Effect of Factor XIII on the fibrinogen and its degradation products:Differentiation of the origin of FDP by gel chromatography
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A method was described in which the origin of FDP could be differentiated by gel chromatography. In the preliminary experiment the stabilized fibrin was digested overnight at 37°C. The resulting fibrin DP was chromatographed on Sephadex G200 column (26×450mm) and the elution volume (Ve) of fibrin DP as well as E was measured by the use of single radial immunodiffusion or antibody-coated latex aggregation test. The Ve of fibrin DP-D was calculated as 98ml and that of E was 141ml. Fibrinogen (Fbg) was digested with plasmin and resulting Fbg DP was chromatographed on the same gel column. The Ve of Fbg DP-D was 128ml and that of E was 140ml. The difference of Ve between both FDPD was explained as the dimer composition of fibrin DP-D of stabilized fibrin wheras the monomer form of fibrinogen DP.The urine containing 10γ/ml of FDP of a case of renal failure was concentrated 20 folds and chromatographed on Biogel A-5m column, which showed that the most of the FDP was early one, as the Ve was slightly larger than that of fibrinogen. The concentrated urine was digested with plasmin and applied on Sephadex G200 column. The chromatography revealed that the resulted FDP-D was monomer, which indicated the FDP in the urine was not originated from stabilized fibrin, but from fibrinogen or non-stabilized fibrin.The factor was examined which interfering the result of this differentiation method. Monodansylcadaverine (MDC) incorporation with F. XIII at various digestive stages of Fbg or fibrin DP was examined basing on the method by Lorand and Urayama. It was found that the early Fbg DP had a slight capability of MDC incorporation while none of fibrin DP. This indicated that the possible crosslinkage of early Fbg DP with F. XIII and if this occurred, the resulting FDP-D could be interpreted as originated from already stabilizedfibrin by the differentiation method.A mixture of purified fibrinogen, plasminogen and urokinase was kept at 37°C and aliquots were taken at different incubation time, then thrombin-calcium was added to each aliquot. SDS-disc electrophoressis after the reduction with DTT of each aliquot revealed that, although Fbg DP-X could be crosslinked with F. XIII and formed subunit-gamma dimer, but most of it, at the presence of fibrinogen, could be incorporated into fibrin by the defibrination procedure. With these result, the interference on the differentiation method by the crosslinkage of Fbg DP was regarded as minimal.
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