The Fragment 1 Region of Prothrombin Facilitates the Favored Binding of Fragment 12 to Zymogen and Enforces Zymogen-like Character in the Proteinase

Abstract

Thrombin is produced from the C-terminal half of prothrombin following its proteolytic activation. The N-terminal half, released as the propiece Fragment 12 (F12), is composed of an N-terminal ␥-carboxyglutamate domain (Gla) followed by two kringles (K1 and K2). The propiece plays essential roles in regulating prothrombin activation and proteinase function. The latter results from the ability of F12 to reversibly bind to the (pro)catalytic domain through K2 with high affinity and highly favorable thermodynamic constants when it is a zymogen in comparison to proteinase. Such discrimination is lost for K2 binding after proteolytic removal of the N-terminal Gla-K1 region of F12. The Ca 2؉ -stabilized structure of the Gla domain is not required for F12 to bind the zymogen form more favorably. Enhanced binding to zymogen versus proteinase correlates with the ability of the propiece to enforce zymogen-like character in the proteinase. This is evident in variants of meizothrombin, an intermediate of prothrombin activation that contains the propiece covalently attached. This phenomenon is also independent of the Gla domain. Thus, the presence of K1 in covalent linkage with K2 in the propiece governs the ability of K2 to bind the (pro)catalytic domain in favor of zymogen, thereby enforcing zymogen-like character in the proteinase.Thrombin, a trypsin-like serine proteinase, plays a preeminent role in hemostasis and the prevention of blood loss (1, 2). It activates platelets and cleaves soluble fibrinogen to form insoluble fibrin, both essential components of the blood clot (3, 4). It acts as a feedback activator of steps in the coagulation cascade to enhance flux toward its own formation (3, 4). When bound to the cofactor, thrombomodulin on the endothelium, it catalyzes the activation of protein C and initiates the anticoagulant reactions responsible for decreasing flux through the coagulation cascade (5). Thus, thrombin functions as a central regulator of coagulation that derives from its ability to act on multiple biological substrates (6).Two anion binding exosites (ABE1 2 and ABE2) play important roles in the binding of substrates and ligands to thrombin (7,8). The role played by ABE1 in ligating a range of thrombin substrates and ligands has been established by numerous biochemical and structural studies (6, 7). In contrast, far fewer ligands have been identified for ABE2, found approximately on the opposite face of the proteinase domain from ABE1 (6). Although ABE2 was originally defined on the basis of heparin binding, the propiece of the zymogen precursor that is cleaved away when thrombin is produced represents an important protein ligand for this site (9, 10).Prothrombin, the zymogen precursor, requires cleavage at two sites to be converted to thrombin (Scheme 1). Cleavage at Arg 271 releases the N-terminal propiece (F12), whereas cleavage at Arg 320 is analogous to the cleavage at Arg 15 in chymotrypsinogen and yields the proteinase. Thrombin is composed of the catalytic domain in disulfide linkage with a short...

Exosite 2-Directed Ligands Attenuate Protein C Activation by the Thrombin–Thrombomodulin Complex

“…52 Another situation in which the interaction between exosite 1 and 2 may be significant is in prothrombin activation. Prothrombin fragment 1.2 binds prethrombin 2 with high affinity and modulates exosite 1, such that it could influence binding of factor Va. 60,61 This suggests that the connection between exosites 1 and 2 may play a role in prothrombin activation.…”

Biophysical exploration of conformational environments in zymogen prothrombin and blood coagulant thrombin.

“…Physiologically, GpIb bound to thrombin ABE II helps in activating the ABE I directed ligand PAR1 80. In addition, allowing zymogen fragment F2 to bind to thrombin ABE II reduces the conversion of fibrinogen to fibrin 167. The first direct evidence for direct allosteric communication between the exosites was reported by Weitz et al using F2 as the ABE II directed ligand and Hirudin as the ABE…”