Summary
Human thrombin with high affinity for fibrin was obtained by subjecting purified thrombin to affinity chromatography on Sepharose insolubilized fibrin monomers, after addition of a radioiodinated subsample of thrombin, molar ratio 1:600. As judged by radioprofiling of the electrophoretic distribution of high-affinity thrombin on 10 per cent polyacrylamide gel containing urea/SDS, the preparation consisted of 70 per cent a-thrombin, 28 per cent β-thrombin and only 2 per cent γ-thrombin. Although a-thrombin was bound more strongly to insolubilized fibrin monomers than the other subfractions, complete separation of the individual components could not be achieved.
High-affinity thrombin was employed for studies on thrombin adsorption to polymerized fibrin, assuming equal behaviour of labelled and unlabelled thrombin.
o avoid passive entrapment of thrombin within the fibrin meshwork at physiological pH, ionic strength and calcium concentration, the optimal fibrinogen concentration was found to be 2.94 umol/1. During such conditions, adsorption of thrombin to polymerized fibrin did not exceed 65 per cent of added thrombin, despite an increasing availability of fibrin-related thrombin binding domains obtained by reducing the thrombin concentration.
Adsorption of thrombin to polymerized fibrin increased by 25 per cent when the ionic strength was reduced to 0.05 mol/1. These findings suggest the presence of thrombin subfractions with different affinities for polymerized fibrin.
Aggregates of high-affinity thrombin formed during its preparation by affinity chromatography, but were prevented by adding polyethylene glycol (m.w. 6,000, final conc. 6.6 g/1). Such aggregates were not inactivated by AT-III, but could still adsorb to polymerized fibrin.
In its monomeric form fibrin-adsorbed thrombin was inactivated by AT-III and could then be extruded from the clot upon syneresis and retraction, but a small fraction remained attached to polymerized fibrin, exhibiting minor amidolytic activity.
Keywords
Thrombin - Fibrin - Antithrombin III - Clot interactions
