Hirudin Fragments and Analogs

Definition
Hirudin is a thrombin-specific inhibitor isolated from the leech Hirudo medicinalis

Hirudin analogs
CX-397 is novel recombinant hirudin analog, composed of the N-terminal half of HV-1 (HV-11-36) combined with the C-terminal half of HV-3 (HV-337-66). Bivalirudin, a synthetic analog of the carboxy-terminus of hirudin, is a reversible thrombin inhibitor.

Structural Characteristics
The thrombin-specific inhibitor hirudin is a polypeptide of 65 or 66 amino acid residues isolated from the leech Hirudo medicinalis. Sequence analysis of different naturally occurring forms of hirudin revealed homologies 80%. The 6 cysteine residues of three disulfide bonds connecting Cys6with Cys14, Cys16 with Cys28, and Cys22 with Cys39 has also been detected. Hirudin consists of an amino-terminal compact domain (residues l-49) held together by the three disulfide linkages and a distorted carboxyl-terminal tail (residues 50- 65) which does not fold back on the rest of the protein. Hirudin reacts rapidly with a-thrombin forming tight, noncovalent 1. The specificity of the interaction of a-thrombin with macromolecular substrates is assumed to reside in interactions at three distinct regions: the primary binding pocket for the P1 residue, an apolar binding site adjacent to the catalytic site, and an anion-binding region which may be responsible for the specific interaction of thrombin with fibrinogen2.

Mode of Action
The hirudin acts as a potent anticoagulant by binding to thrombin with high specificity and affinity. Hirudin is distinguished from conventional protease inhibitors by two unique characteristics. (a) Unlike most protease inhibitors which contain a well-defined reactive site (P1 residue) that interacts with the active site of the target enzyme1. The structural element of hirudin which blocks the active site of thrombin is an extended hydrophobic site including the Hz-terminal residues (Val1, Val2, and Tyr3)3. (b) Unlike most serine protease inhibitors which are compact molecules, hirudin has a tadpole-like shape, consisting of a compact amino-terminal domain and a disordered carboxyl-terminal tail4.

Functions
CX-397, a novel recombinant hirudin analog - A study was conducted using four hirudin analogs in thrombin inhibition, which included CX-397, CX-397R, hirudin variants-1 (HV-11-36) and -3 (HV-337-366).Their anti-thrombin activity was determined by a fluorogenic enzyme assay and compared with that of recombinant HV-1 (rHV-1) and rHV-3. The order of the magnitude of dissociation constants (Ki) of these four hirudin analogs in thrombin inhibition was as follows: CX-397R (0.294 pM) > rHV-1 (0.148 pM) > rHV-3 (0.0593 pM) > CX-397 (0.0433 pM), indicating that CX-397 is the strongest inhibitor among them5.
Reduction of adhesion formation by intraperitoneal administration of a recombinant Hirudin analog - Adhesion formation is a major source of postoperative morbidity and mortality. Therefore, the reduction of postoperative adhesion formation would be of clinical benefit. Various modalities have been shown to reduce adhesion formation, including fibrinolytic enzymes, nonsteroidal anti-inflammatory drugs, and barriers that reduce the apposition of sites of potential adhesion formation. A study was conducted to examine the ability of an inhibitor of thrombin, a recombinant hirudin analog (recHirudin), to reduce the formation of intraperitoneal adhesions in two rabbit models of adhesion formation. In the sidewall and double uterine horn models, recHirudin was administered via Alzet miniosmotic pump for the entire postoperative interval. In both of these models, there was a dose-dependent reduction in adhesion formation as measured by (1) the area of the sidewall injury that was involved in adhesions to the cecum and the bowel or (2) the involvement of the uterine horns to themselves or other peritoneal organs. This implies that postoperative administration of recHirudin to the site of injury reduced the formation of postoperative adhesions in two animal models6.
Influence of bivalirudin on tissue factor-triggered coagulation - Bivalirudin, a synthetic analog of the carboxy-terminus of hirudin, is a reversible thrombin inhibitor. A study was conducted using three in-vitro models (numerical simulations, synthetic coagulation proteome and whole blood) of contact pathway-independent blood coagulation triggered with tissue factor. It was found that increasing concentrations of bivalirudin prolong the initiation phase of thrombin generation in a concentration-dependent manner. At subpharmacologic bivalirudin concentrations (0.5-2 micromol/l), total thrombin generation was significantly increased. At a pharmacologic concentration (5 micromol/l), bivalirudin suppressed thrombin generation in the synthetic coagulation proteome; in numerical simulations and contact pathway-inhibited whole blood, no thrombin generation was detected over 1200-2000 s and platelet activation was inhibited by 80%. The addition of a pharmacologic concentration (9 micromol/l) of a naturally occurring protease inhibitor aprotinin in the presence of at least 0.5 micromol/l bivalirudin provided limited enhancement of the bivalirudin inhibitory effect. This implied that bivalirudin at pharmacologic concentrations is an efficient inhibitor of thrombin generation, platelet activation and clot formation, which acts not as a modulator but as an 'on-off' switch of blood coagulation7.

References
1.Dodt J, Seemüller U, Maschler R, Fritz H (1985). The complete covalent structure of hirudin: Localization of the disulfide bonds. Biol. Chem. Hoppe. Seyler., 366(4), 379-85.
2.John W. Fenton , Frederick A. Ofosu, Diane V. Brezniak, Houria I. Hassouna (1998).Thrombin and Antithrombotics. Semin. Thromb. Hemost., 24, 87-91.
3.TJ Rydel, KG Ravichandran, A Tulinsky, W Bode, R Huber, C Roitsch, and JW Fenton 2nd (1990). The structure of a complex of recombinant hirudin and human alpha-thrombin. Science., 249 (4966), 277-280.
4.Chang JY (1983).The functional domain of hirudin, a thrombin-specific inhibitor. FEBS Lett., 164(2), 307-13.
5.Komatsu Y, Misawa S, Sukesada A, Ohba Y, Hayashi H (1993). CX-397, a novel recombinant hirudin analog having a hybrid sequence of hirudin variants-1 and -3. Biochem. Biophys. Res. Commun., 196(2), 773-9.
6.Rodgers KE, Girgis W, Campeau JD, diZerega GS (1996). Reduction of adhesion formation by intraperitoneal administration of a recombinant Hirudin analog. J. Invest. Surg ., (5), 385-91.
7.Butenas S, Orfeo T, Brummel-Ziedins KE, Mann KG. 2007 Influence of bivalirudin on tissue factor-triggered coagulation. Blood Coagul Fibrinolysis., 18(5), 407-14.