Crystallization and Preliminary Crystallographic Analysis of Antistasin, a Leech-derived Inhibitor of Blood Coagulation Factor Xa

Schreuder, Herman; Arkema, A.; DeBoer, Barry G.; Kalk, Kor H.; Dijkema, R.; Mulders, John W. M.; Theunissen, Henri J.M.; Hól, Wim G. J.

doi:10.1006/jmbi.1993.1360

Cited by 10 publications

(5 citation statements)

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“…Recombinant antistasin, produced in Chinese hamster ovary cells, was prepared and crystallized with ammonium sulfate and sodium chloride as precipitants as described before (Schreuder et al ., 1993; Theunissen et al ., 1994). Antistasin crystallizes in space group I 422 with cell dimensions a = b = 78.3 Å and c = 88.5 Å.…”

Section: Methodsmentioning

confidence: 99%

“…After Recombinant antistasin, produced in Chinese hamster ovary cells, was rigid-body refinement, the R-factor was 36.8%. Seven rounds of refineprepared and crystallized with ammonium sulfate and sodium chloride ment using X-PLOR and subsequent manual rebuilding of the model as precipitants as described before (Schreuder et al, 1993;Theunissen using O (Jones et al, 1991), resulted in a final R-factor of 21. 5% and a et al, 1994).…”

Section: Antistasin Preparation Crystallization and Data Collectionmentioning

confidence: 99%

“…The domains are spatially distant with no inter-and the atomic details of its interaction with factor Xa, we initiated the crystal structure determination of domain backbone-backbone interactions and only few side chain-main chain and side chain-side chain inter-antistasin. A preliminary crystallographic analysis has been published previously (Schreuder et al, 1993). Here, actions.…”

Section: Overall Structurementioning

confidence: 99%

“…To investigate the mechanism of action of antistasin and the atomic details of its interaction with factor Xa, we initiated the crystal structure determination of antistasin. A preliminary crystallographic analysis has been published previously (Schreuder et al ., 1993). Here, we report the 3D‐structure of antistasin at 1.9 Å resolution and suggest a model of the antistasin‐factor Xa complex, based on the antistasin and factor Xa crystal structures.…”

Section: Introductionmentioning

confidence: 99%

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X-ray structure of antistasin at 1.9Aresolution and its modelled complex with blood coagulation factorXa

et al. 1997

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The three-dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 A resolution by X-ray crystallography. The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55-residue protease inhibitor. However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two beta-strands, and does not inhibit factor Xa. The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations. Consequently, the domain shapes are different, the N-terminal domain being wedge-shaped and the C-terminal domain flat. Docking studies suggest that differences in domain shape enable the N-terminal, but not C-terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site. Furthermore, a putative exosite binding region could be defined in the N-terminal domain of antistasin, comprising residues 15-17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa. In the C-terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain.

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Section: Methodsmentioning

confidence: 99%

Section: Antistasin Preparation Crystallization and Data Collectionmentioning

confidence: 99%

Section: Overall Structurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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X-ray structure of antistasin at 1.9Aresolution and its modelled complex with blood coagulation factorXa

et al. 1997

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“…43 Antistasin, a 119 amino acid protein isolated from the salivary glands of the Mexican proboscisfeeding leech Haementeria officinalis is a potent inhibitor of coagulation factor Xa (K i 0.4 nM) and metastasis. [44][45][46][47][48][49] It consists of two homologous domains. Recombinant antistasin has been produced 50 and its anticoagulant potential has been compared with that of hirudin.…”

Section: Coagulation Inhibitorsmentioning

confidence: 99%

Platelet Aggregation and Coagulation Inhibitors in Leech Saliva and Their Roles in Leech Therapy

Rigbi¹,

Orevi²,

Eldor³

1996

Semin Thromb Hemost

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Prolonged bleeding by the host after the leech ceases to feed and several reports that the use of leeches restores blood flow in the microcirculation after plastic surgery led us to search for inhibitors of platelet aggregation in Hirudo medicinalis saliva. Dilute leech saliva was collected by phagostimulating starved leeches with a solution of arginine in saline. The saliva is shown to inhibit human platelet aggregation induced by thrombin, collagen, adenosine diphosphate (ADP), epinephrine, platelet activating factor (1-O-alkyl-2-acetyl-sn-3-glycerophophoryl choline [PAF]), and arachidonic acid. We have isolated the PAF inhibitor and found it to be an amphipathic phosphoglyceride. We have also purified apyrase adenosine triphosphate ([ATP] diphosphohydrolase), which inhibits ADP-induced platelet aggregation, and have described collagenase. Besides well-known hirudin, Hirudo saliva contains a potent inhibitor of coagulation factor Xa. We also report antiaggregant and anticoagulant activities in the crop content of the closely related Nile leech, Limnatis nilotica. Anticoagulants of hematophagous species are surveyed. We have used medicinal leeches in plastic surgery for decompression of skin flaps and in patients with postphlebitic syndrome and peripheral arterial occlusions. Preliminary results indicate certain beneficial effects of leech therapy.

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Structural model of an antistasin/notch-like fusion protein from the cocoon wall of the aquatic leech, Theromyzon tessulatum

2006

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The aquatic leech, Theromyzon tessulatum, secretes a proteinaceous cocoon with extraordinary physical properties (e.g., proteolytic, thermal resiliency). The deduced amino acid sequence of a major protein (Tcp-Theromyzon cocoon protein) from the T. tessulatum cocoon wall has been used to model the endogenous structure of the Tcp protein. The Tcp protein sequence comprises six internal repeats, each containing 12 ordered Cys residues. Amino acid alignments suggest that the region Cys1-->6 is homologous to antistasin, a leech anticoagulant, and Cys7-->12 is homologous to an epidermal growth factor-like domain found in notch-class proteins, which play critical roles in development, signaling, and adhesion throughout the Animalia. Modeling of individual domains (i.e., antistasin and notch) positions multiple hydrophobic and charged residues on the surface. When the antistasin and notch domains were fused, hydrophobic pockets appeared that may facilitate a polymerization mechanism. Collectively, the predicted features of our Tcp model are consistent with the physical properties of the leech cocoon wall.

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Crystallization and Preliminary Crystallographic Analysis of Antistasin, a Leech-derived Inhibitor of Blood Coagulation Factor Xa

Cited by 10 publications

References 0 publications

X-ray structure of antistasin at 1.9Aresolution and its modelled complex with blood coagulation factorXa

X-ray structure of antistasin at 1.9Aresolution and its modelled complex with blood coagulation factorXa

Platelet Aggregation and Coagulation Inhibitors in Leech Saliva and Their Roles in Leech Therapy

Structural model of an antistasin/notch-like fusion protein from the cocoon wall of the aquatic leech, Theromyzon tessulatum

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