Purification and characterization of recombinant antistasin: A leech-derived inhibitor of coagulation factor Xa

Nutt, Elka M.; Jain, Deepak Kumar; Lenny, Albert B.; Schaffer, L. W.; Siegl, P. K. S.; Dunwiddie, Christopher T.

doi:10.1016/0003-9861(91)90325-d

Cited by 71 publications

(53 citation statements)

References 29 publications

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“…This could be achieved through inhibition of factor Xa, the enzyme directly responsible for thrombin activation. Inhibition of factor Xa interrupts both the extrinsic and intrinsic pathways of thrombin production, whilst allowing the thrombin catalyzed activation of protein C. The efficacy of factor Xa inhibition is demonstrated by the anticoagulant properties of the factor Xa specific inhibitors antistasin [6] and tick anticoagulant peptide [7] derived from haematophagous parasites.…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

1995

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Crystal structures of DX9065a and a related bisamidino-aryl inhibitor specific for the blood-clotting factor Xa have been solved in complex with bovine ~-trypsin to a resolution of 1.9 A. Each inhibitor exhibits an extended conformation along the active site, in contrast to the compact folded structures observed for thrombin specific inhibiturs. Few direct contacts (predominantly in the S1 pocke0 are made between trypsin and the inhibitors. Transfer of the inhibitors to the active site of factor Xa suggests a three-site interaction: salt bridge formation at the base of the primary specificity pocket, extensive hydrophobie surface burial and a weak electrostatic interaction between the distal basic component of the inhibitor and an electronegative cavity of factor Xa formed by three backbone carbonyl oxygens. Additivity of these three interactions is the basis for the observed strong inhibition of factor Xa and provides a framework for the design of novel factor Xa inhibitors. A propionic acid group of the inhibitor would clash with the thrombin specific '60-insertion loop', thus conferring selectivity against thrombin.

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Section: Introductionmentioning

confidence: 99%

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

1995

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“…Characterization of Truncated Forms of Antistasin-Fulllength antistasin (ATS-119), an antistasin derivative truncated at residue 116 (ATS-116), and an antistasin derivative truncated at residue 112 (ATS-112) were purified from secreted cell-free products of insect, yeast, and African green monkey kidney cells, respectively, in which recombinant antistasin was expressed (8,10). The molecular weights of the ATS polypeptides and/or the amino acid sequences of carboxyl-terminal tryptic peptides established the identity of each form of ATS (Table I).…”

Section: Resultsmentioning

confidence: 99%

“…Intact antistasin had been successfully expressed in the insect baculovirus host (8). The present study, wherein fulllength and two truncated antistasins (from yeast and an African green monkey kidney cell line) are characterized, suggests that the carboxyl-terminal domain of antistasin, which has little influence on inhibitory potency toward isolated fXa, is important for the anticoagulant activity of antistasin.…”

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confidence: 78%

“…Concentrations of synthetic peptides were determined by quantitative amino acid analysis. Full-length antistasin (ATS-119) and antistasin truncated at residue 116 (ATS-116) were purified using previously described methods from insect and yeast, respectively, that were modified to express recombinant antistasin (8,10). Antistasin truncated at residue 112 (ATS-112) was expressed in the African green monkey kidney CV-1p cell line by transfection with a pBR322-based plasmid containing human immunodeficiency virus-long terminal repeat promoter, immunoglobulin signal, antistasin cDNA, and SV40 * The costs of publication of this article were defrayed in part by the payment of page charges.…”

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confidence: 99%

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Selective Inhibition of Factor Xa in the Prothrombinase Complex by the Carboxyl-terminal Domain of Antistasin

Mao¹,

Przysiecki²,

Krueger³

et al. 1998

Journal of Biological Chemistry

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Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2-6-fold more potently and 2-3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.Factor Xa (fXa), 1 a serine protease that functions at the intersection of the intrinsic and extrinsic pathway for blood coagulation, activates prothrombin to thrombin. Thrombin in turn activates platelets and converts the soluble plasma protein fibrinogen to the insoluble fibrin matrix of blood clots. The central role of fXa in blood coagulation suggests that fXa inhibitors might have therapeutic utility as anticoagulants. It is important to note, however, that fXa functions in the blood coagulation cascade in a complex with calcium ion, factor Va (fVa) and an appropriate phospholipid membrane, and that the catalytic activity toward prothrombin of this prothrombinase complex is several orders of magnitude greater than that of isolated fXa (1, 2). These observations suggest that certain inhibitors might exhibit different inhibitory potencies toward fXa in the prothrombinase complex and isolated fXa.

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“…the literature data from the middle and the end of the 90 ties of last century is abundant with newly discovered, isolated and characterized natural polypeptides and proteins that are inhibitors of the serine proteinases. For example, the year 1995 is rich in literature data about the inhibitors of the coagulation serine proteinases: (69,70,71,85). Kinetic studies reveal that antistasin is a potent, slow, tight-binding Factor Xa inhibitor (37,38,57,58).…”

Section: Synthetic and Natural Peptides As Antithrombotic Agents -A Vmentioning

confidence: 99%

Synthetic and Natural Peptides as Antithrombotic Agents—A View on the Current Development

Atanassov

Tchorbanov

2009

Biotechnology & Biotechnological Equipment

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Purification and characterization of recombinant antistasin: A leech-derived inhibitor of coagulation factor Xa

Cited by 71 publications

References 29 publications

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

Selective Inhibition of Factor Xa in the Prothrombinase Complex by the Carboxyl-terminal Domain of Antistasin

Synthetic and Natural Peptides as Antithrombotic Agents—A View on the Current Development

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