Isolation and autolysis of human meizothrombin in the presence of dansylarginine-N-(3 -ethyl-1,5-pentanediyl)amide

Abstract

Human meizothrombin, a transient intermediate in the activation of prothrombin to thrombin, was isolated in the presence of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA), an active site inhibitor of thrombin. Meizothrombin autolysis to meizothrombin autolysis to meizothrombin des fragment 1 (cleavage at Arg273- Thr274) at different DAPA concentrations was monitoed by gel electrophoresis or by the changed fluorescence intensity of the DAPA-protein complex. Meizothrombin autolysis could be described by apparent pseudo-first order kinetics and was not eliminated even in the presence of a 100-fold molar excess of DAPA. By fitting the autolysis rates observed at different DAPA concentrations to a simple inhibition model, the rate of uninhibited autolysis at zero DAPA concentration, 22 h-1, and the dissociation constant of the DAPA/meizothrombin complex, 5.9 x 10(-8) M, were obtained. The uninhibited autolysis rate so obtained was consistent with an estimate (19 h-1) obtained from gel filtration chromatography experiments. A surprising finding was that the data could not be described adequately without inclusion of a DAPA-insensitive, or background, rate of autolysis. We conclude that the active site of human meizothrombin can never be completely blocked and autolysis can never be completely prevented even in the presence of saturating concentrations of the active site inhibitor DAPA.