Generation of forms of fragment E with differing thrombin-binding properties during digestion of fibrinogen by plasmin.
Two principal forms of fragment E are generated upon digestion of fibrinogen by plasmin, according to the concentration of enzyme used. At a high concentration of plasmin (above 10 micrograms/ml), a form lacking fibrinopeptide A (FpA) at the N-terminus of the A alpha-chain was generated. This form of fragment E caused a dose-dependent increase in thrombin clotting times but had no measurable inhibitory activity towards thrombin cleavage of D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide. At a low concentration of plasmin (less than 1 microgram/ml), fragment E containing 35-40% of the original amount of FpA was present in the terminal digest. The FpA-containing form of fragment E inhibited thrombin cleavage of fibrinogen, inhibited amidolytic activity and bound to the enzyme with an affinity 3-fold tighter than fibrinogen itself (Kd 4.1 +/- 0.3 microM as opposed to 12.7 +/- 1.8 microM). During digestion of fibrinogen at low plasmin concentration, up to 65% of the FpA was cleaved just subsequent to the progressive release of B beta-(1-42)-peptide, and the Arg-16-Gly-17 bond of the A alpha-chain became relatively stable towards plasmin action when present in fragment E (and possibly fragment Y). It is proposed that both forms of fragment E can inhibit clotting by binding to the fibrin(ogen)-recognition site (anion-binding exosite) of thrombin. The FpA-containing form of fragment E can also inhibit binding that occurs distal to the P1 site and thereby interfere with amidolysis of the peptide substrate. Our finding of a lability of the Arg-16-Gly-17 bond in the early phase of digestion may provide an alternative explanation of the increased FpA concentrations observed during thrombolytic therapy.
Goodwin
C A
CA
Thrombosis Research Institute, Chelsea, London, U.K.
Kakkar
V V
VV
Scully
M F
MF
D001665:Binding Sites; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism* / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000378:metabolism; D006801:Humans; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005338:Fibrin Fibrinogen Degradation Products; C018543:fibrinogen fragment E; D005344:Fibrinopeptide A; D005340:Fibrinogen; D013917:Thrombin; D005341:Fibrinolysin