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Novel recombinant fibrinogenase of Agkistrodon acutus venom protects against LPS-induced DIC
Thrombosis Research 123 (2009) 919–924
Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Novel recombinant fibrinogenase of Agkistrodon acutus venom protects against LPS-induced DIC Rongrong Wang 1, Jinlian Cai 1, Yijun Huang, Dong Xu, Hanfei Sang, Guangmei Yan ⁎ Department of Pharmacology, Zhong-shan Medical College, Sun Yat-sen University, Guangzhou, 510080, P.R. China
a r t i c l e
i n f o
Article history: Received 8 August 2008 Received in revised form 8 October 2008 Accepted 29 October 2008 Available online 13 December 2008 Keywords: Disseminated intravascular coagulation Recombinant Fibrinogenase Microthrombi Lipopolysaccharide
a b s t r a c t Introduction: Disseminated intravascular coagulation (DIC) is an acquired syndrome characterized by the widespread activation of coagulation. This leads to failure of multiple organs in the body and finally death. Because there is no effective therapy for DIC, the clinical prognosis is poor and the mortality is high. Materials and Methods: The animals were intravenously injected with Lipopolysaccharide (LPS) for 6 hours and simultaneously infected three doses of recombinant fibrinogenase II (rFII) for 2 hour. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelets count, fibrinogen and fibrin-fibrinogen degradation products (FDP) were determined. The plasma levels of alanine aminotransferase (ALT), creatinine (Cr) and tumor necrosis factor-α (TNF-α) were detected. Liver and kidney samples were stained with hematoxylin-eosin and kidney sections were stained with phosphotungstic acid-hematoxylin. Results: We observed that rFII increased survival rate in LPS-induced DIC rabbits as well as heparin did. Administration of rFII as well as heparin attenuated the increased plasma levels of APTT, PT and FDP and the decreased plasma level of fibrinogen at 6 h. rFII reduced hepatic and renal damages and decreased the levels of ALT and Cr as well as heparin did. rFII also significantly reduced the increased plasma levels of TNF-α. rFII significantly reduced the kidney fibrin deposits with respect to LPS treated animals. Conclusions: Our findings suggest that rFII from Agkistrodon acutus venom could have protective effect on DIC via reducing liver and renal damages and direct degradation of microthrombi. © 2008 Elsevier Ltd. All rights reserved.
Introduction Disseminated intravascular coagulation (DIC) is a life threatening syndrome arising from various causes including disseminated sepsis. It is generally associated with an adverse outcome [1,2]. Although many efforts have been made to develop treatment of DIC, there is no established therapy [3–5]. The basic pathological mechanism of DIC includes the spread of microvascular thrombosis, which prevents adequate blood supply to organs and leads to multiple organ failure and death [1]. Clinical trials aiming at an interruption of “latent coagulation” in sepsis by administration of coagulation inhibitors, such as antithrombin, have so far failed to demonstrate a statistically significant benefit on survival. Heparin blocks endotoxin initiated clotting but is ineffective
Abbreviations: rFII, recombinant fibrinogenase II; DIC, Disseminated intravascular coagulation; LPS, Lipopolysaccharide; ALT, alanine aminotransferase; Cr, creatinine; APTT, activated partial thromboplastin time; PT, prothrombin time; FDP, fibrinfibrinogen degradation products. ⁎ Corresponding author. Department of Pharmacology, Zhong-Shan Medical College, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou, Guangdong, 510089, P.R. China. Tel.: +86 20 87333258; fax: +86 20 87333762. E-mail address: [email protected] (G. Yan). 1 These authors contributed equally to this work. 0049-3848/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.10.017
in preventing organ failure [5]. Likewise, factor Xa blocked with a small molecule in the active site effectively inhibited coagulation induced by E coli in baboons but did not protect from organ failure or prevent death [6]. It is possible that failure of these compounds to protect against DIC may not have prevented the decrease in the blood supply to organs. Furthermore, the safety of anticoagulants in DIC has not been established. Previous use of some proteinases purified from various snake venoms using biochemical methods showed effects on the clinical course of thrombotic disorders [7]. We isolated a novel snake venom fibrinogenase, FIIa, from the Agkistrodon acutus snake [8]. It has the ability to directly degrade fibrin in vitro and dissolves thrombi effectively in vivo [9,10]. In the present study, we cloned the gene, expressed, purified and characterized this novel recombinant protease. We found that this recombinant fibrinogenaseII (rFII) protected against LPS-induced DIC in rabbits. Materials and methods Cloning and preparation of rFII The recombinant fibrinogenase II (rFII) from Agkistrodon acutus venom, was prepared according to the method described by Wang et al [11].
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Animals and drug treatments All animal experiments were conducted in accordance with the National Guide for the Care and Use of Laboratory Animals and were approved by Sun Yat-sen University Animal Care and Use Committee (Guangzhou, China). DIC was induced by infusion with 0.1 mg/kg/h LPS for 6 h in 60 ml (10 ml/h) intravenously through the marginal ear vein. Treatments were started simultaneously with LPS infusion through the contralateral marginal ear vein. Six different groups were established: 1.LPS group: 0.1 mg/kg/h LPS for 6 h; 2. LPS and low dose of rFII group: 0.1 mg/kg/h LPS for 6 h and 0.05 mg/kg/h rFII for 2 h; 3. LPS and high dose of rFII group: 0.1 mg/kg/h LPS for 6 h and 0.1 mg/kg/h rFII for 2 h; 4. LPS and heparin group: 0.1 mg/kg/h LPS for 6 h and 30 IU/kg/h heparin for 6 h; 5. rFII group: 0.1 mg/kg/h rFII for 6 h only; 6. Normal group: saline was infused. Kidneys and livers were removed for subsequent histological studies 24 h after the start of the experiments. Biochemical Analysis Blood samples were taken immediately before LPS infusion and 2 h and 6 h after the start of the infusion. A automatic analyzer (Sysmex SE-9500, Sysmex CA 1500, Japan) was used to determine APTT, PT, platelets count, Fibrinogen and fibrin-fibrinogen degradation products (FDP). 7170A automatic analyzer (HITACHI, Japan) detected the plasma levels of alanine aminotransferase (ALT) and creatinine (Cr).
Table 1 Plasma levels of activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, fibrin degradation product (FDP) and platelet count for normal, LPS-treated, rFII-treated and heparin-treated rabbits. Groups Normal
N
Time (h) PT (s)
10 2 6 LPS 10 2 6 LPS + 10 2 0.025rFII 6 LPS + 10 2 0.05rFII 6 LPS + 10 2 0.1rFII 6 LPS + 10 2 Heparin 6
h h h h h h h h h h h h
7.2 ± 0.8 7.2 ± 0.7 11.0 ± 3.4# 14.9 ± 4.7# 8.5 ± 1.1 10.6 ± 1.8⁎ 7.5 ± 0.5⁎ 9.7 ± 1.3⁎ 7.6 ± 0.8⁎ 9.9 ± 1.3⁎ 7.7 ± 0.6⁎ 8.4 ± 3.5⁎
APTT (s)
Fibrinogen Platelets (g/l) (×109/l)
FDP (μg/ml)
18.2 ± 1.0 18.4 ± 0.7 21.1 ± 5.4# 28.8 ± 5.5# 21.5 ± 3.1 26.3 ± 2.0 21.1 ± 4.5 25.5 ± 7.7⁎ 19.1 ± 1.9 22.2 ± 1.9⁎ 18.6 ± 4.2⁎ 25.6 ± 4.3⁎
3.0 ± 0.8 3.2 ± 0.9 2.7 ± 0.8 1.8 ± 0.2# 2.9 ± 0.6 2.0 ± 0.5 3.1 ± 0.8⁎ 2.4 ± 0.7⁎ 2.2 ± 0.6 2.5 ± 0.4⁎ 3.2 ± 0.6⁎ 2.5 ± 0.9⁎
b 0.05 b 0.05 58.9 ± 16.5# 87.3 ± 28.9# 28.1 ± 8.9⁎ 22.3 ± 7.5⁎ 26.2 ± 5.5⁎ 18.3 ± 6.1⁎ 23.5 ± 3.1⁎ 15.1 ± 4,5⁎ 35.3 ± 6.0⁎ 30.1 ± 5.8⁎
456 ± 15 488 ± 58 304 ± 48# 185 ± 25# 370 ± 53 198 ± 58 345 ± 33 284 ± 14 376 ± 57 257 ± 35 434 ± 25 315 ± 18
#
P b 0.05 as compared to normal group. ⁎P b 0.05 as compared to LPS treatment group. Data are presented as the mean ± S.D. LPS: infusion of 0.1 mg/kg/h LPS for 6 h. LPS+ 0.025rFII: simultaneous infusion of 0.1 mg/kg/h LPS and 0.025 mg/kg rFII. LPS + 0.05r FII: simultaneous infusion of 0.1 mg/kg/h LPS and 0.05 mg /kg rFII. LPS + 0.1rFII: simultaneous infusion of 0.1 mg/kg/h LPS and 0.1 mg/kg rFII. LPS+ heparin: simultaneous infusion of 0.1 mg/kg/h LPS and 30 IU/kg/h heparin.
data are presented as the mean ± S.D. unless otherwise noted. Results of ALT and Cr at 2 h and 6 h were converted to percentages assuming a value of 100% for basal data. Survival curves of LPSinduced DIC were analyzed by the Kaplan-Meyer log-rank test. Results
Measurement of TNF-α concentration The protective effect of rFII on DIC induced by LPS The plasma of rabbits were collected in tube and stored at –20 °C until assayed. The concentrations of TNF-α in animal serum were determined using ELISA KIT (RapidBio Lab. Calabasas, USA). Histopathological Analysis Liver and kidney samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin-eosin. To determine fibrin deposition in microvessels, kidney sections were stained with phosphotungstic acid-hematoxylin. Data Analysis Differences between data groups were evaluated for significance using Student t-test of unpaired data or one-way analysis of variance. Repeated measures analysis of variance was used for multivariate analysis. All experiments were repeated at least three times and the
Fig. 1. The protective effect of rFII on LPS-induced DIC rabbit. DIC was induced by LPS. 0.025, 0.05, 0.1 mg/kg rFII and 50 IU/kg heparin was administered simultaneously with LPS by intravenous injection. Survival was monitored over 24 h.
We investigated the protective effect of rFII in the LPS-induced DIC model in rabbit, a clinically relevant model for human DIC [12,13]. 27.3% (3/11) of rabbits infused with 0.1 mg/kg/h LPS survived by the first 24 h after the start of the experiment. Many bleeding syndromes, such as purpura, venipuncture site bleeding, were observed in some of LPS treatment rabbits. rFII treatment was started simultaneously with LPS induction of DIC. The results showed that the rFII treatment significantly improved survival (Fig. 1). The survival rates of the rFII treatment were significantly increased: seven of eleven rabbits (63.6%) survived after a middle dose of rFII and fourteen of eighteen rabbits (78%) survived after a high dose of rFII respectively. Heparin, the agent clinically used in DIC therapy [14], increased survival rate from 27.3% to 72.7% (Fig. 1). All of animals in the sham group survived (data not shown).
Fig. 2. Effect of rFII on the plasma levels of ALT in rabbits. Blood samples were taken immediately before LPS infusion and 2 h and 6 h after the start of the infusion. An automatic analyzer detected the plasma levels of ALT. Results of ALT at 2 h and 6 h were converted to percentages assuming a value of 100% for basal data. Values are expressed as the mean ± S.D. percent of the initial value before LPS infusion. ⁎P b 0.05 as compared to the LPS group.
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Fig. 3. Effect of rFII on LPS-induced liver injury. The liver tissue specimens were stained with hematoxylin and eosin. Arrows indicate liver necrosis. (Original magnification × 200).
The effects of rFII on biochemical and pathological damages in DIC induced by LPS To further elucidate the effects of rFII on DIC induced by LPS, we systematically investigated biochemical and pathological effects using a rabbit model. Table 1 summarized the plasma levels of activated partial thromboplastin time (APTT), prothrombin time (PT), platelets count, fibrinogen and FDP for normal rabbits, LPS-induced DIC rabbits, rFII-treated rabbits and heparin-treated rabbits. APTT and PT values for LPS-induced DIC rabbits were all significantly higher at 2 h and 6 h than they were for the normal rabbits. However, the values for plasma levels of both fibrinogen and platelets count were significantly lower than those of the normal rabbits. Infusion of 0.1, 0.05 mg/kg rFII and 30 IU/kg/h heparin significantly attenuated the increased plasma levels of both APTT and PT as well as the decreased plasma level of fibrinogen at 6 h. The serum levels of FDP increased significantly after administration of LPS 2 h and 6 h. The increase levels of FDP induced by LPS at 6 hour were significantly inhibited by rFII and heparin. However, rFII and heparin had no effect on platelet levels in LPS-induced DIC rabbits. Bleeding was not observed in any of the rFII treatment rabbits.
Fig. 4. Effect of rFII on the plasma levels of Cr in rabbits. Blood samples were taken immediately before LPS infusion and 2 h and 6 h after the start of the infusion. An automatic analyzer detected the plasma levels of Cr. Results of Cr at 2 h and 6 h were converted to percentages assuming a value of 100% for basal data. Values are expressed as the mean ± S.D. percent of the initial value before LPS infusion. ⁎⁎ P b 0.01 as compared to LPS group.
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Effects of rFII on liver functions in LPS-induced DIC rabbits We investigated the effects of rFII on liver functions in LPS-induced DIC rabbits. Plasma levels of alanine aminotransferase (ALT), an indicator of liver injury, were increased by LPS infusion. However, the levels of ALT were significantly lower in rFII treatment animals as well as in heparin treatment rabbits (Fig. 2). Microscopic assessment of liver tissue revealed severe necrosis with disintegration of hepatic cords and neutrophil infiltrations in LPS treatment animals. rFII and heparin significantly ameliorated liver injury of rabbits after injections of LPS (Fig. 3). Effects of rFII on renal functions in LPS-induced DIC rabbits A similar finding was observed with regard to plasma levels of creatinine (Cr), which is an indicator of renal injury. An increase in plasma Cr levels was observed in the LPS group at 6 h. The levels of Cr were significantly suppressed by rFII as well as by heparin (Fig. 4). Microscopic assessment of renal tissue revealed severe tubular necrosis
and sloughing of tubular cells in LPS treatment groups. rFII and heparin markedly ameliorated these histological changes in kidney (Fig. 5). Moreover, intense deposits of fibrin within glomerular capillaries were observed in LPS treated rabbits and heparin treated rabbits. rFII significantly reduced the kidney fibrin deposits with respect to LPS treated animals (Fig. 6). Therefore, we concluded that rFII had protective effects on liver and renal function in LPS induced DIC in rabbits. Degradation effect of rFII on TNF-α in vivo It has been shown that TNF-α is an important inflammatory marker and generally increases significantly during the early period of DIC. Therefore, we tested whether or not rFII had effect on plasma levels of TNF-α. Rabbits were injected with 0.1 mg/kg/h of LPS and levels of TNF-α were dramatically increased at 0 h, 1 h, 4 h, 8 h and 12 h (Fig. 7). However, infusion of 0.025 mg/kg, 0.05 mg/kg and 0.1 mg/kg of rFII significantly reduced the increased plasma levels of TNF-α at 1 h, 4 h, 8 h and 12 h (Fig. 7).
Fig. 5. Effect of rFII on LPS-induced renal injury. The renal tissue specimens were stained with hematoxylin and eosin. Arrows indicate renal necrosis. (Original magnification × 200).
R. Wang et al. / Thrombosis Research 123 (2009) 919–924
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Fig. 6. Effect of rFII on microthrombi formation in renal glomerulus. The renal tissue specimens were stained with phosphotungstic acid hematoxylin. Arrows indicate microthrombi in renal glomerulus. (Original magnification × 400).
Fig. 7. Effect of rFII on plasma levels of TNF-α in rabbits. n = 6 ⁎P b 0.05 as compared to the LPS group, ⁎⁎ P b 0.01 as compared to LPS group.
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Discussion In this study, we report that rFII from Agkistrodon acutus venom fraction had significantly protective effect against the lethal effects of LPS induced DIC in rabbits. Reduction of LPS-induced biochemical and pathological damage was observed. Infectious disease, in particular septicemia, is the most common clinical condition associated with DIC [1]. The hallmark of the coagulation disorder in sepsis constitutes the imbalance between intravascular fibrin formation and its removal. Severely reduced anticoagulant capacity and inhibited fibrinolysis are opposed to a massive activation of coagulation, finally leading to overwhelming fibrin formation and consumption of clotting factors and inhibitors. Abundant intravascular fibrin formation leads to microvascular thrombosis, which contributes to the development of multiple organ failure that is often life threatening or fatal [15]. Recent results suggested that promoting fibrinolysis might be a promising target for therapy strategies during DIC. Asakura et al. reported that beneficial effects of urokinase on LPS-induced DIC in rats [16]. Protein C inhibited LPS-induced organ injury and lethal effects in animal models [17,18]. In our previous study [11], we observed that fibrin clots were hydrolyzed by rFII more quickly and efficiently than by t-PA in vitro. As a snake venom fibrinolytic enzyme, the mechanism of rFII directly degrading fibrin should be its proteolytic acction. Most importantly, unlike urokinase and t-PA [19,20], rFII lysed fibrin by direct proteolysis without activating intrinsic plasminogen. The direct fibrinolytic effect of rFII on microthrombi without activating plasminogen would reduce the consumption of coagulation factors during the development of DIC. Therefore, we could observe that rFII significantly attenuated the increased plasma levels of APTT and PT as well as heparin did. The direct action of rFII on fibrin may provide a beneficial property compared with heparin and other anticoagulant drugs in use clinically. As the onset of multiorgan dysfunction syndrome has been shown to forecast mortality in DIC, protection of organs particularly liver and kidney function is important in treatment of DIC [1,2]. Autopsy findings have shown that there is diffuse bleeding at various sites with hemorrhagic tissue necrosis and thrombi in small as well as larger blood vessels in DIC [21]. In the present study, we demonstrated that rFII have the protection effect on LPS-induced DIC in rabbits by ameliorating organ dysfunction. Three possible mechanisms are implicated in the favorable results. First, the protective effect of rFII was due mainly to significantly prevent the kidney fibrin deposits induced by LPS. In this study, we observed intense deposits of fibrin within glomerular capillaries in LPS and heparin treated rabbits. We found that rFII significantly prevented the kidney fibrin deposits induced by LPS. It indicated that rFII was ameliorated the reduced blood supply to organs. Secondly, the effect of rFII on biochemical plasma levels may be the consequences of its effect of improving blood flow to organs. We also observed that plasma levels of ALT and Cr, which were increased by LPS infusion, were significantly lower in rFII treated animals. Thirdly, rFII has been shown to have an antiinflammatory effect through directly proteolytic effect on TNF-α which is the critical mediator of LPS-induced organ failure [22]. Although the precise mechanism of the anti-inflammatory effect of rFII has not yet been fully elucidated, in our previous studies, we observed rFII could downregulation the plasma levels of TNF-α without inhibition of its mRNA synthesis [11]. Taken together, these various effects of rFII have a favorable therapeutic effect on LPS induced DIC rabbits.
In conclusion, rFII may have protective effect on LPS-induced DIC through direct degradation of fibrin clots and ameliorating organ dysfunction. These therapeutic effects support the hypothesis that recombinant fibrinogenase may be used for reducing microthrombi and anti-inflammation of DIC. However, we emphasize that the true utility of rFII in combating DIC will require direct testing in clinical trials. Acknowledgments The authors acknowledge financial support by Foundation of Department of Science and Technology of Guangdong Province (2003A10905). The authors declare that they have no competing financial interests. References [1] Levi M, Ten Cate H. Disseminated intravascular coagulation. New Engl J Med 1999;341:586–92. [2] Levi M. Disseminated intravascular coagulation: what's new? Crit Care Clin 2005;21:449–67. [3] Feinstein DI. Diagnosis and management of disseminated intravascular coagulation: the role of heparin therapy. Blood 1982;60:284–7. [4] Lehmann C, Usichenko TI, Pavlovic D. Heparins in sepsis-induced disseminated intravascular coagulation: low weight-high impact? Crit Care Med 2005;33:1455–7. [5] Freeman BD, Zehnbauer BA, Buchman TG. A meta-analysis of controlled trials of anticoagulant therapies in patients with sepsis. Shock 2003;20:5–9. [6] Taylor Jr FB, Chang AC, Peer GT, Mather T, Blick K, Catlett R, et al. DEGR factor Xa blocks disseminated intravascular coagulation initiated by Escherichia coli without preventing shock or organ damage. Blood 1991;78:364–8. [7] Kini RM. Anticoagulant proteins from snake venoms: struture, function and mechanism. Biochem J 2006;397:377–87. [8] Liang XX, Chen JS, Zhou YN, Qiu PX, Yan GM. Purification and biochemical characterization of F II (a), a fibrinolytic enzyme from Agkistrodon acutus venom. Toxicon 2001;39:1133–9. [9] Wang YW, Liang XX, Chen JS, Chen Q, Qiu PX, Lin X, et al. Fibrin(ogen)olytic character of FIIa isolated from Agkistrodon acutus venom. Acta Pharmacol Sin 2005;26:691–5. [10] Liang XX, Zhou YN, Chen JS, Qiu PX, Chen HZ, Sun HH, et al. Enzymological characterization of FIIa, a fibrinolytic enzyme from Agkistrodon acutus venom. Acta Pharmacol Sin 2005;26:1474–8. [11] Wang RR, Qiu PX, Jiang WJ, Cai XF, Ou YQ, Su XW. Recombinant fibrinogenase from Agkistrodon acutus venom protects against sepsis via direct degradation of fibrin and TNF-α. Biochem Pharm 2008;76:620–30. [12] Elsayed YA, Nakagawa K, Ichikawa K, Ohkawara S, Sueishi K. Expression of tissue factor and interleukin-1 beta in a novel rabbit model of disseminated intravascular coagulation induced by carrageenan and lipopolysaccharide. Pathobiology 1995;63:328–40. [13] Levi M, ten Cate H, van der Poll T, van Deventer SJ. Pathogenesis of disseminated intravascular coagulation in sepsis. JAMA 1993;270:975–9. [14] Ibbotson T, Perry CM. Danaparoid: a review of its use in thromboembolic and coagulation disorders. Drugs 2002;62:2283–314. [15] Zeerleder S, Hack CE, Wuillemin WA. Disseminated intravascular coagulation in sepsis. Chest 2005;128:2864–75. [16] Asakura H, Asamura R, Ontachi Y, Hayashi T, Omote M, Arahata M, et al. Beneficial effects of urokinase on lipopolysaccharide-induced disseminated intravascular coagulation in rats: focus on organ function and endothelin levels. Thromb Haemost 2005;93:724–8. [17] Chen CM, Hou CC, Cheng KC, Tian RL, Chang CP, Lin MT. Activated protein C therapy in a rat heat stroke model. Crit Care Med 2006;34:1960–6. [18] Mizutani A, Okajima K, Uchiba M, Noguchi T. Activated protein C reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation. Blood 2000;95:3781–7. [19] Ueshima S, Matsuo O. Development of new fibrinolytic agents. Curr Pharm Des 2006;12:849–57. [20] Dobrovolsky AB, Titaeva EV. The fibrinolysis system: regulation of activity and physiologic functions of its main components. Biochemistry 2002;67:99–108. [21] Shimamura K, Oka K, Nakazawa M, Kojima M. Distribution patterns of microthrombi in disseminated intravascular coagulation. Arch Pathol Lab Med 1983;107:543–7. [22] Cataldegirmen G, Zeng S, Feirt N, Lppagunta N, Dun H, Qu W. RAGE limits regeneration after massive liver injury by coordinated suppression of TNF- and NF-B. J Exp Med 2005;201:473–84.
Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Novel recombinant fibrinogenase of Agkistrodon acutus venom protects against LPS-induced DIC Rongrong Wang 1, Jinlian Cai 1, Yijun Huang, Dong Xu, Hanfei Sang, Guangmei Yan ⁎ Department of Pharmacology, Zhong-shan Medical College, Sun Yat-sen University, Guangzhou, 510080, P.R. China
a r t i c l e
i n f o
Article history: Received 8 August 2008 Received in revised form 8 October 2008 Accepted 29 October 2008 Available online 13 December 2008 Keywords: Disseminated intravascular coagulation Recombinant Fibrinogenase Microthrombi Lipopolysaccharide
a b s t r a c t Introduction: Disseminated intravascular coagulation (DIC) is an acquired syndrome characterized by the widespread activation of coagulation. This leads to failure of multiple organs in the body and finally death. Because there is no effective therapy for DIC, the clinical prognosis is poor and the mortality is high. Materials and Methods: The animals were intravenously injected with Lipopolysaccharide (LPS) for 6 hours and simultaneously infected three doses of recombinant fibrinogenase II (rFII) for 2 hour. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelets count, fibrinogen and fibrin-fibrinogen degradation products (FDP) were determined. The plasma levels of alanine aminotransferase (ALT), creatinine (Cr) and tumor necrosis factor-α (TNF-α) were detected. Liver and kidney samples were stained with hematoxylin-eosin and kidney sections were stained with phosphotungstic acid-hematoxylin. Results: We observed that rFII increased survival rate in LPS-induced DIC rabbits as well as heparin did. Administration of rFII as well as heparin attenuated the increased plasma levels of APTT, PT and FDP and the decreased plasma level of fibrinogen at 6 h. rFII reduced hepatic and renal damages and decreased the levels of ALT and Cr as well as heparin did. rFII also significantly reduced the increased plasma levels of TNF-α. rFII significantly reduced the kidney fibrin deposits with respect to LPS treated animals. Conclusions: Our findings suggest that rFII from Agkistrodon acutus venom could have protective effect on DIC via reducing liver and renal damages and direct degradation of microthrombi. © 2008 Elsevier Ltd. All rights reserved.
Introduction Disseminated intravascular coagulation (DIC) is a life threatening syndrome arising from various causes including disseminated sepsis. It is generally associated with an adverse outcome [1,2]. Although many efforts have been made to develop treatment of DIC, there is no established therapy [3–5]. The basic pathological mechanism of DIC includes the spread of microvascular thrombosis, which prevents adequate blood supply to organs and leads to multiple organ failure and death [1]. Clinical trials aiming at an interruption of “latent coagulation” in sepsis by administration of coagulation inhibitors, such as antithrombin, have so far failed to demonstrate a statistically significant benefit on survival. Heparin blocks endotoxin initiated clotting but is ineffective
Abbreviations: rFII, recombinant fibrinogenase II; DIC, Disseminated intravascular coagulation; LPS, Lipopolysaccharide; ALT, alanine aminotransferase; Cr, creatinine; APTT, activated partial thromboplastin time; PT, prothrombin time; FDP, fibrinfibrinogen degradation products. ⁎ Corresponding author. Department of Pharmacology, Zhong-Shan Medical College, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou, Guangdong, 510089, P.R. China. Tel.: +86 20 87333258; fax: +86 20 87333762. E-mail address: [email protected] (G. Yan). 1 These authors contributed equally to this work. 0049-3848/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.10.017
in preventing organ failure [5]. Likewise, factor Xa blocked with a small molecule in the active site effectively inhibited coagulation induced by E coli in baboons but did not protect from organ failure or prevent death [6]. It is possible that failure of these compounds to protect against DIC may not have prevented the decrease in the blood supply to organs. Furthermore, the safety of anticoagulants in DIC has not been established. Previous use of some proteinases purified from various snake venoms using biochemical methods showed effects on the clinical course of thrombotic disorders [7]. We isolated a novel snake venom fibrinogenase, FIIa, from the Agkistrodon acutus snake [8]. It has the ability to directly degrade fibrin in vitro and dissolves thrombi effectively in vivo [9,10]. In the present study, we cloned the gene, expressed, purified and characterized this novel recombinant protease. We found that this recombinant fibrinogenaseII (rFII) protected against LPS-induced DIC in rabbits. Materials and methods Cloning and preparation of rFII The recombinant fibrinogenase II (rFII) from Agkistrodon acutus venom, was prepared according to the method described by Wang et al [11].
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Animals and drug treatments All animal experiments were conducted in accordance with the National Guide for the Care and Use of Laboratory Animals and were approved by Sun Yat-sen University Animal Care and Use Committee (Guangzhou, China). DIC was induced by infusion with 0.1 mg/kg/h LPS for 6 h in 60 ml (10 ml/h) intravenously through the marginal ear vein. Treatments were started simultaneously with LPS infusion through the contralateral marginal ear vein. Six different groups were established: 1.LPS group: 0.1 mg/kg/h LPS for 6 h; 2. LPS and low dose of rFII group: 0.1 mg/kg/h LPS for 6 h and 0.05 mg/kg/h rFII for 2 h; 3. LPS and high dose of rFII group: 0.1 mg/kg/h LPS for 6 h and 0.1 mg/kg/h rFII for 2 h; 4. LPS and heparin group: 0.1 mg/kg/h LPS for 6 h and 30 IU/kg/h heparin for 6 h; 5. rFII group: 0.1 mg/kg/h rFII for 6 h only; 6. Normal group: saline was infused. Kidneys and livers were removed for subsequent histological studies 24 h after the start of the experiments. Biochemical Analysis Blood samples were taken immediately before LPS infusion and 2 h and 6 h after the start of the infusion. A automatic analyzer (Sysmex SE-9500, Sysmex CA 1500, Japan) was used to determine APTT, PT, platelets count, Fibrinogen and fibrin-fibrinogen degradation products (FDP). 7170A automatic analyzer (HITACHI, Japan) detected the plasma levels of alanine aminotransferase (ALT) and creatinine (Cr).
Table 1 Plasma levels of activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, fibrin degradation product (FDP) and platelet count for normal, LPS-treated, rFII-treated and heparin-treated rabbits. Groups Normal
N
Time (h) PT (s)
10 2 6 LPS 10 2 6 LPS + 10 2 0.025rFII 6 LPS + 10 2 0.05rFII 6 LPS + 10 2 0.1rFII 6 LPS + 10 2 Heparin 6
h h h h h h h h h h h h
7.2 ± 0.8 7.2 ± 0.7 11.0 ± 3.4# 14.9 ± 4.7# 8.5 ± 1.1 10.6 ± 1.8⁎ 7.5 ± 0.5⁎ 9.7 ± 1.3⁎ 7.6 ± 0.8⁎ 9.9 ± 1.3⁎ 7.7 ± 0.6⁎ 8.4 ± 3.5⁎
APTT (s)
Fibrinogen Platelets (g/l) (×109/l)
FDP (μg/ml)
18.2 ± 1.0 18.4 ± 0.7 21.1 ± 5.4# 28.8 ± 5.5# 21.5 ± 3.1 26.3 ± 2.0 21.1 ± 4.5 25.5 ± 7.7⁎ 19.1 ± 1.9 22.2 ± 1.9⁎ 18.6 ± 4.2⁎ 25.6 ± 4.3⁎
3.0 ± 0.8 3.2 ± 0.9 2.7 ± 0.8 1.8 ± 0.2# 2.9 ± 0.6 2.0 ± 0.5 3.1 ± 0.8⁎ 2.4 ± 0.7⁎ 2.2 ± 0.6 2.5 ± 0.4⁎ 3.2 ± 0.6⁎ 2.5 ± 0.9⁎
b 0.05 b 0.05 58.9 ± 16.5# 87.3 ± 28.9# 28.1 ± 8.9⁎ 22.3 ± 7.5⁎ 26.2 ± 5.5⁎ 18.3 ± 6.1⁎ 23.5 ± 3.1⁎ 15.1 ± 4,5⁎ 35.3 ± 6.0⁎ 30.1 ± 5.8⁎
456 ± 15 488 ± 58 304 ± 48# 185 ± 25# 370 ± 53 198 ± 58 345 ± 33 284 ± 14 376 ± 57 257 ± 35 434 ± 25 315 ± 18
#
P b 0.05 as compared to normal group. ⁎P b 0.05 as compared to LPS treatment group. Data are presented as the mean ± S.D. LPS: infusion of 0.1 mg/kg/h LPS for 6 h. LPS+ 0.025rFII: simultaneous infusion of 0.1 mg/kg/h LPS and 0.025 mg/kg rFII. LPS + 0.05r FII: simultaneous infusion of 0.1 mg/kg/h LPS and 0.05 mg /kg rFII. LPS + 0.1rFII: simultaneous infusion of 0.1 mg/kg/h LPS and 0.1 mg/kg rFII. LPS+ heparin: simultaneous infusion of 0.1 mg/kg/h LPS and 30 IU/kg/h heparin.
data are presented as the mean ± S.D. unless otherwise noted. Results of ALT and Cr at 2 h and 6 h were converted to percentages assuming a value of 100% for basal data. Survival curves of LPSinduced DIC were analyzed by the Kaplan-Meyer log-rank test. Results
Measurement of TNF-α concentration The protective effect of rFII on DIC induced by LPS The plasma of rabbits were collected in tube and stored at –20 °C until assayed. The concentrations of TNF-α in animal serum were determined using ELISA KIT (RapidBio Lab. Calabasas, USA). Histopathological Analysis Liver and kidney samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin-eosin. To determine fibrin deposition in microvessels, kidney sections were stained with phosphotungstic acid-hematoxylin. Data Analysis Differences between data groups were evaluated for significance using Student t-test of unpaired data or one-way analysis of variance. Repeated measures analysis of variance was used for multivariate analysis. All experiments were repeated at least three times and the
Fig. 1. The protective effect of rFII on LPS-induced DIC rabbit. DIC was induced by LPS. 0.025, 0.05, 0.1 mg/kg rFII and 50 IU/kg heparin was administered simultaneously with LPS by intravenous injection. Survival was monitored over 24 h.
We investigated the protective effect of rFII in the LPS-induced DIC model in rabbit, a clinically relevant model for human DIC [12,13]. 27.3% (3/11) of rabbits infused with 0.1 mg/kg/h LPS survived by the first 24 h after the start of the experiment. Many bleeding syndromes, such as purpura, venipuncture site bleeding, were observed in some of LPS treatment rabbits. rFII treatment was started simultaneously with LPS induction of DIC. The results showed that the rFII treatment significantly improved survival (Fig. 1). The survival rates of the rFII treatment were significantly increased: seven of eleven rabbits (63.6%) survived after a middle dose of rFII and fourteen of eighteen rabbits (78%) survived after a high dose of rFII respectively. Heparin, the agent clinically used in DIC therapy [14], increased survival rate from 27.3% to 72.7% (Fig. 1). All of animals in the sham group survived (data not shown).
Fig. 2. Effect of rFII on the plasma levels of ALT in rabbits. Blood samples were taken immediately before LPS infusion and 2 h and 6 h after the start of the infusion. An automatic analyzer detected the plasma levels of ALT. Results of ALT at 2 h and 6 h were converted to percentages assuming a value of 100% for basal data. Values are expressed as the mean ± S.D. percent of the initial value before LPS infusion. ⁎P b 0.05 as compared to the LPS group.
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Fig. 3. Effect of rFII on LPS-induced liver injury. The liver tissue specimens were stained with hematoxylin and eosin. Arrows indicate liver necrosis. (Original magnification × 200).
The effects of rFII on biochemical and pathological damages in DIC induced by LPS To further elucidate the effects of rFII on DIC induced by LPS, we systematically investigated biochemical and pathological effects using a rabbit model. Table 1 summarized the plasma levels of activated partial thromboplastin time (APTT), prothrombin time (PT), platelets count, fibrinogen and FDP for normal rabbits, LPS-induced DIC rabbits, rFII-treated rabbits and heparin-treated rabbits. APTT and PT values for LPS-induced DIC rabbits were all significantly higher at 2 h and 6 h than they were for the normal rabbits. However, the values for plasma levels of both fibrinogen and platelets count were significantly lower than those of the normal rabbits. Infusion of 0.1, 0.05 mg/kg rFII and 30 IU/kg/h heparin significantly attenuated the increased plasma levels of both APTT and PT as well as the decreased plasma level of fibrinogen at 6 h. The serum levels of FDP increased significantly after administration of LPS 2 h and 6 h. The increase levels of FDP induced by LPS at 6 hour were significantly inhibited by rFII and heparin. However, rFII and heparin had no effect on platelet levels in LPS-induced DIC rabbits. Bleeding was not observed in any of the rFII treatment rabbits.
Fig. 4. Effect of rFII on the plasma levels of Cr in rabbits. Blood samples were taken immediately before LPS infusion and 2 h and 6 h after the start of the infusion. An automatic analyzer detected the plasma levels of Cr. Results of Cr at 2 h and 6 h were converted to percentages assuming a value of 100% for basal data. Values are expressed as the mean ± S.D. percent of the initial value before LPS infusion. ⁎⁎ P b 0.01 as compared to LPS group.
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Effects of rFII on liver functions in LPS-induced DIC rabbits We investigated the effects of rFII on liver functions in LPS-induced DIC rabbits. Plasma levels of alanine aminotransferase (ALT), an indicator of liver injury, were increased by LPS infusion. However, the levels of ALT were significantly lower in rFII treatment animals as well as in heparin treatment rabbits (Fig. 2). Microscopic assessment of liver tissue revealed severe necrosis with disintegration of hepatic cords and neutrophil infiltrations in LPS treatment animals. rFII and heparin significantly ameliorated liver injury of rabbits after injections of LPS (Fig. 3). Effects of rFII on renal functions in LPS-induced DIC rabbits A similar finding was observed with regard to plasma levels of creatinine (Cr), which is an indicator of renal injury. An increase in plasma Cr levels was observed in the LPS group at 6 h. The levels of Cr were significantly suppressed by rFII as well as by heparin (Fig. 4). Microscopic assessment of renal tissue revealed severe tubular necrosis
and sloughing of tubular cells in LPS treatment groups. rFII and heparin markedly ameliorated these histological changes in kidney (Fig. 5). Moreover, intense deposits of fibrin within glomerular capillaries were observed in LPS treated rabbits and heparin treated rabbits. rFII significantly reduced the kidney fibrin deposits with respect to LPS treated animals (Fig. 6). Therefore, we concluded that rFII had protective effects on liver and renal function in LPS induced DIC in rabbits. Degradation effect of rFII on TNF-α in vivo It has been shown that TNF-α is an important inflammatory marker and generally increases significantly during the early period of DIC. Therefore, we tested whether or not rFII had effect on plasma levels of TNF-α. Rabbits were injected with 0.1 mg/kg/h of LPS and levels of TNF-α were dramatically increased at 0 h, 1 h, 4 h, 8 h and 12 h (Fig. 7). However, infusion of 0.025 mg/kg, 0.05 mg/kg and 0.1 mg/kg of rFII significantly reduced the increased plasma levels of TNF-α at 1 h, 4 h, 8 h and 12 h (Fig. 7).
Fig. 5. Effect of rFII on LPS-induced renal injury. The renal tissue specimens were stained with hematoxylin and eosin. Arrows indicate renal necrosis. (Original magnification × 200).
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Fig. 6. Effect of rFII on microthrombi formation in renal glomerulus. The renal tissue specimens were stained with phosphotungstic acid hematoxylin. Arrows indicate microthrombi in renal glomerulus. (Original magnification × 400).
Fig. 7. Effect of rFII on plasma levels of TNF-α in rabbits. n = 6 ⁎P b 0.05 as compared to the LPS group, ⁎⁎ P b 0.01 as compared to LPS group.
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Discussion In this study, we report that rFII from Agkistrodon acutus venom fraction had significantly protective effect against the lethal effects of LPS induced DIC in rabbits. Reduction of LPS-induced biochemical and pathological damage was observed. Infectious disease, in particular septicemia, is the most common clinical condition associated with DIC [1]. The hallmark of the coagulation disorder in sepsis constitutes the imbalance between intravascular fibrin formation and its removal. Severely reduced anticoagulant capacity and inhibited fibrinolysis are opposed to a massive activation of coagulation, finally leading to overwhelming fibrin formation and consumption of clotting factors and inhibitors. Abundant intravascular fibrin formation leads to microvascular thrombosis, which contributes to the development of multiple organ failure that is often life threatening or fatal [15]. Recent results suggested that promoting fibrinolysis might be a promising target for therapy strategies during DIC. Asakura et al. reported that beneficial effects of urokinase on LPS-induced DIC in rats [16]. Protein C inhibited LPS-induced organ injury and lethal effects in animal models [17,18]. In our previous study [11], we observed that fibrin clots were hydrolyzed by rFII more quickly and efficiently than by t-PA in vitro. As a snake venom fibrinolytic enzyme, the mechanism of rFII directly degrading fibrin should be its proteolytic acction. Most importantly, unlike urokinase and t-PA [19,20], rFII lysed fibrin by direct proteolysis without activating intrinsic plasminogen. The direct fibrinolytic effect of rFII on microthrombi without activating plasminogen would reduce the consumption of coagulation factors during the development of DIC. Therefore, we could observe that rFII significantly attenuated the increased plasma levels of APTT and PT as well as heparin did. The direct action of rFII on fibrin may provide a beneficial property compared with heparin and other anticoagulant drugs in use clinically. As the onset of multiorgan dysfunction syndrome has been shown to forecast mortality in DIC, protection of organs particularly liver and kidney function is important in treatment of DIC [1,2]. Autopsy findings have shown that there is diffuse bleeding at various sites with hemorrhagic tissue necrosis and thrombi in small as well as larger blood vessels in DIC [21]. In the present study, we demonstrated that rFII have the protection effect on LPS-induced DIC in rabbits by ameliorating organ dysfunction. Three possible mechanisms are implicated in the favorable results. First, the protective effect of rFII was due mainly to significantly prevent the kidney fibrin deposits induced by LPS. In this study, we observed intense deposits of fibrin within glomerular capillaries in LPS and heparin treated rabbits. We found that rFII significantly prevented the kidney fibrin deposits induced by LPS. It indicated that rFII was ameliorated the reduced blood supply to organs. Secondly, the effect of rFII on biochemical plasma levels may be the consequences of its effect of improving blood flow to organs. We also observed that plasma levels of ALT and Cr, which were increased by LPS infusion, were significantly lower in rFII treated animals. Thirdly, rFII has been shown to have an antiinflammatory effect through directly proteolytic effect on TNF-α which is the critical mediator of LPS-induced organ failure [22]. Although the precise mechanism of the anti-inflammatory effect of rFII has not yet been fully elucidated, in our previous studies, we observed rFII could downregulation the plasma levels of TNF-α without inhibition of its mRNA synthesis [11]. Taken together, these various effects of rFII have a favorable therapeutic effect on LPS induced DIC rabbits.
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