Anticoagulant peptides. Nature of the interaction of the C-terminal region of hirudin with a noncatalytic binding site on thrombin

Krstenansky, John L.; Owen, Thomas J.; Yates, Mark T.; Mao, Simon J.T.

doi:10.1021/jm00392a030

Cited by 60 publications

(28 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…4), but also in several other thrombin-inhibiting peptides like anophelin of Anopheles mosquitos (Figueiredo et al 2012) and variegin of Amblyomma variegatum, the tropical bont tick ). In addition to Phe56, the nonpolar residues Ile59, Pro60, and Leu64 are crucial for hydrophobic interactions between the C-terminal tail of hirudin and thrombin (Krstenansky et al 1987;Betz et al 1991;Priestle et al 1993). None of these nonpolar amino acid residues is present in any of the HLFs (Fig.…”

Section: Discussionmentioning

confidence: 99%

More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech, Hirudo medicinalis

et al. 2015

View full text Add to dashboard Cite

Blood-sucking leeches like the medicinal leech, Hirudo medicinalis, have been used for medical purposes since ancient times. During feeding, medicinal leeches transfer a broad range of bioactive substances into the host's wound to prevent premature hemostasis and blood coagulation. Hirudin is probably the best known of these substances. Despite its long history of investigation, recombinant production and clinical use, there still exist conflicting data regarding the primary structure of hirudin. Entirely unclear is the potential biological significance of three different subtypes and many isoforms of hirudins that have been characterized so far. Furthermore, there is only incomplete information on their cDNA sequences and no information at all on gene structures and DNA sequences are available in the databases. Our efforts to fill these gaps revealed the presence of multiple hirudin-encoding genes in the genome of Hirudo medicinalis. We have strong evidence for the expression of all three subtypes of hirudin within individual leeches and for the expression of additional hirudins or hirudin-like factors that may have different biological functions and may be promising candidates for new drugs.

show abstract

Section: Discussionmentioning

confidence: 99%

More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech, Hirudo medicinalis

et al. 2015

View full text Add to dashboard Cite

show abstract

“…42 These indicate that the role of residues 5419 / 8k20$$5419 03-18-97 19:51:34 bpal W: Biopolymers 50 ps, respectively, were performed in order to anastructural changes occur. Nonbonded interactions energies, i.e., the sum of electrostatic and van der lyze the structures, and to estimate the precision of the structure determination.…”

Section: Dynamics Analysismentioning

confidence: 98%

“…8,10 Mutagenesis studies indicate that the residues Phe56 to the Gln65 of hirudin are most important for interaction with thrombin. 42 These features are structural determinants of the implicated different mode of action of the class of hirudin inhibitors.…”

Section: Implications To Activitymentioning

confidence: 99%

NMR solution structure of a novel hirudin variant HM2, N-terminal 1-47 and N64 → V + G mutant

Nicastro¹,

Baumer²,

Bolis³

et al. 1997

Biopolymers

View full text Add to dashboard Cite

The 64 amino acid hirudin‐like peptide HM2 (Hirudinaria manillensis) is one of the agents known to specifically block the blood‐clotting enzyme thrombin, and therefore is used as a potential pharmacological tool for the treatment of arterial and venous thrombosis. This peptide and its derivatives provide a new set of probes for studies aimed at elucidating the structural basis of the inhibition of α‐thrombin. We used 581, 699, and 492 nmr‐derived constraints respectively in a protocol employing simulated annealing, followed by restrained molecular dynamics and restrained energy minimization to derive the three‐dimensional structures of HM2 and its mutants the HM2(V + G) and the HM2(1–47). HM2 consists of a well‐defined core region of two double‐stranded β‐sheet and a disordered C‐terminus. These features are shared by other members of the hirudin family. The same type of folding has also been observed for recombinant hirudins whose structure has been determined in solution by nmr spectroscopy and in the structure of the complex hirudin‐thrombin determined by x‐ray diffraction. Molecular dynamics (MD) simulation methods were applied in the study of the structural and dynamic fluctuation properties of the hirudin derivatives solvated by 1625 and 1276 water molecules with periodic boundary conditions for HM2 and HM2(1–47), respectively. Trajectories of 100 and 50 ps for the two unconstrained systems were generated at constant temperature and pressure. Analysis of the MD simulation shows that the structure of the peptide core is fairly rigid and stable in itself while the conformation of the C‐terminal tail, which is involved in the inhibitory mechanism of thrombin, fluctuates and appears as a disordered region. © 1997 John Wiley & Sons, Inc. Biopoly 41: 731–749, 1997

show abstract

“…Many variants of the natural hirudin mol ecule have been introduced [10][11][12], Beside the complete hirudin molecule, peptides, hirudin fragments, and mutants are being developed. Recombinant technology has greatly added to these developments.…”

Section: Variants Of the Hirudin Moleculementioning

confidence: 99%

Laboratory Assays for the Evaluation of Recombinant Hirudin

Walenga

Hoppensteadt²,

Koza³

et al. 1991

Pathophysiol Haemos Thromb

View full text Add to dashboard Cite

Several laboratory methods are available to measure the anticoagulant activity of recombinant hirudin (r-hirudin), a potent thrombin inhibitor. These assays include clot-based, amidolytic, immunologic and physicochemical techniques. Although r-hirudin, like heparin, is an effective anticoagulant, the mechanism of action of the two agents is different. Thus it is not surprising that the global tests, such as the prothrombin time (PT), partial thromboplastin time (APTT) and the Heptest® (Haemachem, Inc., St. Louis, Mo., USA), do not show adequate responses to r-hirudin. In the range of 0.5–10.0 μg/ml, where full anti-coagulation is achieved, as determined by animal models of thrombosis, these assays show little to no prolongation of the time to clot. In order to find a more suitable assay system, modifications of the above assays were evaluated. The diluted APTT and diluted Heptest showed linear concentration-dependent responses to lower levels of r-hirudin with an enhanced sensitivity than that of the classical assays. On the other hand, the diluted thrombin time was too sensitive. Whole-blood clotting assays, ACT® and thrombelastograph, effectively measured r-hirudin levels up to 25 μg/ml. The amidolytic anti-factor IIa assay, specific for evaluating direct thrombin inhibition, was very effective particularly when modified to decrease the sample:thrombin ratio. This assay may be useful in quality control since it is biochemically defined, and reagents are easily standardized. The relevance of the results of the anti-IIa assay to clinical conditions, however, remains to be determined. Thrombin generation assays have limited value in monitoring the anticoagulant effect of r-hirudin since the effect of thrombin inhibition by r-hirudin on coagulation feedback mechanisms, and thus the effect on thrombin generation, appears to be minimal. Immunologic methods such as ELISA and RIA are under development, but they may only be useful for the direct quantitation of absolute levels of r-hirudin and not for monitoring the clinical anticoagulant action. Furthermore, these assays are only sensitive to sub-μg/ml levels. Therefore, thrombin-based clotting and amidolytic assays may at present be the best choice for evaluating the functional, clinical antithrombotic effects of r-hirudin.

show abstract

Anticoagulant peptides. Nature of the interaction of the C-terminal region of hirudin with a noncatalytic binding site on thrombin

Cited by 60 publications

References 0 publications

More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech, Hirudo medicinalis

More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech, Hirudo medicinalis

NMR solution structure of a novel hirudin variant HM2, N-terminal 1-47 and N64 → V + G mutant

Laboratory Assays for the Evaluation of Recombinant Hirudin

Contact Info

Product

Resources

About