Binding of alpha-thrombin to fibrin depends on the quality of the fibrin network.
Binding of human alpha-thrombin to fibrin was studied in a purified system at pH 7.35, I 0.08 and 37 degrees C. Binding experiments with active thrombin resulted in fibrin clots of variable quality, depending on the thrombin concentration: opaque gels composed of 'coarse' network were produced at low thrombin concentrations, while increasing concentrations of thrombin led to more translucent 'fine' gels. Scatchard analysis showed a non-linear dependence of thrombin binding to fibrin, suggesting the existence in fibrin(ogen) of multiple classes of binding sites for thrombin. Binding of catalytic-site-inhibited thrombin was investigated in clots of defined quality produced with three different concentrations of a thrombin-like enzyme, batroxobin (EC 3.4.21.29). Straight lines of different slopes were established by Scatchard analysis of binding data at each fixed batroxobin concentration. These results favour a model according to which binding affinity for thrombin depends on the thickness of fibrin bundles. Labelled active-site-inactivated thrombin incorporated in batroxobin-induced clots was only sparingly released during incubation for 24 h in the presence of a 200-fold excess of unlabelled thrombin, indicating that thrombin binding to fibrin is not reversible and that Scatchard analysis is not appropriate for quantification of binding parameters. Irreversible binding of thrombin appears to reflect trapping of thrombin molecules within fibrin fibres. The amount of trapped thrombin depends on the quality of the fibrin fibres, which in turn is determined by the concentration of the clotting enzyme.
Bänninger
H
H
Central Haematology Laboratory, The University, Inselspital, Bern, Switzerland.
Lämmle
B
B
Furlan
M
M
D000590:Amino Acid Chloromethyl Ketones; D000595:Amino Acid Sequence; D012103:Batroxobin / Q000494:pharmacology; D001665:Binding Sites; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D013917:Thrombin / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D007457:Iodine Radioisotopes; D005337:Fibrin; D012103:Batroxobin; D013917:Thrombin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone