Jia-shu Chen

Enzymological characterization of FIIa, a fibrinolytic enzyme from Agkistrodon acutus venom1

AbstractAim:To study the enzymological characterization of a fibrinolytic enzyme (FIIa) from Agkistrodon acutus venom.Methods:The fibrinogenolytic effect and the influences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The metal content of FIIa was determined by atomic absorption spectroscopy.Results:After incubation with FIIa (0.25 g/L), Aα-, Bβ- and γ-chains of fibrinogen disappeared within 5 min, 30 min, and 8 h, respectively. The molecular weights of major degradation products were 45 000 and 41 000, which were different from those bands produced by plasmin. The fibrinogenolytic activity of FIIa was strongly inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine, but not by phenylmethyl-sulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171±25 mg/kg), potassium (489±17 mg/kg) and calcium (319±13 mg/kg) were found in FIIa. Zn2+, Ca2+ and Mg2+ could recover the fibrinogenolytic activity of FIIa, which was inhibited by EDTA. Only Ca2+ could recover the fibrinogenolytic activity inhibited by EGTA.Conclusion:FIIa can degrade the Aα-, Bβ- and γ-chains of fibrinogen. FIIa is a metalloproteinase, and Zn2+, Ca2+, and disulfide bonds are necessary for its fibrinogenolytic activity.

Recombinant fibrinogenase from Agkistrodon acutus venom protects against sepsis via direct degradation of fibrin and TNF-α

Severe sepsis remains a leading cause of death and disability because of less effective therapy available for this disease. A complex interplay between the inflammatory factors and the coagulation pathways seems to be the fundamental mechanisms for the pathogenesis of sepsis. Here we report that recombinant fibrinogenase II (rF II) from Agkistrodon acutus plasmin-independently degraded the thrombi, and inhibited inflammatory responses by direct and specific degradation of tumor necrosis factor alpha (TNF-alpha) induced by lipopolysaccharide (LPS) without showing proteolytic activities on interleukin-1 (IL-1), cluster of differentiation 68 (CD68) and some other serum proteins. We also report that rF II effectively protected against LPS induced sepsis in a rabbit model. Administration of rF II reduced hepatic and renal damage, decreased the levels of alanine aminotransferase (ALT) and blood urea nitrogen (BUN), and increased survival rate in LPS-induced sepsis rabbits. We further confirmed the rescue effect of rF II on severe sepsis in rat caecal ligation and puncture (CLP) model. Our findings suggest that rF II could effectively protect against sepsis via direct degradation of microthrombi and inflammatory factor TNF-alpha as well as provide a novel strategy to develop a single proteinase molecule for targeting the main pathological processes of this disease.

Fibrin(ogen)olytic character of FIIa isolated from Agkistrodon acutus venom.

AbstractAim:To investigate the fibrin(ogen)olytic character of FIIa isolated from Agkistrodon acutus venom in vitro and in vivo.Methods:125I-labeled human plasma clot lysis was measured in vitro and rabbit carotid artery thrombosis was as an in vivo model.Results:In vitro, urokinase (UK) at 25, 35, 40, 45, 60 kU/L and FIIa at 0.08, 0.23, 0.4, 0.5, and 0.7 g/L resulted an equivalent clot lysis (20%, 40%, 50%, 60%, and 80%). UK at 25-60 kU/L induced 27.3%±3.6%, 35.2%±2.3%, 39.3%±2.4%, 44.2%±4.6%, and 51.1%±1.2% fibrinogen degradation. But FIIaat 0.08–0.7 g/L induced 95.4%±0.3%, >95.6%, >95.6%, >95.6%, >95.6% fibrinogen degradation respectively. In vivo, UK 40 kU/kg and FIIa 1.0 mg/kg reduced the weight of residual thrombus to 9.0±2.5 mg and 7.8±3.5 mg compared with negative control group (30.0±5.4 mg). But the fibrinogen degradation rate after UK 40 kU/kg and FIIa 1.0 mg/kg treatment was 24.4%±6.2% and 4.1%±7.8%, respectively (P < 0.05, n=6). The order of the lysis speed after UK 125 kU/L treatment was platelet poor plasma (PPP) clots>the whole blood clots>platelet rich plasma (PRP) clots. The sequence for FIIa 0.4 g/L was PRP>PPP>whole blood clots.Conclusion:At the same percentage of clot lysis, FIIa degraded more fibrinogen than UK did in vitro but less fibrinogen than UK did in vivo. The order of the lysis speed was PPP>whole blood clots>PRP clots for UK and PRP>PPP>whole blood clots for FIIa.

A novel fibrinogenase from Agkistrodon acutus venom protects against DIC via direct degradation of thrombosis and activation of protein C

The incidence of disseminated intravascular coagulation (DIC), which leads to multiple organ dysfunction and high mortality, has remained constant in recent years. At present, treatments of DIC have focused on preventing cytokine induction, inhibiting coagulation processes and promoting fibrinolysis. Recent clinical trials have supported the use of antithrombin and activated protein C supplementation in DIC. To better understand the mechanism of treatment on DIC, we here report a novel fibrinogenase from Agkistrodon acutus (FIIa) that effectively protected against LPS-induced DIC in a rabbit model, and detected the tissue factors expression in HUVE cells after using FIIa. In vivo, administration of FIIa reduced hepatic and renal damage, increased the concentration of fibrinogen, the activities of protein C, the platelet count, APTT, PT, FDP, the level of AT-III and t-PA, decreased the level of PAI-1, and increased survival rate in LPS-induced DIC rabbits. In vitro experiments, we further confirmed that FIIa up-regulated the expression of t-PA and u-PA, down-regulated the expression of PAI-1, and directly activated protein C. Our findings suggest that FIIa could effectively protect against DIC via direct degradation of microthrombi and activation of protein C as well as provide a novel strategy to develop a single proteinase molecule for targeting the main pathological processes of this disease.

A novel recombinant fibrinogenase of Agkistrodon acutus venom protects against hyperacute rejection via degradation of complements

Hyperacute rejection (HAR) is a main barrier in xenotransplantation, which is mediated by the combination of natural antibody to the xenograft and complement activation. Current therapies have focus on the inhibition of complement by development of complement inhibitor and transgenic animal organ. Here, we investigated the effects of rFII, a recombinant fibrinogenase from Agkistrodon acutus venom, on complement and HAR. The degradation effect of rFII on complement was tested by SDS-PAGE, CH50 examination, ELISA Kit and cofocal immunofluorescence microscopy in vitro and in vivo. An ex-vivo rat-to-human perfusion model and a vivo guinea-pig-to-rat heat HAR model were used to determine the protection of rFII against HAR. Our investigation indicated that rFII could significantly degrade human C5, C6, and C9, decrease the activity of complement, and inhibit the MAC deposition on HUVECs membrane in vitro. In addition, serum levels of C1q, C3 and C4 in rat were gradually reduced after infusion of rFII. Importantly, in an ex vivo rat-to-human perfusion model, the survival of rat hearts perfused with human serum treated with rFII (83.36 ± 16.63 min) were significantly longer than that of hearts perfused with fresh human serum(15.94 ± 4.75 min). At the time of 15 minutes after perfusion, functions of hearts added with 50 ug/ml rFII sustained well with heart rates at 283 ± 65.32 beats/minute and LVDP at 13.70 ± 5.45 Kpa, while that of hearts perfused with fresh human serum were severely damaged by HAR with heart rates at 107.77 ± 40.31 beats/minute and LVDP at 1.01 ± 0.83 Kpa. We also found that rFII significantly decreased the levels of C1q, C3 and C4 in human fresh serum perfusate. In a vivo guinea-pig-to-rat heat HAR model, the survival of rat hearts treated with rFII were significantly longer than that of hearts perfused with normal saline; and relieved heart damage by complete activation. Our finding demonstrates the anti-complement property of rFII and its protection against HAR, indicating that rFII might be as a potential therapeutic agent for xenotransplantation.

The effect of the fibrinolytic enzyme FIIa from Agkistrodon acutus venom on acute pulmonary thromboembolism

AbstractAim:To evaluate the effects of the fibrinolytic enzyme FIIa from Agkistrodon acutus venom on acute pulmonary thromboembolism (APT) in animal models.Methods:Both rabbit and dog APT models were used. For the rabbit APT model, the thrombi weight before and after administration was measured. Central venous pressure (CVP) and mean arterial pressure (MAP) were measured before and 15, 30, 60, and 120 min after the injection of the blood clot. Partial thromboplastin time (APTT), prothrombin time (PT), platelet count, and fibrinogen concentration were measured using auto analyzers. Plasminogen activity was measured based on chromogenic substrates. In the dog APT model, pulmonary blood flow was recorded using pulmonary angiography.Results:Intravenous administration of FIIa (0.1–5.0 mg/kg) improved the APT-induced hemodynamic derangements and reduced thrombi weight. The angiography evidence also showed that the pulmonary emboli had almost disappeared after FIIa infusion. FIIa (0.1, 0.5, or 1.0 mg/kg) did not impair the coagulation pathways, although very high doses of FIIa (5.0 mg/kg) could stimulate the production of plasminogen and result in impairment of the pathways.Conclusion:FIIa could effectively protect against APT via degradation of thrombi with less activation of plasminogen, and may provide a novel fibrinolytic enzyme for targeting the main pathological processes of the disease.

Effects of temperature, pH, and inhibitors on the procoagulant characterization of FIa, a factor X activator from the venom of Daboia russellii siamensis (Myanmar).

FIa, a factor X activator, was isolated from the venom of Daboia russellii siamensis (Myanmar) after a series of chromatographic separations. FIa displayed procoagulant activity by shortening plasma recalcification time and converted human factor X (FX) to activated human factor X (FXa) by cleaving the heavy FX chain, possibly at the Arg51-Ile52 peptide. FIa was positive in a glycoprotein staining test, demonstrating that it is a glycoprotein. Optimal temperature and pH values were important for FIa procoagulant activity. Procoagulant activity was maintained above 85% of the initial activity at pH 7.0∼8.0, and showed equally maximum activity at temperatures ranging from 30 to 50°C. In addition, FIa procoagulant activity was completely inhibited by EDTA (5 mM), but not by PMSF (10 mM), suggesting that it is a metalloproteinase.