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Enhancement of plasma fibrinolysis in vitro by jararhagin, the main haemorrhagic metalloproteinase in Bothrops jararaca venom
Pergamon
0041-0101(95)00102-6
Toxicon, Vol. 33, No. 12, pp. 1605-1617, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0041-0101/95 $9.50 + 0.00
E N H A N C E M E N T OF P L A S M A FIBRINOLYSIS IN VITRO BY J A R A R H A G I N , THE M A I N H A E M O R R H A G I C M E T A L L O P R O T E I N A S E IN B O T H R O P S J A R A R A C A V E N O M M A S A H I K O SUGIKI, I M A S U G I M A R U Y A M A , 1 ETSUO YOSHIDA, I HISASHI M I H A R A , 1 A U R A S. K A M I G U T I 2 and R. DAVID. G. T H E A K S T O N 3 'Department of Physiology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-16, Japan; 2Department of Haematology,Universityof Liverpool,Liverpool,U.K.; and 3VenomResearch Unit, Liverpool School of Tropical Medicine, Liverpool L3 5QA, U.K. (Received 22 May 1995; accepted 4 July 1995)
M. Sugiki, M. Maruyama, E. Yoshida, H. Mihara, A. S. Kamiguti and R. D. G. Theakston. Enhancement of plasma fibrinolysis in vitro by jararhagin, the main haemorrhagic metalloproteinase in Bothropsjararaca venom. Toxicon 33, 1605 1617, 1995.--Jararhagin, a haemorrhagic metalloproteinase from Bothrops jararaca venom, plays an important role in systemic as well as local haemorrhage. In this study, the effect of jararhagin on the fibrinolytic system was investigated. The fibrinolytic activity of various kinds of animal plasmas was measured by the fibrin plate method. No activity was detected in plasma alone. However, after mixing plasma with jararhagin, strong fibrinolytic activity was recorded in guinea-pig, horse, dog, rabbit and human plasmas. The mechanism of the increase of fibrinolytic activity by jararhagin was studied further in guinea-pig plasma. Fibrin-zymographic studies indicated that jararhagin increased tissue-type plasminogen activator (tPA) activity by the dissociation of a complex of tPA with type 1 plasminogen activator inhibitor (PAI-1). c(2-Plasmin inhibitor (~2-PI) activity in the plasma was measured using a synthetic chromogenic substrate method after incubation with jararhagin. The c(2-PI activity in the plasma decreased in both time-dependent and dose-dependent manners. These in vitro results suggest that, in some animal plasmas, jararhagin increases plasma fibrinolytic activity by causing dissociation of the tPA/PAI-I complex and by the inactivation of c(2-PI. It is possible that this direct action of jararhagin on the enhancement of plasma fibrinolytic activity may contribute to the aetiology of systemic haemorrhage frequently observed in human victims of B. jararaca envenoming.
INTRODUCTION The main haemorrhagic factor from Bothrops jararaca venom has been purified and is termed HF2 (Mandelbaum et al., 1976), JFI (Maruyama et al., 1992) or jararhagin (Paine et al., 1992). Jararhagin is a 52,000 mol. wt metalloproteinase which possesses a Zn 2+dependent catalytic site, a disintegrin-like region and a cysteine-rich carboxy-terminal region (Paine et al., 1992). It possesses fibrinolytic activity, has an inhibitory effect on platelet aggregation in vitro and induces disruption of both vascular endothelial cells and 1605
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the v a s c u l a r b a s e m e n t m e m b r a n e in vivo ( K a m i g u t i et al., 1991, 1994). S u b c u t a n e o u s injection o f j a r a r h a g i n has also been shown to cause systemic h a e m o r r h a g e t o g e t h e r with t h r o m b o c y t o p e n i a in e x p e r i m e n t a l a n i m a l s ( K a m i g u t i e t al., 1991). These findings, t o g e t h e r with the o b s e r v a t i o n o f the ineffectiveness o f j a r a r h a g i n inhibition by p l a s m a inhibitors ( K a m i g u t i et al., 1994), suggest that this m e t a l l o p r o t e i n a s e p a r t i c i p a t e s in the d e v e l o p m e n t o f not only local, b u t also systemic h a e m o r r h a g e . The v e n o m also c o n t a i n s a t h r o m b i n - l i k e enzyme a n d o t h e r p r o c o a g u l a n t c o m p o n e n t s that m a y p r o v o k e d i s s e m i n a t e d i n t r a v a s c u l a r c o a g u l a t i o n ( N a h a s e t al., 1979), which is a l m o s t always associated with a c t i v a t i o n o f the fibrinolytic system (Collen, 1980). In fact, fibrinolysis in e n v e n o m e d victims has been r e p o r t e d to be due to intrinsic t h r o m b i n f o r m a t i o n and c o n s e q u e n t high titres o f cross-linked fibrin f r a g m e n t D, together with t h r o m b o c y t o p e n i a a n d / o r d i m i n i s h e d levels o f factor V a n d / o f factor V I I I ( M a r u y a m a e t al., 1990). In a d d i t i o n , e n h a n c e d fibrinolysis was evident from the low level o f ~2-plasmin i n h i b i t o r (~2-PI) f o u n d in these patients. Interference in regulation o f fibrinolysis results in a tendency to t h r o m b o s i s or bleeding. P l a s m i n o g e n a c t i v a t o r s (PAs) trigger the fibrinolytic process (Collen, 1980). T w o different types o f PAs, tissue t y p e - P A (tPA) a n d u r o k i n a s e t y p e - P A (uPA), have been identified in m a m m a l s . These are serine proteinases which c o n v e r t the p l a s m a inactive p r o e n z y m e , p l a s m i n o g e n , to the active enzyme, plasmin, which then causes the d e g r a d a t i o n o f its n a t u r a l substrate, fibrin. The fibrinolytic system is regulated by several m e c h a n i s m s such as the release o f P A from the vascular e n d o t h e l i u m , the fibrin-associated activation o f p l a s m i n o g e n by the P A and the inhibition o f P A and plasmin by c o m p l e x i n g with their specific physiological inhibitors [type 1 p l a s m i n o g e n a c t i v a t o r i n h i b i t o r ( P A l - l ) a n d ~2-PI, respectively] (Sprengers a n d Kluft, 1987). In the present in v i t r o study, we f o u n d a c o n s i d e r a b l e increase o f fibrinolytic activity in h u m a n a n d some o t h e r a n i m a l p l a s m a s treated with purified j a r a r h a g i n . This suggested that it affected some c o m p o n e n t s in the fibrinolytic system causing e n h a n c e m e n t o f fibrinolysis. W e p r o p o s e to investigate the m e c h a n i s m o f activation o f fibrinolysis in v i t r o to find out w h e t h e r j a r a r h a g i n influences the relationship within the t P A / P A I - 1 complex. MATERIALS AND METHODS Materials
Materials included phenyl-Superose (HR 5/5, 1 ml), Mono Q (HR 5/5, 1 ml), HiTrap Blue (1 nal) columns and Sephadex G-25, DEAE-Sephadex (Pharmacia Biotech Ltd, Uppsala, Sweden); bovine fibrinogen (75% w/w clonable; Miles Inc., Kankakee, IL, U.S.A.); amiloride (Sigma Chemical Co., Poole, U.K.); ethylenediaminetetraacetate (EDTA), e-aminocaproic acid (EACA) and polyethylene glycol (average mol. mass 20 kDa; Wako Pure Chemical Industries Ltd, Osaka, Japan); human urokinase (Green Cross Co., Osaka, Japan). Other reagents used were of analytical reagent grade. Puroqcation of.jararhagin Pooled lyophilized B.jararaca venom, obtained from about 100 snakes, was donated by the Instituto Butantan
(S~_o Paulo, Brazil). Jararhagin was purified from the whole venom using a phenyl-Superose hydrophobic interaction column and a Mono Q anion-exchange column on a Pharmacia FPLC system according to the method of Paine et al. (1992). The active fraction was then dialysed with 5 mM CaC12 at 4'C and applied to a HiTrap Blue affinity column equilibrated with 5 mM CaCI2 on the FPLC system to remove any contaminant clotting activity. This procedure was performed because it is known that the coagulant activity in B. jararaca venom is strongly absorbed by this type of column; 2 M NaCI was necessary to elute this activity (Maruyama et al., 1992). The column was washed with 20 ml of 5 mM CaCI2 and eluted with 20 mM Tris-HC1 buffer containing l0 mM CaCI2, pH 8.0, at a flow rate of I ml/min. The eluted fraction was concentrated using polyethylene glycol and dialysed with 20 mM Tris- HC1 buffer containing 2 mM CaCI2, pH 7.4 (Tris~CaCl 2buffer) at 4"C. Protein concentrations were measured using the BioRad Protein Assay (BioRad, Hemel Hempstead, U.K.) according to the manufacturer's instructions with bovine serum albumin as the standard. SDS-PAGE
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(Laemmli, 1970) of the purified protein was carried out using a 10% polyacrylamide gel under non-reducing conditions. Aliquots of the final product were stored at - 4 0 ° C .
Measurement of haemorrhagic activity Haemorrhagic activity was estimated according to the method of Theakston and Reid (1983). One-hundred microlitres of each sample was injected intradermally into the dorsal shaved skin of male C F W strain mice under light halothane/O 2 anaesthesia. The area of the haemorrhagic lesion was measured 24 hr after injection.
Preparation of plasma samples H u m a n blood was collected by puncture of the median cubital vein. Guinea-pigs (Dunkin Hartley), rats (Spragu~Dawley), mice ( T F W strain) and hamsters (Syrian) were anaesthetized by halothane/O 2 inhalation and blood was collected by cardiac puncture in 3.13% w/v trisodium citrate (9 parts blood: 1 part citrate) and then centrifuged at 3000 rpm for 15 min at 4~C. Citrated animal plasmas (dog, horse, goat, sheep, pig and bovine) were purchased from Sigma Chemical Co. (Poole, U.K.).
Measurement qf fibrinolytic activity' Fibrinolytic activity was estimated by the fibrin plate method of Astrup and Mfillertz (1952). The fibrin plates were prepared using 6 mg/ml of either plasminogen (plg)-rich or pig-poor fibrinogen which was clotted using 2 U/ml bovine thrombin (Mochida Pharmaceutical Co., Tokyo, Japan). Plg-poor fibrinogen was prepared by passing plg-rich fibrinogen through a lysine-Sepharose column (Matsuda et al., 1972).
Purification of guinea-pig and human plasminogen Guinea-pig and h u m a n plasminogens were prepared. The former was purified using the method of Deutsch and Mertz (1970). Ten millilitres of freshly prepared guinea-pig plasma was applied to a lysine-Sepharose column (1.4 x 7.5 cm, gel volume 2 ml) equilibrated with 0.1 M Tris HC1 buffer, pH 7.4, and the non-absorbing yellow fraction was collected (pig-poor plasma). After washing the column with 30 ml of equilibrating buffer, elution was performed with 0.1 M Tris HC1 buffer, pH 7.4, containing 0.2 M EACA. The E A C A was removed from the plasminogen by gel filtration on a Sephadex G-25 column (1.7 x 9.5 cm), equilibrated and eluted with 60 m M Tris HC1 buffer, pH 8.5, containing 65 m M NaC1. H u m a n plasminogen was purified from fresh frozen plasma by affinity chromatography on a lysine-Sepharose column followed by a m m o n i u m sulphate precipitation and anion-exchange chromatography on a DEAE-Sephadex column according to the method of Robbins and Summaria (1976).
Fibrin -zymography Fibrin-zymography was carried out according to the method of Granelli-Piperno and Reich (1978). After S D S - P A G E (10% polyacrylamide gel, non-reducing conditions) of sample mixtures prepared as above, the gels were washed twice for 1 hr each with 500 ml of 10 m M phosphate buffer, pH 7.4, containing 0.15 M NaCI and 0.2% v/v Triton X-100 at room temperature with gentle agitation. Following a brief wash with distilled water, the gels were overlaid on fibrin-agar plates. Each plate was prepared by pouring 8.5 ml of 1.2% w/v bovine fibrinogen in 0.1 M phosphate buffer, pH 7.4, 8.5 ml of 1.2% w/v agar in water and 1 ml of 20 U/ml thrombin into a plastic plate (13.5 x 9.5 cm) at 42°C. After incubation of the plates in a moist chamber at 37°C, fibrinolytic activities of enzymes were visualized as lytic bands against a black background. The mol. wts of the bands were estimated by comparison with the mol. wt standards in gels stained with Coomassie Blue R-250.
Measurement of ~e-PI activity Twenty microlitres of the guinea-pig plg-poor plasma was incubated at 37°C with 20#1 of various concentrations of jararhagin for times ranging between 0 and 30 min. E D T A (10/~1 of a 50 m M solution) was then added to the mixture which was allowed to stand for 10 min at room temperature to inactivate jararhagin. To each test plasma sample, 370 #1 of saline was added. The antiplasmin activity of ~2-PI in the samples was measured using the synthetic chromogenic substrate provided in the antiplasmin assay kit (Stachrom Antiplasrain, Diagnostica Stago, Asnieres-sur-Seine, France) according to the manufacturer's instructions.
SDS-PAGE of ot2-Pl after incubation with jararhagin Purified ~z-PI from h u m a n plasma (Celsus Laboratories, Cincinnati, OH, U.S.A.) was incubated at 37°C with jararhagin at a molar ratio of 20 : 1 in 50 m M Tris-HCl buffer, pH 7.4, containing 0.1 M NaC1 and 5 m M CaCI 2 for periods ranging between 0 and 80 min. Reactions were stopped by adding the same volume of sample buffer
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(125 mM Tris-HC1 buffer, pH 6.8, containing 70 mM SDS, 20% v/v glycerol, 0.012% w/v bromophenol blue and 10% v/v 2-mercaptoethanol) and boiling the mixtures for 3 min. Samples containing 1.5 ttg of ~2-PI were then immediately analysed by SDS PAGE (Laemmli, 1970). Proteins were stained with Coomassie Blue R-250 and prestained mol. wt markers (Gibco BRL, Gaithersburg, MD, U.S.A.) were used to estimate the electrophoretic mobilities of the 72-PI proteins.
RESU LTS
Purification o f jararhagin T h e p u r i t y o f the i s o l a t e d e n z y m e was c o n f i r m e d by a n a l y t i c a l S D S P A G E . T h e i s o l a t e d j a r a r h a g i n r e v e a l e d a single p r o t e i n b a n d w i t h a tool. wt o f 52,000 (Fig. 1). It p o s s e s s e d h a e m o r r h a g i c a c t i v i t y a n d n o r e s i d u a l c o a g u l a n t a c t i v i t y a f t e r m i x i n g w i t h the g u i n e a - p i g o r o t h e r p l a s m a s o r f i b r i n o g e n at a final p r o t e i n c o n c e n t r a t i o n o f up to 1 0 0 / z g / m l o f plasma.
kDa --205 --116 "97 m
66
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29
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Fig. 1. SDS PAGE of jararhagin. Purified jararhagin (I.3 pg protein) was analyse d by SDS PAGE. Electrophoresis was performed at 10 20 mA/gel for 150 min and then proteins were stained with Coomassie Blue R-250. Molecular weight markers used were myosin, fl-galactosidase, phosphorylase B, bovine serum albumin, ovalbumin and carbonic anhydrase (Sigma Chemical Co., Poole, U.K.).
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Table I. Fibrinolytic activity ofjararhagin in plasminogenrich fibrin plates Plasma source Fibrinolytic activity (mm2) Human 68.3 _+5.1 Guinea-pig 174.3 __+5.5 Horse 163.1 ___5.1 Dog 161.3 _+ 12.4 Rabbit 103.7 _+ 11.6 Rat 0 Mouse 0 Hamster 0 Goat 0 Sheep 0 Pig 0 Bovine 0 Control 48.0 + 1.7 Twenty microlitres of plasma was mixed with 20 ~1 of hararhagin (160/~g/ml) and 30/~1of the mixture was applied on the fibrin plate. After 18 hr at 37°C, the lytic area was measured. Fibrinolytic activity was expressed as the area of lysis (mm2) by multiplying two perpendicular diameters of the lysed area. Instead of plasma, 150 mM NaC1 containing 2 mM CaC12was mixed with jararhagin as the control. Data show the mean _ S.D. (n = 3).
Fibrinolytic activity of jararhagin in different animal plasmas on plasminogen-rich fibrin plates The effect o f jararhagin on the fibrinolytic system was investigated by measuring its fibrinolytic activity after mixing with various animal plasmas (Table 1). N o fibrinolytic activity was detected in the different plasmas mixed with T r i s ~ a C l 2 buffer alone. However, after mixing the plasmas with jararhagin, strong fibrinolytic activity was recorded in the plasmas o f guinea-pig, horse, dog, rabbit and h u m a n (Table 1, Fig. 2). The fibrinolytic activity in these plasmas containing jararhagin was m o r e p r o n o u n c e d than the original fibrinolytic activity o f j a r a r h a g i n alone (Table 1). N o activity was detected in rat, mouse, hamster, goat, sheep, pig and bovine plasmas even after mixing with jararhagin (Table 1, Fig. 2).
Effect of jararhag& on plasminogen activation I n c u b a t i o n o f purified guinea-pig or h u m a n plasminogen gave no evidence o f plasmin formation as shown by the absence o f fibrinolytic activity on the plg-poor fibrin plate (Fig. 3). Activation o f both plasminogens by urokinase (controls) was observed simultaneously.
Fibrinolytic activity in normal and plasminogen-poor guinea-pig plasma after incubation with jararhagin As jararhagin caused the highest increase in fibrinolytic activity in guinea-pig plasma, this plasma type was selected in order to clarify the mechanism o f jararhagin-induced increase in fibrinolysis. This rapid increase in activity on both plg-rich and plg-poor fibrin plates is shown in Fig. 4. The activity 30 min after the incubation was 89 ___ 18 m m z on the plg-rich fibrin plates and 7 2 _ 1 0 m m 2 on the pig-poor fibrin plates (Fig. 4A). Both activities gradually increased until 120 min incubation. The activity on plg-rich plates was
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increased to a greater extent than that on the pig-poor fibrin plates. It is known that fibrin in the plg-rich plate is lysed by PAs, plasmin and plasmin-like enzymes, while that of the pig-poor plate is lysed by plasmin and plasmin-like enzymes (Etoh et al., 1992). Therefore, the difference in the activity of these enzymes on each type of fibrin plate represents the PA activity in the samples. Figure 4B shows the fibinolytic activity in the pig-poor plasma after incubation with jararhagin. The difference in the activity between pig-rich and pig-poor fibrin plates was similar to that in the normal plasma (Fig. 4A). However, no activity was detected on the pig-poor plates, indicating that plasminogen was essential for the enhancement of fibrinolysis by jararhagin. These data therefore indicate that jararhagin increases both plasma PA activity and also plasmin activity in vitro. Fibrin-zymography o [ p l a s m a after incubation with .jararhagin Figure 5 shows plg-rich fibrin-zymographic patterns of the fibrinolytic enzyme in guinea-pig plasma at different times of incubation with jararhagin. One principal lyric band of tool. wt 105,000 and two closely located minor lytic bands of 56,000 and 52,000, respectively, were observed in the plasma without incubation (lane 1). After treatment with jararhagin, the 105,000 tool. wt band gradually disappeared, while the intensities of the 56,000 and 52,000 tool. wt bands increased (lanes 2 4). A new lytic band of tool. wt 47,000
Fig. 2. Fibrinolytic activity of jararhagin in plasminogen-rich fibrin plates. Jararhagin incubated in: 1, human plasma; 2, guinea-pig plasma; 3, horse plasma; 4, rabbit plasma: 5, dog plasma; 6, rat plasma; 7, 150ram Nacl containing 2mM CaC12alone as the control. The same mixtures were used as described in Table 1.
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Fig. 3. Effect of jararhagin on plasminogen. Fifty microlitres of purified human plasminogen (300#g/ml) or guinea-pig plasminogen (200 #g/ml) was incubated at 37C with 50 #1 ofjararhagin (160/~g/ml) in 20 mM Tris-HCl buffer, pH 7.4, containing 5 mM CaC12. After 30 min, 25/11 of 50 mM EDTA was added to the mixture and left at room temperature for 10 min to inactivate jararhagin. The sample (30 #1) was applied to the pig-poor fibrin plate to determine the level of plasmin formation. For controls, plasminogen of both origins was incubated with urokinase (UK, l0 U/ml) instead of jararhagin. 1, Human plasminogen + jararhagin; 2, human plasminogen + UK; 3, guinea-pig plasminogen + jararhagin; 4, guinea-pig plasminogen + UK; 5, buffer + UK.
a p p e a r e d after 60 min i n c u b a t i o n (lane 4). A f t e r i n c u b a t i o n o f the p l a s m a with Tris CaCI2 buffer a l o n e (control), the 105,000 lytic b a n d still r e m a i n e d a n d b o t h 56,000 a n d 52,000 mol. wt lyric b a n d s d i s a p p e a r e d (lanes 5 7). N o lyric b a n d was detected using p l g - p o o r f i b r i n - a g a r plates, i n d i c a t i n g t h a t these lytic b a n d s were due to PA(s). M o r e o v e r , all lyric b a n d s did n o t d i m i n i s h on z y m o g r a p h y using pig-rich f i b r i n - a g a r plates c o n t a i n i n g 1 m M a m i l o r i d e , a p o t e n t i n h i b i t o r o f u P A (Vassalli a n d Belin, 1987). This i n d i c a t e d that all lytic b a n d s were due to t P A ( d a t a n o t shown).
Inactivation of %-plasmin inhibitor by jararhagin It was t h o u g h t that a n y free p l a s m i n is r a p i d l y b o u n d a n d i n a c t i v a t e d by its specific inhibitor, ~2-PI, in p l a s m a ( M o r o i a n d A o k i , 1976). H o w e v e r , we f o u n d strong p l a s m i n activity in g u i n e a - p i g p l a s m a i n c u b a t e d with j a r a r h a g i n (Fig. 4A), suggesting that j a r a r h a g i n m i g h t have s o m e effects on ~2-PI in the p l a s m a . W e used the p l g - p o o r p l a s m a for e s t i m a t i n g the direct effect o f j a r a r h a g i n on ~2-PI to a v o i d the interference o f p l a s m i n TOX 33/12
D
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which could be formed in normal plasma in the assay system for 0~2-PI activity. In preliminary experiments, it was confirmed that there was no significant difference in c~2-PI activity between normal and pig-poor plasma, and no plasmin activity was detected in the plg-poor plasma 30 min after incubation with jararhagin, using this assay system. Figure 6 shows c~2-PI activity in the plasma at different times of incubation with different concentrations o f j a r a r h a g i n (0-80/~g/ml). The ~2-PI activity in the plasma was decreased both time and dose dependently. Proteolysis of purified human ~2-PI with jararhagin was visualized using S D S - P A G E (Fig. 7). The principal band ofmol, wt 67,000 of ~2-PI (lane 2) was degraded by jararhagin in a time-dependent manner, yielding two major degradation products of mol. wt 59,000 and 50,000 (lanes 3-7). DISCUSSION Here we have demonstrated that a venom haemorrhagic factor, jararhagin, increases in vitro fibrinolysis directly in both human and in some other animal plasmas. The
enhanced fibrinolysis in plasma was due to the dissociation of the tPA/PAI-1 complex by jararhagin. Furthermore, jararhagin rapidly inactivates c~2-PI. Jararhagin increases the in vitro fibrinolytic activity in plasma and the extent of this increase varies depending on the mammalian species involved (Table 1). Since jararhagin is proteolytically active in plasma, these observed differences could reflect different jararhagin affinity for the substrate in the various plasma species. Another possibility may be the varying inhibitory capacity of plasma proteinase inhibitors against jararhagin in different species.
B
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0 30 60 120 3'0 60 1½0 Incubation time (min) Incubation time (rain) Fig. 4. Fibrinolytic activity in normal guinea-pig plasma (A) and in pig-poor plasma (B) after incubation with jararhagin. Forty microlitres of plasma was incubated at 37"C with 40 #1 of jararhagin (160 pg/ml). After various times, 20 pl of 50 mM EDTA was added to stop the reaction. The fibrinolytic activity in the plasma was measured using the pig-rich ( O--) and pig-poor (--Q--) fibrin plates. Each point represents the mean of three experiments. Horizontal bars are standard deviations of the means. 0
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kDa
1
2
3
4
5
6
--
105
---
56
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47
7
Fig. 5. Fibrin-zymography of plasma after incubation with jararhagin. Guinea-pig plasma (10/d) was incubated at 37'C with 10/ll of jararhagin (1601Lg/ml) or Tris~ CaC12 buffer. After different times, 5/11 of 50 mM EDTA was added. After 10 min at room temperature, 25/d of sample buffer (125mM Tris HC1 buffer containing 70mM SDS. 20% glycerol and 0.012% bromophenoi blue, pH 6.8) was added and 15/~1 of the sample was applied. SDS PAGE and fibrin-zymography were carried out as described in Materials and Methods. Plasma was incubated with jararhagin for 0 rain (lane 1), 15 min (lane 2), 30 min (lane 3), 60 min (lane 4) or with buffer for 0 min (lane 5), 30 min (lane 6), 60 min (lane 7).
By using z y m o g r a p h y o f plasma (Fig. 5) it is shown that jararhagin decreases the a m o u n t o f the principal 105,000 mol. wt t P A / P A I - I complex. In h u m a n plasma this complex appears as a band o f 110,000 which is a SDS-stable complex o f tPA and PAl-1 by z y m o g r a p h y ( K r u i t h o f et al., 1984; Booth et al., 1987). This lytic band gradually disappeared during incubation o f guinea-pig plasma with jararhagin, whereas the activity o f 56,000 and 52,000 lyric bands increased and a new lytic band o f 47,000 appeared after 60 min incubation (lanes 1-4). Z y m o g r a p h i c studies using plg-poor fibrin-agar plates or plg-rich plates containing a u P A inhibitor indicated that these lyric bands were probably due to tPA. P A I - I is structurally related to the serine protease inhibitor (serpin) superfamily (Pannekoek et al., 1986) and reacts with tPA followed by the formation o f a stable complex with a higher tool. wt (Sprengers and Kluft, 1987). In addition, guinea-pig sinusoidal endothelial cells (Rieder et al., 1993) and megakaryocytes (Konkle et al., 1993) p r o d u c e PAI-1 whose tool. wts are 43,000 and 45,000, respectively. These findings therefore confirm that the lyric band o f mol. wt 105,000 in our experiments is a t P A / P A I - l complex (Fig. 5). I n c u b a t i o n o f the plasma with buffer alone produced no change in the lytic band corresponding to the t P A / P A I - I complex and rapid disappearance o f the lytic bands o f free tPAs (Fig. 5, lanes 5 7) due to instability o f the tPA at 3 T C . These control data therefore indicate that jararhagin actually interacts with this complex decreasing tPA activity in the complex by releasing free tPA from the complex as observed in lanes 2 - 4 . The interaction o f jararhagin with this complex m a y result in a transition from the complexed state to the free forms o f t P A which is also p r o b a b l y modified by jararhagin.
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Recently, it has been reported that dissociation o f the tPA/PAI-1 complex, resulting in the appearance o f biologically active free forms o f tPA and PAI-1, occurs with SDS treatment (Gaussem et al., 1993) and with activated protein C (Grailhe et al., 1993), suggesting that this could represent a potential source o f fibrinolytic activity. Indeed, our results show that some free tPA was present in guinea-pig plasma, but substantial a m o u n t s appeared after jararhagin treatment. This suggests that jararhagin releases tPA from the complex and that this free tPA is responsible for the increase in plasma fibrinolytic activity. The precise mechanism o f dissociation o f the complex still remains to be established. Dissociation o f other enzyme/serpin complexes have already been shown to occur [e.g. thrombin/ antithrombin lII (AT III) and plasmin/~2-PI] (Griffith and Lundblad, 1981; Y a n et al., 1993). The cq-PI activity in jararhagin-treated plasma has been shown to decrease in time- and dose-dependent manners (Fig. 6). Moreover, purified h u m a n ez-PI was also degraded by jararhagin time-dependently (Fig. 7). cq-PI is a m e m b e r o f serpin superfamily and rapidly inactivates the proteolytic activity o f plasmin (Holmes et al., 1987). It has been reported that the only plasma inhibitor which reacts with jararhagin is cq-macroglobulin, but the inhibition is not complete even with a large molar excess o f this inhibitor (Kamiguti et al., 1994). In addition, progressive degradation o f c~2-PI results in a gradual decrease in its capacity to react with plasmin (Clemmensen et al., 1981). The increase o f plasmin activity in plasma caused by jararhagin (Fig. 4A) suggests that plasma cq-PI m a y be inactivated 100
"
.~.
.~, t..
50
=. E i ¢-1
0 0
1;
2'0
3'0
I n c u b a t i o n time (min) Fig. 6. a2-PI activity in plasma after incubation with jararhagin. Guinea-pig pig-poor plasma was incubated with various concentrations of jararhagin as described in Materials and Methods. Final concentrations of jararhagin were - - x - - , 0 p g/ml; IN--, 10/~g/ml; I - - , 20 #g/ml; --C) ,40 #g/ml; - - O - - , 80 #g/ml. The residual a2-PI activity in the plasma is expressed as percentage activity of the standards. Standards (100% activity) were prepared using the same procedure described in Materials and Methods, except that the plasma was mixed with TrisqZaCl2 buffer instead of iararhagin and was not incubated. A 50:50 dilution of this standard with saline was designated as 50% standard activity and saline alone was designated as 0% activity.
Snake Venom and Fibrinolysis
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kDa m67 m59 ---50
1
2
3
45
67
8
Fig. 7. Degradation of ~2-PI by jararhagin. SDS-PAGE (10% polyacrylamide gel, reducing conditions) of purified human cq-PI (7.5/IM) treated with jararhagin (0.37/~M) at 3TC for different lengths of time (lane 2, 0 min; lane 3, 5 min; lane 4, 10 min; lane 5, 20 min; lane 6, 40 min; lane 7, 80 min). Under the conditions applied, jararhagin (0.37t~M) was not detected by Coomassie Blue stain (lane l). c~2-PI (7.5/~M) was incubated at 3TC for 80 min without jararhagin for the control (lane 8).
by degradation of this inhibitor by jararhagin. Such degradation of other serpins by snake venom enzymes has been reported in other studies involving cq-proteinase inhibitor (Kress et al., 1979) and AT III (Janssen et al., 1992). Jararhagin directly increases the in vitro fibrinolytic activity in plasma by decreasing the activities of PAI-1 and cq-PI, both of which are key physiological inhibitors in the regulation of fibrinolysis. This increase in fibrinolysis may be responsible for the disturbance of the haemostatic mechanism observed in some patients following electroshock or complicated labour (Bennett et al., 1990). Excess plasmin formation would be expected to result in premature lysis of haemostatic plugs, thus causing abnormal haemorrhage. In fact, congenital deficiency of either PAI-1 (William et al., 1992) or ch-PI (Aoki et al., 1979) has been reported to show lifelong severe haemorrhagic diathesis. Therefore, with the important reservation that this is an in vitro study based mainly on results with guinea-pig plasma, it is possible that this direct action of jararhagin on the enhancement of fibrinolysis may contribute to the systemic haemorrhage frequently observed in B. j a r a r a c a envenoming in humans.
Acknowledgements--The authors thank the team of the Venom Research Unit, Liverpool School of Tropical
Medicine, for its excellent technical cooperation.
REFERENCES Aoki, N., Saito, H., Kamiya, T., Koie, K. and Sakata, Y. (1979) Congenital deficiency of ch-plasmin inhibitor associated with severe hemorrhagic tendency. J. clin. Invest. 63, 877 884. Astrup, T. and Mfillertz, S. (1952) The fibrin plate method for estimating fibrinolytic activity. Arch. biochem. Biophys. 40, 346-351.
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Bennett, B., Croll, A., Ferguson, K. and Booth, N. A. (1990) Complexing of tissue plasminogen activator with PAl-l, ~2-macroglobulin, and Cl-inhibitors: studies in patients with defibrination and a fibrinolytic state after electroshock or complicated labor. Blood 75, 671-676. Booth, N. A., Walker, E., Maughan, R. and Bennett, B. (1987) Plasminogen activator in normal subjects after exercise and venous occlusion: t-PA circulates as complexes with Cl-inhibitor and PAl-1. Blood69, 1600 1604. Clemmensem I., Thorsen, S., Mfillertz, S. and Petersen, L. C. (1981) Properties of three different molecular forms of the ~2 plasmin inhibitor. Eur. J. Biochem. 120, 105 112. Collen, D. (1980) On the regulation and control of fibrinolysis. Thromb. Haemost. 43, 77 89. Deutsch, D. G. and Mertz, E. T. (1970) Plasminogen: purification from human plasma by affinity chromatography. Science 170, 1095 1096. Etoh, Y., Sumi, H., Tsushima, H., Maruyama, M. and Mihara, H. (1992) Fibrinolytic enzymes in ascites during experimental acute pancreatitis in rats. Int. J. Pancreatol. 12, 127 137. Gaussem, P., Grailhe, P. and Angl~s-Cano, E. (1993) Sodium dodecyl sulfate-induced dissociation of complexes between human tissue plasminogen activator and its specific inhibitor. J. biol. Chem. 268, 12,150 12,155. Grailhe, P., Bezeaud, A., Gaussem, P., Guillin, M. C. and Angl6s-Cano, E. (1993) Neutralization of plasminogen activator inhibitor-I (PAI-1) by activated protein C is species-dependent. Fibrinolysis 7, 123 133. Granelli-Piperno, A. and Reich, E. (1978) A study of proteases and protease inhibitor complexes in biological fluids. J. e,cp. Med. 148, 223 234. Griffith, M. J. and Lundblad, R. L. (1981) Dissociation of antithrombin I l l thrombin complex. Formation of active and inactive antithrombin III. Biochemi~'try 20, 105 I10. Holmes, W. E., Nelles, L., Lijnen, H. R. and Collen, D. (1987) Primary structure of human :q-antiplasmin, a serine protease inhibitor (serpin). J. biol. Chem. 262, 1659 1664. Janssen, M., Meier, J. and Freyvogel, T. A. (1992) Purification and characterization of an antithrombin 11I inactivating enzyme from the venom of the African night adder (Causus rhombeatus). Toxicon 30, 985 999. Kamiguti, A. S., Theakston, R. D. G., Desmond, H. and Hutton, R. A. (1991) Systemic haemorrhage in rats induced by a haemorrhagic fraction from Bothropsjararaca venom. Toxicon 29, 1097 1105. Kamiguti, A. S., Desmond, H. P., Theakston, R. D. G., Hay, C. R. M. and Zuzel, M. (1994) Ineffectiveness of the inhibition of the main haemorrhagic metalloproteinase from Bothropsjararaca venom by its only plasma inhibitor, :~2-macroglobulin. Biochim. biophys. Acta 1200, 307 314. Konkle, B. A., Schick, P. K., He, X., Liu, R. J. and Mazur, E. M. (1993) Plasminogen activator inhibitor-I mRNA is expressed in platelets and megakaryocytes and the megakaryoblastic cell line CHRF-288. Arteriosclcr. Thromb. 13, 669 674. Kress, L. F., Kurecki, T., Chan, S. K. and Laskowski, M., Sr (1979) Characterization of the inactive fragment resulting from limited proteolysis of human alpha~-proteinase inhibitor by Crotalus adamanteus proteinase II. J. biol. Chem. 254, 5317 5320. Kruithof, E. K. O., Tran-Thang, C., Ransijn, A. and Bachmann, F. (1984) Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 64, 907 913. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685. Mandelbaum, F. R., Reichl, A. P. and Assakura, M. T. (1976) Some physical and biochemical characteristics of HF2, one of the haemorrhagic factors in the venom of Bothrops jararaca. In: Animal, Plant, and Microbial Toxins, Vol. 1, pp. i11 121 (Ohsaka, A., Hayashi, K. and Sawai, Y., Eds). London: Plenum Press. Maruyama, M., Kamiguti, A. S., Cardoso, J. L. C., Sano-Martins, I. S., Chudzinski, A. M., Santoro, M. L., Morena, P., Tomy, S. C., Antonio, L. C., Mihara, H. and Kelen, E. M. A. (1990) Studies on blood coagulation and fibrinolysis in patients bitten by Bothrops jararaca (jararaca). Thromb. Haemost. 63, 449 453. Maruyama, M., Sugiki, M., Yoshida, E., Mihara, H. and Nakajima, N. (1992) Purification and characterization of two fibrinolytic enzymes from Bothrops jararaca (jararaca) venom. Toxicon 30, 853 864. Matsuda, M., Iwanaga, S. and Nakamura, S. (1972) A simple, large scale method for preparation of plasminogen-free fibrinogen. Tbromb. Res. l, 619-630. Moroi, M. and Aoki, N. (1976) Isolation and characterization of :q-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis. J. biol. Chem. 251, 5956-5965. Nahas, L., Kamiguti, A. S. and Barros, M. A. R. (1979) Thrombin-like and factor X-activator components of Bothrops snake venoms. Thromh. Haemost. 41, 314 328. Paine. M. J. 1., Desmond, H. P., Theakston, R. D. G. and Crampton, J. M. (1992) Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops ]araraca venom. J. biol. Chem. 267, 22,869 22,876. Pannekoek, H., Veerman, H., Lambers, H., Diegaarde, P., Verweij, C. L., van Zonneveld, A. J. and van Mourik, J. A. (1986) Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family. E M B O J. 5, 2539 2544. Rieder, H., Armbrust, T., Meyer zum Bfishenfelde, K. H. and Ramadori, G. (1993) Contribution of sinusoidal endothelial liver cells to liver fibrosis: Expression of transforming growth factor-fl I receptors and modulation of plasmin-generation enzymes by transforming growth factor-/:t I. Hepatology 18, 937 944. Robbins, K. C. and Summaria, L. (1976) Plasminogen and plasmin. Meth. Enzymol. 45, 257 273. Sprengers, E. D. and Kluft, C. (1987) Plasminogen activator inhibitors. Blood 69, 381 387.
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Theakston, R. D. G. and Reid, H. A. (1983) Development of simple standard assay procedures for the characterization of snake venoms. Bull. WHO 61, 949-956. Vassalli, J. D. and Belin, D. (1987) Amiloride selectively inhibits the urokinase-type plasminogen activator. FEBS Lett. 214, 187 191. William, P. F., Shapiro, A. D., Shih, J. L., Schleef, R. R. and Ginsburg, D. (1992) Brief report: Complete deficiency of plasminogen-activator inhibitor type 1 due to a frame-shift mutation. N. Engl. J. Med. 327, 1729-1733. Yah, D., Urano, T., Takada, Y. and Takada, A. (1993) Dissociation of cc2-plasmin-inhibitor-plasmin complex and regeneration of plasmin activity by SDS treatment. Thromb. Res. 69, 491 499.
0041-0101(95)00102-6
Toxicon, Vol. 33, No. 12, pp. 1605-1617, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0041-0101/95 $9.50 + 0.00
E N H A N C E M E N T OF P L A S M A FIBRINOLYSIS IN VITRO BY J A R A R H A G I N , THE M A I N H A E M O R R H A G I C M E T A L L O P R O T E I N A S E IN B O T H R O P S J A R A R A C A V E N O M M A S A H I K O SUGIKI, I M A S U G I M A R U Y A M A , 1 ETSUO YOSHIDA, I HISASHI M I H A R A , 1 A U R A S. K A M I G U T I 2 and R. DAVID. G. T H E A K S T O N 3 'Department of Physiology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-16, Japan; 2Department of Haematology,Universityof Liverpool,Liverpool,U.K.; and 3VenomResearch Unit, Liverpool School of Tropical Medicine, Liverpool L3 5QA, U.K. (Received 22 May 1995; accepted 4 July 1995)
M. Sugiki, M. Maruyama, E. Yoshida, H. Mihara, A. S. Kamiguti and R. D. G. Theakston. Enhancement of plasma fibrinolysis in vitro by jararhagin, the main haemorrhagic metalloproteinase in Bothropsjararaca venom. Toxicon 33, 1605 1617, 1995.--Jararhagin, a haemorrhagic metalloproteinase from Bothrops jararaca venom, plays an important role in systemic as well as local haemorrhage. In this study, the effect of jararhagin on the fibrinolytic system was investigated. The fibrinolytic activity of various kinds of animal plasmas was measured by the fibrin plate method. No activity was detected in plasma alone. However, after mixing plasma with jararhagin, strong fibrinolytic activity was recorded in guinea-pig, horse, dog, rabbit and human plasmas. The mechanism of the increase of fibrinolytic activity by jararhagin was studied further in guinea-pig plasma. Fibrin-zymographic studies indicated that jararhagin increased tissue-type plasminogen activator (tPA) activity by the dissociation of a complex of tPA with type 1 plasminogen activator inhibitor (PAI-1). c(2-Plasmin inhibitor (~2-PI) activity in the plasma was measured using a synthetic chromogenic substrate method after incubation with jararhagin. The c(2-PI activity in the plasma decreased in both time-dependent and dose-dependent manners. These in vitro results suggest that, in some animal plasmas, jararhagin increases plasma fibrinolytic activity by causing dissociation of the tPA/PAI-I complex and by the inactivation of c(2-PI. It is possible that this direct action of jararhagin on the enhancement of plasma fibrinolytic activity may contribute to the aetiology of systemic haemorrhage frequently observed in human victims of B. jararaca envenoming.
INTRODUCTION The main haemorrhagic factor from Bothrops jararaca venom has been purified and is termed HF2 (Mandelbaum et al., 1976), JFI (Maruyama et al., 1992) or jararhagin (Paine et al., 1992). Jararhagin is a 52,000 mol. wt metalloproteinase which possesses a Zn 2+dependent catalytic site, a disintegrin-like region and a cysteine-rich carboxy-terminal region (Paine et al., 1992). It possesses fibrinolytic activity, has an inhibitory effect on platelet aggregation in vitro and induces disruption of both vascular endothelial cells and 1605
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M. SUGIKI et al.
the v a s c u l a r b a s e m e n t m e m b r a n e in vivo ( K a m i g u t i et al., 1991, 1994). S u b c u t a n e o u s injection o f j a r a r h a g i n has also been shown to cause systemic h a e m o r r h a g e t o g e t h e r with t h r o m b o c y t o p e n i a in e x p e r i m e n t a l a n i m a l s ( K a m i g u t i e t al., 1991). These findings, t o g e t h e r with the o b s e r v a t i o n o f the ineffectiveness o f j a r a r h a g i n inhibition by p l a s m a inhibitors ( K a m i g u t i et al., 1994), suggest that this m e t a l l o p r o t e i n a s e p a r t i c i p a t e s in the d e v e l o p m e n t o f not only local, b u t also systemic h a e m o r r h a g e . The v e n o m also c o n t a i n s a t h r o m b i n - l i k e enzyme a n d o t h e r p r o c o a g u l a n t c o m p o n e n t s that m a y p r o v o k e d i s s e m i n a t e d i n t r a v a s c u l a r c o a g u l a t i o n ( N a h a s e t al., 1979), which is a l m o s t always associated with a c t i v a t i o n o f the fibrinolytic system (Collen, 1980). In fact, fibrinolysis in e n v e n o m e d victims has been r e p o r t e d to be due to intrinsic t h r o m b i n f o r m a t i o n and c o n s e q u e n t high titres o f cross-linked fibrin f r a g m e n t D, together with t h r o m b o c y t o p e n i a a n d / o r d i m i n i s h e d levels o f factor V a n d / o f factor V I I I ( M a r u y a m a e t al., 1990). In a d d i t i o n , e n h a n c e d fibrinolysis was evident from the low level o f ~2-plasmin i n h i b i t o r (~2-PI) f o u n d in these patients. Interference in regulation o f fibrinolysis results in a tendency to t h r o m b o s i s or bleeding. P l a s m i n o g e n a c t i v a t o r s (PAs) trigger the fibrinolytic process (Collen, 1980). T w o different types o f PAs, tissue t y p e - P A (tPA) a n d u r o k i n a s e t y p e - P A (uPA), have been identified in m a m m a l s . These are serine proteinases which c o n v e r t the p l a s m a inactive p r o e n z y m e , p l a s m i n o g e n , to the active enzyme, plasmin, which then causes the d e g r a d a t i o n o f its n a t u r a l substrate, fibrin. The fibrinolytic system is regulated by several m e c h a n i s m s such as the release o f P A from the vascular e n d o t h e l i u m , the fibrin-associated activation o f p l a s m i n o g e n by the P A and the inhibition o f P A and plasmin by c o m p l e x i n g with their specific physiological inhibitors [type 1 p l a s m i n o g e n a c t i v a t o r i n h i b i t o r ( P A l - l ) a n d ~2-PI, respectively] (Sprengers a n d Kluft, 1987). In the present in v i t r o study, we f o u n d a c o n s i d e r a b l e increase o f fibrinolytic activity in h u m a n a n d some o t h e r a n i m a l p l a s m a s treated with purified j a r a r h a g i n . This suggested that it affected some c o m p o n e n t s in the fibrinolytic system causing e n h a n c e m e n t o f fibrinolysis. W e p r o p o s e to investigate the m e c h a n i s m o f activation o f fibrinolysis in v i t r o to find out w h e t h e r j a r a r h a g i n influences the relationship within the t P A / P A I - 1 complex. MATERIALS AND METHODS Materials
Materials included phenyl-Superose (HR 5/5, 1 ml), Mono Q (HR 5/5, 1 ml), HiTrap Blue (1 nal) columns and Sephadex G-25, DEAE-Sephadex (Pharmacia Biotech Ltd, Uppsala, Sweden); bovine fibrinogen (75% w/w clonable; Miles Inc., Kankakee, IL, U.S.A.); amiloride (Sigma Chemical Co., Poole, U.K.); ethylenediaminetetraacetate (EDTA), e-aminocaproic acid (EACA) and polyethylene glycol (average mol. mass 20 kDa; Wako Pure Chemical Industries Ltd, Osaka, Japan); human urokinase (Green Cross Co., Osaka, Japan). Other reagents used were of analytical reagent grade. Puroqcation of.jararhagin Pooled lyophilized B.jararaca venom, obtained from about 100 snakes, was donated by the Instituto Butantan
(S~_o Paulo, Brazil). Jararhagin was purified from the whole venom using a phenyl-Superose hydrophobic interaction column and a Mono Q anion-exchange column on a Pharmacia FPLC system according to the method of Paine et al. (1992). The active fraction was then dialysed with 5 mM CaC12 at 4'C and applied to a HiTrap Blue affinity column equilibrated with 5 mM CaCI2 on the FPLC system to remove any contaminant clotting activity. This procedure was performed because it is known that the coagulant activity in B. jararaca venom is strongly absorbed by this type of column; 2 M NaCI was necessary to elute this activity (Maruyama et al., 1992). The column was washed with 20 ml of 5 mM CaCI2 and eluted with 20 mM Tris-HC1 buffer containing l0 mM CaCI2, pH 8.0, at a flow rate of I ml/min. The eluted fraction was concentrated using polyethylene glycol and dialysed with 20 mM Tris- HC1 buffer containing 2 mM CaCI2, pH 7.4 (Tris~CaCl 2buffer) at 4"C. Protein concentrations were measured using the BioRad Protein Assay (BioRad, Hemel Hempstead, U.K.) according to the manufacturer's instructions with bovine serum albumin as the standard. SDS-PAGE
Snake Venom and Fibrinolysis
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(Laemmli, 1970) of the purified protein was carried out using a 10% polyacrylamide gel under non-reducing conditions. Aliquots of the final product were stored at - 4 0 ° C .
Measurement of haemorrhagic activity Haemorrhagic activity was estimated according to the method of Theakston and Reid (1983). One-hundred microlitres of each sample was injected intradermally into the dorsal shaved skin of male C F W strain mice under light halothane/O 2 anaesthesia. The area of the haemorrhagic lesion was measured 24 hr after injection.
Preparation of plasma samples H u m a n blood was collected by puncture of the median cubital vein. Guinea-pigs (Dunkin Hartley), rats (Spragu~Dawley), mice ( T F W strain) and hamsters (Syrian) were anaesthetized by halothane/O 2 inhalation and blood was collected by cardiac puncture in 3.13% w/v trisodium citrate (9 parts blood: 1 part citrate) and then centrifuged at 3000 rpm for 15 min at 4~C. Citrated animal plasmas (dog, horse, goat, sheep, pig and bovine) were purchased from Sigma Chemical Co. (Poole, U.K.).
Measurement qf fibrinolytic activity' Fibrinolytic activity was estimated by the fibrin plate method of Astrup and Mfillertz (1952). The fibrin plates were prepared using 6 mg/ml of either plasminogen (plg)-rich or pig-poor fibrinogen which was clotted using 2 U/ml bovine thrombin (Mochida Pharmaceutical Co., Tokyo, Japan). Plg-poor fibrinogen was prepared by passing plg-rich fibrinogen through a lysine-Sepharose column (Matsuda et al., 1972).
Purification of guinea-pig and human plasminogen Guinea-pig and h u m a n plasminogens were prepared. The former was purified using the method of Deutsch and Mertz (1970). Ten millilitres of freshly prepared guinea-pig plasma was applied to a lysine-Sepharose column (1.4 x 7.5 cm, gel volume 2 ml) equilibrated with 0.1 M Tris HC1 buffer, pH 7.4, and the non-absorbing yellow fraction was collected (pig-poor plasma). After washing the column with 30 ml of equilibrating buffer, elution was performed with 0.1 M Tris HC1 buffer, pH 7.4, containing 0.2 M EACA. The E A C A was removed from the plasminogen by gel filtration on a Sephadex G-25 column (1.7 x 9.5 cm), equilibrated and eluted with 60 m M Tris HC1 buffer, pH 8.5, containing 65 m M NaC1. H u m a n plasminogen was purified from fresh frozen plasma by affinity chromatography on a lysine-Sepharose column followed by a m m o n i u m sulphate precipitation and anion-exchange chromatography on a DEAE-Sephadex column according to the method of Robbins and Summaria (1976).
Fibrin -zymography Fibrin-zymography was carried out according to the method of Granelli-Piperno and Reich (1978). After S D S - P A G E (10% polyacrylamide gel, non-reducing conditions) of sample mixtures prepared as above, the gels were washed twice for 1 hr each with 500 ml of 10 m M phosphate buffer, pH 7.4, containing 0.15 M NaCI and 0.2% v/v Triton X-100 at room temperature with gentle agitation. Following a brief wash with distilled water, the gels were overlaid on fibrin-agar plates. Each plate was prepared by pouring 8.5 ml of 1.2% w/v bovine fibrinogen in 0.1 M phosphate buffer, pH 7.4, 8.5 ml of 1.2% w/v agar in water and 1 ml of 20 U/ml thrombin into a plastic plate (13.5 x 9.5 cm) at 42°C. After incubation of the plates in a moist chamber at 37°C, fibrinolytic activities of enzymes were visualized as lytic bands against a black background. The mol. wts of the bands were estimated by comparison with the mol. wt standards in gels stained with Coomassie Blue R-250.
Measurement of ~e-PI activity Twenty microlitres of the guinea-pig plg-poor plasma was incubated at 37°C with 20#1 of various concentrations of jararhagin for times ranging between 0 and 30 min. E D T A (10/~1 of a 50 m M solution) was then added to the mixture which was allowed to stand for 10 min at room temperature to inactivate jararhagin. To each test plasma sample, 370 #1 of saline was added. The antiplasmin activity of ~2-PI in the samples was measured using the synthetic chromogenic substrate provided in the antiplasmin assay kit (Stachrom Antiplasrain, Diagnostica Stago, Asnieres-sur-Seine, France) according to the manufacturer's instructions.
SDS-PAGE of ot2-Pl after incubation with jararhagin Purified ~z-PI from h u m a n plasma (Celsus Laboratories, Cincinnati, OH, U.S.A.) was incubated at 37°C with jararhagin at a molar ratio of 20 : 1 in 50 m M Tris-HCl buffer, pH 7.4, containing 0.1 M NaC1 and 5 m M CaCI 2 for periods ranging between 0 and 80 min. Reactions were stopped by adding the same volume of sample buffer
M. SUGIKI et al.
1608
(125 mM Tris-HC1 buffer, pH 6.8, containing 70 mM SDS, 20% v/v glycerol, 0.012% w/v bromophenol blue and 10% v/v 2-mercaptoethanol) and boiling the mixtures for 3 min. Samples containing 1.5 ttg of ~2-PI were then immediately analysed by SDS PAGE (Laemmli, 1970). Proteins were stained with Coomassie Blue R-250 and prestained mol. wt markers (Gibco BRL, Gaithersburg, MD, U.S.A.) were used to estimate the electrophoretic mobilities of the 72-PI proteins.
RESU LTS
Purification o f jararhagin T h e p u r i t y o f the i s o l a t e d e n z y m e was c o n f i r m e d by a n a l y t i c a l S D S P A G E . T h e i s o l a t e d j a r a r h a g i n r e v e a l e d a single p r o t e i n b a n d w i t h a tool. wt o f 52,000 (Fig. 1). It p o s s e s s e d h a e m o r r h a g i c a c t i v i t y a n d n o r e s i d u a l c o a g u l a n t a c t i v i t y a f t e r m i x i n g w i t h the g u i n e a - p i g o r o t h e r p l a s m a s o r f i b r i n o g e n at a final p r o t e i n c o n c e n t r a t i o n o f up to 1 0 0 / z g / m l o f plasma.
kDa --205 --116 "97 m
66
---
45
--
29
-,-
Dye
front
Fig. 1. SDS PAGE of jararhagin. Purified jararhagin (I.3 pg protein) was analyse d by SDS PAGE. Electrophoresis was performed at 10 20 mA/gel for 150 min and then proteins were stained with Coomassie Blue R-250. Molecular weight markers used were myosin, fl-galactosidase, phosphorylase B, bovine serum albumin, ovalbumin and carbonic anhydrase (Sigma Chemical Co., Poole, U.K.).
Snake Venom and Fibrinolysis
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Table I. Fibrinolytic activity ofjararhagin in plasminogenrich fibrin plates Plasma source Fibrinolytic activity (mm2) Human 68.3 _+5.1 Guinea-pig 174.3 __+5.5 Horse 163.1 ___5.1 Dog 161.3 _+ 12.4 Rabbit 103.7 _+ 11.6 Rat 0 Mouse 0 Hamster 0 Goat 0 Sheep 0 Pig 0 Bovine 0 Control 48.0 + 1.7 Twenty microlitres of plasma was mixed with 20 ~1 of hararhagin (160/~g/ml) and 30/~1of the mixture was applied on the fibrin plate. After 18 hr at 37°C, the lytic area was measured. Fibrinolytic activity was expressed as the area of lysis (mm2) by multiplying two perpendicular diameters of the lysed area. Instead of plasma, 150 mM NaC1 containing 2 mM CaC12was mixed with jararhagin as the control. Data show the mean _ S.D. (n = 3).
Fibrinolytic activity of jararhagin in different animal plasmas on plasminogen-rich fibrin plates The effect o f jararhagin on the fibrinolytic system was investigated by measuring its fibrinolytic activity after mixing with various animal plasmas (Table 1). N o fibrinolytic activity was detected in the different plasmas mixed with T r i s ~ a C l 2 buffer alone. However, after mixing the plasmas with jararhagin, strong fibrinolytic activity was recorded in the plasmas o f guinea-pig, horse, dog, rabbit and h u m a n (Table 1, Fig. 2). The fibrinolytic activity in these plasmas containing jararhagin was m o r e p r o n o u n c e d than the original fibrinolytic activity o f j a r a r h a g i n alone (Table 1). N o activity was detected in rat, mouse, hamster, goat, sheep, pig and bovine plasmas even after mixing with jararhagin (Table 1, Fig. 2).
Effect of jararhag& on plasminogen activation I n c u b a t i o n o f purified guinea-pig or h u m a n plasminogen gave no evidence o f plasmin formation as shown by the absence o f fibrinolytic activity on the plg-poor fibrin plate (Fig. 3). Activation o f both plasminogens by urokinase (controls) was observed simultaneously.
Fibrinolytic activity in normal and plasminogen-poor guinea-pig plasma after incubation with jararhagin As jararhagin caused the highest increase in fibrinolytic activity in guinea-pig plasma, this plasma type was selected in order to clarify the mechanism o f jararhagin-induced increase in fibrinolysis. This rapid increase in activity on both plg-rich and plg-poor fibrin plates is shown in Fig. 4. The activity 30 min after the incubation was 89 ___ 18 m m z on the plg-rich fibrin plates and 7 2 _ 1 0 m m 2 on the pig-poor fibrin plates (Fig. 4A). Both activities gradually increased until 120 min incubation. The activity on plg-rich plates was
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M. SUGIKI et al.
increased to a greater extent than that on the pig-poor fibrin plates. It is known that fibrin in the plg-rich plate is lysed by PAs, plasmin and plasmin-like enzymes, while that of the pig-poor plate is lysed by plasmin and plasmin-like enzymes (Etoh et al., 1992). Therefore, the difference in the activity of these enzymes on each type of fibrin plate represents the PA activity in the samples. Figure 4B shows the fibinolytic activity in the pig-poor plasma after incubation with jararhagin. The difference in the activity between pig-rich and pig-poor fibrin plates was similar to that in the normal plasma (Fig. 4A). However, no activity was detected on the pig-poor plates, indicating that plasminogen was essential for the enhancement of fibrinolysis by jararhagin. These data therefore indicate that jararhagin increases both plasma PA activity and also plasmin activity in vitro. Fibrin-zymography o [ p l a s m a after incubation with .jararhagin Figure 5 shows plg-rich fibrin-zymographic patterns of the fibrinolytic enzyme in guinea-pig plasma at different times of incubation with jararhagin. One principal lyric band of tool. wt 105,000 and two closely located minor lytic bands of 56,000 and 52,000, respectively, were observed in the plasma without incubation (lane 1). After treatment with jararhagin, the 105,000 tool. wt band gradually disappeared, while the intensities of the 56,000 and 52,000 tool. wt bands increased (lanes 2 4). A new lytic band of tool. wt 47,000
Fig. 2. Fibrinolytic activity of jararhagin in plasminogen-rich fibrin plates. Jararhagin incubated in: 1, human plasma; 2, guinea-pig plasma; 3, horse plasma; 4, rabbit plasma: 5, dog plasma; 6, rat plasma; 7, 150ram Nacl containing 2mM CaC12alone as the control. The same mixtures were used as described in Table 1.
Snake Venom and Fibrinolysis
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Fig. 3. Effect of jararhagin on plasminogen. Fifty microlitres of purified human plasminogen (300#g/ml) or guinea-pig plasminogen (200 #g/ml) was incubated at 37C with 50 #1 ofjararhagin (160/~g/ml) in 20 mM Tris-HCl buffer, pH 7.4, containing 5 mM CaC12. After 30 min, 25/11 of 50 mM EDTA was added to the mixture and left at room temperature for 10 min to inactivate jararhagin. The sample (30 #1) was applied to the pig-poor fibrin plate to determine the level of plasmin formation. For controls, plasminogen of both origins was incubated with urokinase (UK, l0 U/ml) instead of jararhagin. 1, Human plasminogen + jararhagin; 2, human plasminogen + UK; 3, guinea-pig plasminogen + jararhagin; 4, guinea-pig plasminogen + UK; 5, buffer + UK.
a p p e a r e d after 60 min i n c u b a t i o n (lane 4). A f t e r i n c u b a t i o n o f the p l a s m a with Tris CaCI2 buffer a l o n e (control), the 105,000 lytic b a n d still r e m a i n e d a n d b o t h 56,000 a n d 52,000 mol. wt lyric b a n d s d i s a p p e a r e d (lanes 5 7). N o lyric b a n d was detected using p l g - p o o r f i b r i n - a g a r plates, i n d i c a t i n g t h a t these lytic b a n d s were due to PA(s). M o r e o v e r , all lyric b a n d s did n o t d i m i n i s h on z y m o g r a p h y using pig-rich f i b r i n - a g a r plates c o n t a i n i n g 1 m M a m i l o r i d e , a p o t e n t i n h i b i t o r o f u P A (Vassalli a n d Belin, 1987). This i n d i c a t e d that all lytic b a n d s were due to t P A ( d a t a n o t shown).
Inactivation of %-plasmin inhibitor by jararhagin It was t h o u g h t that a n y free p l a s m i n is r a p i d l y b o u n d a n d i n a c t i v a t e d by its specific inhibitor, ~2-PI, in p l a s m a ( M o r o i a n d A o k i , 1976). H o w e v e r , we f o u n d strong p l a s m i n activity in g u i n e a - p i g p l a s m a i n c u b a t e d with j a r a r h a g i n (Fig. 4A), suggesting that j a r a r h a g i n m i g h t have s o m e effects on ~2-PI in the p l a s m a . W e used the p l g - p o o r p l a s m a for e s t i m a t i n g the direct effect o f j a r a r h a g i n on ~2-PI to a v o i d the interference o f p l a s m i n TOX 33/12
D
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M. SUG1KI et al.
which could be formed in normal plasma in the assay system for 0~2-PI activity. In preliminary experiments, it was confirmed that there was no significant difference in c~2-PI activity between normal and pig-poor plasma, and no plasmin activity was detected in the plg-poor plasma 30 min after incubation with jararhagin, using this assay system. Figure 6 shows c~2-PI activity in the plasma at different times of incubation with different concentrations o f j a r a r h a g i n (0-80/~g/ml). The ~2-PI activity in the plasma was decreased both time and dose dependently. Proteolysis of purified human ~2-PI with jararhagin was visualized using S D S - P A G E (Fig. 7). The principal band ofmol, wt 67,000 of ~2-PI (lane 2) was degraded by jararhagin in a time-dependent manner, yielding two major degradation products of mol. wt 59,000 and 50,000 (lanes 3-7). DISCUSSION Here we have demonstrated that a venom haemorrhagic factor, jararhagin, increases in vitro fibrinolysis directly in both human and in some other animal plasmas. The
enhanced fibrinolysis in plasma was due to the dissociation of the tPA/PAI-1 complex by jararhagin. Furthermore, jararhagin rapidly inactivates c~2-PI. Jararhagin increases the in vitro fibrinolytic activity in plasma and the extent of this increase varies depending on the mammalian species involved (Table 1). Since jararhagin is proteolytically active in plasma, these observed differences could reflect different jararhagin affinity for the substrate in the various plasma species. Another possibility may be the varying inhibitory capacity of plasma proteinase inhibitors against jararhagin in different species.
B
A e~
E
100-
,~ 100°,,°~,,Jl
,-°'°
.~ ¢.)
.2 ~
e., .:,
50-
2
50-
+
0
0 30 60 120 3'0 60 1½0 Incubation time (min) Incubation time (rain) Fig. 4. Fibrinolytic activity in normal guinea-pig plasma (A) and in pig-poor plasma (B) after incubation with jararhagin. Forty microlitres of plasma was incubated at 37"C with 40 #1 of jararhagin (160 pg/ml). After various times, 20 pl of 50 mM EDTA was added to stop the reaction. The fibrinolytic activity in the plasma was measured using the pig-rich ( O--) and pig-poor (--Q--) fibrin plates. Each point represents the mean of three experiments. Horizontal bars are standard deviations of the means. 0
Snake Venom and Fibrinolysis
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kDa
1
2
3
4
5
6
--
105
---
56
--
52
--
47
7
Fig. 5. Fibrin-zymography of plasma after incubation with jararhagin. Guinea-pig plasma (10/d) was incubated at 37'C with 10/ll of jararhagin (1601Lg/ml) or Tris~ CaC12 buffer. After different times, 5/11 of 50 mM EDTA was added. After 10 min at room temperature, 25/d of sample buffer (125mM Tris HC1 buffer containing 70mM SDS. 20% glycerol and 0.012% bromophenoi blue, pH 6.8) was added and 15/~1 of the sample was applied. SDS PAGE and fibrin-zymography were carried out as described in Materials and Methods. Plasma was incubated with jararhagin for 0 rain (lane 1), 15 min (lane 2), 30 min (lane 3), 60 min (lane 4) or with buffer for 0 min (lane 5), 30 min (lane 6), 60 min (lane 7).
By using z y m o g r a p h y o f plasma (Fig. 5) it is shown that jararhagin decreases the a m o u n t o f the principal 105,000 mol. wt t P A / P A I - I complex. In h u m a n plasma this complex appears as a band o f 110,000 which is a SDS-stable complex o f tPA and PAl-1 by z y m o g r a p h y ( K r u i t h o f et al., 1984; Booth et al., 1987). This lytic band gradually disappeared during incubation o f guinea-pig plasma with jararhagin, whereas the activity o f 56,000 and 52,000 lyric bands increased and a new lytic band o f 47,000 appeared after 60 min incubation (lanes 1-4). Z y m o g r a p h i c studies using plg-poor fibrin-agar plates or plg-rich plates containing a u P A inhibitor indicated that these lyric bands were probably due to tPA. P A I - I is structurally related to the serine protease inhibitor (serpin) superfamily (Pannekoek et al., 1986) and reacts with tPA followed by the formation o f a stable complex with a higher tool. wt (Sprengers and Kluft, 1987). In addition, guinea-pig sinusoidal endothelial cells (Rieder et al., 1993) and megakaryocytes (Konkle et al., 1993) p r o d u c e PAI-1 whose tool. wts are 43,000 and 45,000, respectively. These findings therefore confirm that the lyric band o f mol. wt 105,000 in our experiments is a t P A / P A I - l complex (Fig. 5). I n c u b a t i o n o f the plasma with buffer alone produced no change in the lytic band corresponding to the t P A / P A I - I complex and rapid disappearance o f the lytic bands o f free tPAs (Fig. 5, lanes 5 7) due to instability o f the tPA at 3 T C . These control data therefore indicate that jararhagin actually interacts with this complex decreasing tPA activity in the complex by releasing free tPA from the complex as observed in lanes 2 - 4 . The interaction o f jararhagin with this complex m a y result in a transition from the complexed state to the free forms o f t P A which is also p r o b a b l y modified by jararhagin.
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M. SUGIKI et al.
Recently, it has been reported that dissociation o f the tPA/PAI-1 complex, resulting in the appearance o f biologically active free forms o f tPA and PAI-1, occurs with SDS treatment (Gaussem et al., 1993) and with activated protein C (Grailhe et al., 1993), suggesting that this could represent a potential source o f fibrinolytic activity. Indeed, our results show that some free tPA was present in guinea-pig plasma, but substantial a m o u n t s appeared after jararhagin treatment. This suggests that jararhagin releases tPA from the complex and that this free tPA is responsible for the increase in plasma fibrinolytic activity. The precise mechanism o f dissociation o f the complex still remains to be established. Dissociation o f other enzyme/serpin complexes have already been shown to occur [e.g. thrombin/ antithrombin lII (AT III) and plasmin/~2-PI] (Griffith and Lundblad, 1981; Y a n et al., 1993). The cq-PI activity in jararhagin-treated plasma has been shown to decrease in time- and dose-dependent manners (Fig. 6). Moreover, purified h u m a n ez-PI was also degraded by jararhagin time-dependently (Fig. 7). cq-PI is a m e m b e r o f serpin superfamily and rapidly inactivates the proteolytic activity o f plasmin (Holmes et al., 1987). It has been reported that the only plasma inhibitor which reacts with jararhagin is cq-macroglobulin, but the inhibition is not complete even with a large molar excess o f this inhibitor (Kamiguti et al., 1994). In addition, progressive degradation o f c~2-PI results in a gradual decrease in its capacity to react with plasmin (Clemmensen et al., 1981). The increase o f plasmin activity in plasma caused by jararhagin (Fig. 4A) suggests that plasma cq-PI m a y be inactivated 100
"
.~.
.~, t..
50
=. E i ¢-1
0 0
1;
2'0
3'0
I n c u b a t i o n time (min) Fig. 6. a2-PI activity in plasma after incubation with jararhagin. Guinea-pig pig-poor plasma was incubated with various concentrations of jararhagin as described in Materials and Methods. Final concentrations of jararhagin were - - x - - , 0 p g/ml; IN--, 10/~g/ml; I - - , 20 #g/ml; --C) ,40 #g/ml; - - O - - , 80 #g/ml. The residual a2-PI activity in the plasma is expressed as percentage activity of the standards. Standards (100% activity) were prepared using the same procedure described in Materials and Methods, except that the plasma was mixed with TrisqZaCl2 buffer instead of iararhagin and was not incubated. A 50:50 dilution of this standard with saline was designated as 50% standard activity and saline alone was designated as 0% activity.
Snake Venom and Fibrinolysis
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kDa m67 m59 ---50
1
2
3
45
67
8
Fig. 7. Degradation of ~2-PI by jararhagin. SDS-PAGE (10% polyacrylamide gel, reducing conditions) of purified human cq-PI (7.5/IM) treated with jararhagin (0.37/~M) at 3TC for different lengths of time (lane 2, 0 min; lane 3, 5 min; lane 4, 10 min; lane 5, 20 min; lane 6, 40 min; lane 7, 80 min). Under the conditions applied, jararhagin (0.37t~M) was not detected by Coomassie Blue stain (lane l). c~2-PI (7.5/~M) was incubated at 3TC for 80 min without jararhagin for the control (lane 8).
by degradation of this inhibitor by jararhagin. Such degradation of other serpins by snake venom enzymes has been reported in other studies involving cq-proteinase inhibitor (Kress et al., 1979) and AT III (Janssen et al., 1992). Jararhagin directly increases the in vitro fibrinolytic activity in plasma by decreasing the activities of PAI-1 and cq-PI, both of which are key physiological inhibitors in the regulation of fibrinolysis. This increase in fibrinolysis may be responsible for the disturbance of the haemostatic mechanism observed in some patients following electroshock or complicated labour (Bennett et al., 1990). Excess plasmin formation would be expected to result in premature lysis of haemostatic plugs, thus causing abnormal haemorrhage. In fact, congenital deficiency of either PAI-1 (William et al., 1992) or ch-PI (Aoki et al., 1979) has been reported to show lifelong severe haemorrhagic diathesis. Therefore, with the important reservation that this is an in vitro study based mainly on results with guinea-pig plasma, it is possible that this direct action of jararhagin on the enhancement of fibrinolysis may contribute to the systemic haemorrhage frequently observed in B. j a r a r a c a envenoming in humans.
Acknowledgements--The authors thank the team of the Venom Research Unit, Liverpool School of Tropical
Medicine, for its excellent technical cooperation.
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Bennett, B., Croll, A., Ferguson, K. and Booth, N. A. (1990) Complexing of tissue plasminogen activator with PAl-l, ~2-macroglobulin, and Cl-inhibitors: studies in patients with defibrination and a fibrinolytic state after electroshock or complicated labor. Blood 75, 671-676. Booth, N. A., Walker, E., Maughan, R. and Bennett, B. (1987) Plasminogen activator in normal subjects after exercise and venous occlusion: t-PA circulates as complexes with Cl-inhibitor and PAl-1. Blood69, 1600 1604. Clemmensem I., Thorsen, S., Mfillertz, S. and Petersen, L. C. (1981) Properties of three different molecular forms of the ~2 plasmin inhibitor. Eur. J. Biochem. 120, 105 112. Collen, D. (1980) On the regulation and control of fibrinolysis. Thromb. Haemost. 43, 77 89. Deutsch, D. G. and Mertz, E. T. (1970) Plasminogen: purification from human plasma by affinity chromatography. Science 170, 1095 1096. Etoh, Y., Sumi, H., Tsushima, H., Maruyama, M. and Mihara, H. (1992) Fibrinolytic enzymes in ascites during experimental acute pancreatitis in rats. Int. J. Pancreatol. 12, 127 137. Gaussem, P., Grailhe, P. and Angl~s-Cano, E. (1993) Sodium dodecyl sulfate-induced dissociation of complexes between human tissue plasminogen activator and its specific inhibitor. J. biol. Chem. 268, 12,150 12,155. Grailhe, P., Bezeaud, A., Gaussem, P., Guillin, M. C. and Angl6s-Cano, E. (1993) Neutralization of plasminogen activator inhibitor-I (PAI-1) by activated protein C is species-dependent. Fibrinolysis 7, 123 133. Granelli-Piperno, A. and Reich, E. (1978) A study of proteases and protease inhibitor complexes in biological fluids. J. e,cp. Med. 148, 223 234. Griffith, M. J. and Lundblad, R. L. (1981) Dissociation of antithrombin I l l thrombin complex. Formation of active and inactive antithrombin III. Biochemi~'try 20, 105 I10. Holmes, W. E., Nelles, L., Lijnen, H. R. and Collen, D. (1987) Primary structure of human :q-antiplasmin, a serine protease inhibitor (serpin). J. biol. Chem. 262, 1659 1664. Janssen, M., Meier, J. and Freyvogel, T. A. (1992) Purification and characterization of an antithrombin 11I inactivating enzyme from the venom of the African night adder (Causus rhombeatus). Toxicon 30, 985 999. Kamiguti, A. S., Theakston, R. D. G., Desmond, H. and Hutton, R. A. (1991) Systemic haemorrhage in rats induced by a haemorrhagic fraction from Bothropsjararaca venom. Toxicon 29, 1097 1105. Kamiguti, A. S., Desmond, H. P., Theakston, R. D. G., Hay, C. R. M. and Zuzel, M. (1994) Ineffectiveness of the inhibition of the main haemorrhagic metalloproteinase from Bothropsjararaca venom by its only plasma inhibitor, :~2-macroglobulin. Biochim. biophys. Acta 1200, 307 314. Konkle, B. A., Schick, P. K., He, X., Liu, R. J. and Mazur, E. M. (1993) Plasminogen activator inhibitor-I mRNA is expressed in platelets and megakaryocytes and the megakaryoblastic cell line CHRF-288. Arteriosclcr. Thromb. 13, 669 674. Kress, L. F., Kurecki, T., Chan, S. K. and Laskowski, M., Sr (1979) Characterization of the inactive fragment resulting from limited proteolysis of human alpha~-proteinase inhibitor by Crotalus adamanteus proteinase II. J. biol. Chem. 254, 5317 5320. Kruithof, E. K. O., Tran-Thang, C., Ransijn, A. and Bachmann, F. (1984) Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 64, 907 913. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685. Mandelbaum, F. R., Reichl, A. P. and Assakura, M. T. (1976) Some physical and biochemical characteristics of HF2, one of the haemorrhagic factors in the venom of Bothrops jararaca. In: Animal, Plant, and Microbial Toxins, Vol. 1, pp. i11 121 (Ohsaka, A., Hayashi, K. and Sawai, Y., Eds). London: Plenum Press. Maruyama, M., Kamiguti, A. S., Cardoso, J. L. C., Sano-Martins, I. S., Chudzinski, A. M., Santoro, M. L., Morena, P., Tomy, S. C., Antonio, L. C., Mihara, H. and Kelen, E. M. A. (1990) Studies on blood coagulation and fibrinolysis in patients bitten by Bothrops jararaca (jararaca). Thromb. Haemost. 63, 449 453. Maruyama, M., Sugiki, M., Yoshida, E., Mihara, H. and Nakajima, N. (1992) Purification and characterization of two fibrinolytic enzymes from Bothrops jararaca (jararaca) venom. Toxicon 30, 853 864. Matsuda, M., Iwanaga, S. and Nakamura, S. (1972) A simple, large scale method for preparation of plasminogen-free fibrinogen. Tbromb. Res. l, 619-630. Moroi, M. and Aoki, N. (1976) Isolation and characterization of :q-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis. J. biol. Chem. 251, 5956-5965. Nahas, L., Kamiguti, A. S. and Barros, M. A. R. (1979) Thrombin-like and factor X-activator components of Bothrops snake venoms. Thromh. Haemost. 41, 314 328. Paine. M. J. 1., Desmond, H. P., Theakston, R. D. G. and Crampton, J. M. (1992) Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops ]araraca venom. J. biol. Chem. 267, 22,869 22,876. Pannekoek, H., Veerman, H., Lambers, H., Diegaarde, P., Verweij, C. L., van Zonneveld, A. J. and van Mourik, J. A. (1986) Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family. E M B O J. 5, 2539 2544. Rieder, H., Armbrust, T., Meyer zum Bfishenfelde, K. H. and Ramadori, G. (1993) Contribution of sinusoidal endothelial liver cells to liver fibrosis: Expression of transforming growth factor-fl I receptors and modulation of plasmin-generation enzymes by transforming growth factor-/:t I. Hepatology 18, 937 944. Robbins, K. C. and Summaria, L. (1976) Plasminogen and plasmin. Meth. Enzymol. 45, 257 273. Sprengers, E. D. and Kluft, C. (1987) Plasminogen activator inhibitors. Blood 69, 381 387.
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Theakston, R. D. G. and Reid, H. A. (1983) Development of simple standard assay procedures for the characterization of snake venoms. Bull. WHO 61, 949-956. Vassalli, J. D. and Belin, D. (1987) Amiloride selectively inhibits the urokinase-type plasminogen activator. FEBS Lett. 214, 187 191. William, P. F., Shapiro, A. D., Shih, J. L., Schleef, R. R. and Ginsburg, D. (1992) Brief report: Complete deficiency of plasminogen-activator inhibitor type 1 due to a frame-shift mutation. N. Engl. J. Med. 327, 1729-1733. Yah, D., Urano, T., Takada, Y. and Takada, A. (1993) Dissociation of cc2-plasmin-inhibitor-plasmin complex and regeneration of plasmin activity by SDS treatment. Thromb. Res. 69, 491 499.