Thrombin generation assays

Thrombin generation assays (TGA) first evolved from standard clotting tests such as the PT, aPTT and TT, as they are essentially measuring the formation of thrombin through the formation of a fibrin clot. With the advancement of colorimetric assays and thrombin-specific peptide substrates, thrombin could be measured directly, eliminating interference from biological variables that often affect clot-based assays. In addition, clot-based assays are only truly reflective of the first burst of thrombin, whereas a measurement of the amount of thrombin is a better marker of clotting function. 

As with the case of prothrombin, Factor X can be activated by both physiological and nonphysiological activators. For substrate assays, both types of activators have been employed. An example of the former is the use of thromboplastin (A6, V5) an example of the latter is the employment of Russell s viper venom (RW) in the presence of calcium ions (Al, B7, V4). In general, the latter activator appears to be favored because it does not require phospholipids for activation hence thrombin generation is retarded (A6, Dl), and decarboxylated Factor X is not activated by RW. The most frequently used substrate has been S-2222, but S-2337, which has a lower K, may become more widely used (A8, 816). 

There are many other coagulation tests described in the literature which investigators have used to evaluate the lipid materials. These include the whole blood clotting time in glass or silicone-treated tubes, the thrombin generation test, and the prothrombin consumption test using clotted blood or platelet-free native plasma. In general they are modifications or variations of the basic principles involved in the assays mentioned above. 

Since both these proteins are cofactors and not proteases, the assay system for synthetic substrates involves a complicated method that requires the availability of purified coagulation factors. For Factor VIII, the assay is based on the rate of Factor X activation, and the conditions must be chosen so as to make the test proportional only to the Factor VIII, activity. In 1979, Segat-chian reported on a two-stage test method that utilized the chromogenic substrate S-2222 (S12). In this assay the inhibitor hirudin is added to block thrombin s activity so the generation of Factor Xa is the critical parameter. Recently Rosen has reported on a modified assay (R4). 

Figure 8.3 Sandwich assay developed for thrombin using two different aptamers, magnetic particles, and enzymatic amplification. Two selected aptamers binding thrombin in two different, nonoverlapping sites are used. The protein captured by the first aptamer fixed onto magnetic particles is detected after addition of the second biotinylated aptamer and of streptavidin labeled with an enzyme (alkaline phosphatase). Detection of the product generated by the enzymatic reaction is achieved by differential pulse voltammetry onto screen-printed electrodes onto which the magnetic nanoparticles are deposited and kept in contact through a magnet. [From (Centi et al., 2007b).]...

Weber et al. also applied a genetic algorithm to a modular reaction scheme, but optimized directly against an experimental enzyme assay as the fitness function, rather than a theoretical QSAR model. In 20 generations of parallel synthesis of 20 Ugi reactions, thrombin inhibition was increased from 1000 fiM to 0.22 iiM. 

Ikebukuro et al. [12] first reported an electrochemical aptasensor for the detection of thrombin based on a sandwich-based assay. Two different aptamers specific for thrombin were used the 29-mer thiolated aptamer and the 15-mer aptamer labeled with glucose dehydrogenase [GDH]. The thiolated aptamer was immobilized onto gold electrodes thrombin at different concentrations and then the enzyme-labeled aptamer was added to the aptamer-modified electrodes. The electric current generated by the addition of glucose was measured at 0.1 V vs. Ag/AgCl in a buffer containing... 

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