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European Journal of Biochemistry May 1992The carbohydrate side chains of the thrombin-like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic...
Carbohydrate structure of a thrombin-like serine protease from Agkistrodon rhodostoma. Structure elucidation of oligosaccharides by methylation analysis, liquid secondary-ion mass spectrometry and proton magnetic resonance.
The carbohydrate side chains of the thrombin-like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. Glycans obtained were characterized by digestion with exoglycosidases, methylation analysis and, in part, by liquid secondary-ion mass spectrometry and 1H-NMR spectroscopy. The results reveal that this snake venom glycoprotein contains partially truncated di-, tri- and tetraantennary complex type N-glycans carrying Fuc(alpha 1-6) residues at the innermost N-acetylglucosamine and solely (alpha 2-3)-linked sialic acid substituents. As a characteristic feature, ancrod oligosaccharides comprise mainly sialylated Gal beta 3GlcNAc beta lactosamine antennae. Furthermore, a small proportion of the sugar chains were found to carry a NeuAc alpha 3GalNAc beta 4GlcNAc beta antenna exclusively linked to C-2 of Man(alpha 1-3) residues of the pentasaccharide core. Thus, many of the glycans found represent novel glycoprotein-N-glycan structures.
Topics: Carbohydrate Sequence; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Magnetic Resonance Spectroscopy; Mass Spectrometry; Methylation; Molecular Sequence Data; Oligosaccharides; Protons; Serine Endopeptidases; Thrombin; Viper Venoms
PubMed: 1315684
DOI: 10.1111/j.1432-1033.1992.tb16863.x -
FEBS Letters Feb 1992The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity...
The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanase-treated peptides. Monoclonal antibodies raised against the denatured protein and HF-deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.
Topics: Amino Acid Sequence; Amino Acids; Ancrod; Blotting, Western; Molecular Sequence Data; Sequence Alignment; Viper Venoms
PubMed: 1544412
DOI: 10.1016/0014-5793(92)80559-y -
International Journal of Experimental... Dec 1991We have compared the effects of ancrod and recombinant tissue plasminogen activator (rtPA) on nephrotoxic nephritis induced in pre-immunized rabbits by the...
We have compared the effects of ancrod and recombinant tissue plasminogen activator (rtPA) on nephrotoxic nephritis induced in pre-immunized rabbits by the administration of nephrotoxic globulin (NTG; sheep anti-rabbit glomerular basement membrane). We used three different doses of NTG: in each experiment three groups of six rabbits were preimmunized with normal sheep globulin and given NTG: group A received no further treatment; group B received rtPA, 2 mg/kg 12 hourly; group C received ancrod 2 U/kg 12 hourly. Animals were bled daily for estimation of plasma fibrinogen and serum creatinine, then killed on day 5 and kidneys removed for histology. 1 ml/kg of NTG caused massive glomerular necrosis, all three groups having severe renal failure. With 0.5 ml/kg of NTG, ancrod and rtPA both effectively prevented fibrin deposition in Bowman's space, but all animals had severe proliferative glomerulonephritis and marked renal failure. With 0.25 ml/kg of NTG, control animals developed severe proliferative nephritis and advanced renal failure, ancrod provided almost complete protection, and the rtPA group had renal injury and functional impairment intermediate between the other two groups. We conclude that renal failure in severe nephrotoxic nephritis is fibrin-independent, but in less fulminant nephritis renal function can be protected by defibrination with ancrod. rtPA is capable of reducing glomerular fibrin accumulation as effectively as ancrod, but provides inferior protection of renal function.
Topics: Ancrod; Animals; Fibrin; Kidney; Kidney Glomerulus; Male; Nephritis; Rabbits; Tissue Plasminogen Activator
PubMed: 1768613
DOI: No ID Found -
Blood Nov 1991In order to determine the efficacy and safety of ancrod, a rapid acting defibrinogenating drug, for patients with heparin-induced thrombocytopenia, 11 consecutive...
In order to determine the efficacy and safety of ancrod, a rapid acting defibrinogenating drug, for patients with heparin-induced thrombocytopenia, 11 consecutive patients who required anticoagulant therapy because of venous thromboembolism and who developed acute heparin-induced thrombocytopenia or had a history of heparin-induced thrombocytopenia were treated with ancrod. Heparin therapy was discontinued (in patients receiving heparin) and ancrod started at a dose of 1 to 2 U/kg every 24 hours with subsequent daily doses adjusted to maintain fibrinogen levels between 0.5 and 1.0 g/L. Ancrod was continued until warfarin had become effective. The platelet count increased to more than 150 x 10(9)/L within 2 to 10 days in all thrombocytopenic patients. Two patients with a history of heparin-induced thrombocytopenia maintained normal platelet counts while receiving ancrod. Two patients had recurrent venous thrombosis while receiving warfarin, 10 days after ancrod was discontinued: one of these patients had metastatic pancreatic carcinoma and developed phlegmasia cerulea dolens and the other patient developed a venographically proven extension of her deep venous thrombosis. One patient suffered a bleeding episode into the thigh with a 16-g/L decrease in her hemoglobin level while receiving ancrod therapy. No other side effects were noted. Our experience indicates that ancrod therapy is a reasonable approach for patients with heparin-induced thrombocytopenia who require anticoagulant therapy.
Topics: Aged; Aged, 80 and over; Ancrod; Anticoagulants; Female; Heparin; Humans; Male; Middle Aged; Thrombocytopenia; Thrombophlebitis
PubMed: 1932741
DOI: No ID Found -
British Journal of Cancer Oct 1991Flavone acetic acid (FAA) is a novel antitumour agent that has a profound effect on the vasculature in murine tumour models. Previously we have shown that FAA induces a...
Flavone acetic acid (FAA) is a novel antitumour agent that has a profound effect on the vasculature in murine tumour models. Previously we have shown that FAA induces a coagulopathy and thrombocytopaenia in tumour-bearing mice, and the purpose of the present study was to determine the significance of the FAA-induced intravascular coagulation in the antitumour action of FAA. Several anticoagulant agents were tested for their effectiveness in altering ex vivo coagulation of murine plasma; heparin and ancrod were found to be most effective. These agents were administered to tumour-bearing mice prior to FAA and TNF treatment with little effect on the induced regrowth delay. However: the FAA-induced consumption of platelets in tumour-bearing mice was not blocked by anticoagulant treatment. These data suggest that platelet consumption occurs independently of the normal coagulation pathway, and further that fibrin deposition may not be a major factor in the antitumour action of FAA.
Topics: Adenocarcinoma; Animals; Anticoagulants; Antineoplastic Agents; Blood Coagulation; Flavonoids; Male; Mice; Mice, Inbred CBA; Platelet Count; Prothrombin Time
PubMed: 1911218
DOI: 10.1038/bjc.1991.382 -
The American Journal of Pathology Dec 1990Recently evidence was provided for a pathway whereby circulating fibrinogen enters megakaryocyte granules by an endocytic mechanism. Synthesis of fibrinogen by...
Recently evidence was provided for a pathway whereby circulating fibrinogen enters megakaryocyte granules by an endocytic mechanism. Synthesis of fibrinogen by megakaryocytes has been reported. To determine the relationship between plasma fibrinogen and alpha-granule fibrinogen in megakaryocytes and platelets, the fibrinogen content of these cells was studied in rats defibrinated by use of Ancrod, a thrombinlike enzyme purified from the venom of Agkistrodon rhodostoma. Unlike thrombin, Ancrod does not induce platelet secretion. Rats were injected with Ancrod (50 units/kilogram body weight) at 8-hour intervals for 5 days. There were no significant changes in platelet counts. Blood from the treated rats failed to clot, and plasma fibrinogen levels were less than 15 mg/dl. Bone marrow from defibrinated rats and untreated control rats was stained immunohistochemically for fibrinogen and two other alpha-granule proteins, albumin and platelet factor 4 (PF4), in plastic-embedded sections. The presence of these three proteins in platelets was detected by Western blots. Only trace amounts of fibrinogen were detected in megakaryocytes and platelets from defibrinated rats, but fibrinogen in control megakaryocytes and platelets was readily demonstrated. However defibrinated and control rats did not differ in albumin and PF4 content in megakaryocytes and platelets. It is concluded that a major portion of rat platelet fibrinogen is derived from plasma by endocytosis by megakaryocytes.
Topics: Ancrod; Animals; Blood Platelets; Blotting, Western; Fibrin; Fibrinogen; Megakaryocytes; Platelet Count; Platelet Factor 4; Rats; Rats, Inbred Strains; Serum Albumin
PubMed: 2260627
DOI: No ID Found -
Kidney International Jan 1990The effect of ancrod, a defibrinating agent, on murine lupus glomerulonephritis in the male BXSB mouse was studied to determine the relationship between macrophage...
The effect of ancrod, a defibrinating agent, on murine lupus glomerulonephritis in the male BXSB mouse was studied to determine the relationship between macrophage procoagulant activity (PCA), fibrin deposition and glomerulonephritis. Marked renal disease and fibrin deposition were noted by three months of age in control mice, whereas little or no disease was seen in ancrod treated mice until five months of age. Similar high titers of anti-DNA antibodies and renal deposition of IgG were seen in both groups of mice. PCA rose with age in both ancrod treated and untreated mice, although it was significantly higher in control animals than in the ancrod treated group. Furthermore, ancrod therapy resulted in a decrease in plasma PCA inducing activity (PIF) and a decrease in the effectiveness of PIF to induce PCA in peritoneal macrophages in vitro. No mortality was observed in the 20 ancrod treated mice, whereas 10 of 20 control animals died. We conclude that defibrination with ancrod delays the development of renal fibrin deposition and glomerulonephritis and improves survival in BXSB mice. This was associated with a decrease in plasma PCA inducing activity and with an inhibitory effect on PCA induction. These results suggest that PCA contributes to injury in murine lupus glomerulonephritis by promoting fibrin deposition.
Topics: Ancrod; Animals; Blood Coagulation Factors; Fibrin; Fibrinogen; Lupus Nephritis; Macrophages; Male; Mice; Time Factors
PubMed: 2299806
DOI: 10.1038/ki.1990.4 -
Anesthesiology Dec 1989Heparin is the anticoagulant used during cardiopulmonary bypass (CPB). Both the use of heparin and the reversal of its effect with protamine have well-documented... (Comparative Study)
Comparative Study
Heparin is the anticoagulant used during cardiopulmonary bypass (CPB). Both the use of heparin and the reversal of its effect with protamine have well-documented complications. Ancrod is a defibrinogenating enzyme that has been used as an anticoagulant in humans, but its use as an anticoagulant for CPB has been limited to studies in animals. Twenty patients for elective aortocoronary bypass surgery were anticoagulated by means of an intravenous infusion of ancrod pre-operatively. Target plasma fibrinogen concentrations of 0.40-0.80 g/l were achieved within 13.3 +/- 2.5 h using an average dose of ancrod of 1.65 +/- 0.55 U/g. All perfusions were without incident. Postoperative blood loss (2286 +/- 1311 cc) was compared to that of 20 matched controls (1737 +/- 973 cc), as was blood product use; 4.1 +/- 2.1 U of packed cells versus 2.5 +/- 2.3 U (P less than 0.05) and 5.6 +/- 3.1 U of plasma versus 2.6 +/- 2.9 U (P less than 0.05) in the ancrod and heparin-treated groups, respectively. There were no differences in the postoperative courses or recovery periods of the ancrod-treated and control patients. This study confirms the efficacy and feasibility of ancrod as an alternative form of anticoagulation for CPB.
Topics: Adult; Aged; Ancrod; Anticoagulants; Cardiopulmonary Bypass; Female; Fibrinogen; Heparin; Humans; Male; Middle Aged; Platelet Count
PubMed: 2589675
DOI: 10.1097/00000542-198912000-00010 -
The Journal of Biological Chemistry Aug 1989Heparin cofactor II (HCII) is a highly specific serine proteinase inhibitor, which complexes covalently with thrombin in a reaction catalyzed by heparin and other...
Heparin cofactor II (HCII) is a highly specific serine proteinase inhibitor, which complexes covalently with thrombin in a reaction catalyzed by heparin and other polyanions. The molecular basis for the thrombin specificity may be explained by the identification here of a segment of HCII including residues 54-75 that binds to thrombin. A synthetic peptide, HCII(54-75), based on this segment of HCII, Gly-Glu-Glu-Asp-Asp-Asp-Tyr-Leu-Asp-Leu-Glu- Lys-Ile-Phe-Ala-Glu-Asp-Asp-Asp-Tyr-Ile-Asp inhibited thrombin's cleavage of fibrinogen. Clotting activity of thrombin was inhibited 50% at a concentration of 28 microM. Polyacrylamide gel electrophoresis showed that HCII(54-75) inhibited thrombin's cleavage of both the A alpha and B beta polypeptides in fibrinogen. However, the peptide did not block thrombin's active site, as hydrolysis of chromogenic substrates was not inhibited. HCII(54-75) probably binds to the same site on thrombin as do carboxyl-terminal residues of hirudins, thrombin inhibitors of leeches. HCII(54-75) inhibited binding of thrombin to a synthetic peptide corresponding to residues 54-66 of hirudin PA, but the hirudin peptide was about 30-fold more potent in binding and clotting assays. Both synthetic peptides, as a result of their polyanionic character, might be expected to stimulate the reaction of HCII with thrombin. However, the hirudin-related peptide inhibited this reaction, suggesting that it blocked a site on thrombin required for interaction with HCII. HCII(54-75) had a net stimulatory effect on the thrombin-HCII reaction as a consequence of its lower affinity for thrombin and greater negative charge relative to the hirudin-related peptide. These studies suggest that residues 54-75 of HCII interact with a noncatalytic binding site on thrombin and that this interaction contributes to efficient inhibition of thrombin by HCII.
Topics: Amino Acid Sequence; Ancrod; Antithrombins; Binding Sites; Drug Interactions; Fibrinogen; Glycoproteins; Heparin Cofactor II; Hirudins; Molecular Sequence Data; Peptide Fragments; Thrombin; Thrombin Time
PubMed: 2760054
DOI: No ID Found -
British Journal of Cancer Aug 1988Coumarins inhibit metastasis in a number of animal models, but the mechanism of this effect remains unclear. We have investigated the relationship between the...
Coumarins inhibit metastasis in a number of animal models, but the mechanism of this effect remains unclear. We have investigated the relationship between the coagulation system and metastasis using a new model system, involving i.v. injection of Mtln3 rat mammary carcinoma cells into Fischer 344 rats, and subsequent estimation of pulmonary seeding. Injection of factors II, VII, IX and X elevated the median number of surface pulmonary seedlings per animal to 182, and injection of factors II, IX and X to 181, compared with a median for control animals of 12 (P less than 0.001). Injection of factor VII alone, or of bovine serum albumin did not significantly affect pulmonary seeding. In a second experiment, arvin defibrination reduced the mean plasma fibrinogen concentration to 76.8 mg dl-1 from a control value of 228 mg dl-1. This degree of defibrination had no significant effects on pulmonary seeding, nor on the enhancing effects of factor complex injection (median numbers of seedlings per animal; control 15, arvin 21, arvin plus factors II, VII, IX and X 170, factors II, VII, IX and X only, 157). Factor complex injections did not detectably shorten thrombotest clotting times. In vitro testing suggested that Mtln3 cells contain little or no conventional factor X activating cancer procoagulant. The complex of coagulation factors II, IX and X appears to contain a component which greatly enhances metastasis in this model. This may explain the previously reported antimetastatic effect of coumarin anticoagulants, which suppress factors II, VII, IX and X. The enhancing effect of the factor complex does not appear to be altered by significant reductions in fibrin forming capacity, and defibrination itself has no effect on metastasis. These findings suggest the possibility that the effect of this factor complex on metastasis may be mediated via mechanisms other than the formation of a fibrin clot.
Topics: Ancrod; Animals; Anticoagulants; Blood Coagulation Factors; Female; Fibrin; Fibrinogen; Lung Neoplasms; Mammary Neoplasms, Experimental; Neoplasm Metastasis; Rats; Rats, Inbred F344; Tumor Cells, Cultured
PubMed: 3166906
DOI: 10.1038/bjc.1988.184