ISOLATION AND SOME PROPERTIES OF THROMBIN-E AND OTHER PROTHROMBIN DERIVATIVES

Multiple forms of thrombin zymogen occurred as prothrombin complex, prothrombin, abnormal prothrombin, prethrombin and prethrombin-E. Prothrombin less the PR fragment represented prethrombin and was the stage beyond which degradation of prothrombin did not readily go with thrombin. By removing the O fragment from prethrombin, esterase activity developed, due to a structure called prethrombin-E. This enzyme was 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 formed and consisted of the A chain held to the B chain by a disulfide bond. It had esterase and proteolytic activity, and basically classical thrombin occurred only in this 1 form. Commonly, thrombin preparations consisted of thrombin and thrombin degraded to thrombin-E and B1 chain. During autolysis at 4.degree. C and pH 8.0, proteolytic activity of thrombin was lost, while the specific esterase activity increased. The B1 portion of B chain split off at an Arg-Lys bond. It precipitated in pure form, leaving in solution thrombin-E which had B2 chain attached to A chain by a disulfide bond and had only esterase activity. Breaking the disulfide bond of thrombin-E enabled the isolation of A and B2 chains. Either thrombin or autoprothrombin C removed the PR fragment from prothrombin. The R fragment was isolated and probably contained all the carbohydrate of prothrombin which was not associated with the B1 chain of thrombin. Thrombin-E was free of carbohydrate. The 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 was confirmed, namely, esterase activity .fwdarw. esterase + proteolytic activity .fwdarw. esterase activity. The respective associated structures were prethrombin-E .fwdarw. thrombin .fwdarw. thrombin-E, and in these the condition of B1 chain is as follows: bound, free at NH2-terminal end and absent. An antecedent in the form of prethrombin was easily obtained as a degradation product of prothrombin. The fragments aligned in prothrombin as follows: P + R + O + A chain + B1 chain + B2 chain. Digestion of prothrombin with thrombin stopped with PR removal which was the prethrombin stage, but continued if prothrombin was first denatured. Autolysis of thrombin stopped at the thrombin-E stage (A chain + B2 chain), but if thrombin-E was denatured, thrombin degraded it further. [Studies were carried out on purified bovine prothrombin complex.].