Thrombin preparation

Initially human thrombin obtained from the blood transfusion service was used. However, although immediate thrombosis of the aneurysms was observed, recurrence occurred at follow up CECT (Fig. 7.10). This is not surprising in patients with on going pancreatitis. Repeat thrombin injections were required - in one case on three separate occasions. Though eventually permanent occlusion was obtained and the patients are still alive and well, subsequent patients were treated with autologous thrombin prepared from 50 cc of their own venous blood. This was done to avoid potential anaphylaxis. 

Thrombin has been purified by chromatographic techniques on IRC 50 columns. The amino acid composition of some thrombin preparations has been studied. Thrombin prepared from citrate-activated type I prothrombin lacks tryptophan and half cystine whereas thrombin prepared from extrinsic activation of type I prothrombin contains the 19 amino acids found in prothrombin. One molecule of thrombin contains one isoleucine N-terminal and one aspartic C-terminal [9]. 

Considerable advance has recently been made in the purification of bovine thrombin with the demonstration by Rasmussen (1955) that most of the active component of commercial thrombin preparations can be separated from the inactive protein impurities by chromatography on columns of Amberlite IRC-50 resin. This enzyme can be inhibited with diisopropylfluorophosphate (DFP) (Gladner and Laki, 1956 Miller and Van Vunakis, 1956) and reactivated by nucleophilic reagents such as hydroxamic acid (Gladner and Laki, 1956). On the assumption that DFP and thrombin combine in a mole ratio of 1 1 a molecular weight of 13,700 can be calculated (Gladner el al., 1958). The DFP is bound to a seryl residue, the sequence around which bears remarkable similarities to those of other enzymes which combine with DFP, as shown in Table XIII. 

Thrombin (from bovine blood plasma) [9002-04-4] Mj 32,600 [EC 3.4.4.13]. Purified by chromatography on a DEAE-cellulose column, while eluting with O.IM NaCl, pH 7.0, followed by chromatography on Sephadex G-200. Final preparation was free from plasminogen and plasmin. [Yin and Wessler J Biol Chem 243 112 796S.]... 

Anticoagulants. Figure 3 Relative effects of UFH, LMWH, and fondaparinux on AT-mediated inhibition of factor Xa and thrombin (lla). Whereas UFH catalyzes inhibition of Xa and thrombin equally well, only LMWH chains of 18 saccharide units or longer catalyze thrombin inhibition thus, the anti-Xa/anti-lla ratio of LMWH preparations ranges from 2 1 to 4 1. In contrast, fondaparinux exclusively inhibits Xa. (Modified from [3], with permission from Chest.)... 

The marine natural product dynosin A (92) is a new member of the aerugi-nosin family and a novel inhibitor of thrombin and Factor Vila. In Hanessian s total synthesis of 92 [66], both the dihydroxyoctahydroindole 88 and the A3 pyrroline moiety 91 were prepared by RCM-based routes (Scheme 17). 

This is a dry sponge of human fibrin prepared by elotting a foam of human fibrinogen solution with human thrombin. It is then freeze-dried, cut into shapes and sterilized by dry heat at 130°C for 3 hours. Before use, it is saturated with thrombin solution. Blood coagulation occurs in contact with the thrombin in the interstices of the foam. 

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]. 

V Caciagli, F Cardinali, F Bonelli, P Lombardi. Large-scale production of peptides using the solid phase continuous flow method. Part 2 Preparative synthesis of a 26-mer peptide thrombin inhibitor. J Pept Sci 4, 327, 1998. 

Compared with monocyclic piperazine scaffolds, bicyclic piperazines often provide platforms with even more points of diversity compared to monocyclic piperazines. Condensed bicyclic piperazines enlarge the chemical space of piperazine scaffolds, as well as the family of target proteins. For example, bicyclic piperazinones were prepared and evaluated in vitro and in vivo as thrombin inhibitors [49]. The cocrystal structure of a bicyclic piperazinone inhibitor 154 with thrombin was shown in Fig. 9. So far, chemists have developed many MCR methods to synthesize a variety of bicyclic piperazine scaffolds. 

Most Ru-catalysed oxidations of secondary alcohol to ketone steps involve TPAP one that does not is a stage in the industrial preparation of the inhibitor thrombin, which used RuClj/aq. Na(Br03)/CH3CN [166],... 

In contrast direct-acting preparations have the ability to inhibit fibrin-bound thrombin and include hirudin derived from medicinal leeches but now produced by recombinant technology. Particular interest centres on ximelagatran which may be a possible... 

For the inhibition of AT Ill-mediated thrombin activity, derivatives of 8 were prepared containing a flexible spacer of around 50 atoms plus a sulfated maltooligosaccharide or another AT III binding pentasaccharide [35]. 

Bicyclic pyridones have been prepared (Scheme 73) and found to be potent orally bioavailable thrombin inhibitors <2000BML1069>. 

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