ABSTRACT
A novel assay for factor XIII is described that utilizes exclusively small synthetic peptides as substrates for the cross-linking reaction catalyzed by activated factor XIII (FXIIIa). The acyl donor substrate (selection peptide) is immobilized on a microplate via biotin while the acyl acceptor substrate (detection peptide) is labeled with the fluorochrome Oregon green to allow sensitive detection without the need for secondary enzyme systems for signal amplification. Starting with an amino acid sequence from the fibrin gamma-chain (GQQHHLGGAKQAGDV) as a prototype peptide, the influence of amino acid exchanges were investigated with respect to their impact on the FXIIIa-catalyzed reaction. It was found that FXIIIa readily accepts a broad range of substrate peptides, with a proline neighboring the essential lysine having the most detrimental effect. The assay appears to be valuable for the molecular characterization of factor XIII and may be used for a deeper investigation into the substrate requirements of this final enzyme of wound repair, and eventually also for the characterization of other transglutaminases.
Subject(s)
Cross-Linking Reagents/chemistry , Factor XIII/analysis , Factor XIII/chemistry , Peptides/chemistry , Cross-Linking Reagents/chemical synthesis , Ethylmaleimide/pharmacology , Factor XIII/antagonists & inhibitors , Humans , Peptides/chemical synthesis , Sensitivity and Specificity , Substrate SpecificityABSTRACT
Prothrombin complex concentrates (PCC) were compared in an in vitro test system for thrombogenicity (thrombin generation assay) employing plasma from coumarin-treated patients. Among these concentrates one had a proven history of thrombogenicity, whereas the remainder did not cause such fatal casualties in the past. Investigations into the thrombogenic component were performed by spiking experiments in which we biased a typical PCC without reported thromboembolic complications into one with a performance in the thrombin generation assay like that with a proven history of thrombogenicity. Hereby, it was possible to identify prothrombin as the most plausible thrombogenic component. Additional experiments performed with anticoagulant components (antithrombin together with heparin) resulted in a perfect reversal of the observed in vitro thrombogenicity. Our in vitro observations corroborate on an experimental basis the widespread medicinal usage of antithrombin administration as a regimen for the avoidance of thromboembolic complications during treatment with PCC and related products, and vice versa. Our observation casts doubts upon the widely accepted idea of activated factor IX as the thromboembolic agent in PCC. Also, our finding may be taken as an example for the feasibility of this test system as an in vitro model for thrombogenicity.
Subject(s)
Blood Coagulation Factors/chemistry , Prothrombin/chemistry , Thrombin/chemistry , Blood Coagulation Factors/adverse effects , Blood Coagulation Factors/therapeutic use , Factor IXa/chemistry , Hemophilia B/therapy , Humans , Plasma/chemistryABSTRACT
A novel assay for the determination of factor VIII (FVIII) is described. The assay uses a fluorescence-based detection system comparable with the common chromogenic test. At the same time, the assay is analogous to the clotting test as it does not involve pre-activation of FVIII. The assay was adjusted to perform equally well with patient's plasma and FVIII concentrates as samples. The combined employment of two different FVIII-deficient plasmas turned out to be of crucial importance in order to render the matrices as similar as possible to patient's plasma and, simultaneously, to obtain maximum sensitivity. Samples with a FVIII content down to 0.01 IU/ml are readily measured as are samples with a FVIII content of 1 IU/ml or somewhere in between. Upon dilution of samples, concentrates and plasma exhibited the same dose-response characteristics.
