Regulation Of Fibrin Formation

B Blombäck, B Kudryk, M Okada, S Söderman, L Therkildsen
1981 Thrombosis and Haemostasis  
Nossel et al. have shown that fibrin formation in vivo may be regulated by release of the Bβ 1-42 fragment from fibrinogen by plasmin. We have studied the effect of trypsin on the fibrin formation in vitro. Fibrinogen solutions (1 g/1) in Tris-imidazole buffer, pH 7.4, were incubated with trypsin (1:2,000, w/w) for 1-10 min. After incubation soybean trypsin inhibitor was added and clottable fibrinogen recovered by adding thrombin. Non-clottable fibrinogen in the supernatant was recovered by
more » ... as recovered by adding equal volume of ethanol. SDS-PAGE, before and after reduction, was performed on the above protein fractions as well as N-terminal analysis on N-DSK obtained from them by treatment with CNBr. Clot-supernatants were used in radioimmunoassay (RIA) for estimation of the following fragments: FPA, FPB, Bβ 15-42 and Hi2-DSK (peptide from COOH-terminal part of α achain). It was found that during incubation the fibrinogen progressively lost its coagulability. The clottable fraction appeared to have the same SDS-PAGE patterns as for intact fibrinogen. The non-clottable fraction showed progressive diminution of intact A α-chains but no degradation products could be observed. The B γ-chain on SDS-PAGE appeared to be shifted towards the Y-chain. The RIA assays showed that concomitant with loss of coagulability there was release of peptides containing Bβ 15-42. Release of FPA and FPB occurred during tryptic digestion but no clotting occurred. Only small release of Hi2-DSK immunoreactivity occurred. NH2-terminal analysis of N-DSK suggested that Bβ 42-43, Bβ 54-55 and a third, as yet unidentified bond, had been split by trypsin. These results suggest that tryptic cleavages in the Bβ and Aα chains destroy the polymerization sites which are unfolded in the molecule by release of FPA and FPB. Similar studies employing plasmin are now in progress.
doi:10.1055/s-0038-1652442 fatcat:2u3t2f5xivhpjoik3ttheddmoa