A heparin binding site in antithrombin III. Identification, purification, and amino acid sequence.

Chemical modification of antithrombin III with the tryptophan reagent, dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl (HNB) moiety per molecule of antithrombin III. Heparin protects against tryptophan modification, particularly at low reagent concentrations. Unlike native antithrombin, which has high affinity for heparin, HNB-anti- thrombin does not bind to a heparin-agarose affinity column. Furthermore, the heparin-induced increase in tryptophan fluorescence, obtained with native antithrombin, is not observed with the singly modified inhibitor. HNB-anti- thrombin does not exhibit heparin-promoted rate enhancement in the inactivation of thrombin and Factor Xa. However, in the absence of heparin, HNB-antithrombin and native antithrombin possess progressive antithrombin activity, inactivating these proteases at identical rates. These results indicate that the integrity of a specific tryptophan residue is required for the binding of heparin to antithrombin III. Chemical and enzymatic cleavage techniques have been used to isolate peptides containing this tryptophan from both HNB-labeled and native antithrombin and to identify this critical tryptophan residue within the amino acid sequence of the antithrombin molecule.

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Crotalase, a Fibrinogen-Clotting Snake Venom Enzyme: Primary Structure and Evidence for a Fibrinogen Recognition Exosite Different from Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614423 ◽

1999 ◽

Vol 81 (01) ◽

pp. 81-86 ◽

Cited By ~ 19

Author(s):

Agnes Henschen-Edman ◽

Ida Theodor ◽

Brian Edwards ◽

Hubert Pirkle

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

Amino Acid Sequence ◽

Spatial Modeling ◽

Glycosylation Site ◽

Amino Sugars ◽

Edman Degradation ◽

Heparin Binding ◽

Heparin Binding Site ◽

Total Molecular Weight

SummaryCrotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradation. The resulting amino acid sequence, VIGGDEC NINEHRFLVALYDYWSQLFLCGGTLINNEWVLTAAHCDRTHI LIYVGVHDRSVQFDKEQRRFPKEKYFFDCSNNFTKWDKDIM LIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRAMGWGQTTSPQET LPDVPHCANINLLDYEVCRTAHPQFRLPATSRTLCAGVLEG GIDTCNRDSGGPLICNGQFQGIVFWGPDPCAQPDKPGLYTK VFDHLDWIQSIIAGEKTVNCP, is characteristic of a serine protein-ase. Comparison with thrombin, the physiological fibrinogen-clotting enzyme, showed that thrombin’s fibrinogen-recognition exosite (FRE) is poorly represented in crotalase. Hirudin, a FRE-dependent inhibitor, had no effect on crotalase. Spatial modeling of crotalase yielded a possible alternative fibrinogen-recognition site comprised of Arg 60F, Lys 85, Lys 87, and Arg 107 (underlined in the sequence above). Crotalase also lacks thrombin’s YPPW loop, as well as its functionally important ETW 146-148, and its heparin-binding site. The enzyme contains a single asparagine-linked glycosylation site, NFT, bearing neutral and amino sugars that account for 8.3% of the enzyme’s total molecular weight of 29,027. The calculated absorbance of crotalase at 280 nm, 1%, cm-1is 15.2.

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Heparin binding affinity of normal and genetically modified antithrombin III measured using a monoclonal antibody to the heparin binding site of antithrombin III

Biochemistry ◽

10.1021/bi00079a027 ◽

1993 ◽

Vol 32 (28) ◽

pp. 7286-7293 ◽

Cited By ~ 15

Author(s):

Jane Watton ◽

C. Longstaff ◽

D. A. Lane ◽

T. W. Barrowcliffe

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Binding Affinity ◽

Antithrombin Iii ◽

Genetically Modified ◽

Heparin Binding ◽

Heparin Binding Site

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The Asp272–Glu282Region of Platelet Glycoprotein Ibα Interacts with the Heparin-binding Site of α-Thrombin and Protects the Enzyme from the Heparin-catalyzed Inhibition by Antithrombin III

Journal of Biological Chemistry ◽

10.1074/jbc.275.6.3887 ◽

2000 ◽

Vol 275 (6) ◽

pp. 3887-3895 ◽

Cited By ~ 38

Author(s):

Raimondo De Cristofaro ◽

Erica De Candia ◽

Sergio Rutella ◽

Jeffrey I. Weitz

Keyword(s):

Binding Site ◽

Antithrombin Iii ◽

Platelet Glycoprotein ◽

Heparin Binding ◽

Heparin Binding Site

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Pulmonary arterial thrombosis in a neonate with homozygous deficiency of antithrombin III: successful outcome following pulmonary thrombectomy and infusions of antithrombin III concentrate

Cardiology in the Young ◽

10.1017/s1047951100009240 ◽

2000 ◽

Vol 10 (3) ◽

pp. 275-278 ◽

Cited By ~ 5

Author(s):

K. Roman ◽

E. Rosenthal ◽

R. Razavi

Keyword(s):

Binding Site ◽

Arterial Thrombosis ◽

Antithrombin Iii ◽

Pulmonary Trunk ◽

Successful Outcome ◽

Heparin Binding ◽

The Third ◽

Heparin Binding Site ◽

Surgical Thrombectomy ◽

Pulmonary Arterial

AbstractWe report a newborn male who presented with severe central cyanosis on the third day of life. Partial thrombotic obstruction of the pulmonary trunk secondary to Antithrombin III (homozygous defect of heparin binding site) deficiency was subsequently diagnosed. Surgical thrombectomy, and infusions of Antithrombin III concentrate, led to a successful outcome. We postulate that intrauterine thrombosis occurred to give this unusual presentation.

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