ 2 - A N T I P L A S M I N T H A T D E C R E A S E BY 5 0 % T H E F I B R I N - B I N D I N G O F P L A S M I N O G E N F R A G M E N T S K l + 2 + 3 a , d e f i n e d in T a b l e I. (*) T h i s c o n c e n t r a t i o n ( c 6 2 ) d e c r e a s e d fibrin-binding by 62% ( * * ) 6.8 pM ~2-antiplasmLn d i d n o t d e c r e a s e the fibrin-binding ( T a b l e I I I ) . T h e v a l u e s of c 50 ( c 6 2 ) axe f r o m Figs. 2 a n d 3, a n d T a b l e II. ( T h e c l o t t i n g m i x t u r e s c o n t a i n e d 9 . 2 /~M f i b r i n o g e n a n d 1.2 pM p l a s m i n o g e n d e r i v a t i v e ) . T h e v a l u e s of K d axe f r o m t h e l i t e r a t u r e a n d t h e o n e s f r o m r e f e r e n c e [ 8 ] axe d e t e r m i n e d by using the s a m e f r a g m e n t s as u s e d in the present s t u d y . T h e v a l u e s in p a r e n t h e s e s axe K d v a l u e s w h i c h axe calcul a t e d f r o m t h e c50 ( c 6 2 ) values. T h e s e c a l c u l a t i o n s axe b a s e d o n : (1) the t o t a l a m o u n t s o f p l a s m i n o g e n d e r i v a t i v e a n d a m i n o a c i d or c~2 - a n t i p l a s m i n in the c l o t t i n g m i x t u r e axe k n o w n , (2) the a m o u n t o f fibrinb o u n d p l a s m i n o g e n d e r i v a t i v e is m e a s u r e d , a n d (3) t h e free a m o u n t of p l a s m i n o g e n d e r i v a t i v e ( a n d c o n s e q u e n t l y the a m o u n t s o f free a n d b o u n d a m i n o a c i d or c~2 - a n t i p l a s m i n ) is c a l c u l a t e d b y using the r a t i o , ( a m o u n t of free p l a s m i n o g e n d e r i v a t i v e ) / ( a m o u n t of fibrin-bound p l a s m i n o g e n derivative), w h i c h is c o n s t a n t at varying c o n c e n t r a t i o n s o f d e r i v a t i v e ( T a b l e I). It is a s s u m e d t h a t a m i n o a c i d or ~ 2 - a n t i p l a s m i n d o e s n o t bind to fibrin ( e i t h e r d i r e c t l y or via the p l a s m i n o g e n d e r i v a t i v e s ) a n d that o n l y o n e binding site is i n v o l v e d . It is also a s s u m e d that equilibrium c o n d i t i o n s e x i s t . Fragment
K1+2+3 a
6 - A m i n o h e x a n o i c acid
T r a n e x a m i e acid
~2"Antiplasmin
c50
c50
Kd
c50
Kd
10 *
1 [81 (4.8)
1.2
0.18 [12] (0.50)
10-100
6.3 [ 8 ]
**
9 [12]
Kd
30 (25)
K4
30-300
Miniplasminogen
340
Lys-Plasminogen
130
36 [ 7 ]
70 (187) 35,260 and 10,000 [33] (37)
** (38)
18
2.2, 36 a n d 1,035 [34] (5.0)
5
0.63 [12] (1.3)
Discussion Markus and co-workers [32--34] demonstrated by an equilibrium ultrafiltration technique using 6~aminohexanoic acid or tranexamic acid as a ligand that plasminogen contains at least five lysine-binding sites, one of which is a strong binding-site. The purification of the three major native plasminogen fragments, that are obtained from Glu-plasminogen by limited proteolysis with elastase, involves affinity chromatography on lysine-Sepharose [1]. The conditions under which they are eluted led us to conclude when they were first prepared that K1+2+3 and K4 each contains at least one lysine-binding site, whereas miniplasminogen does not contain any [1]. Recently, Markus et al. [8] studied the binding of tranexamic acid to these same fragments by their equilibrium ultrafiltration technique. They showed that K1+2+3 actually contains at least two lysine-binding sites, one of which is the strong binding site, found earlier in both Glu-and Lys-plasminogen [32--34]. They also showed that K4 contains a single weaker lysine-binding site, whereas no ligand binding could be demonstrated to miniplasminogen. The strong lysine-binding site on K1+2+3 has recently been attributed to the K1 part [7 ]. The binding of fibrin and a2-antiplasmin to plasmin(ogen) plays an important role in the regulation of fibrinolysis [25,35]. Wiman and co-workers [12, 13,35] have proposed that plasmin(ogen) binds to fibrin and a2-antiplasmin
385 through its lysine-binding sites and that the strong lysine-binding site plays a key role in this binding. Their proposal is based on the finding [13] that plasminogen and plasminogen fragments that bind to lysine-Sepharose also bind to fibrin-Sepharose and can be eluted by less than 5 mM 64minohexanoic acid, while plasminogen fragments that do not bind to lysine-Sepharose also do not bind to fibrin~epharose. It is also based on the fact that ~ concentrations of 6-aminohexanoic acid inhibit the reversible first reaction step between the a2antiplasmin and the Lys-plasmin but not the first reversible reaction step between the a2-antiplasmin and the miniplasmin because the latter lacks the strong lysine-binding site [6,11]. The K1+2+3 b variant which has a glucosamine-based oligosaccharide attached to Asn-288 binds only very weakly to fibrin (Table I) and was therefore not studied in detail. The Kl+2+3a variant, with no oligosaccharide attached to Asn-288 binds somewhat more strongly. The finding that pM concentrations of 6-aminohexanoic acid, tranexamic acid and a2-antiplasmin decrease the fibrin-binding of Kl+2+3a (Tables II and V, Fig. 3) indicates a specific effect and agrees with the fact that K1+2+3 contains the strong lysine-binding site [7,8] and an important a2-antiplasmin-binding site [12]. The discrepancy in our K d values indicated in parentheses in Table V and those reported in the literature may be due to different assay systems and preparations as well as to the fact that the assumptions made in the text of Table V may be wrong. Our results are compatible with either of two hypotheses. (a) K1+2+3a binds to fibrin through its high,affinity lysine-binding site and is removed by competitive binding of 6-aminohexanoic acid or tranexamic acid. (b) Kl+2+3a binds to fibrin through a different binding site, specific for fibrin, and 6-aminohexanoic acid or tranexamic acid when bound to the high-affinity lysine-binding site acts as an allosteric effector to change the conformation of the fibrin-binding site, thus decreasing the fibrin-binding. The effect of a2-antiplasmin on the fibrinbinding of Kl+2+3a can be interpreted in the same way. The fibrin-binding of K1+2+3 is markedly lower in a plasma clot than in a purified fibrin clot (Table IV). This can be readily explained by an effect in the plasma clot of a2-antiplasmin alone, or by a combined effect of this inhibitor, and the histidine-rich glycoprotein which has recently been shown to bind strongly to K1+2+3 [36]. The fibrin-binding of K4 is relatively w e a k ( T a b l e I). The concentrations of 6-aminohexanoic acid or tranexamic acid required for 50% decrease in this binding is less well defined than with K1+2+3 a (Table V) because a large change in the concentration of amino acid causes only a small change in fibrin-binding (Table II). The effects of the amino acids on the fibrin-binding of K4 can be explained in either of the two ways described for K1+2+3 a above and are compatible with the dissociation constants reported in the literature for the single binding site on K4 (Table V). No effect of a2-antiplasmin at 6.8 pM could be demonstrated on the fibrin-binding of K4 (Table III). This agrees with the fact that K4 has only a relatively weak binding site for a2-antiplasmin (Table V). Miniplasminogen (12SI-labelled as well as unlabelled) was found to bind more strongly to fibrin than any of the other fragments (Table I). It is not possible to say from the present data whether the fibrin-binding site of miniplasminogen is located in its K5-part or in its serine proteinase zymogen part. Wiman
386 and Wall~n [13], were unable to demonstrate an affinity between fibrinogenSepharose and the plasmin-light chain obtained by partial reduction. It is possible that their light chain may have lost a fibrin(ogen)-binding site due to cleavage of intra-chain disulphide bridges. It has been shown that the light chain prepared under mild reducing conditions looses most of its proteolytic activity (casein substrate) and most of its ability to incorporate [aH]diisopropyl phosphorofluoridate [37]. It was a surprising finding that 6~minohexanoic acid and tranexamic acid decrease the fibrin-binding of miniplasminogen (Fig. 2, Table V), since previous results indicate that this fragment does not contain lysine-binding sites [ 1,8]. Furthermore, 6-aminohexanoic acid has only a slight effect on the kinetics of interaction between miniplasmin and a2-antiplasmin [6,11]. One possible explanation for this finding is that the interaction observed between fibrin and miniplasminogen could expose a hidden lysinebinding site in the latter. Another possibility is that the binding site for miniplasminogen in fibrin could be affected by 6-aminohexanoic acid. a2-Antiplasmin at 6.8 plVI has no effect on the fibrin-binding of miniplasminogen (Table III). This agrees with the observation that the strength of binding in the first reversible step of the reaction between a2~antiplasmin and plasmin is weaker with miniplasmin than with Lys-plasmin [6,11]. The fibrin-binding of miniplasminogen is somewhat lower in a plasma clot than in a purified fibrin clot (Table IV) indicating that one or more plasma proteins may interfere with the reaction between this fragment and fibrin. The fibrin-binding of Glu- and Lys-plasminogen observed in the present study confirms previous results [21,38] showing that the removal of residues 1--76 from Glu-plasminogen increases the extent of fibrin-binding. The present study also confirms that 6~aminohexanoic acid, tranexamic acid and a2~antiplasmin decrease the fibrin-binding of Lys-plasminogen [21,38,39] and that the fibrin-binding of Lys-plasminogen is lower in a plasma clot than in a purified fibrin clot [38]. The values of c50 and Kd in Table V indicate that the decrease in the fibrin-binding of Lys-plasminogen caused by 6-aminohexanoic acid or tranexamic acid is mainly related to saturation of the strong and intermediate lysine-binding sites in the K1--K4 part of the plasminogen molecule. The 6~minohexanoic acid- or tranexamic acid-induced gross change in conformation of Glu-plasminogen associated with the exposure of the activator-susceptible Args60-Va1561 bond is mainly related to saturation of the weak lysine binding sites [33,34]. Acknowledgements Supported by the Danish Medical Research Council (ST and IC) and the Danish Hospital Foundation for Medical Research; Region of Copenhagen, the Faroe Islands and Greenland (ST). It was also supported b y the National Heart, Lung and Blood Institute, NIH, grant no. HL-16238, Bethesda, MD, U.S.A. (SM). The valuable technical assistance of Jannie Bank and Inge Schuster is appreciated.
387
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C h e m . 2 4 4 , 3 5 9 0 - - 3 5 9 7 16 T h o r s e n , S., C l e m m e n s e n , I., S o t t r u p - J e n s e n , L. a n d M a g n u s s o n , S. ( 1 9 8 0 ) in P r o t i d e s o f t h e Biological F l u i d s ( P e e t e r s , H., e d . ) , Vol. 2 8 , p p . 3 6 5 - - 3 6 9 , P e r g a m o n Press, O x f o r d 17 H a r b o e , N. a n d Inglld, A. ( 1 9 7 3 ) S c a n d . J. I m m u n o l . 2, S u p p l . 1 , 1 6 1 - - 1 6 4 1 8 Mfillertz, S. a n d C l e m m e n s e n , I. ( 1 9 7 6 ) B i o c h e m . J. 1 5 9 , 5 4 5 - - 5 5 3 19 W e b e r , K., Pringle, J . R . a n d O s b o r n , M. ( 1 9 7 2 ) M e t h o d s E n z y m o l . 2 6 , 3 - - 2 7 2 0 T h o r s e n , S. a n d M~lllertz, S. ( 1 9 7 4 ) S c a n d . J. Clin. L a b . Invest. 3 4 , 1 6 7 - - 1 7 6 21 T h o r s e n , S. ( 1 9 7 5 ) B i o c h i m . B i o p h y s , A c t a 3 9 3 , 5 5 - - 6 5 2 2 W i m a n , B. a n d C o l l e n , D. ( 1 9 7 7 ) E u r . J. B i o e h e m . 7 8 , 1 9 - - 2 6 23 L a u r e l l , C.-B. ( 1 9 7 2 ) S c a n d . J. Clin. L a b . Invest. 2 9 , S u p p l . 1 2 4 , 2 1 - - 3 7 2 4 N o r 6 n , I., R a m s t r S m , G. a n d Wall6n, P. ( 1 9 7 5 ) H a e m o s t a s i s 4, 1 1 0 - - 1 2 4 2 5 T h o r s e n , S. ( 1 9 7 7 ) D a n i s h Med. Bull. 2 4 , 1 8 9 - - 2 0 6 26 Wall~n, P. a n d W i m a n , B. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 5 7 , 1 2 2 - - 1 3 4 2 7 G r o s , C. a n d L a b o u e s s e . B. ( 1 9 6 9 ) E u r . J. B i o c h e m . 7 , 4 6 3 - - - 4 7 0 2 8 C h r i s t e n s e n , U. a n d C l e m m e n s e n , I. ( 1 9 7 8 ) B i o c h e m . J. 1 7 5 , 6 3 5 - - - 6 4 1 29 M c F a r l a n e , A.S. ( 1 9 5 8 ) N a t u r e 1 8 2 , 5 3 3 0 C o l l e n , D. ( 1 9 7 4 ) in P l a s m i n o g e n a n d P r o t h r o m b i n M e t a b o l i s m in M a n , A c e o , L e u v e n 31 J o h a n s s o n , B.G. ( 1 9 7 2 ) S c a n d . J. Clin. L a b . I n v e s t . 2 9 , S u p p l . 1 2 4 , 7 - - 1 9 3 2 M a r k u s , G., D e P a s q u a l e , J . 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Biochimica et Biophysica Acta, 6 6 8 ( 1 9 8 1 ) 3 7 7 - - 3 8 7 E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 38670
ADSORPTION TO FIBRIN OF NATIVE FRAGMENTS OF KNOWN PRIMARY STRUCTURE FROM HUMAN PLASMINOGEN
S I X T U S T H O R S E N a, I N G E C L E M M E N S E N a, L A R S S O T T R U P - J E N S E N b a n d STAFFAN MAGNUSSON b
a Department o f C!inical Chemistry, University o f Copenhagen, Hvidovre Hospital, DK-2650 Hvidovre and b Department o f Molecular Biology and Plant Physiology, University o f Aarhus, DK-8000 Aarhus C (Denmark) (Received October 14th, 1980)
Key words: Plasminogen fragment: Fibrin binding; Lysine-binding site; Aminohexanoic acid; Tranexamic acid; as -Antiplasmin; (Human plasma)
Summary Limited proteolysis o f native Glu-plasminogen with pancreatic elastase produced three major fragments, K1+2+3, K4, K5-1ight chain (miniplasminogen). Fibrin-binding was determined by clotting fibrinogen in the presence of 12sIlabelled fragments and measuring l~sI in the washed fibrin and in the supernatant. Of the fragments miniplasminogen showed the highest fibrin-binding, the strength o f which was intermediate between those of Glu-plasminogen and Lysplasminogen. The fibrin-binding of all three f~agments was decreased by 6-aminohexanoic acid or tranexamic acid. This decrease was most pronounced with K1+2+3. The fibrin-binding o f K1+2+3, but not that of K4 and miniplasminogen was decreased by a2-antiplasmin. The fibrin-binding of K1+2+3 and miniplasminogen was lower in a plasma clot than in a purified fibrin clot. Our results indicate that each of the three fragments can bind to fibrin. They confirm that an a2-antiplasmin-binding site is located on K1+2+3. Furthermore two of the fragments, namely K4 and K1+2+3 contain lysine-binding site(s).
Introduction Native human Glu-plasminogen is a single peptide chain of known primary structure [1--3]. It consists of three parts. (a) The N-terminal part comprising Abbreviations: SDS, sodium dodecyl sulfate; GalNH 2, Galactosamine; GleNH2, Glueosamine; KI unit, kallil~xein inhibitor unit; NIH unit, National Institutes of Health unit; tranexamic acid, trans-4-aminomethylcyelohexane-l-caxboxylic acid; D-Val-Leu-Lys-pNA, D-valyl-L-leucyl-L-lysine 4-nitroanilide. 0 0 0 5 - 2 7 9 5 / 8 1 / 0 0 0 0 - - 0 0 0 0 / $ 0 2 . 5 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
378 residues 1--76 (M~ = 8800). The removal of this part appreciably alters the conformation of plasminogen [4]. (b) The intermediate part comprising residues 77--560 (Mr = 57 000), which contains five kringle structures (K1--K5) that are homologous with the two kringle structures in prothrombin [5 ]. This part of the molecule contains sites that bind to fibrin and a~-antiplasmin and sites, denoted lysine-binding sites, that bind a number of co-aminocarboxylic acids, such as L-lysine, 6-aminohexanoic acid and tranexamic acid [6--14]. These sites are to some extent functionally interdependent but their exact structural bases have not been elucidated. (c) The C-terminal part, residues 561--790 (Mr = 25 700), which upon activation becomes the light chain and is the serine proteinase part of plasmin. It shows extensive homology with other serine proteinases such as thrombin, trypsin, chymotrypsin and elastase [1,3, 15]. Limited proteolysis of Glu-plasminogen with elastase (pancreatic or leucocyte) produces three major fragments with known primary structure [1]. They are K1+2+3, K4 and K5-1ight chain (neoplasminogen-Val-442, miniplasminogen). The present work describes the fibrin-binding of these fragments and the effect of 6-aminohexanoic acid, tranexamic acid or a2-antiplasmin on this binding. Preliminary data on this work have been presented [16]. Materials and Methods
Buffers. Buffer 1, 0.05 M Tris-HC1/0.10 M NaC1, adjusted to pH 7.70 (20°C) with NaOH, ionic strength 0.15. B u f f e r 2 , 0.05 M Tris-HC1/0.25 M NaC1, adjusted to pH 7.70 (20°C) with NaOH, ionic strength 0.30.6-Aminohexanoic acid, tranexamic acid and D-Val-Leu-Lys-pNA (S-2251) were from Kabi Ltd., Stockholm. Bovine thrombin (8 • 104 NIH units/g) was from Leo Pharmaceuticals, Copenhagen. Solutions of these reagents were prepared in Buffer 1. Plasma was obtained from human blood stabilized by 0.1 vol. 0.11 M trisodium citrate. Specific rabbit immunoglobulins against human plasminogen were from Dakopatts, Copenhagen. Specific rabbit immunoglobulins against a2-antiplasmin were isolated from antiserum [17] which was produced as described [18]. SDS-polyacrylamide gel electrophoresis was performed as described [19]. Gel slabs (2.7 X 82 × 82 mm) contained 7.5% or 10% acrylamide with 6 M urea. Concentration of proteins. The concentration (absorbance) of fibrinogen, clottable fibrinogen or purified Glu- or Lys-plasminogen was determined at 280 nm [20,21]. The concentration of K1+2+3, K4 or miniplasminogen was determined by amino acid analysis. The concentration (active site) of Glu- or Lysplasminogen or miniplasminogen was determined by titration with aprotinin (Trasylol) after its conversion to plasmin [20]. The plasmin activity was measured at 25°C by rate assay on D-Val-Leu-Lys-pNA (substrate concentration, 0.36 M in Buffer 1) [6]. The concentrations (active site) were only used when stated. The concentration of purified a~-antiplasmin was determined at 280 nm using nlcrnA!% 6.7 and M~ = 67 000 [22] or by titration with plasmin [18]. The residual plasmin activity was measured on D-Val-Leu-Lys-pNA. Titrated concentrations were used unless otherwise stated. The concentration of Glu-plasminogen or a2-antiplasmin in plasma was determined by electroimmuno assay [23] using purified Glu-plasminogen or a2-antiplasmin as standard. Preparation of proteins. Human fibrinogen from IMCO Corporation, Ltd., =
379 Stockholm. Contaminating plasminogen was removed b y specific binding to lysine-Sepharose 4B (Pharmacia, Uppsala) as previously described [24], except that fibrinogen was dialysed against Buffer 2 and passed through a lysineSepharose column equilibrated and eluted with the same buffer. Prior to use the stock solution of fibrinogen was diluted with I vol. distilled water and further diluted with Buffer 1. The coagulability (mean -+ S.E. (n)), expressed as the ratio, (clottable fibrinogen concentration)/(fibrinogen concentration), was 0.98 + 0.019 (11). SDS-polyacrylamide gel electrophoresis of a reduced fibrinogen sample indicated that the A(a)-chain was intact in a large fraction of the fibrinogen molecules (Fig. 1). Plasminogen and plasminogen fragments. Procedures used were as follows. Glu-plasminogen was purified from outdated Acid-Citrate-Dextrose stabilized plasma b y affinity chromatography on lysine-Sepharose 4B [20]. Spontaneous proteolytic activity was removed by treatment o f the plasminogen eluate from 2000 ml plasma with 20 ml aprotinin-Sepharose 4B (400 nmol aprotinin per ml gel) for 20 h at 4°C [25]. This was followed b y gel filtration on Ultrogel AcA 44 (LKB, Stockholm) and ion exchange chromatography on DEAESephadex A-50 (Pharmacia) [26]. Lys-plasminogen was prepared as described [25]. The Glu-plasminogen eluate from the affinity chromatography step was allowed to spontaneously convert to the modified form and spontaneous proteolytic activity was removed b y aprotinin-Sepharose as above. This was followed b y gel filtration on Ultrogel AcA 44. K1+2+3, K4 and miniplasmi-
D
° 8 2
3
4
5
6
7
'"
Fig. 1. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in u r e a o f r e d u c e d s a m p l e s o f (1) h u m a n f i b r i n o g e n f r o m I M C O ; (2) G l u - p l a s m i n o g e n ; (3) L y s - p l a s m i n o g e n ; (4) K l + 2 + 3 a ; (5) K l + 2 + 3 b ; (6) K 4 ; (7) m i n i p l a s m i n o geM; (8) c~2 - a n t i p l a s m i n . T h e K 1 + 2 + 3 v a r i a n t s axe d e f i n e d in T a b l e I. E l e c t r o p h o r e s i s w a s c a r r i e d o u t in gel slabs (2,7 X 82 X 82 r a m ) c o n t a i n i n g 10% ( r u n 6) or 7.5% a c z y l a m i d e in 6 M u r e a a n d 0.1 M s o d i u m p h o s p h a t e , p H 7.0. T h e a m o u n t o f p r o t e i n a p p l i e d to t h e gel w a s 37 p g i n (1), 72 p g in (6), a n d 1 3 - - 2 0 p g in t h e o t h e r r u n s . (2) to (5) w e r e r u n in t h e s a m e gel slab.
380 nogen (defined in Table I) were prepared b y specific limited proteolysis of Gluplasminogen using porcine elastase (pancreatic) and isolated b y gel filtration on Sephadex G-75 (Pharmacia) followed b y affinity chromatography on lysineSepharose using a linear gradient of 6-aminohexanoic acid [1]. The identities of the Glu- and Lys-plasminogen were verified by N-terminal amino acid analysis [27] and by molecular weight determination b y SDS-polyacrylamide gel electrophoresis. K1+2+3, K4 and miniplasminogen were identified by their primary structures [1]. The ratio, (concentration (active site))/(concentration (absorbance)), ranged between 0.77 and 1.0 with preparations of Glu-plasminogen, Lys-plasminogen and miniplasminogen. The spontaneous catalytic amount of plasmin as fraction of the total amount of plasmin + activatable plasminogen was less than 0.004 in all preparations. Glu-plasminogen and its derivatives are depicted in SDS-polyacrylamide gel electrophoresis in Fig. 1. ~2-Antiplasmin was purified in a five-step procedure from plasma stabilized by AcidCitrate-Dextrose. Step 1. Adsorption of plasminogen to lysine-Sepharose using 0.2 vol. packed gel. Step 2. Fractional (NH4)2SO4-precipitation of the plasminogen-depleted supernatant [18]. Step 3. Affinity chromatography on plasminogen~Sepharose. This was performed as described [22 ], except that ~2-antiplasmin was eluted with 0 . 0 4 M sodium phosphate, pH 7.4 containing I mM tranexamic acid. Setp 4. Ion exchange chromatography. This was performed as described [22], but at pH 7.4. Step 5. Gel filtration on Ultrogel AcA 44, equilibrated and eluted with 0.05 M Tris-HC1/0.55 M NaC1, adjusted to pH 7.70 (20°C) with NaOH. The eluted ~2-antiplasmin was concentrated and dialysed against Buffer 1. Dilutions were made with the same buffer. The ratio, (concentration (titrated))/(concentration (absorbance)), was 0.81. Most of the inhibitor had retained its ability to bind to plasminogen-Sepharose [28]. SDS-polyacrylamide gel electrophoresis of the purified inhibitor is shown in Fig. 1. Radioiodination of proteins. This was performed according to McFarlane [29] with some modifications [30] by using iodine-125 (Na~2SI, carrier free, in NaOH solution, pH 8--11, free from reducing agent) from the Radiochemical Centre, Amersham. U n b o u n d iodine and protein in the iodination solution was separated on Sephadex G-25 (Pharmacia) equilibrated and eluted with Buffer 2 (miniplasminogen) or Buffer 1 (other proteins). Prior to use the labelled miniplasminogen solution was diluted with 1 vol. distilled water and further diluted with Buffer 1. The other labelled protein solutions were diluted with Buffer 1. The iodine substitution level ranged from 0.008 to 0.3 atoms of iodine per mole of protein. The specific radioactivity ranged from 0.04 to 3.3 mCi per ~mol of protein. Lysine-Sepharose affinity chromatography of a mixture of unlabelled and labelled K1+2+3, K4 or miniplasminogen showed that radioactivity and protein (monitored by measuring absorbance at 280 nm) were eluted in the same peak, when a linear gradient of 6~aminohexanoic acid was used [1]. Agarose gel electrophoresis at pH 8.6 [31] of a mixture of unlabelled and labelled Glu- or Lys-plasminogen followed by autoradiography on Kodak X-Omat duplicating film showed that radioactivity and protein had the same mobility (fi- or 3,-mobility, respectively). The ratio, (concentration (active site))/(concentration absorbance)) did not change after iodination of Gluplasminogen, Lys-plasminogen or miniplasminogen. Fibrin-binding of labelled plasminogen fragments. This was determined in
381 fibrin or plasma clots (vol. = 500 pl) by clotting a mixture of fibrinogen or plasma and 125I-labelled fragment with a t h r o m b i n solution (final t h r o m b i n co n c e n tr atio n , 4 • 103 NIH U/l). In assays o f the effect of 6-aminohexanoic acid, tranexamic acid or a2-antiplasmin the substance was present during fibrin f o r m a t io n . The fibrin threads were separated from the liquid phase by winding them up on a glass rod at r o o m t e m p e r a t u r e (20°C) over a period of 30 min [21]. The fibrin was squeezed free of liquid and washed briefly in 5 ml 0.15 M NaC1. Finally th e fibrin was dried by blotting on a piece of filter paper and then dissolved in 1 ml 2.5 M NaOH by incubation for 20 min at 100°C followed by dilution with 9 vols. distilled water. Radioactivity was measured in the resulting 0.25 M NaOH-solution, in the liquid remaining after removal of fibrin, and in a control with the same composition as the clotting mixture, except that it did n o t contain th r om bi n. Radioactivity of the control differed little from the sum o f the radioactivities of the fibrin and the liquid phase. The complete recovery o f fibrin on the glass rod was checked by measuring the absorbance at 280 nm of the 0.25 M NaOH solution. Fibrin-binding was expressed as, (radioactivity o f fibrin)/(radioactivity of control). Fibrin-binding of unlabelled miniplasminogen. This was determined in fibrin clots as previously r e p o r t e d for Glu- or Lys-plasminogen [21], except that miniplasminogen after its conversion to miniplasmin was determined by using D-Val-Leu-Lys-pNA as a substrate. Results
Table I shows that a large fraction o f the labelled Lys-plasminogen (0.71-0.74) or labelled miniplasminogen (0.44--0.45), in the respective clotting mixtures, was b o u n d to fibrin. Separate experiments showed that this also applied to unlabelled miniplasminogen. The fraction b o u n d was 0.55 (mean of two determinations) in a clotting mixture containing the same concentrations of fibrinogen and fragment as in Table I. In the case o f the two labelled variants of K1+2+3 (Table I), the variant with an oligosaccharide group attached to Thr345 (K1+2+3a) was found to bind to fibrin to the same e x t e n t (0.17--0.19) as labelled Glu-plasminogen (0.17). The second variant ( K 1 + 2 + 3 b ) w i t h oligosaccharide groups attached to Asn-288 and to Thr-345, and the labelled K4 fragment exhibited the weakest fibrin-binding (0.033--0.090). The fraction o f each fragment b o u n d to fibrin was essentially independent of its concent rat i on in the clotting mixture. Similar results were obtained with 2 or 3 different preparations of each fragment (results with all preparations are included in Table I). Inclusion o f 6oaminohexanoic acid or tranexamic acid decreased the fibrint)inding o f the labelled plasminogen fragments (Fig. 2, Table II). If az-antiplasmin was included only the binding of Kl+2+3a and Lys-plasminogen was decreased (Fig. 3). There was no effect of a2-antiplasmin on the binding o f K4 or miniplasminogen (Table III). The fibrin-binding of labelled Kl+2+3a, miniplasminogen or Lys-plasminogen in a fibrin clot was 1.6 to 2.6 times higher than that of the same fragment in a plasma clot (Table IV). Inclusion of the same amounts o f Glu-plasminogen and a2-antiplasmin in the fibrin clots as were present in the plasma clots
382 TABLE
I
FIBRIN-BINDING
OF 125I.LABELLED
The clotting mixture
contained
PLASMINOGEN
FRAGMENTS
9.2 ~M clottable fibrinogen.
Fragment (Sequence; Mz)
Concentration in c l o t t i n g mixture (#M)
Fraction of labelled fzagment bound to fibrin Mean
S.E. (n)
K1 + 2 + 3 a (73/79--353;
34 000) *
1.8 0.5 0.2
0.19 0.19 0.17
0.0067 0.013 0.0058
( 6) ( 6) ( 3)
K1 + 2 + 3 b (73/79--353;
39000)
1.9
0.033
0.0029
( 9)
1.2 0.4 0.2
0.065 0.090 0.082
0.0049 0.0054 0.0056
(6) (6) ( 7)
1.2 0.3 0.1
0.45 0.44 0.44
0.014 0.0089 0.0058
(17) (12) (3)
1.2 0.2
0.71 0.74
0.0081 0.012
(14) ( 3)
1.2
0.17
0.0082
( 6)
K4 (354--439;
**
9701)
Miniplasminogen
(442--790;
38 000)
Lys-plasminogen
(77--790;
Glu-plasminogen
(1--790 ; 90 000)
82 000)
* GalNH2-based oligosaccharide gzoup attached to Thr-345 in the K3-K4 connecting strand. * * S a m e as ( * ) e x c e p t t h a t a n a d d i t i o n a l G l c N H 2 - b a s e d o l i g o s a c c h a z i d e g ~ o u p is a t t a c h e d t o A s n - 2 8 8 i n K3.
"~ 0.8 3 R
-~ 0,B
e-
o~0.6
E
® ~0.6
~'gE
oo ._ c
EE
_~ -r -d 0A (3.(3-
"'------o
,.tm
._ m
"5
g
h
~
0.2
<,, 10 °
,
. 10~
.
.
.
, 10 z
10~
,
, 10"
6 - A H A or tr~mexamic acid c o n c . ( p M )
02
u_
I 2 3 4 5 6 7 c(z-Antiplasmin conc. ( p M )
F i g . 2. F i b r i n - b i n d i n g o f 1 2 5 i . l a b e l l e d m i n i p l a s m i n o g e n o r 1 2 S i _ l a b e l l e d L y s - p l a s m i n o g e n as a f u n c t i o n o f the concentration of 6-aminohexanoic acid (6-AHA) or tranexamic acid. Miniplasminogen with 6-AHA ( e ) ; m i n i p l a s m i n o g e n w i t h t r a n e x a m i c a c i d (A); L y s - p l a s m i n o g e n w i t h 6 - A H A (©); L y s - p l a s m i n o g e n w i t h txanexamic acid (a). The clotting mLxture contained 1.2 pM plasminogen derivative and 9.2 #M clottablc fibrinogen. Each point on the cuxves represents the mean of 2--5 determinations with S.E. ~ 0.020. F i g . 3. F i b r i n - b i n d i n g o f 125 I - l a b e l l e d K 1 + 2 + 3 a ( d e f i n e d i n T a b l e I ) o r 125 I - l a b e l l e d L y s - p l a s m i n o g e n as a function of a2-antiplasmin concentration. K1+2+3 a (e); Lys-plasminogen (c). The clotting mixture contained 1.2 pM plasminogen derivative and 9.2 #M clottable fibrinogen. Each point on the curves represents the mean of 2--4 determinations with S.E. ~ 0.0074 with K1+2+3 a and S.E. ~ 0.015 with Lysplasminogen.
383 decreased the fibrin-binding of Kl+2+3a to the same level as was observed in the plasma clot. The fibrin-binding of miniplasminogen decreased insignificantly (P < 0.1) while the fibrin-binding of Lys-plasminogen decreased to a level which was intermediate between that in the fibrin clot and that in the plasma clot. Elimination of Glu-plasminogen from the fibrin clots did not change the results (not shown).
T A B L E II I N F L U E N C E O F 6 - A M I N O H E X A N O I C A C I D OR T R A N E X A M I C A C I D ON F I B R I N - B I N D I N G OF 1 2 5 I . L A B E L L E D K 1 + 2 + 3 a ( D E F I N E D IN T A B L E I) OR 1 2 S I . L A B E L L E D K 4 T h e c l o t t i n g m i x t u r e c o n t a i n e d 9.2 #M c l o t t a b l e f i b r i n o g e n a n d 1.2 pM f r a g m e n t . A m i n o acid concentration (~M)
A m i n o acid
None 6 - A m i n o h e x a n o i c acid 6 - A m i n o h e x a n o i c acid T r a n e x a m i c acid T r a n e x a m i c acid
F r a c t i o n of labelled f r a g m e n t b o u n d to fibrin, m e a n S.E. (n = 4)
0 30 300 10 100
K1+2+3 a
K4
0.17 0,083 0.046 0,065 0,040
0.058 0.041 0.021 0.051 0.020
0.0075 0.0013 0.0047 0.0029 0.0021
0.0023 0.0028 0.0015 0.0024 0.0018
TABLE III I N F L U E N C E O F ~ 2 " A N T I P L A S M I N ON F I B R I N - B I N D I N G O F 1 2 5 1 - L A B E L L E D K 4 OR 125 I - L A B E L LED M1NIPLASMINOGEN T h e c l o t t i n g m i x t u r e c o n t a i n e d 9.2 /sM c l o t t a b l e f i b r i n o g e n a n d 1.2 pM f r a g m e n t . c~2 - A n t i p l a s m i n concentration (#M)
F r a c t i o n o f labelled f r a g m e n t b o u n d to fibrin, m e a n S.E. (n) K4
0 6,8
0.079 0.072
Miniplasminogen 0 . 0 0 5 5 (2) 0 . 0 0 2 0 (47
0.50 0.50
0 . 0 2 3 (47 0 . 0 2 0 (4)
TABLE IV F I B R I N B I N D I N G O F 1 2 5 I _ L A B E L L E D P L A S M I N O G E N F R A G M E N T S IN P L A S M A C L O T S , F I B R I N C L O T S C O N T A I N I N G G l u - P L A S M I N O G E N A N D ~ 2 - A N T I P L A S M I N , A N D IN F I B R I N C L O T S K 1 + 2 + 3 a is d e f i n e d in T a b l e I. T h e c l o t t i n g m i x t u r e c o n t a i n e d 6.1 pM f i b r i n o g e n (all s y s t e m s ) , 1.8 /aM G l u - p l a s m i n o g e n (PC a n d FC 1), 1.0 pM c~2 - a n t i p l a s m i n (PC and FC 1) a n d 0.2 pM labelled f r a g m e n t (all s y s t e m s ) . T h e P l a s m a vol. f r a c t i o n = 0.8 in PC. PC, p l a s m a clots; FC 1, fibrin clots c o n t a i n i n g Glu-plasmin o g e n a n d c~2 - a n t i p l a s m i n ; FC 2, fibrin clots. System
F r a c t i o n of labelled f r a g m e n t b o u n d to fibrin, m e a n S,E. (n) K1+2+3 a
PC FC 1 FC 2
0.038 0.041 0.10
0 . 0 0 1 7 (4) 0 . 0 0 1 5 (4) 0 . 0 0 7 4 (47
Miniplasminogen
Lys-plasminogen
0.24 0.36 0.39
0.27 0.44 0.56
0 . 0 2 7 (6) 0 . 0 1 5 (4) 0 . 0 0 4 2 (6)
0 . 0 2 2 (6) 0 . 0 1 9 (4) 0 . 0 1 6 (6)
384 TABLE V C O R R E L A T I O N B E T W E E N D I S S O C I A T I O N C O N S T A N T S ( K d ) IN p M , A N D C O N C E N T R A T I O N S (c50) I N pM O F 6 - A M I N O H E X A N O I C A C I D , T R A N E X A M I C A C I D A N D c ~ 2 - A N T I P L A S M I N T H A T D E C R E A S E BY 5 0 % T H E F I B R I N - B I N D I N G O F P L A S M I N O G E N F R A G M E N T S K l + 2 + 3 a , d e f i n e d in T a b l e I. (*) T h i s c o n c e n t r a t i o n ( c 6 2 ) d e c r e a s e d fibrin-binding by 62% ( * * ) 6.8 pM ~2-antiplasmLn d i d n o t d e c r e a s e the fibrin-binding ( T a b l e I I I ) . T h e v a l u e s of c 50 ( c 6 2 ) axe f r o m Figs. 2 a n d 3, a n d T a b l e II. ( T h e c l o t t i n g m i x t u r e s c o n t a i n e d 9 . 2 /~M f i b r i n o g e n a n d 1.2 pM p l a s m i n o g e n d e r i v a t i v e ) . T h e v a l u e s of K d axe f r o m t h e l i t e r a t u r e a n d t h e o n e s f r o m r e f e r e n c e [ 8 ] axe d e t e r m i n e d by using the s a m e f r a g m e n t s as u s e d in the present s t u d y . T h e v a l u e s in p a r e n t h e s e s axe K d v a l u e s w h i c h axe calcul a t e d f r o m t h e c50 ( c 6 2 ) values. T h e s e c a l c u l a t i o n s axe b a s e d o n : (1) the t o t a l a m o u n t s o f p l a s m i n o g e n d e r i v a t i v e a n d a m i n o a c i d or c~2 - a n t i p l a s m i n in the c l o t t i n g m i x t u r e axe k n o w n , (2) the a m o u n t o f fibrinb o u n d p l a s m i n o g e n d e r i v a t i v e is m e a s u r e d , a n d (3) t h e free a m o u n t of p l a s m i n o g e n d e r i v a t i v e ( a n d c o n s e q u e n t l y the a m o u n t s o f free a n d b o u n d a m i n o a c i d or c~2 - a n t i p l a s m i n ) is c a l c u l a t e d b y using the r a t i o , ( a m o u n t of free p l a s m i n o g e n d e r i v a t i v e ) / ( a m o u n t of fibrin-bound p l a s m i n o g e n derivative), w h i c h is c o n s t a n t at varying c o n c e n t r a t i o n s o f d e r i v a t i v e ( T a b l e I). It is a s s u m e d t h a t a m i n o a c i d or ~ 2 - a n t i p l a s m i n d o e s n o t bind to fibrin ( e i t h e r d i r e c t l y or via the p l a s m i n o g e n d e r i v a t i v e s ) a n d that o n l y o n e binding site is i n v o l v e d . It is also a s s u m e d that equilibrium c o n d i t i o n s e x i s t . Fragment
K1+2+3 a
6 - A m i n o h e x a n o i c acid
T r a n e x a m i e acid
~2"Antiplasmin
c50
c50
Kd
c50
Kd
10 *
1 [81 (4.8)
1.2
0.18 [12] (0.50)
10-100
6.3 [ 8 ]
**
9 [12]
Kd
30 (25)
K4
30-300
Miniplasminogen
340
Lys-Plasminogen
130
36 [ 7 ]
70 (187) 35,260 and 10,000 [33] (37)
** (38)
18
2.2, 36 a n d 1,035 [34] (5.0)
5
0.63 [12] (1.3)
Discussion Markus and co-workers [32--34] demonstrated by an equilibrium ultrafiltration technique using 6~aminohexanoic acid or tranexamic acid as a ligand that plasminogen contains at least five lysine-binding sites, one of which is a strong binding-site. The purification of the three major native plasminogen fragments, that are obtained from Glu-plasminogen by limited proteolysis with elastase, involves affinity chromatography on lysine-Sepharose [1]. The conditions under which they are eluted led us to conclude when they were first prepared that K1+2+3 and K4 each contains at least one lysine-binding site, whereas miniplasminogen does not contain any [1]. Recently, Markus et al. [8] studied the binding of tranexamic acid to these same fragments by their equilibrium ultrafiltration technique. They showed that K1+2+3 actually contains at least two lysine-binding sites, one of which is the strong binding site, found earlier in both Glu-and Lys-plasminogen [32--34]. They also showed that K4 contains a single weaker lysine-binding site, whereas no ligand binding could be demonstrated to miniplasminogen. The strong lysine-binding site on K1+2+3 has recently been attributed to the K1 part [7 ]. The binding of fibrin and a2-antiplasmin to plasmin(ogen) plays an important role in the regulation of fibrinolysis [25,35]. Wiman and co-workers [12, 13,35] have proposed that plasmin(ogen) binds to fibrin and a2-antiplasmin
385 through its lysine-binding sites and that the strong lysine-binding site plays a key role in this binding. Their proposal is based on the finding [13] that plasminogen and plasminogen fragments that bind to lysine-Sepharose also bind to fibrin-Sepharose and can be eluted by less than 5 mM 64minohexanoic acid, while plasminogen fragments that do not bind to lysine-Sepharose also do not bind to fibrin~epharose. It is also based on the fact that ~ concentrations of 6-aminohexanoic acid inhibit the reversible first reaction step between the a2antiplasmin and the Lys-plasmin but not the first reversible reaction step between the a2-antiplasmin and the miniplasmin because the latter lacks the strong lysine-binding site [6,11]. The K1+2+3 b variant which has a glucosamine-based oligosaccharide attached to Asn-288 binds only very weakly to fibrin (Table I) and was therefore not studied in detail. The Kl+2+3a variant, with no oligosaccharide attached to Asn-288 binds somewhat more strongly. The finding that pM concentrations of 6-aminohexanoic acid, tranexamic acid and a2-antiplasmin decrease the fibrin-binding of Kl+2+3a (Tables II and V, Fig. 3) indicates a specific effect and agrees with the fact that K1+2+3 contains the strong lysine-binding site [7,8] and an important a2-antiplasmin-binding site [12]. The discrepancy in our K d values indicated in parentheses in Table V and those reported in the literature may be due to different assay systems and preparations as well as to the fact that the assumptions made in the text of Table V may be wrong. Our results are compatible with either of two hypotheses. (a) K1+2+3a binds to fibrin through its high,affinity lysine-binding site and is removed by competitive binding of 6-aminohexanoic acid or tranexamic acid. (b) Kl+2+3a binds to fibrin through a different binding site, specific for fibrin, and 6-aminohexanoic acid or tranexamic acid when bound to the high-affinity lysine-binding site acts as an allosteric effector to change the conformation of the fibrin-binding site, thus decreasing the fibrin-binding. The effect of a2-antiplasmin on the fibrinbinding of Kl+2+3a can be interpreted in the same way. The fibrin-binding of K1+2+3 is markedly lower in a plasma clot than in a purified fibrin clot (Table IV). This can be readily explained by an effect in the plasma clot of a2-antiplasmin alone, or by a combined effect of this inhibitor, and the histidine-rich glycoprotein which has recently been shown to bind strongly to K1+2+3 [36]. The fibrin-binding of K4 is relatively w e a k ( T a b l e I). The concentrations of 6-aminohexanoic acid or tranexamic acid required for 50% decrease in this binding is less well defined than with K1+2+3 a (Table V) because a large change in the concentration of amino acid causes only a small change in fibrin-binding (Table II). The effects of the amino acids on the fibrin-binding of K4 can be explained in either of the two ways described for K1+2+3 a above and are compatible with the dissociation constants reported in the literature for the single binding site on K4 (Table V). No effect of a2-antiplasmin at 6.8 pM could be demonstrated on the fibrin-binding of K4 (Table III). This agrees with the fact that K4 has only a relatively weak binding site for a2-antiplasmin (Table V). Miniplasminogen (12SI-labelled as well as unlabelled) was found to bind more strongly to fibrin than any of the other fragments (Table I). It is not possible to say from the present data whether the fibrin-binding site of miniplasminogen is located in its K5-part or in its serine proteinase zymogen part. Wiman
386 and Wall~n [13], were unable to demonstrate an affinity between fibrinogenSepharose and the plasmin-light chain obtained by partial reduction. It is possible that their light chain may have lost a fibrin(ogen)-binding site due to cleavage of intra-chain disulphide bridges. It has been shown that the light chain prepared under mild reducing conditions looses most of its proteolytic activity (casein substrate) and most of its ability to incorporate [aH]diisopropyl phosphorofluoridate [37]. It was a surprising finding that 6~minohexanoic acid and tranexamic acid decrease the fibrin-binding of miniplasminogen (Fig. 2, Table V), since previous results indicate that this fragment does not contain lysine-binding sites [ 1,8]. Furthermore, 6-aminohexanoic acid has only a slight effect on the kinetics of interaction between miniplasmin and a2-antiplasmin [6,11]. One possible explanation for this finding is that the interaction observed between fibrin and miniplasminogen could expose a hidden lysinebinding site in the latter. Another possibility is that the binding site for miniplasminogen in fibrin could be affected by 6-aminohexanoic acid. a2-Antiplasmin at 6.8 plVI has no effect on the fibrin-binding of miniplasminogen (Table III). This agrees with the observation that the strength of binding in the first reversible step of the reaction between a2~antiplasmin and plasmin is weaker with miniplasmin than with Lys-plasmin [6,11]. The fibrin-binding of miniplasminogen is somewhat lower in a plasma clot than in a purified fibrin clot (Table IV) indicating that one or more plasma proteins may interfere with the reaction between this fragment and fibrin. The fibrin-binding of Glu- and Lys-plasminogen observed in the present study confirms previous results [21,38] showing that the removal of residues 1--76 from Glu-plasminogen increases the extent of fibrin-binding. The present study also confirms that 6~aminohexanoic acid, tranexamic acid and a2~antiplasmin decrease the fibrin-binding of Lys-plasminogen [21,38,39] and that the fibrin-binding of Lys-plasminogen is lower in a plasma clot than in a purified fibrin clot [38]. The values of c50 and Kd in Table V indicate that the decrease in the fibrin-binding of Lys-plasminogen caused by 6-aminohexanoic acid or tranexamic acid is mainly related to saturation of the strong and intermediate lysine-binding sites in the K1--K4 part of the plasminogen molecule. The 6~minohexanoic acid- or tranexamic acid-induced gross change in conformation of Glu-plasminogen associated with the exposure of the activator-susceptible Args60-Va1561 bond is mainly related to saturation of the weak lysine binding sites [33,34]. Acknowledgements Supported by the Danish Medical Research Council (ST and IC) and the Danish Hospital Foundation for Medical Research; Region of Copenhagen, the Faroe Islands and Greenland (ST). It was also supported b y the National Heart, Lung and Blood Institute, NIH, grant no. HL-16238, Bethesda, MD, U.S.A. (SM). The valuable technical assistance of Jannie Bank and Inge Schuster is appreciated.
387
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