CHARACTERIZATION OF A STABLE FORM OF HUMAN MEIZOTHROMBIN DERIVED FROMRECOMBINANT PROTHROMBIN (R155A, R271A, AND R284A)

Meizothrombin is a transient intermediate produced during the activati on of prothrombin by the prothrombinase complex. Because meizothrombin is very sensitive to further activation and autolysis, its isolation is possible only in the presence of active site thrombin inhibitors. T his complicates studies of the activities and functions of meizothromb in. As a model, we have expressed a mutant human prothrombin cDNA (R15 5A, R271A, R284A) with three of the cleavage sites modified so that th ey are no longer cleaved by factor Xa or thrombin. Several stable baby hamster kidney cell lines were isolated that secreted up to 20 mug/ml of carboxylated mutant prothrombin. After purification, the mutant pr othrombin was activated by the prothrombinase complex or by ecarin, re sulting in the formation of a meizothrombin-like molecule. Electrophor etic analysis and NH2-terminal sequence analysis were consistent with cleavage of a single bond between Arg320-Ile321 and proper processing of the prepropeptide. The meizothrombin was stable for weeks at 4-degr ees-C. Activation in the presence of dansylarginine N-(3-ethyl-1,5-pen tanediyl) amide confirmed the conversion of prothrombin via meizothrom bin. Compared with human plasma-derived thrombin, recombinant meizothr ombin demonstrated approximately 7% clotting activity, 100% p-toluene- sulfonylarginine methyl ester esterase activity, and approximately 35% S2238 amidolytic activity, and could attenuate fibrinolysis.