Meizothrombin: Zymogen or Proteinase? Slow, Ligand-Dependent Equilibration Between Equally Populated Zymogen-Like and Proteinase-Like Forms Explains Its Selectively Anticoagulant Function
Authors: Bradford Harlan , Krishnaswamy Sriram 
Published: October 2019

Abstract
Abstract 533
Prothrombinase activates prothrombin by catalyzing its ordered proteolysis at Arg320 followed by cleavage at Arg271. Initial cleavage at Arg320 yields the proteinase meizothrombin (mIIa), which accumulates abundantly as an intermediate before its conversion to thrombin (IIa). Although mIIa is a proteinase, it only acts on a limited subset of substrates cleaved by IIa. mIIa is considered an anticoagulant proteinase because it functions efficiently in protein C activation while exhibiting poor clotting activity or reactivity towards antithrombin III. This limited substrate repertoire of mIIa has remained enigmatic and is generally considered to lie in the retention of covalent linkage to the fragment 1.2 (F12) domain allowing for membrane binding. Instead, our recent findings with IIa, illustrating its ligand dependent interconversions between zymogen-like and proteinase-like states, predict that covalent linkage of F12 to the proteinase domain in mIIa would impart it with zymogen-like properties. We produced stable and active mIIa (mIIaQQQ) using a recombinant prothrombin variant in which the bonds susceptible to autolysis were rendered uncleavable by substitution of Arg at 155, 271 and 284 with Gln. Fluorescence stopped flow studies were pursued with the probes dansyl arginine 3-ethyl piperidine amide (DAPA) or Nα-dansyl-(p-guanidino)-L-phenylalanine-piperidide (I-2581) to characterize the binding of ligands to the active site of mIIaQQQ and IIa. Binding to IIa was rapid and consistent with a rate limiting, bimolecular interaction between probe and the active site of the proteinase. In contrast, traces with mIIaQQQ were distinctly biphasic with ∼50% of the fluorescence change occurring on the millisecond timescale followed by a slow second phase (∼50%) that occurred over several seconds. Global fitting indicated that the findings were consistent with a pre-equilibrium between two forms of mIIa, one which binds the active site probe with μM affinity and a second that binds with nM affinity. The two forms interconvert with forward and reverse rate constants of ∼2 s−1. We surmise that these reflect zymogen-like and proteinase-like forms that are equally populated and interconvert slowly with each other in a ligand-dependent fashion. Accordingly, the distributions of the two forms could be altered by ligands established to affect the transition of IIa between zymogen-like and proteinase-like states. The equilibrium distribution was altered to favor the zymogen-like form by decreasing Na+ to 0 at constant ionic strength. In contrast, soluble thrombomodulin (sTM) drove the equilibrium towards the proteinase-like state in a manner consistent with a 1:1 interaction between mIIaQQQ and sTM. Surprisingly, the pre-equilibrium was heavily dependent on covalent linkage with fragment 1 (F1) or its structural integrity. Proteolytic removal of F1, chelation of Ca2+ with EDTA or elimination of 4-carboxyglutamic acid modifications had a profound effect on forcing the enzyme into the proteinase-like state. Thus, the equilibrium distribution of mIIa between zymogen-like and proteinase-like forms is affected by F1 and its Ca2+-stabilized conformation despite the fact that this domain is expected to be distant from the catalytic site. Our findings shed unexpected light into the mechanisms underlying the peculiar activity profile of mIIa relative to IIa. Its ability to interconvert slowly and reversibly between equally populated zymogen-like and proteinase-like states lies at the heart of its properties. By driving it to proteinase, thrombomodulin imparts full activity to mIIa allowing for efficient function in the anticoagulant pathway. In contrast, more weakly binding substrates, inhibitors or ligands will be less effective at perturbing the equilibrium thereby allowing mIIa to persist in blood with reduced activity towards procoagulant substrates. The F1 domain participates in an unexpected way in enforcing these unique features of mIIa. By virtue of its essential role in modulating the equilibrium distribution between zymogen-like and proteinase-like states, we document a new function for F1 in its role as a zymogenizer of mIIa.

Disclosures:
No relevant conflicts of interest to declare.