Thrombin-specific inhibition by and slow cleavage of hirulog-1

Witting, Joyce I.; Bourdon, Paul; Brezniak, Diane V.; Maraganore, John M.; Fenton, John W.

doi:10.1042/bj2830737

Cited by 108 publications

(74 citation statements)

References 53 publications

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“…Thrombin inhibitors are exposed to three types of proteases in vivo (1) thrombin, which forms complexes with the inhibitors and may hydrolyze them (DiMaio et al, 1990;Witting et al, 1992a;Szewczuk et al, 1993), (2) plasma proteases, which the inhibitors encounter in the circulation, and (3) proteases of the kidney, which are involved in the clearance of the peptides. The proteolytic stability of the inhibitor P448, dansyl-Arg-(D-Pip)-pAdodyAbu-DFEEIPEEYLQ-OH, was examined.…”

Section: Activementioning

confidence: 99%

“…The crystal structure of PPACK-thrombin (Bode et al, 1989) suggested that D-PhePro-Arg in the bivalent inhibitors binds to the thrombin active site in the substrate binding mode, where Arg-X is the scissile peptide bond. In fact, the active site blocking moiety, D-Phe-Pro-Arg-Pro, of the bivalent inhibitors was hydrolyzed slowly at Arg-Pro peptide bond (DiMaio et al, 1990;Witting et al, 1992a;Szewczuk et al,1993). The arginine and benzamidine-based thrombin inhibitors such as MD-805, NAPAP, and TAPAP are other types of active site inhibitors (Okamoto et al, 1980(Okamoto et al, , 1981Kikumoto et al, 1980aKikumoto et al, ,b, 1984Stiirzebecher et al, 1983Stiirzebecher et al, , 1984.…”

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confidence: 99%

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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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Section: Activementioning

confidence: 99%

mentioning

confidence: 99%

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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“…The Ki and Kd values were determined for a-thrombin by tight binding and consumptive inhibitor methods, as described (22,23).…”

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confidence: 99%

High-affinity alpha-thrombin binding to platelet glycoprotein Ib alpha: identification of two binding domains.

Gralnick¹,

Williams²,

McKeown³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The platelet thrombin binding site(s) has been a controversial issue. Vu and coworkers (4-6) have cloned a platelet thrombin receptor which is a member of the 7-transmembrane loop-receptor family. This receptor is cleaved by thrombin between residues R41 and S42, exposing a new amino-terminal peptide, which serves as a tethered ligand whose binding site resides within the first six amino acids. This tethered ligand peptide binds to an undefined site that induces receptor activation (7-9).Prior to the discovery and cloning of the seven-transmembrane loop receptor, several studies had defined platelet glycoprotein lb (GPIb) as an a-thrombin receptor. These studies described the binding of thrombin to GPIb and the inhibition of a-thrombin binding to GPIb by glycocalicin (a proteolytic fragment of GPIba) and monoclonal antibodies directed against . The decrease or absence of high-affinity a-thrombin binding to Bernard-Soulier platelets, which lack GP~ba (17), provided further evidence that GPIb was a thrombin receptor. In experiments using crosslinking of a-thrombin to human platelets, the only identifiable site of binding was GPIb (18,19).The proposed roles of GPIba in thrombin binding include acting as a high-affinity receptor for localizing a-thrombin near the cell surface or serving as a site for excess thrombin binding (20). In this scheme, GPIba acts primarily as a neutralizing site for excess thrombin generation or as an independent modulator of platelet function through an undefined mechanism. We have studied the high-affinity binding of a-thrombin to human platelets and have examined the inhibitory effects of synthetic peptides derived from portions of the sequence of the GPIba chain and from the seventransmembrane loop thrombin receptor in order to assess the site(s) of high-and moderate-affinity thrombin binding to platelets and thrombin-induced platelet aggregation.MATERIALS AND METHODS Platelet Preparation. Blood was obtained by venipuncture using a two-syringe technique. The blood from the second syringe was placed into polypropylene tubes containing 0.1 ml of sodium citrate anticoagulant, final concentration 10.9 pM. Platelet-rich plasma was prepared from whole blood after centrifugation at 750 x g for 3 min at 250C. Then 6-10 ml of platelet-rich plasma was placed on a discontinuous Larcoll (Sigma) gradient, 20%o (3 ml) and 10% (5 ml), and the platelets were separated from the plasma proteins by centrifugation at 2000 x g for 30 min. The platelets were then resuspended in Hepes buffer at pH 7.35. All platelets were used within 2 hr of being obtained.Thrombin Lalin. Human a-thrombin was prepared and radiolabeled as described (21). Two hundred fifty microliters of a-thrombin solution (1 pg/2 units of activity) was treated with 5 Ad oflactoperoxidase beads, 5 td of0.5 mM KI, 0.5 mCi (18.5 MBq) of Na125I, and 1 A4 of 0.015% H202 for 5 min at room temperature. Free 125I was separated from bound 125I on a 10-ml column of Sephadex G-25 fine equilibrated with 0.05 M Tris/0.1 M NaCl at pH 7.35. Fractions were ...

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“…98 Unlike hirudin, bivalirudin produces only transient inhibition of the active site of thrombin, because, once bound, thrombin cleaves the Pro-Arg bond within the amino-terminal of bivalirudin. 99,100 Without its aminoterminal segment, the carboxy-terminal portion of bivalirudin bound to thrombin's substrate recognition site is a much weaker thrombin inhibitor. 99 Consequently, substrates can compete with cleaved bivalirudin for access to exosite 1.…”

Section: Bivalirudinmentioning

confidence: 99%

“…99,100 Without its aminoterminal segment, the carboxy-terminal portion of bivalirudin bound to thrombin's substrate recognition site is a much weaker thrombin inhibitor. 99 Consequently, substrates can compete with cleaved bivalirudin for access to exosite 1. Bivalirudin's plasma half-life after intravenous infusion is 25 minutes.…”

Section: Bivalirudinmentioning

confidence: 99%