Thrombin

Thrombin is a serine protease in the blood coagulation cascade. The enzyme liberates fibrinopeptide A and B from fibrinogen by selectively cleaving two Arg-Gly bonds. Fibrin, the remaining portion of fibrinogen, polymerizes, forming a clot. This key role for thrombin makes inhibition of the enzyme an important target for cardiovascular research. 

The authors used the information gleaned from these studies to design the closely related chloromethyl ketone inhibitor of thrombin FPAM (68). Molecular modeling of this proposed inhibitor docked in the thrombin active site indicated that interactions similar to those important to fibrinopeptide binding could be obtained. Subsequent X-ray crystal structure analysis of the 68-thrombin complex was supportive of the peptidomimetic hypothesis, 

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Thrombin DRWVLTAAHC LLYPPWDKNF TVDDLLVRIG KHSRTRYERK VEKISMLDKI... 

Thrombin YIHPRYNWKE irLDRDIALLK LKRPIELSDY IHPVCLPDKQ TAAKLLHAGF... 

Thrombin KGRVTGWGNR RETWTTSVAE VQPSVLQWN LPLVERPVCK ASTRIRITDN... 

Thrombin 14FCAGYKPGE GKRGDACEGD SGCPF /MKEP YNNRWYQMGI VSWGEGCDRD... 

Fig. 10.12 Sequence alignment of trypsin, chymotrypsin and thrombin (bovine). The active sites histidine, aspartic acid and serine are highlighted.

D, H W Hoeffken, D Crosse, J Stuerzebecher, P D Martin, B F P Edwards and W Bode 1992. Refined 2.3 Angstroms X-Ray Crystal Structure of Bovine Thrombin Complexes Formed witli he 3 Benzamidine and Arginine-Based Thrombin Inhibitors NAPAP, 4-TAPAP and MQPA A Starting Point for Improving Antithrombotics. Journal of Molecular Biology 226 1085-1099. 

D, J Sturzebecher and WBode 1991. Geometry of Binding of the N-Alpha-Tosylated Piperidides of weffl-Amidino-Phenylalanine, Para Amidino-Phenylalanine and para-Guanidino-Phenylalanine to Thrombin and Trypsin - X-ray Crystal Structures of Their Trypsin Complexes and Modeling of their Thrombin Complexes. FEBS Letters 287 133-138. 

Jones-Hertzog D K and W L Jorgensen 1997. Binding Affinities for Sulphonamide Inhibitors witl Human Thrombin Using Monte Carlo Simulations with a Linear Response Method. Journal o Medicinal Chemistry 40 1539-1549. 

Cell—Cell Interactions. Older generations of leukocyte filters depended partly on the formation of platelet—leukocyte—thrombin formations. It is not clear whether this mechanism plays a role in third-generation filters. 

By similar logic, protein affinity Hbraries have been constmcted to identify protein—protein combining sites, as in antibody—antigen interaction (19) and recombinant Hbraries have been made which produce a repertoire of antibodies in E. coli (20). In another case, a potential DNA-based therapeutic strategy has been studied (21). DNAs from a partially randomized Hbrary were selected to bind thrombin in vitro. Oligonucleotides, termed aptamers that bound thrombin shared a conserved sequence 14—17 nucleotides long. 

Dismption of the endothehal surface of blood vessels expose coUagen fibers and connective tissue. These provide surfaces that promote platelet adherence, platelet release reaction, and subsequent platelet aggregation. Substances Hberated from the platelets stimulate further platelet aggregation, eg, adenosine diphosphate maintain vasoconstriction, eg, serotonin and participate in blood coagulation, eg, platelet Factors III and IV. In addition, the release reaction modifies platelet membranes in a manner that renders phosphoHpid available for coagulation. The thrombin [9002-04-4] elaborated by the coagulation mechanism is a potent agent in the induction of the platelet release reaction. 

Thrombin, the two-chain derivative of the prothrombin molecule, has a molecular weight of approximately 37,000 daltons. Its proteolytic properties induce the conversion of fibrinogen to fibrin to produce the initial visible manifestation of coagulation, the soluble fibrin clot. In addition, thrombin influences the activity of Factors V, VIII, and XIII and plasmin. Thrombin affects platelet function by inducing viscous metamorphosis and the release reaction with subsequent aggregation. 

Coagulation Factors II, III, VII, IX, X, XI, and Xlla fragments, thrombin, and plasmin are classified as serine proteases because each possesses a serine residue with neighboring histidine and asparagine residues at its enzymatically active site (Table 3). Factors II, VII, IX, and X, Protein C, Protein S, and Protein Z are dependent on the presence of vitamin K [84-80-0] for their formation as biologically functionally active procoagulant glycoproteins. 

Factor II. Prothrombin is a vitamin K-dependent compound synthesized by the Hver. When prothrombin is activated it is cleaved at two sites, resulting in a two-chain molecule linked by a disulfide bond that has a molecular weight of 37,000 daltons. Thrombin is the serine protease that initiates the conversion of soluble fibrinogen into fibrin. 

Factor V. High in sialic acid content. Factor V is a large asymmetric single-chain glycoprotein that becomes an active participant in the coagulation cascade when it is converted to its active form by a-thrombin. Approximately 25% of human Factor V is found in the whole blood associated with platelets. Factor V is an essential cofactor along with Factor Xa plus phosphohpid plus Ca " in the conversion of prothrombin to thrombin. 

Factor XIII. Factor XIII circulates in the blood as a zymogen composed of two pairs of different polypeptide chains designated A and B. Inert Factor XIII has a molecular weight of 350,000 daltons and is converted to its active transglutaminase form in the presence of thrombin and calcium. Activated Factor XIII, Xllla, induces an irreversible amide exchange reaction between the y-glutamine and S-lysine side chains of adjacent fibrin... 

Protein G. This vitamin K-dependent glycoproteia serine protease zymogen is produced ia the Hver. It is an anticoagulant with species specificity (19—21). Proteia C is activated to Proteia by thrombomodulin, a proteia that resides on the surface of endothefial cells, plus thrombin ia the presence of calcium. In its active form, Proteia selectively iaactivates, by proteolytic degradation. Factors V, Va, VIII, and Villa. In this reaction the efficiency of Proteia is enhanced by complex formation with free Proteia S. la additioa, Proteia activates tissue plasminogen activator, which... 

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. 

Argatroban [74863-84-6] ((2R,4R)-4-methyl-l-[A/ -)(3 methyl l,2,3,4-tetrahydto-8-quiaoIiaesulfonyl)-L-atgiayl]-2-piperidiaecatboxyhc acid monohydrate) is a potent inhibitor of thrombin formation and activity (49). This agent has been studied in vitro and ia a few animal models. Its toxicity and activity ia humans ate unknown. 

Calcium is essential to several steps in the enzyme cascade of the blood clotting process, such as the conversion of prothrombin to thrombin (23). Clotting can be inhibited in stored blood suppHes by addition of complexing agents such as EDTA or citrate which reduce the levels of the free ion, Ca(Il). 

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