Hirudin and other thrombin inhibitors experimental results and potential clinical applications

Badimón, Lina; Merino, Álvaro; Badimón, Juan J.; Chesebro, James H.; Fuster, Valentín

doi:10.1016/1050-1738(91)90032-a

Cited by 19 publications

(11 citation statements)

References 20 publications

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“…Unfortunately, the thrombus-bound thrombin is poorly accessible to heparin (Hogg & Jackson, 1989;Weitz et al, 1990). In contrast, small active site inhibitors such as MD-805l (Kikumoto et al, 1980b) and benzamidine derivatives Hauptmann et al, 1980;Krupinsky et al, 1989) effectively inhibit thrombin trapped in the thrombus (Badimon et al, 1991). A major disadvantage that applies to most of these active-site-directed synthetic inhibitors is their short half-life of a few minutes (Badimon et al, 1991).…”

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confidence: 99%

“…In addition, it has regulatory functions in the activation of several proteases such as factor V, factor VIII, factor XIII, and protein C in the coagulation cascade (Fenton, 1981) as well as platelets (Fenton & Bing, 1986). However, beside these important physiological roles, thrombin is known to have severe adverse effects, specially in myocardial infarctus disorders (Badimon et al, 1991). Effectively an acute blockage of coronary arteries by a thrombus almost always leads to myocardial infarction.…”

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confidence: 99%

“…A major disadvantage that applies to most of these active-site-directed synthetic inhibitors is their short half-life of a few minutes (Badimon et al, 1991). Since thrombolytic therapy activates the coagulation cascade resulting in continuous production of thrombin, the inhibitors Abbreviations: y Abu, 4-arrlinobutyric acid; Ac, acetyl; Acha, 1 -aminocyclohexanecarboxylic acid; pAdod, 12-aminododecanoic acid; AMC, 7-amind-4-methylcoumarin; Bbs,Boc,Cha,Fmoc,renylmethoxycarbonyl; FRE, fibrinogen recognition exo site; HPLC, high-performance liquid chromatography; MD-805, (2R,4R)-4-methyll-[Nu-(3-methyl-l,2,3,4-tetrahydro-8-quinolinesulfonyl)-~-arginyl]-2-piperidinecarboxylic acid; NAPAP, Nu-(2-naphthylsulfonylglycyl)-~,~-p-amidinophenylaliinylpiperidine; 1-Nas, 1-naphthalenesulfonyl; 2-Nas, 2-naphthalenesulfonyl; Nle, norleucine; OBzl, benzyl ester; Pip, pipecolic acid; PPACK, D-Phe-Pro-Arg chloromethyketone; 3-TAPAP, Na- with short half-lives may not be effective agents (Badimon et al, 1991).…”

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confidence: 99%

“…Hirudin is a small protein of 65 amino acids that has following potential advantages over other anticoagulants: (1) it is the most potent and specific thrombin inhibitor with a K, value of 2.2 x M (Stone & Hofsteenge, 1986), (2) it blocks the active site and the FRE of thrombin simultaneously (Rydel et al, 1990(Rydel et al, & 1991, (3) it inhibits thrombusbound thrombin as well as free thrombin in solution (Badimon et al, 1991), (4) it has a long half-life of 50 min when given intravenously or subcutaneously (Markwardt et al, 1984), (5) its antigenicity is very weak, and (6) it has no acute side effects.…”

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Design of Potent Bivalent Thrombin Inhibitors Based on Hirudin Sequence: Incorporation of Nonsubstrate-Type Active Site Inhibitors

Tsuda¹,

Cygler²,

Gibbs³

et al. 1994

Biochemistry

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Hirudin from medicinal leech is the most potent and specific thrombin inhibitor from medicinal leech with a K, value of 2.2 x M. It consists of an active site blocking moiety, hirudin'-48, a fibrinogen-recognition exo-site binding moiety, hir~din~~-~~, and a linker, hir~din~~-~~, connecting these inhibitor moieties. Synthetic inhibitors were designed based on the C-terminal portion of hirudin. The bulky active site blocking moiety, was replaced by small nonsubstrate-type active site inhibitors of thrombin, e.g., dansyl-Arg-(D-pipecolic acid). The linker moiety was replaced by o-amino acids of (12-aminododecanoic acid)-(4-aminobutyric acid), and hirudi~P-~~ was used as a fibrinogen-recognition exo-site binding moiety in most of the inhibitors. The crystal structure of the inhibitor in complex with human a-thrombin showed that dansyl, Arg, and D-pipecolic acid of the active site blocking moiety occupy S3, S1, and S2 subsites of thrombin, respectively, and were therefore designated as P3, P1, and P2 residues.The use of dansyl-Arg-(D-pipecolic acid) improved the affinity (KJ of the inhibitor 10-100-fold (down to 1.70 x lo-" M) compared to that of the similar compounds having D-Phe-Pro-Arg as their substratetype inhibitor moiety (K = 10-9-10-10 M). The linker connected to P2 residue eliminated the scissile peptide bond. The inhibitor was also stable against human plasma proteases. Further inhibitor design revealed that the toxic dansyl group could be replaced by 4-tert-butylbenzenesulfonyl group and 1-or 2-naphthalenesulfonyl group for in vivo studies. In addition, the replacement of hir~din~~-~~ with [TY~~~,-

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Design of Potent Bivalent Thrombin Inhibitors Based on Hirudin Sequence: Incorporation of Nonsubstrate-Type Active Site Inhibitors

Tsuda¹,

Cygler²,

Gibbs³

et al. 1994

Biochemistry

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“…As anticoagulant we chose hirudin which was linked to polyethyleneglycol (PEG-hirudin) to increase its molecular weight from 10 000 to 17 000 and therefore to prevent it from passing across the dialysis membrane and being excreted rapidly by the kidneys (33). Furthermore, it was shown that hirudin achieves more stable and reproducible plasma levels with less inter-individual variability than heparin (38). Hirudin is a homogenous substance of one chemical structure which acts at a single target (thrombin), whereas heparin is a heterogeneous mixture which exerts its anticoagulant effect through catalysis of a number of enzymatic reactions.…”

Section: Resultsmentioning

confidence: 99%

Continuous Analysis in Extracorporeally Circulating Blood - A Rat Model Applying Flow-Through Ion-Selective Electrodes for the Measurement of Ca2+, K+, Na+ and pH

Heidrich¹,

Niemeier²,

Seyfarth³

1998

CCLM

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A rat model is introduced which enables investigations in anticoagulated blood with continuous measurements by a flow-through electrode system. In the present study, a potentiometric ion-selective electrode (ISE)-system was used for measuring Ca2+, K+, Na+ and pH in rats. The setup was adjusted to an extracorporeal blood-volume of 0.750 ml. This permits indirect measurements of the analytes via a dialysis membrane, with electrical separation of the ISE's and the animal. The flow-rates of blood and dialysis-solution were adjusted in such a way that water diffusing from the aqueous dialysis solution into the blood, across the dialysis membrane, does not alter the haematocrit. Polyethyleneglycol-hirudin was used for anticoagulation, since it was superior to heparin. The assembly enables continuous measurements in the living anaesthetized rat over a time period of at least 3 hours.

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Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human α‐thrombin

et al. 1996

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The crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human alpha-thrombin have been determined at 2.0 A resolution and refined to crystallographic R-factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl-Arg-(D)Pipecolic acid (Dns-Arg-(D)Pip), reminiscent of the active-site directed inhibitors MD-805 and MQPA, rendering them resistant to thrombin-induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55-65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin-MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring-ring system interactions. Unlike in the substrate-like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well-ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C-terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes.

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Hirudin and other thrombin inhibitors experimental results and potential clinical applications

Cited by 19 publications

References 20 publications

Design of Potent Bivalent Thrombin Inhibitors Based on Hirudin Sequence: Incorporation of Nonsubstrate-Type Active Site Inhibitors

Design of Potent Bivalent Thrombin Inhibitors Based on Hirudin Sequence: Incorporation of Nonsubstrate-Type Active Site Inhibitors

Continuous Analysis in Extracorporeally Circulating Blood - A Rat Model Applying Flow-Through Ion-Selective Electrodes for the Measurement of Ca2+, K+, Na+ and pH

Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human α‐thrombin

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