Thrombin activity assay

The oleosin fusion procedure was used for the purification of the commercially valuable plant-based blood anticoagulant hirudin in transgenic Brassica carinata and Brassica napus. Hirudin, a natural protein from the medicinal leech Hirudo medicinalis, is superior to other anticoagulants such as heparin. Recombinant hirudin was cleaved from oil-bodies using endoproteinase Factor Xa. Released hirudin was biologically active, as determined by a colorimetric thrombin inhibition assay. 

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. 

AMPK can also be activated by a Ca2+-mediated pathway involving phosphorylation at Thr-172 by the Ca2+/calmodulin-dependent protein kinase, CaMKK 3. CaMKKa and CaMKK 3 were discovered as the upstream kinase for the calmodulin-dependent protein kinases-1 and -IV they both activate AMPK in a Ca2+/ calmodulin-dependent manner in cell-free assays, although CaMKK 3 appears to much more active against AMPK in intact cells. Expression of CaMKKa and CaMKK(3 primarily occurs in neural tissues, but CaMKKp is also expressed in some other cell types. Thus, the Ca2+-mediated pathway for AMPK activation has now been shown to occur in response to depolarization in rat neuronal tissue, in response to thrombin (acting via a Gq-coupled receptor) in endothelial cells, and in response to activation of the T cell receptor in T cells. 

The concept that different structural domains on the heparin chains are principally involved for optimal activity in the foregoing interactions could not be perceived in early work on structure-activity correlations, because the activity of heparin has been most frequently evaluated only with whole-blood-clotting tests (such as the U.S.P. assay). Development of assays for specific clotting-factors (especially Factor Xa and thrombin) has permitted a better insight into the mechanism of action of heparin at different levels of the coagulation cascade. 

The appearance of anionic phospholipids, particularly phosphatidylserine, on the cell siuface activates prothrombinase complex culminating in the formation of thrombin (Bevers et al., 1982 Connor et al., 1989). The assay can be performed with pure coagulation proteins and specific chromogenic substtates to produce a very sensitive test to detect the appearance of phosphatidylserine on ceU siufaces. Nevertheless, it has been shown that changes in the disposition of phosphatidylethanolamine and sphingomyelin may interfere with the ability of phosphatidylserine-containing membranes to activate prothrombinase (Smeets et al., 1996). 

One molecule of lepirudin binds to one molecule of thrombin and thereby blocks the thrombogenic activity of thrombin. As a result, all thrombin-dependent coagulation assays are affected (eg, aPTT values increase in a dose-dependent fashion). Pharmacokinetics ... 

Fig. 11.6. Icmt inhibition impairs activation of RhoA and Racl GTPases and impacts on RhoA- and Racl-mediated cell migration. A, thrombin-mediated activation of RhoA. Cells were treated with thrombin (-f) or vehicle (—) for 15 min, whereupon levels of bound and total RhoA were determined. B, EGF-mediated activation of Racl. Cells were treated with EGF (-f) or vehicle (—) for 15 min, whereupon lysates were prepared and levels of bound and total Racl were determined. C and D, RhoA rescues directed migration. MDA-MB-231 cells were treated for 3 days with cysmethynil Cysmeth) or vehicle Cont) and recombinant adenovirus carrying HA-tagged RhoA or GFP was introduced as indicated. Cells were harvested and transwell migration assays were conducted. E and F, Racl rescues random migration. MDA-MB-231 cells were treated as described above, except that recombinant adenovirus carrying HA-tagged Racl or GFP was introduced. This figure is reproduced from Ref. [28].

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