Isolation and some properties of thrombin-E and other prothrombin derivatives

Abstract

Multiple forms of thrombin zymogen occur as prothombin complex, prothrombin, abnormal prothrombin, prethrombin, and prethrombin-E. Prothrombin less PR fragment represents prethrombin and is the stage beyond which degradation of prothrombin does not readily go with thrombin. By removing O fragment from prethrombin, esterase activity develops, due to a structure called prethrombin-E. This enzyme is a single chain structure with molecular weight, amino acid composition and carbohydrate content the same as thrombin. By breaking an Arg-Ile bond in prethrombin-E with autoprothrombin C, thrombin forms and consists of the A chain held to the B chain by a disulfide bond. It has esterase and proteolytic activity, and basically classical thrombin occurs only in this one form. Commonly, thrombin preparations consist of thrombin and thrombin degraded to thrombin-E and B1 chain. During autolysis at 4° C and pH 8.0, proteolytic activity of thrombin is lost while the specific esterase activity increases. The B1 portion of B chain splits off at an Arg-Lys bond. It precipitates in pure form leaving in solution thrombin-E which has B2 chain attached to A chain by a disulfide bond, and has only esterase activity. Breaking the disulfide bond of thrombin-E enables the isolation of A and B2 chains. Either thrombin or autoprothrombin C can remove PR fragment from prothrombin. R fragment was also isolated and probably contains all the carbohydrate of prothrombin which is not associated with B1 chain of thrombin. Thrombin-E is free of carbohydrate. O fragment retarded fibrin formation by thrombin. It enhanced the esterase activity of thrombin. An unidentified procoagulant, probably a form of autoprothrombin C, was closely associated with O fragment, but was removed. The prothrombin activation sequence described by Seegers and Landaburu in 1957 has been confirmed; namely, esterase activity → esterase + proteolytic activity → esterase activity. The respective associated structures are prethrombin-E → thrombin → thrombin-E; and in these, the condition of B1 chain is as follows: bound, free at NH 2-terminal end, and absent. In addition, an antecedent in the form of prethrombin is easily obtained as a degradation product of prothrombin. The fragments align in prothrombin as follows: P + R + O + A chain + B1 chain + B2 chain. Digestion of prothrombin with thrombin stops with PR removal which is the prethrombin stage, but continues if prothrombin is first denatured. Likewise, autolysis of thrombin stops at the thrombin-E stage (A chain + B2 chain), but if thrombin-E is denatured, thrombin can degrade it further.