Meizothrombin Is Unexpectedly Zymogen-Like: Its Slow Conversion to Proteinase Dominates Thrombin Production by Prothrombinase
Authors: Bradford Harlan , Krishnaswamy Sriram 
Published: October 2019

Abstract
Abstract 2215
The proteolytic activation of prothrombin catalyzed by prothrombinase is a paradigm for zymogen activation resulting from ordered cleavage at multiple sites. Initial cleavage at Arg320 forms meizothrombin (mIIa). The associated conversion of zymogen to proteinase is instrumental in facilitating further processing at Arg271 to yield thrombin. A full kinetic explanation for this process remains obscure and controversial because of the limitations of standard kinetic approaches. We now uncover novel facets of this reaction by rapid kinetic studies to approximate single catalytic turnover. Product formation was measured continuously by stopped flow using Dansyl arginine (3-ethyl-1,5-pentanediyl) amide (DAPA) or discontinuously using rapid quenching and analysis by SDS-PAGE or peptidyl substrate cleavage following rapid mixing of preformed prothrombinase (0.3 μM) with prothrombin (0.3 μM). Prothrombin cleavage, assessed discontinuously, was essentially complete within 0.2 seconds. The results indicated initial cleavage at Arg320 to form mIIa followed by subsequent cleavage at Arg271 to produce thrombin. However, product formation measured continuously with DAPA, yielded a pronounced lag and proceeded ∼20-30-fold more slowly. The intermediate, mIIa, which is expected to bind DAPA was invisible to the continuous measurements. Analysis was further simplified using a recombinant prothrombin variant, which is exclusively cleaved at Arg320 to produce mIIa and not processed further. Continuous detection of proteinase formation by stopped flow proceeded ∼30-fold more slowly than cleavage at Arg320 measured discontinuously. These findings indicate that rapid cleavage at the Arg320 site is followed by an unexpectedly slow reaction (t½ ∼ 0.5–1 s) in which the cleaved product matures to form a competent active site. This conclusion was further tested employing stable mIIa prepared by the action of Ecarin on recombinant prothrombin variants that were not degraded further even without occluding the active site. Stopped flow kinetic studies for the binding of DAPA to these variants revealed markedly biphasic traces. The data could be globally analyzed according to a two step mechanism with an initial slow equilibrium between zymogen-like and proteinase forms in which only the proteinase form can bind the active site ligand. The zymogen- like and proteinase forms were approximately equally populated and interconverted slowly (t½ ∼ 0.5 s). These findings independently corroborate the conclusions from the kinetic studies of prothrombin cleavage. Accordingly, inclusion of this slow step could explain profiles of prothrombin depletion, transient formation of mIIa and the final appearance of thrombin seen in the action of prothrombinase on prothrombin. Zymogen-like mIIa accumulates at much higher concentrations than would be predicted from knowledge of the kinetics of the individual cleavage steps because its slow maturation to proteinase is required for further cleavage at Arg271. Thus, the rate-limiting maturation of mIIa to proteinase plays a dominant role in regulating thrombin formation. Furthermore, while mIIa is a poor catalyst for many of substrates of thrombin, it retains the ability to bind thrombomodulin and function in the anticoagulant pathway. As a result of its unexpectedly zymogen-like character and slow conversion to proteinase, mIIa produced as an intermediate by prothrombinase would be resistant to inhibition in plasma and thereby potentially be dispersed by flowing blood to exert its selectively anticoagulant functions distant from its site of production.

Disclosures:
No relevant conflicts of interest to declare.