Kinetics of conversion of prothrombin to thrombin by biological activators. I. Demonstration of a prothrombin derivative which reacts with proteolytic enzyme inhibitors.

Purified prothrombin can be converted into thrombin in different ways (2), namely, by the use of biological activators, by the action of high concentrations of sodium citrate, and by treatment with trypsin. Biological activation of prothrombin requires lipoprotein tissue-factor(s) that have prothrombin-converting or thromboplastic activity, protein serum-factor(s) which augment thromboplastic activity, and ionic calcium. Thrombin formed in the presence of these quasi-physiological activators has been referred to as “biothrombin.” Most experimental evidence indicates that biothrombin is formed enaymatically from prothrombin; but the evidence has been interpreted variously as indicating that the enzymatic process is autocatalytic (3a), first order with respect to prothrombin (4), or involves a more complex sequence of reactions (2, 5). Prothrombin conversion in citrate (6) resembles autocatalysis in that the time course of thrombin formation is a sigmoid curve and preformed thrombin must be added to initiate the reaction (5, 7, 8). The process of thrombin formation in citrate may involve extensive degradation or dissociation of prothrombin, for before thrombin formation, much of the carbohydrate and nitrogen content of the original prothrombin preparation becomes nonprecipitable in trichloroacetic acid; and prothrombin initially present becomes refractory to biological activators (9). “Citrate thrombin,” which subsequently forms, has a lower molecular weight than prothrombin (4). These observations have led to the conclusion that one or more prothrombin derivatives may form before formation of “citrate thrombin.” However, recent studies by Alexander (10) and Goldstein et al. (11) indicate that purified prothrombin that can be converted to thrombin by citrate is contaminated with other clotting factors, and therefore the significance of initial changes in physical properties of prothrombin preparations exposed to citrate is not clear. Tryptic digestion of prothrombin results in release of large amounts of acid soluble material and formation of thrombin which has a lower molecular weight than prothrombin (4). It appears that “trypsin-thrombin” is an incidental consequence of nonspecific proteolysis. There is little, if any, change in the physical properties of prothrombin when “biothrombin” forms; for acid soluble material is not released in significant amounts (2) and “biothrombin” ap-