Inhibition of fibrin polymerization by fragment d is affected by calcium, Gly-Pro-Arg and Gly-His-Arg.
Fibrinopeptides A and B were removed from purified human fibrinogen by bovine thrombin, whereas the snake venom protease batroxobin only split fibrinopeptide A from fibrinogen. Aggregation of the resulting desAB- and desA-fibrin monomers was evaluated by recording the turbidity of incubation mixtures. Fibrin assembly was strongly accelerated by increasing the calcium concentration from 10(-5) to 10(-3) M. Fragment D was obtained from fibrinogen by proteolytic degradation with plasmin in the presence of Ca2+. At a 4-fold molar concentration relative to fibrinogen, fragment D dramatically inhibited fibrin polymerization at up to 10(-4) M Ca2+. This anticlotting activity was, however, much less pronounced at 10(-3) M Ca2+. The thrombin clotting time, measured on human plasma, was prolonged by fragment D in a dose-dependent manner. In citrate-containing plasma, the fibrinogen clotting was significantly delayed by an equimolar concentration of fragment D. In barium sulfate-adsorbed oxalated plasma, containing 2.5 mM Ca2+, the same amount of fragment D hardly affected fibrin polymerization. We conclude that fragment D has no important anticlotting effect under physiological conditions. The synthetic peptide Gly-Pro-Arg, corresponding to the amino-terminal sequence of the fibrin alpha-chain, inhibited aggregation of both desA-fibrin and desAB-fibrin at 10(-3) M Ca2+. The inhibition of desAB-fibrin polymerization by Gly-Pro-Arg was abolished at 10(-5) M Ca2+. In addition, Gly-Pro-Arg depressed the anticlotting activity of fragment D at low calcium concentration. An analogue of the amino-terminus of fibrin beta-chain, Gly-His-Arg, strongly accelerated aggregation of desA-fibrin monomers, but only moderately enhanced polymerization of desAB-fibrin monomers at 10(-5) M Ca2+, both in the presence and in the absence of fragment D. This activating effect of Gly-His-Arg was abolished at 10(-3) M Ca2+. It is suggested that the binding of calcium, Gly-His-Arg, and possibly also Gly-Pro-Arg, induces a conformational change in fibrin monomers and thus accelerates the polymerization process.
D000818:Animals; D001777:Blood Coagulation; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D005337:Fibrin / Q000378:metabolism*; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D005344:Fibrinopeptide A / Q000302:isolation & purification; D005345:Fibrinopeptide B / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009842:Oligopeptides / Q000494:pharmacology*
D005338:Fibrin Fibrinogen Degradation Products; D046911:Macromolecular Substances; D009842:Oligopeptides; C036309:fibrin fragment D; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; C035024:glycyl-prolyl-arginine; C035023:glycyl-histidyl-arginine; D005337:Fibrin; D005340:Fibrinogen; D002118:Calcium