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Coagulation factor XIII (FXIII) is converted by thrombin and Ca 2 + into an active transglutaminase (FXIIIa) in the fi nal phase of coagulation cascade. Its main function is the mechanical stabilization of fi brin clot and its protection from fi brinolysis by cross-linking of fi brin chains and α 2 -plasmin inhibitor to fi brin. In non-substituted patients FXIII defi ciency is a severe hemorrhagic diathesis, not infrequently with fatal consequences. The main reason for using FXIII assays is the diagnosis of FXIII defi ciency. The aim of this review is to provide a comprehensive critical evaluation of the methods reported for the determination of FXIII activity in the plasma. Such methods are based on two principles: 1) measurement of labeled amines incorporated by FXIIIa into a glutamine residue of a substrate protein, 2) monitoring ammonia released from a peptide bound glutamine residue by FXIIIa using NAD(P)H dependent glutamate dehydrogenase indicator reaction. The incorporation assays are sensitive, but cumbersome and time-consuming, they are diffi cult to standardize and cannot be automated. The ammonia release assays are less sensitive, but quick, well standardized, and can be automated; this type of assay is recommended for the screening of FXIII defi ciency. The traditional clot solubility assay should not be used for this purpose.

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ABSTRACT:The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.