Amino acid thrombin

Himdin [8001-27-2] is a polypeptide of 66 amino acids found ia the saUvary gland secretions of the leech Himdo medicinalis (45). It is a potent inhibitor of thrombin and biads to y-thrombia with a dissociation constant of 0.8 x 10 ° M to 2.0 x lO " M. Himdin forms a stable noncovalent complex with free and bound thrombin completely iadependent of AT-III. This material has now been cloned and expressed ia yeast cells (46,47). Its antigenic poteatial ia humans remains to be estabUshed. 

The serine proteases are the most extensively studied class of enzymes. These enzymes are characterized by the presence of a unique serine amino acid. Two major evolutionary families are presented in this class. The bacterial protease subtilisin and the trypsin family, which includes the enzymes trypsin, chymotrypsin, elastase as well as thrombin, plasmin, and others involved in a diverse range of cellular functions including digestion, blood clotting, hormone production, and complement activation. The trypsin family catalyzes the reaction ... 

Amino acid receptors Monoamine receptors Lipid receptors Purine receptors Neuropeptide receptors Peptide hormone receptors Chemokine receptors Glycoprotein receptors Protease receptors Metabotropic glutamate and GABAb receptors Adrenoceptors, dopamine and 5-HT receptors, muscarinic and histamine receptors Prostaglandin, thromboxane and PAF receptors Adenosine and ATP (P2Y) receptors Neuropeptide Y, opiate, cholecystokinin VIP, etc. Angiotensin, bradykinin, glucagon, calcitonin, parathyroid, etc. Interleukin-8 TSH, LH/FSH, chorionic gonadotropin, etc. Thrombin... 

The most potent thrombin inhibitor is hirudin, originally isolated from the salivary glands of the medicinal leech Hirudo medicinalis. Its inhibition constant is in the femtomolar (10-15 M) range (57). It is a 65-amino-acid tyrosine-sulfated single-chain polypeptide. Recombinant hirudin differs from native hirudin by the absence of the sulfate group on tyrosine 63 (Tyr-63) and is referred to as desulfato hirudin. The loss of this sulfate group reduces the thrombin inhibitory potency by 10-fold. 

Bivalent inhibitors of thrombin have been synthesized to bind the anion-binding exosite and active (catalytic) site of thrombin simultaneously. By coupling the carboxy terminal fragment of hirudin to a tripeptide (D-Phe-Pro-Arg) by including a spacer molecule, both the anion exosite and the catalytic site are blocked. An example of such a molecule is Hirulog, which has 20 amino acids and has a Kj of 2 nM (61). Its ability to block the active site has been questioned, since thrombin has been shown to cleave the Arg-Pro bond of Hirulog slowly in vivo (58). In addition to hirudin and hirudin-like compounds, three other classes of site-directed thrombin inhibitors deserve mention. 

Synthetic heterocyclic and modified amino acid derivatives have been grouped in a class of thrombin inhibitors called peptidomimetics. An example of such a compound is argatroban, with a molecular mass of 532 Da. It blocks thrombin s active catalytic site by binding to the adjacent apolar binding site. This selective reversible inhibitor of thrombin has a K of 19 nM and blocks thrombin s role in coagulation and fibrinolysis (62). 

Prothrombin (factor II) is a 582 amino acid, 72.5 kDa glycoprotein, which represents the circulating zymogen of thrombin (Ha). It contains up to six y-carboxyglutamate residues towards its N-terminal end, via which it binds several Ca2+ ions. Binding of Ca2+ facilitates prothrombin binding to factor Xa at the site of vascular injury. The factor Xa complex then proteolytically... 

Figure 12.5 Proteolytic cleavage of prothrombin by factor Xa, yielding active thrombin. Although prothrombin is a single-chain glycoprotein, thrombin consists of two polypeptides linked by what was originally the prothrombin intrachain disulfide bond. The smaller thrombin polypeptide fragment consists of 49 amino acid residues, and the large polypeptide chain contains 259 amino acids. The N-terminal fragment released from prothrombin contains 274 amino acid residues. Activation of prothrombin by Xa does not occur in free solution, but at the site of vascular damage...

Antithrombin, already mentioned in the context of heparin, is the most abundantly occurring natural inhibitor of coagulation. It is a single-chain 432 amino acid glycoprotein displaying four oligosaccharide side chains and an approximate molecular mass of 58 kDa. It is present in plasma at concentrations of 150 pig ml 1 and is a potent inhibitor of thrombin (factor Ha), as well as of factors IXa and Xa. It inhibits thrombin by binding directly to it in a 1 1 stoichiometric complex. 

Replacement of the p-aminoacyl moiety with an a-aminoacid derivative such as isoleucyl or cyclohexylglycyl led to a 2- to 4-fold decrease in potency. This was the first indication that SAR between this series and the a-amino acid series was distinct. Early on it was discovered that the "right hand side" amide could be replaced with an ester or acid moiety. This result led to a more systematic exploration of acid substitutions. Ortho-, meta- and para-substituted phenylacetic acid derivatives were prepared, and the latter analog (11, Figure 5) proved to be the first submicromolar inhibitor prepared in this series (IC50 = 510 nM). Grati-fyingly, 11 was devoid of thrombin inhibitory activity [31]. 

De Filippis, V., DeBoni, S., DeDea, E., Dalzoppo, D., Grandi, C., and Fontana, A. Incorporation of the fluorescent amino acid 7-azatryptophan into the core domain 1-47 of hirudin as a probe of hirudin folding and thrombin recognition, Protein Sci., 13 1489-1502,2004. 

Other non-covalent interactions such as C=0 F-C type, between a fluorine atom and the carbonyl of an amino acid, may take place for the stabilisation of enzyme-inhibitor supramolecular structures [28,30]. It is why the 4-fluorophenyl group is an important motif for binding pocket, as shown by the enhancement of one order of magnitude of the K by introducing one fluorine atom on thrombin inhibitor (Fig. 5) [30],... 

A. General description Lepirudin is a recombinant hirudin, a highly specific direct inhibitor of thrombin, derived from yeast cells. The polypeptide is composed of 65 amino acids and has a molecular weight of 7 kD. Natural hirudin is produced in trace amounts as a family of highly homologous isopolypeptides by the medicinal leech. Lepirudin is identical to natural hirudin except for the substitution of leucine for isoleucine at the N-terminal end... 

A. General description Bivalirudin, a 20 amino-acid synthetic peptide, is a direct thrombin inhibitor. It is an analogue of recombinant hirudin (Refludan), a 65 amino-acid anticoagulant derived from the leech. The molecular weight of bivalirudin is about 2.2kDa (anhydrous free base peptide). 

Examination of models of BPTI-mutants bound to Factor Xa show the L-amino acids in the P3 position project into solvent. In the Factor Xa cleavage sites in thrombin these residues are polar and acidic (Glu, Asp) they are polar and basic in antistasin (Arg), and polar and neutral in Ecotin (Ser). The exception is TFPI- II, with this position occupied by lie. The BPTI random mutant results are consistent with the TFPI-II case and show a preference for aliphatics or aromatics in this position. There is a hydrophobic pocket in the enzyme, formed by Trp215, Tyr"5 and Phe174, that would be accessible to a D-residue in this position. 

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