Fibrinogen thrombin activation

A second example of protease inhibitor design that properly illustrates the peptide scaffold-based approach is that of thrombin inhibitors. Work with these compounds led to the identification of highly potent, selective, and in vivo-effective lead compounds. A member of the serine protease family, thrombin cleaves a number of substrates (e.g., fibrinogen) and activates its platelet receptor (a G-protein-coupled receptor) by proteolysis of the extracellular N-terminal domain which results in self-activation (for a review see Reference 66). Initial lead inhibitors of thrombin were substrate-based, including the fibrinogen P3-Pi Phe-Pro-Arg sequence [67] and simple Arg derivatives such as Tos-Arg-OMe [68]. 

Studies of plasma clots made under a variety of conditions suggest that the network is established early as a result of the activation process (Blomback et al., 1994) and that the thrombin activation pathway modulates clot structure (Torbet, 1995). Light scattering studies, combined with other physical chemical techniques, have substantiated this conclusion for purified fibrinogen and provided more information about the structure and properties of the assembling clot (Bernocco et al., 2000 Ferri et al., 2001, 2002 Profumo et al., 2003). 

Thrombin activates platelets, converts fibrinogen to fibrin, activates factor XIII, which stabilizes fibrin, and activates factors V and VIE, which accelerate the generation of prothrombinase. Therefore, the inhibition of thrombin is essential in preventing and treating thromboembolic disorders. 

The presence of markers of thrombin generation, thrombin activity, and fibrin (fibrinogen) degradation indicates that... 

Irrespective of whether factor X is activated by extrinsic or intrinsic pathways, it catalyzes the activation of thrombin from prothrombin (Fig. 11.6c). Initially, activated factor X (factor Xa) produces small amounts of thrombin that cleave factor V, which is more sensitive to thrombin than even fibrinogen, thrombin s major substrate. Factor Va is a non-proteolytic cofactor accelerator. It binds to factor Xa and greatly increases the... 

Xa-factor Va (prothrombinase). Tenase activates factor X and pro-thrombinase converts prothrombin to thrombin. Thrombin activates platelets and catalyzes the conversion of fibrinogen to fibrin. Thrombin also amplifies coagulation by activating factors V, VIII, and XL... 

The choice of which enzyme to monitor is important. Some workers prefer to select Factor Xa because it does not react with fibrinogen or cause the platelet release reaction as thrombin does. Wahl and co-workers prefer to test for thrombin activity since they found S-2238 to be more sensitive than S-2222 (W6). This objection could be overcome by using a substrate (S-2337) which is claimed to be more sensitive for Factor Xa (S16). As of now, the synthetic substrate assay for PF4 is unlikely to replace the radioimmunoassay because of lower sensitivity. 

The covalent modification of human a-thrombin with pyridoxal 5 -phosphate involves phosphopyridoxylation at two sites.Attachment of the cofactor to the first site was accompanied by a loss in the fibrinogen clotting activity of the thrombin, whereas attachment of the cofactor to the second site resulted in a decrease in the sensitivity to heparin in the anti-(thrombin III)-thrombin reaction. 

S Thrombin Activation of Fibrinogen Provides for Hydrophobic Association to Form the Soft Fibrin Clot... 

Unfortunately, many investigations of the reaction between fibrinogen and thrombin have been carried out in whole plasma, with modification of the normal clotting process by the addition of heparin or oxalate, for example. The presence of such materials is an unnecessary complication, and in plasma it may be expected that other specific substances normally present, such as antithrombin, may also complicate the process. For unambiguous results, it is desirable to study the reaction with purified preparations of known fibrinogen content and thrombin activity, respectively, and in which any additional protein present is inert with respect to the clotting process. 

III. A degree of inhibition almost similar to that of heparin was shown by SCM-DAC-70 through its inhibition of fibrinogen transformation, and the hydrolytic activity for the synthetic substrate, the unit activity of SCM-DAC-70 would be one-third that of heparin for the inhibition of thrombin activity by the AT-III complex (Fig. 6). The maximum adsorption number of SCM-DAC-70 to AT-III was also confirmed by fluorescence titration [28]. The specificity of SCM-DAC-70 was investigated by applying other coagulation factors, and it had the highest affinity for AT-III. 

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