Inhibition of fibrin polymerization by fragment D is affected by calcium, Gly-Pro-Arg and Gly-His-Arg

Abstract

Fibrinopeptides A and B were removed from purified human fibrinogen by bovine thrombin, whereas the snake venom protease hatroxobin 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 Ca 2+. At a 4-fold molar concentration relative to fibrinogen, fragment D dramatically inhibited fibrin polymerization at up to 10 −4 M Ca 2+. This anticlotting activity was, however, much less pronounced at 10 −3 M Ca 2+. 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 Ca 2+, 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 α-chain, inhibited aggregation of both desA-fibrin and desAB-fibrin at 10 −3 M Ca 2+. The inhibition of desAB-fibrin polymerization by Gly-Pro-Arg was abolished at 10 −5 M Ca 2+. In addition, Gly-Pro-Arg depressed the anticlotting activity of fragment D at low calcium concentration. An analogue of the amino-terminus of fibrin β-chain, Gly-His-Arg, strongly accelerated aggregation of desA-fibrin monomers, but only moderately enhanced polymerization of desAB-fibrin monomers at 10 −5 M Ca 2+, both in the presence and in the absence of fragment D. This activating effect of Gly-His-Arg was abolished at 10 −3 M Ca 2+. 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.